AU2015201796B2 - Rationally designed, synthetic antibody libraries and uses therefor - Google Patents

Abstract

Procedure to overcome the inadequacies inherent in the known methods for generating libraries of antibody-encoding polynucleotides by specifically designing the libraries with directed sequence and length diversity. The libraries are designed to reflect the preimmune repertoire naturally created by the human immune system and are based on rational design informed by examination of publicly available databases of human antibody sequences.

Description

RATIONALLY DESIGNED. SYNTHETIC ANTIBODY LIBRARIES AND USES THEREFOR

RELATED APPLICATIONS

This application claims priority to U.S. provisional application serial number 60/993,785, filed on September 14, 2007, incorporated herein in its entirety by this reference.

This application is a divisional of Australian Patent Application No. 2008298603, the entire contents of which are herein incorporated by reference.

BACKGROUND OF THE INVENTION

Antibodies have profound relevance as research tools and in diagnostic and therapeutic applications. However, the identification of useful antibodies is difficult and once identified, antibodies often require considerable redesign or 'humanization' before they are suitable for therapeutic applications.

Previous methods for identifying desirable antibodies have typically involved phage display of representative antibodies, for example human libraries derived by amplification of nucleic acids from B cells or tissues, or, alternatively, synthetic libraries. However, these approaches have limitations. For example, most human libraries known in the art contain only the antibody sequence diversity that can be experimentally captured or cloned from the source (e.g., B cells). Accordingly, the human library may completely lack or under-represent certain useful antibody sequences. Synthetic or consensus libraries known in the art have other limitations, such as the potential to encode non-naturally occurring (e.g., non-human) sequences that have the potential to be immunogenic. Moreover, certain synthetic libraries of the art suffer from at least one of two limitations: (1) the number of members that the library can theoretically contain (/.<?., theoretical diversity) may be greater than the number of members that can actually be synthesized, and (2) the number of members actually synthesized may be so great as to preclude screening of each member in a physical realization of the library, thereby decreasing the probability that a library member with a particular property may be isolated.

For example, a physical realization of a library (e.g., yeast display, phage display, ribosomal display, etc.) capable of screening 1012 library members will only sample about 10% of the sequences contained in a library with 1013 members. Given a median CDRH3 length of about 12.7 amino acids (Rock et al., J. Exp. Med., 1994, - 1 - 179:323-328), the number of theoretical sequence variants in CDRH3 alone is about 2012'7, or about 3.3 x 1016 variants. This number does not account for known variation that occurs in CDRH1 and CDRH2, heavy chain framework regions, and pairing with different light chains, each of which also exhibit variation in their respective CDRL1, 2015201796 09 Apr 2015 5 CDRL2, and CDRL3. Finally, the antibodies isolated from these libraries are often not amenable to rational affinity maturation techniques to improve the binding of the candidate molecule.

Accordingly, a need exists for smaller (i.e., able to be synthesized and physically realizable) antibody libraries with directed diversity that systematically represent 10 candidate antibodies that are non-immunogenic (i.e., more human) and have desired properties (e.g., the ability to recognize a broad variety of antigens). However, obtaining such libraries requires balancing the competing objectives of restricting the sequence diversity represented in the library (to enable synthesis and physical realization, potentially with oversampling, while limiting the introduction of non-human 15 sequences) while maintaining a level of diversity sufficient to recognize a broad variety of antigens. Prior to the instant invention, it was known in the art that “[although libraries containing heavy chain CDR3 length diversity have been reported, it is impossible to synthesize DNA encoding both the sequence and the length diversity found in natural heavy chain CDR3 repertoires” (Hoet et al., Nat. Biotechnol., 2005, 23: 20 344, incorporated by reference in its entirety).

Therefore, it would be desirable to have antibody libraries which (a) can be readily synthesized, (b) can be physically realized and, in certain cases, oversampled, (c) contain sufficient diversity to recognize all antigens recognized by the preimmune human repertoire (i.e., before negative selection), (d) are non-immunogenic in humans 25 (i.e., comprise sequences of human origin), and (e) contain CDR length and sequence diversity, and framework diversity, representative of naturally-occurring human antibodies. Embodiments of the instant invention at least provide, for the first time, antibody libraries that have these desirable features.

30 SUMMARY OF THE INVENTION

The present invention is relates to, at least, synthetic polynucleotide libraries, methods of producing and using the libraries of the invention, kits and computer readable forms including the libraries of the invention. In some embodiments, the -2- libraries of the invention are designed to reflect the preimmune repertoire naturally created by the human immune system and are based on rational design informed by examination of publicly available databases of human antibody sequences. It will be appreciated that certain non-limiting embodiments of the invention are described below. 2015201796 09 Apr 2015 5 As described throughout the specification, the invention encompasses many other embodiments as well.

In certain embodiments, the invention comprises a library of synthetic polynucleotides, wherein said polynucleotides encode at least 106 unique antibody CDRH3 amino acid sequences comprising: 10 20 25 (i) an N1 amino acid sequence of 0 to about 3 amino acids, wherein each amino acid of the N1 amino acid sequence is among the 12 most frequently occurring amino acids at the corresponding position inNl amino acid sequences of CDRH3 amino acid sequences that are functionally expressed by human B cells; (ii) a human CDRH3 DH amino acid sequence, N- and C-terminal truncations thereof, or a sequence of at least about 80% identity to any of them; (iii) an N2 amino acid sequence of 0 to about 3 amino acids, wherein each amino acid of the N2 amino acid sequence is among the 12 most frequently occurring amino acids at the corresponding position in N2 amino acid sequences of CDRH3 amino acid sequences that are functionally expressed by human B cells; and (iv) a human CDRH3 H3-JH amino acid sequence, N-terminal truncations thereof, or a sequence of at least about 80% identity to any of them.

In other embodiments, the invention comprises a library of synthetic polynucleotides, wherein said polynucleotides encode at least about 106 unique antibody CDRH3 amino acid sequences comprising: (i) an N1 amino acid sequence of 0 to about 3 amino acids, wherein: -3 - 2015201796 09 Apr 2015 5 10 15 20 25 (a) the most N-terminal N1 amino acid, if present, is selected from a group consisting of R, G, P, L, S, A, Y, K, I, Q, T and D; (b) the second most N-terminal N1 amino acid, if present, is selected from a group consisting of G, P, R, S, L, V, E, A, D, I, T and K; and (c) the third most N-terminal N1 amino acid, if present, is selected from the group consisting of G, R, P, S, L, A, V, T, E, D, K and F; (ii) a human CDRH3 DH amino acid sequence, N- and C-terminal truncations thereof, or a sequence of at least about 80% identity to any of them; (iii) an N2 amino acid sequence of 0 to about 3 amino acids, wherein: (a) the most N-terminal N2 amino acid, if present, is selected from a group consisting of G, P, R, L, S, A, T, V, E, D, F and H; (b) the second most N-terminal N2 amino acid, if present, is selected from a group consisting of G, P, R, S, T, L, A, V, E, Y, D and K; and (c) the third most N-terminal N2 amino acid, if present, is selected from the group consisting of G, P, S, R, L, A, T, V, D, E, W and Q; and (iv) a human CDRH3 H3-JH amino acid sequence, N-terminal truncations thereof, or a sequence of at least about 80% identity to any of them.

In still other embodiments, the invention comprises a library of synthetic polynucleotides, wherein said polynucleotides encode at least about 106 unique antibody CDRH3 amino acid sequences that are at least about 80% identical to an amino acid sequence represented by the following formula: 30 [X]-[N1]-[DH]-[N2]-[H3-JH], wherein: -4- (i) X is any amino acid residue or no amino acid residue; (ii) N1 is an amino acid sequence selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV, D, E, F, Η, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, WGT, and combinations thereof; (iii) DH is an amino acid sequence selected from the group consisting of all possible reading frames that do not include a stop codon encoded by IGHD1-1, IGHD1-20, IGHD1-26, IGHD1-7, IGHD2-15, IGHD2-2, IGHD2-21, IGHD2-8, IGHD3-10, IGHD3-16, IGHD3-22, IGHD3-3, IGHD3-9, IGHD4-17, IGHD4-23, IGHD4-4, IGHD-4-11, IGHD5-12, IGHD5-24, IGHD5-5, IGHD-5-18, IGHD6-13, IGHD6-19, IGHD6-25, IGHD6-6, and IGHD7-27, and N- and C-terminal truncations thereof; (iv) N2 is an amino acid sequence selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV, D, E, F, Η, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, WGT, and combinations thereof; and -5 - (v) H3-JH is an amino acid sequence selected from the group consisting of AEYFQH, EYFQH, YFQH, FQH, QH, H, YWYFDL, WYFDL, YFDL, FDL, DL, L, AFDV, FDV, DV, V, YFDY, FDY, DY, Y, NWFDS, WFDS, FDS, DS, S, 2015201796 09 Apr 2015 5 YYYYYGMDV, YYYYGMDV, YYYGMDV, YYGMDV, YGMDV, GMDV, and MDV, or a sequence of at least 80% identity to any of them.

In still another embodiment, the invention comprises wherein said library consists essentially of a plurality of polynucleotides encoding CDRH3 amino acid 10 sequences that are at least about 80% identical to an amino acid sequence represented by the following formula: [X]-[N 1 ]-[DH]-[N2]-[H3-JH], wherein: (i) X is any amino acid residue or no amino acid residue; 15 20 25 30 (ii) N1 is an amino acid sequence selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, YP, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV, D, E, F, Η, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, WGT, and combinations thereof; (iii) DH is an amino acid sequence selected from the group consisting of all possible reading frames that do not include a stop codon encoded by IGHD1-1, IGHD1-20, IGHD1-26, IGHD1-7, IGHD2-15, IGHD2-2, IGHD2-21, IGHD2-8, IGHD3-10, IGHD3-16, IGHD3-22, IGHD3-3, IGHD3-9, IGHD4-17, IGHD4-23, IGHD4-4, IGHD-4-11, IGHD5-12, IGHD5-24, IGHD5-5, IGHD-5-18, -6- IGHD6-13, IGHD6-19, IGHD6-25, IGHD6-6, and IGHD7-27, and N- and C-terminal truncations thereof; 2015201796 09 Apr 2015 (iv) N2 is an amino acid sequence selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, 5 PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV, D, E, F, Η, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, 10 LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, WGT, and combinations thereof; and 15 (v) H3-JH is an amino acid sequence selected from the group consisting of AEYFQH, EYFQH, YFQH, FQH, QH, H, YWYFDL, WYFDL, YFDL, FDL, DL, L, AFDV, FDV, DV, V, YFDY, FDY, DY, Y, NWFDS, WFDS, FDS, DS, S, YYYYYGMDV, YYYYGMDV, YYYGMDV, YYGMDV, 20 YGMDV, GMDV, and MDV, or a sequence of at least 80% identity to any of them.

In another embodiment, the invention comprises a library of synthetic polynucleotides, wherein said polynucleotides encode one or more full length antibody heavy chain sequences, and wherein the CDRH3 amino acid sequences of the heavy 25 chain comprise: (i) an N1 amino acid sequence of 0 to about 3 amino acids, wherein each amino acid of the N1 amino acid sequence is among the 12 most frequently occurring amino acids at the corresponding position inNl amino acid sequences of CDRH3 amino acid 30 sequences that are functionally expressed by human B cells; -7- (ii) a human CDRH3 DH amino acid sequence, N- and C-terminal truncations thereof, or a sequence of at least about 80% identity to any of them; 2015201796 09 Apr 2015 5 (iii) an N2 amino acid sequence of 0 to about 3 amino acids, wherein each amino acid of the N2 amino acid sequence is among the 12 most frequently occurring amino acids at the corresponding position in N2 amino acid sequences of CDRH3 amino acid sequences that are functionally expressed by human B cells; and (iv) a human CDRH3 H3-JH amino acid sequence, N-terminal truncations thereof, or a sequence of at least about 80% identity to any of them.

The following embodiments may be applied throughout the embodiments of the instant invention. In one aspect, one or more CDRH3 amino acid sequences further comprise an N-terminal tail residue. In still another aspect, the N-terminal tail residue is 15 selected from the group consisting of G, D, and E.

In yet another aspect, the N1 amino acid sequence is selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, 20 GGP, GGR, GGA, GGS, GGL, GGT, GGV, D, E, F, Η, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, WGT, and 25 combinations thereof. In certain other aspects, the N1 amino acid sequence may be of about 0 to about 5 amino acids.

In yet another aspect, the N2 amino acid sequence is selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP, GGG, GPG, 30 GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV, D, E, F, Η, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, -8- QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, WGT, and combinations thereof. In certain other aspects, the N2 sequence may be of about 0 to 5 about 5 amino acids. 2015201796 09 Apr 2015

In yet another aspect, the H3-JH amino acid sequence is selected from the group consisting of AEYFQH, EYFQH, YFQH, FQH, QH, H, YWYFDL, WYFDL, YFDL, FDL, DL, L, AFDV, FDV, DV, V, YFDY, FDY, DY, Y, NWFDS, WFDS, FDS, DS, S, YYYYYGMDV, YYYYGMDV, YYYGMDV, YYGMDV, YGMDV, GMDV, and 10 MDV.

In other embodiments, the invention comprises a library of synthetic polynucleotides encoding a plurality of antibody CDRH3 amino acid sequences, wherein the percent occurrence within the central loop of the CDRH3 amino acid sequences of at least one of the following i - i+1 pairs in the library is within the ranges specified below: 15 Tyr-Tyr in an amount from about 2.5% to about 6.5%;

Ser-Gly in an amount from about 2.5% to about 4.5%;

Ser-Ser in an amount from about 2% to about 4%;

Gly-Ser in an amount from about 1.5% to about 4%;

Tyr-Ser in an amount from about 0.75% to about 2%; 20 Tyr-Gly in an amount from about 0.75% to about 2%; and

Ser-Tyr in an amount from about 0.75% to about 2%.

In still other embodiments, the invention comprises a library of synthetic polynucleotides encoding a plurality of antibody CDRH3 amino acid sequences, wherein the percent occurrence within the central loop of the CDRH3 amino acid sequences of at 25 least one of the following i - i+2 pairs in the library is within the ranges specified below:

Tyr-Tyr in an amount from about 2.5% to about 4.5%;

Gly-Tyr in an amount from about 2.5% to about 5.5%;

Ser-Tyr in an amount from about 2% to about 4%;

Tyr-Ser in an amount from about 1.75% to about 3.75%; -9-

Ser-Gly in an amount from about 2% to about 3.5%; 2015201796 09 Apr 2015

Ser-Ser in an amount from about 1.5% to about 3%;

Gly-Ser in an amount from about 1.5% to about 3%; and

Tyr-Gly in an amount from about 1% to about 2%. 5 In another embodiment, the invention comprises a library of synthetic polynucleotides encoding a plurality of antibody CDRH3 amino acid sequences, wherein the percent occurrence within the central loop of the CDRH3 amino acid sequences of at least one of the following i - i+3 pairs in the library is within the ranges specified below:

Gly-Tyr in an amount from about 2.5% to about 6.5%; 10 Ser-Tyr in an amount from about 1% to about 5%;

Tyr-Scr in an amount from about 2% to about 4%;

Ser-Ser in an amount from about 1% to about 3%;

Gly-Ser in an amount from about 2% to about 5%; and

Tyr-Tyr in an amount from about 0.75% to about 2%. 15 In one aspect of the invention, at least 2, 3, 4, 5, 6, or 7 of the specified i - i+1 pairs in the library are within the specified ranges. In another aspect, the CDRH3 amino acid sequences are human. In yet another aspect, the polynucleotides encode at least about 106 unique CDRH3 amino acid sequences.

In other aspects of the invention, the polynucleotides further encode one or more 20 heavy chain chassis amino acid sequences that are N-terminal to the CDRH3 amino acid sequences, and the one or more heavy chain chassis sequences are selected from the group consisting of about Kabat amino acid 1 to about Kabat amino acid 94 encoded by IGHV1-2, IGHV1-3, IGHV1-8, IGHV1-18, IGHV1-24, IGHV1-45, IGHV1-46, IGHV1-58, IGHV1-69, IGHV2-5, IGHV2-26, IGHV2-70, IGHV3-7, IGHV3-9, 25 IGHV3-11, IGHV3-13, IGHV3-15, IGHV3-20, IGHV3-21, IGHV3-23, IGHV3-30, IGHV3-33, IGHV3-43, IGHV3-48, IGHV3-49, IGHV3-53, IGHV3-64, IGHV3-66, IGHV3-72, IGHV3-73, IGHV3-74, IGHV4-4, IGHV4-28, IGHV4-31, IGHV4-34, IGHV4-39, IGHV4-59, IGHV4-61, IGHV4-B, IGHV5-51, IGHV6-1, and IGHV7-4-1, or a sequence of at least about 80% identity to any of them. - 10-

In another aspect, the polynucleotides further encode one or more FRM4 amino acid sequences that are C-terminal to the CDRH3 amino acid sequences, wherein the one or more FRM4 amino acid sequences are selected from the group consisting of a FRM4 amino acid sequence encoded by IGHJ1, IGHJ2, IGHJ3, IGHJ4, IGHJ5, and 5 IGHJ6, or a sequence of at least about 80% identity to any of them. In still another aspect, the polynucleotides further encode one or more immunoglobulin heavy chain constant region amino acid sequences that are C-terminal to the FRM4 sequence. 2015201796 09 Apr 2015

In yet another aspect, the CDRH3 amino acid sequences are expressed as part of full-length heavy chains. In other aspects, the full-length heavy chains are selected from 10 the group consisting of an IgGl, IgG2, IgG3, and IgG4, or combinations thereof. In one embodiment, the CDRH3 amino acid sequences are from about 2 to about 30, from about 8 to about 19, or from about 10 to about 18 amino acid residues in length. In other aspects, the synthetic polynucleotides of the library encode from about 106 to about 1014, from about 107 to about 1013, from about 108 to about 1012, from about 109 to about 1012, 15 or from about 1010 to about 1012 unique CDRH3 amino acid sequences.

In certain embodiments, the invention comprises a library of synthetic polynucleotides, wherein said polynucleotides encode a plurality of antibody VKCDR3 amino acid sequences comprising about 1 to about 10 of the amino acids found at Rabat positions 89, 90, 91, 92, 93, 94, 95, 95A, 96, and 97, in selected VKCDR3 amino acid 20 sequences derived from a particular IGKV or IGKJ germline sequence.

In one aspect, the synthetic polynucleotides encode one or more of the amino acid sequences listed in Table 33 or a sequence at least about 80% identical to any of them.

In some embodiments, the invention comprises a library of synthetic 25 polynucleotides, wherein said polynucleotides encode a plurality of unique antibody VKCDR3 amino acid sequences that are of at least about 80% identity to an amino acid sequence represented by the following formula: [VKChassis] - [L3 - VK]- [X]- [ JR* ], wherein: (i) VK Chassis is an amino acid sequence selected from the group 30 consisting of about Kabat amino acid 1 to about Rabat amino acid 88 encoded by IGKV1-05, IGKV1-06, IGKV1-08, IGKV1-09, IGKV1-12, IGKV1-13, IGKV1-16, IGKV1-17, IGKV1-27, - 11 - IGKV1-33, IGKV1-37, IGKV1-39, IGKV1D-16, IGKV1D-17, IGKV1D-43, IGKV1D-8, IGKV2-24, IGKV2-28, IGKV2-29, IGKV2-30, IGKV2-40, IGKV2D-26, IGKV2D-29, IGKV2D-30, IGKV3-11, IGKV3-15, IGKV3-20, IGKV3D-07, IGKV3D-11, IGKV3D-20, IGKV4-1, IGKV5-2, IGKV6-21, and IGKV6D-41, or a sequence of at least about 80% identity to any of them; 2015201796 09 Apr 2015 5 (ii) L3-VK is the portion of the VKCDR3 encoded by the IGKV gene segment; and (iii) X is any amino acid residue; and (iv) JK* is an amino acid sequence selected from the group consisting of sequences encoded by IGJK1, IGJK2, IGJK3, IGJK4, and IGJK5, wherein the first residue of each IGJK sequence is not present.

In still other aspects, X may be selected from the group consisting of F, L, I, R, 15 W,Y, andP.

In certain embodiments, the invention comprises a library of synthetic polynucleotides, wherein said polynucleotides encode a plurality of VXCDR3 amino acid sequences that are of at least about 80% identity to an amino acid sequence represented by the following formula: 20 [VX_Chassis]-[L3-\^.]-[JX], wherein: (i) VX Chassis is an amino acid sequence selected from the group consisting of about Kabat amino acid 1 to about Kabat amino acid 88 encoded by IGXVl-36, IGXVl-40, Ιϋλνΐ-44, Ιϋλνΐ-47, IGXV1-51, Κ3λν 10-54, IGXV2-11, IGXV2-14, IGXV2-18, 25 IGXV2-23, IGXV2-8, IGXV3-1, IGXV3-10, IGXV3-12, IGXV3- 16, IGXV3-19, IGXV3-21, IGXV3-25, IGXV3-27, IGXV3-9, IGXV4-3, Κ3λν4-60, Ιϋλν4-69, IGXV5-39, IGXV5-45, IGXV6-57, IGXV7-43, IGlV7-46, IGXV8-61, IGXV9-49, and IGXVIO-54, or a sequence of at least about 80% identity to any of them; - 12- (ii) L3-VX is the portion of the VXCDR3 encoded by the IGXV segment; and 2015201796 09 Apr 2015 (iii) JX is an amino acid sequence selected from the group consisting of sequences encoded by IG)J 1-01, IG?J2-01, IGXJ3-01, IG/J3- 5 02, IG/J6-01, IG/J7-01, and IG/J7-02, and wherein the first residue of each IG-Ιλ sequence may or may not be deleted.

In further aspects, the invention comprises a library of synthetic polynucleotides, wherein said polynucleotides encode a plurality of antibody proteins comprising: (i) a CDRH3 amino acid sequence of claim 1; and 10 (ii) a VKCDR3 amino acid sequence comprising about 1 to about 10 of the amino acids found at Kabat positions 89, 90, 91, 92, 93, 94, 95, 95A, 96, and 97, in selected VKCDR3 sequences derived from a particular IGKV or IGKJ germline sequence.

In still further aspects, the invention comprises a library of synthetic 15 polynucleotides, wherein said polynucleotides encode a plurality of antibody proteins comprising: (i) a CDRH3 amino acid sequence of claim 1; and (ii) a VKCDR3 amino acid sequences of at least about 80% identity to an amino acid sequence represented by the following formula: 20 [VKChassis]-[L3- VK]- [X]- [ JK*], wherein: (a) VK Chassis is an amino acid sequence selected from the group consisting of about Kabat amino acid 1 to about Kabat amino acid 88 encoded by IGKV1-05, IGKV1-06, IGKV1-08, IGKV 1-09, IGKV1-12, IGKV1-13, IGKV1-25 16, IGKV1-17, IGKV1-27, IGKV1-33, IGKV1-37, IGKV1-39, IGKV ID-16, IGKV1D-17, IGKV1D-43, IGKV1D-8, IGKV2-24, IGKV2-28, IGKV2-29, IGKV2-30, IGKV2-40, IGKV2D-26, IGKV2D-29, IGKV2D-30, IGKV3-11, IGKV3-15, IGKV3-20, IGKV3D-07, 30 IGKV3D-11, IGKV3D-20, IGKV4-1, IGKV5-2, IGKV6- -13 - 21, and IGKV6D-41, or a sequence of at least about 80% identity to any of them; 2015201796 09 Apr 2015 (b) L3-VK is the portion of the VKCDR3 encoded by the IGKV gene segment; and 5 (c) X is any amino acid residue; and (d) JK* is an amino acid sequence selected from the group consisting of sequences encoded by IGJK1, IGJK2, IGJK3, IGJK4, and IGJK5, wherein the first residue of each IGJK sequence is not present. 10 In some aspects, the VKCDR3 amino acid sequence comprises one or more of the sequences listed in Table 33 or a sequence at least about 80% identical to any of them. In other aspects, the antibody proteins are expressed in a heterodimeric form. In yet another aspect, the human antibody proteins are expressed as antibody fragments. In still other aspects of the invention, the antibody fragments are selected from the group 15 consisting of Fab, Fab', F(ab’)2, Fv fragments, diabodies, linear antibodies, and singlechain antibodies.

In certain embodiments, the invention comprises an antibody isolated from the polypeptide expression products of any library described herein.

In still other aspects, the polynucleotides further comprise a 5’ polynucleotide 20 sequence and a 3 ’ polynucleotide sequence that facilitate homologous recombination.

In one embodiment, the polynucleotides further encode an alternative scaffold.

In another embodiment, the invention comprises a library of polypeptides encoded by any of the synthetic polynucleotide libraries described herein.

In yet another embodiment, the invention comprises a library of vectors 25 comprising any of the polynucleotide libraries described herein. In certain other aspects, the invention comprises a population of cells comprising the vectors of the instant invention.

In one aspect, the doubling time of the population of cells is from about 1 to about 3 hours, from about 3 to about 8 hours, from about 8 to about 16 hours, from about 30 16 to about 20 hours, or from 20 to about 30 hours. In yet another aspect, the cells are yeast cells. In still another aspect, the yeast is Saccharomyces cerevisiae. - 14-

In other embodiments, the invention comprises a library that has a theoretical total diversity of N unique CDRH3 sequences, wherein N is about 106 to about 1015; and wherein the physical realization of the theoretical total CDRH3 diversity has a size of at least about 3N, thereby providing a probability of at least about 95% that any individual 5 CDRH3 sequence contained within the theoretical total diversity of the library is present in the actual library. 2015201796 09 Apr 2015

In certain embodiments, the invention comprises a library of synthetic polynucleotides, wherein said polynucleotides encode a plurality of antibody V7.CDR3 amino acid sequences comprising about 1 to about 10 of the amino acids found at Kabat 10 positions 89, 90, 91, 92, 93, 94, 95, 95A, 95B, 95C, 96, and 97, in selected VXCDR3 sequences encoded by a single germline sequence.

In some embodiments, the invention relates to a library of synthetic polynucleotides encoding a plurality of antibody CDRH3 amino acid sequences, wherein the library has a theoretical total diversity of about 106 to about 1015 unique CDRH3 15 sequences.

In still other embodiments, the invention relates to a method of preparing a library of synthetic polynucleotides encoding a plurality of antibody VK amino acid sequences, the method comprising: (i) providing polynucleotide sequences encoding: 20 (a) one or more VKChassis amino acid sequences selected from the group consisting of about Kabat amino acid 1 to about Kabat amino acid 88 encoded by IGKV1-05, IGKV1-06, IGKV1-08, IGKV1-09, IGKV1-12, IGKV1-13, IGKV1-16, IGKV1-17, IGKV1-27, IGKV1-33, IGKV1-37, IGKV1-39, IGKV1D-16, IGKV1D-17, IGKV1D-43, IGKV1D-8, IGKV2-24, IGKV2-28, IGKV2-29, IGKV2-30, IGKV2-40, 25 IGKV2D-26, IGKV2D-29, IGKV2D-30, IGKV3-11, IGKV3-15, IGKV3-20, IGKV3D-07, IGKV3D-11, IGKV3D-20, IGKV4-1, IGKV5-2, IGKV6-21, and IGKV6D-41, or a sequence at least about 80% identical to any of them; (b) one or more L3-VK amino acid sequences, wherein L3-VK the portion of the VKCDR3 amino acid sequence encoded by the IGKV gene segment; 30 (c) one or more X residues, wherein X is any amino acid residue; and - 15 - (d) one or more JK* amino acid sequences, wherein JK* is an 2015201796 09 Apr 2015 amino acid sequence selected from the group consisting amino acid sequences encoded by IGKJ1, IGKJ2, IGKJ3, IGKJ4, and IGKJ5, wherein the first amino acid residue of each sequence is not present; and 5 (ii) assembling the polynucleotide sequences to produce a library of synthetic polynucleotides encoding a plurality of human VK sequences represented by the following formula: [VKChassis] - [L3 -VK]- [X]- [ JK* ].

In some embodiments, the invention relates to a method of preparing a library of 10 synthetic polynucleotides encoding a plurality of antibody light chain CDR3 sequences, the method comprising: (i) determining the percent occurrence of each amino acid residue at each position in selected light chain CDR3 amino acid sequences derived from a single germline polynucleotide sequence; 15 (ii) designing synthetic polynucleotides encoding a plurality of human antibody light chain CDR3 amino acid sequences, wherein the percent occurrence of any amino acid at any position within the designed light chain CDR3 amino acid sequences is within at least about 30% of the percent occurrence in the selected light chain CDR3 amino acid sequences derived from a single germline polynucleotide sequence, as 20 determined in (i); and (iii) synthesizing one or more polynucleotides that were designed in (ii).

In other embodiments, the invention relates to a method of preparing a library of synthetic polynucleotides encoding a plurality of antibody νλ amino acid sequences, the method comprising: 25 (i) providing polynucleotide sequences encoding: (a) one or more Vl_Chassis amino acid sequences selected from the group consisting of about Kabat residue 1 to about Kabat residue 88 encoded by Κ}λνΐ-36, Κ}λνΐ-40, Κ3λνΐ-44, Κϊλνΐ-47, IGXV1-51, IGXV10-54, Κ}λν2-11, Ιϋλν2-14, Ιϋλν2-18, Κϊλν2-23, Ιϋλν2-8, IGXV3-1, IGXV3-10, Κ}λν3-12, IGXV3-16, 30 IGXV3-19, Κ3λΥ3-21, Κ3λΥ3-25, IGXV3-27, IGXV3-9, ΙϋλΥ4-3, IGXV4-60, IGXV4-69, -16- IGXV5-39, IGXV5-45, IGXV6-57, IGXV7-43, IGXV7-46, IGXV8-61, IGXV9-49, and IGXV10-54, or a sequence at least about 80% identical to any of them; 2015201796 09 Apr 2015 (b) one ore more L3-VX sequences, wherein L3-VX is the portion of the VXCDR3 amino acid sequence encoded by the IGXV gene segment; 5 (c) one or more JX sequences, wherein JX is an amino acid sequence selected from the group consisting of amino acid sequences encoded by IGXJ1-01, IGXJ2-01, IGXJ3-01, IGXJ3-02, IGXJ6-01, IGXJ7-01, and IGXJ7-02 wherein the first amino acid residue of each sequence may or may not be present; and (ii) assembling the polynucleotide sequences to produce a library of synthetic 10 polynucleotides encoding a plurality of human VX amino acid sequences represented by the following formula: [VX_Chassis]-[L3-VX]-[JX].

In certain embodiments, the amino acid sequences encoded by the polynucleotides of the libraries of the invention are human. 15 The present invention is also directed to methods of preparing a synthetic polynucleotide library comprising providing and assembling the polynucleotide sequences of the instant invention.

In another aspect, the invention comprises a method of preparing the library of synthetic polynucleotides encoding a plurality of antibody CDRH3 amino acid 20 sequences, the method comprising: (i) providing polynucleotide sequences encoding: 25 30 (a) one or more N1 amino acid sequences of about 0 to about 3 amino acids, wherein each amino acid of the N1 amino acid sequence is among the 12 most frequently occurring amino acids at the corresponding position in N1 sequences of CDRH3 amino acid sequences that are functionally expressed by human B cells; (b) one or more human CDRH3 DH amino acid sequences, island C-terminal truncations thereof, or a sequence of at least about 80% identity to any of them; - 17- (c) one or more N2 amino acid sequences of about 0 to about 3 amino acids, wherein each amino acid of the N1 amino acid sequence is among the 12 most frequently occurring amino acids at the corresponding position in N2 amino 2015201796 09 Apr 2015 5 acid sequences of CDRH3 amino acid sequences that are functionally expressed by human B cells; and (d) one or more human CDRH3 H3-JH amino acid sequences, N-terminal truncations thereof, or a sequence of at least about 80% identity to any of them; and 10 (ii) assembling the polynucleotide sequences to produce a library of synthetic polynucleotides encoding a plurality of human antibody CDRH3 amino acid sequences represented by the following formula: [N1]-[DH]-[N2]-[H3-JH], 15 In one aspect, one or more of the polynucleotide sequences are synthesized via split-pool synthesis.

In another aspect, the method of the invention further comprises the step of recombining the assembled synthetic polynucleotides with a vector comprising a heavy chain chassis and a heavy chain constant region, to form a full-length heavy chain. 20 In another aspect, the method of the invention further comprises the step of providing a 5 ’ polynucleotide sequence and a 3 ’ polynucleotide sequence that facilitate homologous recombination. In still another aspect, the method of the invention further comprises the step of recombining the assembled synthetic polynucleotides with a vector comprising a heavy chain chassis and a heavy chain constant region, to form a full- 25 length heavy chain.

In some embodiments, the step of recombining is performed in yeast. In certain embodiments, the yeast is S. cerevisiae.

In certain other embodiments, the invention comprises a method of isolating one or more host cells expressing one or more antibodies, the method comprising: 30 (i) expressing the human antibodies of any one of claims 40 and 46 in one or more host cells; - 18 - (ii) contacting the host cells with one or more antigens; and 2015201796 17 Mar 2017 (iii) isolating one or more host cells having antibodies that bind to the one or more antigens.

In another aspect, the method of the invention further comprises the step of isolating one or more antibodies from the one or more host cells that present the antibodies which recognize the one or more antigens. In yet another aspect, the method of the invention further comprises the step of isolating one or more polynucleotide sequences encoding one or more antibodies from the one or more host cells that present the antibodies which recognize the one or more antigens.

In certain other embodiments, the invention comprises a kit comprising the library of synthetic polynucleotides encoding a plurality of antibody CDRH3 amino acid sequences, or any of the other sequences disclosed herein.

In still other aspects, the CDRH3 amino acid sequences encoded by the libraries of synthetic polynucleotides described herein, or any of the other sequences disclosed herein, are in computer readable form.

In another aspect, the invention provides a library of synthetic polynucleotides, wherein said polynucleotides encode a plurality of antibody VKCDR3 amino acid sequences comprising about 1 to about 10 of the amino acids found at Kabat positions 89, 90, 91, 92, 93, 94, 95, 95 A, 96, and 97, in selected VKCDR3 amino acid sequences derived from a particular IGKV or IGKJ germline sequence.

In another aspect, the invention provides a library of synthetic polynucleotides, wherein said polynucleotides encode a plurality of unique antibody VKCDR3 amino acid sequences that are of at least about 80% identity to an amino acid sequence represented by the following formula: [VK_Chassis] - [L3 - VK] - [X] - [ JK* ], wherein: (i) VK Chassis is an amino acid sequence selected from the group consisting of about Kabat amino acid 1 to about Kabat amino acid 88 encoded by IGKV1 -05, IGKV1-06, IGKV 1-08, IGKV 1-09, IGKV1-12, IGKV1-13, IGKV1-16, IGKV1-17, IGKV1-27, IGKV1-33, IGKV 1-37, IGKV1-39, IGKV1D-16, IGKV1D-17, IGKV1D-43, IGKV1D-8, IGKV2-24, IGKV2-28, IGKV2-29, IGKV2-30, IGKV2-40, IGKV2D-26, IGKV2D-29, IGKV2D-30, IGKV3-11, IGKV3-15, IGKV3-20, IGKV3D-07, IGKV3D-11, IGKV3D-20, IGKV4-1, IGKV5-2, IGKV6-21, and IGKV6D-41, or a sequence of at least about 80% - 19-identity to any of them; 2015201796 17 Mar 2017 (ii) L3-VK is the portion of the VKCDR3 encoded by the IGKV gene segment; and (iii) X is any amino acid residue; and (iv) JK* is an amino acid sequence selected from the group consisting of amino acid sequences encoded by IGJK1, IGJK2, IGJK3, IGJK4, and IGJK5, wherein the first amino acid residue of each amino acid sequence is not present.

In another aspect, the invention provides a library of synthetic polynucleotides, wherein said polynucleotides encode a plurality of antibody proteins comprising: (i) at least 106 unique antibody CDRH3 amino acid sequences comprising: (a) an N1 amino acid sequence selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV, D, E, F, Η, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, WGT; (b) a human CDRH3 DH amino acid sequence, N- and C-terminal truncations thereof, or a sequence of at least about 80% identity to any of them; (c) an N2 amino acid sequence selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV, D, E, F, Η, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, WGT; and (d) a human CDRH3 H3-JH amino acid sequence or N-terminal truncations thereof; and (ii) a VKCDR3 amino acid sequence comprising about 1 to about 10 amino acids.

In another aspect, the invention provides a library of synthetic polynucleotides, - 19A-wherein said polynucleotides encode a plurality of antibody proteins comprising: 2015201796 17 Mar 2017 (i) at least 106 unique antibody CDRH3 amino acid sequences comprising: (a) an N1 amino acid sequence selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV, D, E, F, Η, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, WGT; (b) a human CDRH3 DH amino acid sequence, N- and C-terminal truncations thereof, or a sequence of at least about 80% identity to any of them; (c) an N2 amino acid sequence selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV, D, E, F, Η, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, WGT; and (d) a human CDRH3 H3-JH amino acid sequence or N-terminal truncations thereof; and (ii) a VKCDR3 amino acid sequence of at least about 80% identity to a amino acid sequence represented by the following formula: [VK_Chassis]-[L3-VK]-[X]-[JK*], wherein: (a) VK Chassis is an amino acid sequence selected from the group consisting of about Kabat amino acid 1 to about Kabat amino acid 88 encoded by IGKV1 -05, IGKV1-06, IGKV1-08, IGKV1-09, IGKV1-12, IGKV1-13, IGKV1- 16, IGKV1-17, IGKV1-27, IGKV1-33, IGKV1-37, IGKV1-39, IGKV1D-16, IGKV1D-17, IGKV1D-43, IGKV1D-8, IGKV2-24, IGKV2-28, IGKV2-29, IGKV2- 30, IGKV2-40, IGKV2D-26, IGKV2D-29, IGKV2D-30, IGKV3-11, IGKV3-15, IGKV3-20, IGKV3D-07, IGKV3D-11, IGKV3D-20, IGKV4-1, IGKV5-2, IGKV6- 21, and IGKV6D-41, or a sequence of at least about 80% identity to any of them; (b) L3-VK is the portion of the VKCDR3 encoded by the IGKV gene - 19B -segment; and 2015201796 13 Apr 2017 (c) X is any amino acid residue; and (d) JK* is an amino acid sequence selected from the group consisting of amino acid sequences encoded by IGJK1, IGJK2, IGJK3 , IGJK4, and IG JK5 , wherein the first residue of each IGJK amino acid sequence is not present.

In another aspect, the invention provides a method of isolating one or more host cells expressing one or more antibodies, the method comprising: (i) expressing the antibodies according to the present disclosure in one or more host cells, (ii) contacting the host cells with one or more antigens; and (iii) isolating one or more host cells having antibodies that bind to the one or more antigens.

In another aspect, the invention provides a kit comprising the library of synthetic polynucleotides according to the present disclosure.

In another aspect, the invention also provides a library of synthetic polynucleotides, wherein said polynucleotides encode a plurality of antibody proteins comprising: (i) at least 106 unique antibody CDRH3 amino acid sequences comprising: (a) an N1 amino acid sequence selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV, D, E, F, Η, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, WGT; (b) a human CDRH3 DH amino acid sequence, N- and C-terminal truncations thereof, or a sequence of at least about 80% identity to any of them; (c) an N2 amino acid sequence selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, - 19C - GGV, D, E, F, Η, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, EH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, WGT; and 2015201796 13 Apr 2017 (d) a human CDRH3 H3-JH amino acid sequence or N-terminal truncations thereof; and (ii) a VKCDR3 amino acid sequence comprising about 1 to about 10 amino acids found at Kabat positions 89, 90, 91, 92, 93, 94, 95, 95 A, 96, and 97, in selected VKCDR3 amino acid sequences derived from a particular IGKV or IGKJ germline sequence.

In another aspect, the invention also provides a library of synthetic polynucleotides, wherein said polynucleotides encode a plurality of antibody proteins comprising: (i) at least 106 unique antibody CDRH3 amino acid sequences comprising: (a) an N1 amino acid sequence selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV, D, E, F, Η, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, WGT; (b) a human CDRH3 DH amino acid sequence, N- and C-terminal truncations thereof, or a sequence of at least about 80% identity to any of them; (c) an N2 amino acid sequence selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV, D, E, F, Η, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, WGT; and -19D- (d) a human CDRH3 H3-JH amino acid sequence or N-terminal truncations 2015201796 13 Apr 2017 thereof; and (ii) a VKCDR3 amino acid sequence of at least about 80% identity to a amino acid sequence represented by the following formula: [VK_Chassis]-[L3-VK]-[X]-[JK*], wherein: (a) VK Chassis is an amino acid sequence selected from the group consisting of about Kabat amino acid 1 to about Kabat amino acid 88 encoded by IGKV1 -05, IGKV1-06, IGKV1-08, IGKV1-09, IGKV1-12, IGKV1-13, IGKV1- 16, IGKV1-17, IGKV1-27, IGKV1-33, IGKV1-37, IGKV1-39, IGKV1D-16, IGKV1D-17,1GKV1D-43, IGKV1D-8, IGKV2-24, IGKV2-28, IGKV2-29, IGKV2- 30, IGKV2-40, IGKV2D-26, IGKV2D-29, IGKV2D-30, IGKV3-11, IGKV3-15, IGKV3-20, IGKV3D-07, IGKV3D-11, IGKV3D-20, IGKV4-1, IGKV5-2, IGKV6- 21, and IGKV6D-41, or a sequence of at least about 80% identity to any of them; (b) L3-VK is the portion of the VKCDR3 encoded by the IGKV gene segment; and (c) X is any amino acid residue; and (d) JK* is an amino acid sequence selected from the group consisting of amino acid sequences encoded by IGJK1, IGJK2, IGJK3 , IGJK4, and IG JK5 , wherein the first residue of each IGJK amino acid sequence is not present.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts a schematic of recombination between a fragment (e.g., CDR3) and a vector (e.g., comprising a chassis and constant region) for the construction of a library. -19E-

Figure 2 depicts the length distribution of the N1 and N2 regions of rearranged human antibody sequences compiled from Jackson et al. (J. Immunol Methods, 2007, 324: 26, incorporated by reference in its entirety). 2015201796 13 Apr 2017

Figure 3 depicts the length distribution of the CDRL3 regions of rearranged human kappa light chain sequences compiled from the NCBI database (Appendix A).

Figure 4 depicts the length distribution of the CDRL3 regions of rearranged human lambda light chain sequences compiled from the NCBI database (Appendix B).

Figure 5 depicts a schematic representation of the 424 cloning vectors used in the synthesis of the CDRH3 regions before and after ligation of the [DH]-[N2]-[JH] segment.

Figure 6 depicts a schematic stincture of a heavy chain vector, prior to recombination with a CDRH3. -19F-

Figure 7 depicts a schematic diagram of a CDRH3 integrated into a heavy chain vector and the polynucleotide and polypeptide sequences of CDRH3. 2015201796 09 Apr 2015

Figure 8 depicts a schematic structure of a kappa light chain vector, prior to recombination with a CDRL3. 5 Figure 9 depicts a schematic diagram of a CDRL3 integrated into a light chain vector and the polynucleotide and polypeptide sequences of CDRL3.

Figure 10 depicts the length distribution of the CDRH3 domain (Rabat positions 95-102) from 96 colonies obtained by transformation with 10 of the 424 vectors synthesized as described in Example 10 (observed), as compared to the expected {i.e., 10 designed) distribution.

Figure 11 depicts the length distribution of the DH segment from 96 colonies obtained by transformation with 10 of the 424 vectors synthesized as described in Example 10 (observed), as compared to the expected {i.e., designed) distribution.

Figure 12 depicts the length distribution of the N2 segment from 96 colonies 15 obtained by transformation with 10 of the 424 vectors synthesized as described in Example 10 (observed), as compared to the expected {i.e., designed) distribution.

Figure 13 depicts the length distribution of the H3-JH segment from 96 colonies obtained by transformation with 10 of the 424 vectors synthesized as described in Example 10 (observed), as compared to the expected {i.e., designed) distribution. 20 Figure 14 depicts the length distribution of the CDRH3 domains from 291 sequences prepared from yeast cells transformed according to the method outlined in Example 10.4, namely the co-transformation of vectors containing heavy chain chassis and constant regions with a CDRH3 insert (observed), as compared to the expected {i.e., designed) distribution. 25 Figure 15 depicts the length distribution of the [Tail]-[N1] region from the 291 sequences prepared from yeast cells transformed according to the protocol outlined in Example 10.4 (observed), as compared to the expected {i.e., designed) distribution.

Figure 16 depicts the length distribution of the DH region from the 291 sequences prepared from yeast cells transformed according to the protocol outlined in 30 Example 10.4 (observed), as compared to the theoretical {i.e., designed) distribution.

Figure 17 depicts the length distribution of the N2 region from the 291 sequences prepared from yeast cells transformed according to the protocol outlined in Example 10.4 (observed), as compared to the theoretical {i.e., designed) distribution. -20-

Figure 18 depicts the length distribution of the H3-JH region from the 291 sequences prepared from yeast cells transformed according to the protocol outlined in Example 10.4 (observed), as compared to the theoretical (i.e., designed) distribution. 2015201796 09 Apr 2015

Figure 19 depicts the familial origin of the JH segments identified in the 291 5 sequences (observed), as compared to the theoretical (i.e., designed) familial origin.

Figure 20 depicts the representation of each of the 16 chassis of the library (observed), as compared to the theoretical (i.e., designed) chassis representation. VH3-23 is represented twice; once ending in CAR and once ending in CAK. These representations were combined, as were the ten variants of VH3-33 with one variant of 10 VH3-30.

Figure 21 depicts a comparison of the CDRL3 length from 86 sequences selected from the VKCDR3 library of Example 6.2 (observed) to human sequences (human) and the designed sequences (designed).

Figure 22 depicts the representation of the light chain chassis amongst the 86 15 sequences selected from the library (observed), as compared to the theoretical (i.e., designed) chassis representation.

Figure 23 depicts the frequency of occurrence of different CDRH3 lengths in an exemplary library of the invention, versus the preimmune repertoire of Lee et al. (Immunogenetics, 2006, 57: 917, incorporated by reference in its entirety). 20 Figure 24 depicts binding curves for 6 antibodies selected from a library of the invention.

Figure 25 depicts binding curves for 10 antibodies selected from a library of the invention binding to hen egg white lysozyme.

25 DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to, at least, synthetic polynucleotide libraries, methods of producing and using the libraries of the invention, kits and computer readable forms including the libraries of the invention. The libraries taught in this application are described, at least in part, in terms of the components from which they 30 are assembled.

In certain embodiments, the instant invention provides antibody libraries specifically designed based on the composition and CDR length distribution in the naturally occurring human antibody repertoire. It is estimated that, even in the absence -21 - of antigenic stimulation, a human makes at least about 107 different antibody molecules. The antigen-binding sites of many antibodies can cross-react with a variety of related but different epitopes. In addition the human antibody repertoire is large enough to ensure that there is an antigen-binding site to fit almost any potential epitope, albeit with low 5 affinity. 2015201796 09 Apr 2015

The mammalian immune system has evolved unique genetic mechanisms that enable it to generate an almost unlimited number of different light and heavy chains in a remarkably economical way, by combinatorially joining chromosomally separated gene segments prior to transcription. Each type of immunoglobulin (Ig) chain (i.e., κ light, λ 10 light, and heavy) is synthesized by combinatorial assembly of DNA sequences selected from two or more families of gene segments, to produce a single polypeptide chain. Specifically, the heavy chains and light chains each consist of a variable region and a constant (C) region. The variable regions of the heavy chains are encoded by DNA sequences assembled from three families of gene segments: variable (IGHV), joining 15 (IGHJ) and diversity (IGHD). The variable regions of light chains are encoded by DNA sequences assembled from two families of gene segments for each of the kappa and lambda light chains: variable (IGLV) and joining (IGLJ). Each variable region (heavy and light) is also recombined with a constant region, to produce a full-length immunoglobulin chain. 20 While combinatorial assembly of the V, D and J gene segments make a substantial contribution to antibody variable region diversity, further diversity is introduced in vivo, at the pre-B cell stage, via imprecise joining of these gene segments and the introduction of non-templated nucleotides at the junctions between the gene segments. 25 After a B cell recognizes an antigen, it is induced to proliferate. During proliferation, the B cell receptor locus undergoes an extremely high rate of somatic mutation that is far greater than the normal rate of genomic mutation. The mutations that occur are primarily localized to the Ig variable regions and comprise substitutions, insertions and deletions. This somatic hypermutation enables the production of B cells 30 that express antibodies possessing enhanced affinity toward an antigen. Such antigen-driven somatic hypermutation fine-tunes antibody responses to a given antigen.

Significant efforts have been made to create antibody libraries with extensive diversity, and to mimic the natural process of affinity maturation of antibodies against -22- various antigens, especially antigens associated with diseases such as autoimmune diseases, cancer, and infectious disease. Antibody libraries comprising candidate binding molecules that can be readily screened against targets are desirable. However, the full promise of an antibody library, which is representative of the preimmune human 5 antibody repertoire, has remained elusive. In addition to the shortcomings enumerated above, and throughout the application, synthetic libraries that are known in the art often suffer from noise (i.e., very large libraries increase the presence of many sequences which do not express well, and/or which misfold), while entirely human libraries that are known in the art may be biased against certain antigen classes (e.g., self-antigens). 2015201796 09 Apr 2015 10 Moreover, the limitations of synthesis and physical realization techniques restrict the functional diversity of antibody libraries of the art. The present invention provides, for the first time, a fully synthetic antibody library that is representative of the human preimmune antibody repertoire (e.g., in composition and length), and that can be readily screened (i.e., it is physically realizable and, in some cases can be oversampled) using, 15 for example, high throughput methods, to obtain, for example, new therapeutics and/or diagnostics

In particular, the synthetic antibody libraries of the instant invention have the potential to recognize any antigen, including self-antigens of human origin. The ability to recognize self-antigens is usually lost in an expressed human library, because self-20 reactive antibodies are removed by the donor’s immune system via negative selection. Another feature of the invention is that screening the antibody library using positive clone selection, for example, by FACS (florescence activated cell sorter) bypasses the standard and tedious methodology of generating a hybridoma library and supernatant screening. Still further, the libraries, or sub-libraries thereof, can be screened multiple 25 times, to discover additional antibodies against other desired targets.

Before further description of the invention, certain terms are defined. 1. Definitions

Unless defined otherwise, all technical and scientific terms used herein have the 30 meaning commonly understood by one of ordinary skill in the art relevant to the invention. The definitions below supplement those in the art and are directed to the embodiments described in the current application. -23-

The term "antibody" is used herein in the broadest sense and specifically encompasses at least monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, human antibodies, and antibody fragments. An antibody is a protein comprising one or 5 more polypeptides substantially or partially encoded by immunoglobulin genes or 2015201796 09 Apr 2015 fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. "Antibody fragments" comprise a portion of an intact antibody, for example, one 10 or more portions of the antigen-binding region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibodies, and multi-specific antibodies formed from intact antibodies and antibody fragments.

An "intact antibody" is one comprising full-length heavy- and light- chains and 15 an Fc region. An intact antibody is also referred to as a “full-length, heterodimeric” antibody or immunoglobulin.

The term "variable" refers to the portions of the immunoglobulin domains that exhibit variability in their sequence and that are involved in determining the specificity and binding affinity of a particular antibody (i.e., the “variable domain(s)”). Variability 20 is not evenly distributed throughout the variable domains of antibodies; it is concentrated in sub-domains of each of the heavy and light chain variable regions.

These sub-domains are called “hypervariable” regions or “complementarity determining regions” (CDRs). The more conserved (i.e., non-hypervariable) portions of the variable domains are called the “framework” regions (FRM). The variable domains of naturally 25 occurring heavy and light chains each comprise four FRM regions, largely adopting a β-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the β -sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRM and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-30 binding site (see Rabat et al. Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991, incorporated by reference in its entirety). The constant domains are not directly involved in antigen -24- binding, but exhibit various effector functions, such as, for example, antibody-dependent, cell-mediated cytotoxicity and complement activation. 2015201796 09 Apr 2015

The “chassis” of the invention represent a portion of the antibody heavy chain variable (IGHV) or light chain variable (IGLV) domains that are not part of CDRH3 or 5 CDRL3, respectively. The chassis of the invention is defined as the portion of the variable region of an antibody beginning with the first amino acid of FRM1 and ending with the last amino acid of FRM3. In the case of the heavy chain, the chassis includes the amino acids including from about Kabat position 1 to about Kabat position 94. In the case of the light chains (kappa and lambda), the chassis are defined as including 10 from about Kabat position 1 to about Kabat position 88. The chassis of the invention may contain certain modifications relative to the corresponding germline variable domain sequences presented herein or available in public databases. These modifications may be engineered (e.g., to remove N-linked glycosylation sites) or naturally occurring (e.g., to account for allelic variation). For example, it is known in 15 the art that the immunoglobulin gene repertoire is polymorphic (Wang et al., Immunol. Cell. Biol., 2008, 86: 111; Collins et al., Immunogenetics, 2008, DOI 10.1007/s00251-008-0325-z, published online, each incorporated by reference in its entirety); chassis, CDRs (e.g., CDRH3) and constant regions representative of these allelic variants are also encompassed by the invention. In some embodiments, the allelic variant(s) used in 20 a particular embodiment of the invention may be selected based on the allelic variation present in different patient populations, for example, to identify antibodies that are non-immunogenic in these patient populations. In certain embodiments, the immunogenicity of an antibody of the invention may depend on allelic variation in the major histocompatibility complex (MHC) genes of a patient population. Such allelic variation 25 may also be considered in the design of libraries of the invention. In certain embodiments of the invention, the chassis and constant regions are contained on a vector, and a CDR3 region is introduced between them via homologous recombination.

In some embodiments, one, two or three nucleotides may follow the heavy chain chassis, forming either a partial (if one or two) or a complete (if three) codon. When a 30 full codon is present, these nucleotides encode an amino acid residue that is referred to as the “tail,” and occupies position 95.

The “CDRH3 numbering system” used herein defines the first amino acid of CDRH3 as being at Kabat position 95 (the “tail,” when present) and the last amino acid -25 - of CDRH3 as position 102. The amino acids following the “tail” are called “Nl” and, when present, are assigned numbers 96, 96A, 96B, etc. The Nl segment is followed by the “DH” segment, which is assigned numbers 97, 97A, 97B, 97C, etc. The DH segment is followed by the “N2” segment, which, when present, is numbered 98, 98A, 2015201796 09 Apr 2015 5 98B, etc. Finally, the most C-terminal amino acid residue of the set of the “H3-JH” segment is designated as number 102. The residue directly before (N-terminal) it, when present, is 101, and the one before (if present) is 100. For reasons of convenience, and which will become apparent elsewhere, the rest of the H3-JH amino acids are numbered in reverse order, beginning with 99 for the amino acid just N-terminal to 100, 99A for 10 the residue N-terminal to 99, and so forth for 99B, 99C, etc. Examples of certain CDRH3 sequence residue numbers may therefore include the following: 13 Amino Acid CDR-H3 with Nl and N2 (95) (96) (96A) (97) (97A) (97B) (97C) (97D) (98) (99) (100) (101) (102) 15 I —|--------I-----------------------I----|------------------|

Tail Nl DH N2 H3-JH 10 Amino Acid CDR-H3 without Nl and N2 (97) (97A) (97B) (97C) (97D) (97E) (97F) (97G) (101) (102)

20 I--------------------------------------I---------I

DH H3-JH

As used herein, the term “diversity” refers to a variety or a noticeable heterogeneity. The term “sequence diversity” refers to a variety of sequences which are 25 collectively representative of several possibilities of sequences, for example, those found in natural human antibodies. For example, heavy chain CDR3 (CDRH3) sequence diversity may refer to a variety of possibilities of combining the known human DH and H3-JH segments, including the Nl and N2 regions, to form heavy chain CDR3 sequences. The light chain CDR3 (CDRL3) sequence diversity may refer to a variety of 30 possibilities of combining the naturally occurring light chain variable region contributing to CDRL3 {i.e., L3-VL) and joining (i.e., L3-JL) segments, to form light chain CDR3 sequences. As used herein, H3-JH refers to the portion of the IGHJ gene contributing to CDRH3. As used herein, L3-VL and L3-JL refer to the portions of the IGLV and IGLJ genes (kappa or lambda) contributing to CDRL3, respectively. -26-

As used herein, the term “expression” includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion. 2015201796 09 Apr 2015

As used herein, the term "host cell" is intended to refer to a cell into which a 5 polynucleotide of the invention. It should be understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. 10

The term “length diversity” refers to a variety in the length of a particular nucleotide or amino acid sequence. For example, in naturally occurring human antibodies, the heavy chain CDR3 sequence varies in length, for example, from about 3 amino acids to over about 35 amino acids, and the light chain CDR3 sequence varies in 15 length, for example, from about 5 to about 16 amino acids. Prior to the instant invention, it was known in the art that it is possible to design antibody libraries containing sequence diversity or length diversity (see, e.g., Hoet et al., Nat. Biotechnol., 2005, 23: 344; Kretzschmar and von Ruden, Curr. Opin. Biotechnol., 2002 13: 598; and Rauchenberger et al., J. Biol. Chem., 2003 278: 38194, each of which is incorporated by 20 reference in its entirety); however, the instant invention is directed to, at least, the design of synthetic antibody libraries containing the sequence diversity and length diversity of naturally occurring human sequences. In some cases, synthetic libraries containing sequence and length diversity have been synthesized, however these libraries contain too much theoretical diversity to synthesize the entire designed repertoire and/or too many 25 theoretical members to physically realize or oversample the entire library.

As used herein, a sequence designed with “directed diversity” has been specifically designed to contain both sequence diversity and length diversity. Directed diversity is not stochastic.

As used herein, “stochastic” describes a process of generating a randomly 30 determined sequence of amino acids, which is considered as a sample of one element from a probability distribution.

The term “library of polynucleotides” refers to two or more polynucleotides having a diversity as described herein, specifically designed according to the methods of -27- the invention. The term “library of polypeptides” refers to two or more polypeptides having a diversity as described herein, specifically designed according to the methods of the invention. The term "library of synthetic polynucleotides" refers to a polynucleotide library that includes synthetic polynucleotides. The term "library of vectors" refers 5 herein to a library of at least two different vectors. As used herein, the term “human 2015201796 09 Apr 2015 antibody libraries,” at least includes, a polynucleotide or polypeptide library which has been designed to represent the sequence diversity and length diversity of naturally occurring human antibodies.

As described throughout the specification, the term “library” is used herein in its 10 broadest sense, and also may include the sub-libraries that may or may not be combined to produce libraries of the invention.

As used herein, the term “synthetic polynucleotide” refers to a molecule formed through a chemical process, as opposed to molecules of natural origin, or molecules derived via template-based amplification of molecules of natural origin (e.g., 15 immunoglobulin chains cloned from populations of B cells via PCR amplification are not “synthetic” used herein). In some instances, for example, when referring to libraries of the invention that comprise multiple components (e.g., Nl, DH, N2, and/or H3-JH), the invention encompasses libraries in which at least one of the aforementioned components is synthetic. By way of illustration, a library in which certain components 20 are synthetic, while other components are of natural origin or derived via template-based amplification of molecules of natural origin, would be encompassed by the invention.

The term "split-pool synthesis" refers to a procedure in which the products of a plurality of first reactions are combined (pooled) and then separated (split) before participating in a plurality of second reactions. Example 9, describes the synthesis of 25 278 DH segments (products), each in a separate reaction. After synthesis, these 278 segments are combined (pooled) and then distributed (split) amongst 141 columns for the synthesis of the N2 segments. This enables the pairing of each of the 278 DH segments with each of the 141 N2 segments. As described elsewhere in the specification, these numbers are non-limiting. 30 “Preimmune” antibody libraries have similar sequence diversities and length diversities to naturally occurring human antibody sequences before these sequences have undergone negative selection or somatic hypermutation. For example, the set of sequences described in Lee et al. (Immunogenetics, 2006, 57: 917, incorporated by -28- reference in its entirety) is believed to represent sequences from the preimmune repertoire. In certain embodiments of the invention, the sequences of the invention will be similar to these sequences (e.g., in terms of composition and length). In certain embodiments of the invention, such antibody libraries are designed to be small enough 5 to chemically synthesize and physically realize, but large enough to encode antibodies with the potential to recognize any antigen. In one embodiment of the invention, an antibody library comprises about 107 to about 1020 different antibodies and/or polynucleotide sequences encoding the antibodies of the library. In some embodiments, the libraries of the instant invention are designed to include ΙΟ3, ΙΟ4, 105, ΙΟ6, 107, 10s, 10 ΙΟ9, ΙΟ10, 1011, 1012, 1013, 1014, 1015, ΙΟ16, ΙΟ17, ΙΟ18, 1019, or 102° different antibodies and/or polynucleotide sequences encoding the antibodies. In certain embodiments, the libraries of the invention may comprise or encode about 103 to about 105, about 105 to about 107, about 107 to about 109, about 109 to about 1011, about 1011 to about 1013, about 1013 to about 1015, about 1015 to about 1017, or about 1017 to about 1020 different 15 antibodies. In certain embodiments of the invention, the diversity of the libraries may be characterized as being greater than or less than one or more of the diversities enumerated above, for example greater than about ΙΟ3, ΙΟ4, ΙΟ5, ΙΟ6, ΙΟ7, ΙΟ8, ΙΟ9, 1010, 1011, ΙΟ12, ΙΟ13, ΙΟ14, ΙΟ15, ΙΟ16, ΙΟ17, ΙΟ18, 1019, or 1020 or less than about ΙΟ3, ΙΟ4, 105, ΙΟ6, ΙΟ7, ΙΟ8, ΙΟ9, ΙΟ10, 1011, ΙΟ12, ΙΟ13, ΙΟ14, ΙΟ15, ΙΟ16, ΙΟ17, ΙΟ18, 1019, or 102°. In 20 certain other embodiments of the invention, the probability of an antibody of interest being present in a physical realization of a library with a size as enumerated above is at least about 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 99.9% (see Library Sampling, in the Detailed Description, for more information on the probability of a particular sequence 25 being present in a physical realization of a library). The antibody libraries of the 2015201796 09 Apr 2015 invention may also include antibodies directed to, for example, self (i.e., human) antigens. The antibodies of the present invention may not be present in expressed human libraries for reasons including because self-reactive antibodies are removed by the donor’s immune system via negative selection. However, novel heavy/light chain 30 pairings may in some cases create self-reactive antibody specificity (Griffiths et al. US Patent 5,885,793, incorporated by reference in its entirety). In certain embodiments of the invention, the number of unique heavy chains in a library may be about 10, 50, 10 , 150, 103, 104, 105,106, 107, 108, 109, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, -29- ΙΟ19, ΙΟ20, or more. In certain embodiments of the invention, the number of unique light chains in a library may be about 5, 10, 25, 50, 102, 150, 500, ΙΟ3, ΙΟ4, 105,106, ΙΟ7, 108, ΙΟ9, ΙΟ10, ΙΟ11, ΙΟ12, ΙΟ13, ΙΟ14, ΙΟ15, ΙΟ16, ΙΟ17, ΙΟ18, ΙΟ19, 1020, or more. 2015201796 09 Apr 2015

As used herein, the term “human antibody CDRH3 libraries,” at least includes, a 5 polynucleotide or polypeptide library which has been designed to represent the sequence diversity and length diversity of naturally occurring human antibodies. “Preimmune” CDRH3 libraries have similar sequence diversities and length diversities to naturally occurring human antibody CDRH3 sequences before these sequences undergo negative selection and somatic hypermutation. Known human CDRH3 sequences are represented 10 in various data sets, including Jackson et al., J. Immunol Methods, 2007, 324: 26;

Martin, Proteins, 1996, 25: 130; and Lee et al., Immunogenetics, 2006, 57: 917, each of which is incorporated by reference in its entirety. In certain embodiments of the invention, such CDRH3 libraries are designed to be small enough to chemically synthesize and physically realize, but large enough to encode CDRH3s with the 15 potential to recognize any antigen. In one embodiment of the invention, an antibody library includes about 106 to about 1015 different CDRH3 sequences and/or polynucleotide sequences encoding said CDRH3 sequences. In some embodiments, the libraries of the instant invention are designed to about ΙΟ3, ΙΟ4, ΙΟ5, ΙΟ6, ΙΟ7, ΙΟ8, 109, ΙΟ10, 1011, ΙΟ12, ΙΟ13, ΙΟ14, 1015, or 1016, different CDRH3 sequences and/or 20 polynucleotide sequences encoding said CDRH3 sequences. In some embodiments, the libraries of the invention may include or encode about 103 to about 106, about 106 to about 108, about 108 to about 1010, about 1010 to about 1012, about 1012 to about 1014, or about 1014 to about 1016 different CDRH3 sequences. In certain embodiments of the invention, the diversity of the libraries may be characterized as being greater than or less 25 than one or more of the diversities enumerated above, for example greater than about ΙΟ3, ΙΟ4, ΙΟ5, ΙΟ6, ΙΟ7, ΙΟ8, ΙΟ9, ΙΟ10, 1011, ΙΟ12, ΙΟ13, ΙΟ14, 1015, or 1016 or less than about 103,104, 105, ΙΟ6, ΙΟ7, ΙΟ8, ΙΟ9, ΙΟ10, 1011, ΙΟ12, ΙΟ13, ΙΟ14, 1015, or 1016. In certain embodiments of the invention, the probability of a CDRH3 of interest being present in a physical realization of a library with a size as enumerated above is at least 30 about 0.0001 %, 0.001 %, 0.01 %, 0.1 %, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 99.9% (see Library Sampling, in the Detailed Description, for more information on the probability of a particular sequence being present in a physical realization of a library). -30-

The preimmune CDRH3 libraries of the invention may also include CDRH3s directed to, for example, self (i.e., human) antigens. Such CDRH3s may not be present in expressed human libraries, because self-reactive CDRH3s are removed by the donor’s immune system via negative selection. 2015201796 09 Apr 2015 5 Libraries of the invention containing “VKCDR3” sequences and “VACDR3” sequences refer to the kappa and lambda sub-sets of the CDRL3 sequences, respectively. These libraries may be designed with directed diversity, to collectively represent the length and sequence diversity of the human antibody CDRL3 repertoire. “Preimmune” versions of these libraries have similar sequence diversities and length diversities to 10 naturally occurring human antibody CDRL3 sequences before these sequences undergo negative selection. Known human CDRL3 sequences are represented in various data sets, including the NCBI database (see Appendix A and Appendix B for light chain sequence data sets) and Martin, Proteins, 1996, 25: 130 incorporated by reference in its entirety. In certain embodiments of the invention, such CDRL3 libraries are designed to 15 be small enough to chemically synthesize and physically realize, but large enough to encode CDRL3s with the potential to recognize any antigen.

In one embodiment of the invention, an antibody library comprises about 105 different CDRL3 sequences and/or polynucleotide sequences encoding said CDRL3 sequences. In some embodiments, the libraries of the instant invention are designed to 20 comprise about 101, ΙΟ2, ΙΟ3, ΙΟ4, ΙΟ6, 107, or 108 different CDRL3 sequences and/or polynucleotide sequences encoding said CDRL3 sequences. In some embodiments, the libraries of the invention may comprise or encode about 101 to about 103, about 103 to about 105, or about 105 to about 108 different CDRL3 sequences. In certain embodiments of the invention, the diversity of the libraries may be characterized as 25 being greater than or less than one or more of the diversities enumerated above, for example greater than about 101, ΙΟ2, ΙΟ3, ΙΟ4, 105, ΙΟ6, 107, or 108 or less than about 101, ΙΟ2, ΙΟ3, ΙΟ4, 105, ΙΟ6, 107, or 108. In certain embodiments of the invention, the probability of a CDRL3 of interest being present in a physical realization of a library with a size as enumerated above is at least about 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 30 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 99.9% (see Library Sampling, in the Detailed Description, for more information on the probability of a particular sequence being present in a physical realization of a library). The preimmune CDRL3 libraries of the invention may also include CDRL3s directed to, -31 - for example, self (i.e., human) antigens. Such CDRL3s may not be present in expressed human libraries, because self-reactive CDRL3s are removed by the donor’s immune system via negative selection. 2015201796 09 Apr 2015

As used herein, the term “known heavy chain CDR3 sequences” refers to heavy 5 chain CDR3 sequences in the public domain that have been cloned from populations of human B cells. Examples of such sequences are those published or derived from public data sets, including, for example, Zemlin et al., JMB, 2003, 334: 733; Lee et al., Immunogenetics, 2006, 57: 917; and Jackson etal. J. Immunol Methods, 2007, 324: 26, each of which are incorporated by reference in their entirety. 10 As used herein, the term “known light chain CDR3 sequences” refers to light chain CDR3 sequences (e.g., kappa or lambda) in the public domain that have been cloned from populations of human B cells. Examples of such sequences are those published or derived from public data sets, including, for example, the NCBI database (see Appendices A and B filed herewith). 15 As used herein the term “antibody binding regions” refers to one or more portions of an immunoglobulin or antibody variable region capable of binding an antigen(s). Typically, the antibody binding region is, for example, an antibody light chain (or variable region or one or more CDRs thereof), an antibody heavy chain (or variable region or one or more CDRs thereof), a heavy chain Fd region, a combined 20 antibody light and heavy chain (or variable regions thereof) such as a Fab, F(ab’)2, single domain, or single chain antibodies (scFv), or any region of a full length antibody that recognizes an antigen, for example, an IgG (e.g., an IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibody.

The term “framework region” refers to the art-recognized portions of an antibody 25 variable region that exist between the more divergent (i.e., hypervariable) CDRs. Such framework regions are typically referred to as frameworks 1 through 4 (FRM1, FRM2, FRM3, and FRM4) and provide a scaffold for the presentation of the six CDRs (three from the heavy chain and three from the light chain) in three dimensional space, to form an antigen-binding surface. 30 The term “canonical structure” refers to the main chain conformation that is adopted by the antigen binding (CDR) loops. From comparative structural studies, it has been found that five of the six antigen binding loops have only a limited repertoire of available conformations. Each canonical structure can be characterized by the torsion -32- angles of the polypeptide backbone. Correspondent loops between antibodies may, therefore, have very similar three dimensional structures, despite high amino acid sequence variability in most parts of the loops (Chothia and Lesk, J. Mol. Biol., 1987, 196: 901; Chothia et al., Nature, 1989, 342: 877; Martin and Thornton, J. Mol. Biol., 2015201796 09 Apr 2015 5 1996, 263: 800, each of which is incorporated by reference in its entirety). Furthermore, there is a relationship between the adopted loop structure and the amino acid sequences surrounding it. The conformation of a particular canonical class is determined by the length of the loop and the amino acid residues residing at key positions within the loop, as well as within the conserved framework (i.e., outside of the loop). Assignment to a 10 particular canonical class can therefore be made based on the presence of these key amino acid residues. The term “canonical structure” may also include considerations as to the linear sequence of the antibody, for example, as catalogued by Kabat (Kabat et al., in “Sequences of Proteins of Immunological Interest,” 5 Edition, U.S. Department of Heath and Human Services, 1992). The Kabat numbering scheme is a widely adopted 15 standard for numbering the amino acid residues of an antibody variable domain in a consistent manner. Additional structural considerations can also be used to determine the canonical structure of an antibody. For example, those differences not fully reflected by Kabat numbering can be described by the numbering system of Chothia et al. and/or revealed by other techniques, for example, crystallography and two or three-dimensional 20 computational modeling. Accordingly, a given antibody sequence may be placed into a canonical class which allows for, among other things, identifying appropriate chassis sequences (e.g., based on a desire to include a variety of canonical structures in a library). Kabat numbering of antibody amino acid sequences and structural considerations as described by Chothia et al., and their implications for construing 25 canonical aspects of antibody structure, are described in the literature.

The terms “CDR”, and its plural “CDRs”, refer to a complementarity determining region (CDR) of which three make up the binding character of a light chain variable region (CDRL1, CDRL2 and CDRL3) and three make up the binding character of a heavy chain variable region (CDRH1, CDRH2 and CDRH3). CDRs contribute to 30 the functional activity of an antibody molecule and are separated by amino acid sequences that comprise scaffolding or framework regions. The exact definitional CDR boundaries and lengths are subject to different classification and numbering systems. CDRs may therefore be referred to by Kabat, Chothia, contact or any other boundary -33- definitions, including the numbering system described herein. Despite differing boundaries, each of these systems has some degree of overlap in what constitutes the so called "hypervariable regions" within the variable sequences. CDR definitions according to these systems may therefore differ in length and boundary areas with 5 respect to the adjacent framework region. See for example Kabat, Chothia, and/or MacCallum et al., (Kabat et al., in “Sequences of Proteins of Immunological Interest,” 5th Edition, U.S. Department of Health and Human Services, 1992; Chothia et al., J. 2015201796 09 Apr 2015

Mol. Biol., 1987, 196: 901; and MacCallum et al., J. Mol. Biol., 1996, 262: 732, each of which is incorporated by reference in its entirety). 10 The term “amino acid” or “amino acid residue” typically refers to an amino acid having its art recognized definition such as an amino acid selected from the group consisting of: alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine (Cys or C); glutamine (Gin or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His or H); isoleucine (lie or I): leucine (Leu or L); lysine 15 (Lys or K); methionine (Met or M); phenylalanine (Phe or F); proline (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Val or V), although modified, synthetic, or rare amino acids may be used as desired. Generally, amino acids can be grouped as having a nonpolar side chain (e.g., Ala, Cys, lie, Leu, Met, Phe, Pro, Val); a negatively charged side chain (e.g., Asp, Glu); a 20 positively charged sidechain (e.g., Arg, His, Lys); or an uncharged polar side chain (e.g.,

Asn, Cys, Gin, Gly, His, Met, Phe, Ser, Thr, Trp, and Tyr).

The term “polynucleotide(s)” refers to nucleic acids such as DNA molecules and RNA molecules and analogs thereof (e.g., DNA or RNA generated using nucleotide analogs or using nucleic acid chemistry). As desired, the polynucleotides may be made 25 synthetically, e.g., using art-recognized nucleic acid chemistry or enzymatically using, e.g., a polymerase, and, if desired, be modified. Typical modifications include methylation, biotinylation, and other art-known modifications. In addition, the nucleic acid molecule can be single-stranded or double-stranded and, where desired, linked to a detectable moiety. 30 The terms “theoretical diversity”, “theoretical total diversity”, or “theoretical repertoire” refer to the maximum number of variants in a library design. For example, given an amino acid sequence of three residues, where residues one and three may each be any one of five amino acid types and residue two may be any one of 20 amino acid -34- types, the theoretical diversity is 5x20x5=500 possible sequences. Similarly if sequence X is constructed by combination of 4 amino acid segments, where segment 1 has 100 possible sequences, segment 2 has 75 possible sequences, segment 3 has 250 possible sequences, and segment 4 has 30 possible sequences, the theoretical total diversity of 5 fragment X would be 100x75x200x30, or 5.6x10s possible sequences. 2015201796 09 Apr 2015

The term “physical realization” refers to a portion of the theoretical diversity that can actually be physically sampled, for example, by any display methodology. Exemplary display methodology include: phage display, ribosomal display, and yeast display. For synthetic sequences, the size of the physical realization of a library depends 10 on (1) the fraction of the theoretical diversity that can actually be synthesized, and (2) the limitations of the particular screening method. Exemplary limitations of screening methods include the number of variants that can be screened in a particular assay (e.g., ribosome display, phage display, yeast display) and the transformation efficiency of a host cell (e.g., yeast, mammalian cells, bacteria) which is used in a screening assay. For 15 the purposes of illustration, given a library with a theoretical diversity of 1012 members, an exemplary physical realization of the library (e.g., in yeast, bacterial cells, ribosome display, etc.; details provided below) that can maximally include 1011 members will, therefore, sample about 10% of the theoretical diversity of the library. However, if less than 1011 members of the library with a theoretical diversity of 1012 are synthesized, and 20 the physical realization of the library can maximally include 1011 members, less than 10% of the theoretical diversity of the library is sampled in the physical realization of the library. Similarly, a physical realization of the library that can maximally include more than 1012 members would "oversample" the theoretical diversity, meaning that each member may be present more than once (assuming that the entire 1012 theoretical 25 diversity is synthesized).

The term “all possible reading frames” encompasses at least the three forward reading frames and, in some embodiments, the three reverse reading frames.

The term “antibody of interest” refers to any antibody that has a property of interest that is isolated from a library of the invention. The property of interest may 30 include, but is not limited to, binding to a particular antigen or epitope, blocking a binding interaction between two molecules, or eliciting a certain biological effect.

The term “functionally expressed” refers to those immunoglobulin genes that are expressed by human B cells and that do not contain premature stop codons. -35-

The term “full-length heavy chain” refers to an immunoglobulin heavy chain that contains each of the canonical structural domains of an immunoglobulin heavy chain, including the four framework regions, the three CDRs, and the constant region. The term “full-length light chain” refers to an immunoglobulin light chain that contains each of 5 the canonical structural domains of an immunoglobulin light chain, including the four framework regions, the three CDRs, and the constant region. 2015201796 09 Apr 2015

The term “unique,” as used herein, refers to a sequence that is different (e.g. has a different chemical structure) from every other sequence within the designed theoretical diversity. It should be understood that there are likely to be more than one copy of many 10 unique sequences from the theoretical diversity in a particular physical realization. For example, a library comprising three unique sequences may comprise nine total members if each sequence occurs three times in the library. However, in certain embodiments, each unique sequence may occur only once.

The term “heterologous moiety” is used herein to indicate the addition of a 15 composition to an antibody wherein the composition is not normally part of the antibody. Exemplary heterologous moieties include drugs, toxins, imaging agents, and any other compositions which might provide an activity that is not inherent in the antibody itself.

As used herein, the term “percent occurrence of each amino acid residue at each 20 position” refers to the percentage of instances in a sample in which an amino acid is found at a defined position within a particular sequence. For example, given the following three sequences:

KVR

KYP 25 KRP, K occurs in position one in 100% of the instances and P occurs in position three in about 67% of the instances. In certain embodiments of the invention, the sequences selected for comparison are human immunoglobulin sequences.

As used herein, the term “most frequently occurring amino acids“ at a specified 30 position of a sequence in a population of polypeptides refers to the amino acid residues that have the highest percent occurrence at the indicated position in the indicated polypeptide population. For example, the most frequently occurring amino acids in each of the three most N-terminal positions in N1 sequences of CDRH3 sequences that are -36- functionally expressed by human B cells are listed in Table 21, and the most frequently occurring amino acids in each of the three most N-terminal positions in N2 sequences of CDRH3 sequences that are functionally expressed by human B cells are listed in Table 22. 2015201796 09 Apr 2015 5 For the purposes of analyzing the occurrence of certain duplets (Example 13) and the information content (Example 14) of the libraries of the invention, and other libraries, a “central loop” of CDRH3 is defined. If the C-terminal 5 amino acids from Rabat CDRH3 (95-102) are removed, then the remaining sequence is termed the “central loop”. Thus, considering the duplet occurrence calculations of Example 13, using a 10 CDRH3 of size 6 or less would not contribute to the analysis of the occurrence of duplets. A CDRH3 of size 7 would contribute only to the i - i+1 data set, a CDRH3 of size 8 would also contribute to the i - i+2 data set, and a CDRH3 of size 9 and larger would also contribute to the i - i+3 data set. For example, a CDR H3 of size 9 may have amino acids at positions 95-96-97-98-99- 100-100A-101-102, but only the first four 15 residues (bolded) would be part of the central loop and contribute to the pair-wise occurrence (duplet) statistics. As a further example, a CDRH3 of size 14 may have the sequence: 95-96-97-98-99-100-100A-100B-100C-100D-100E-1 OOF-101-102. Here, only the first nine residues (bolded) contribute to the central loop.

Library screening requires a genotype-phenotype linkage. The term "genotype-20 phenotype linkage" is used in a manner consistent with its art-recognized meaning and refers to the fact that the nucleic acid (genotype) encoding a protein with a particular phenotype (e.g., binding an antigen) can be isolated from a library. For the purposes of illustration, an antibody fragment expressed on the surface of a phage can be isolated based on its binding to an antigen (e.g., Ladner et al.). The binding of the antibody to 25 the antigen simultaneously enables the isolation of the phage containing the nucleic acid encoding the antibody fragment. Thus, the phenotype (antigen-binding characteristics of the antibody fragment) has been "linked" to the genotype (nucleic acid encoding the antibody fragment). Other methods of maintaining a genotype-phenotype linkage include those of Wittrup et al. (US Patent Nos. 6,300,065, 6,331,391, 6,423,538, 30 6,696,251, 6,699,658, and US Pub. No. 20040146976, each of which is incorporated by reference in its entirety), Miltenyi (US Patent No. 7,166,423, incorporated by reference in its entirety), Fandl (US Patent No. 6,919,183, US Pub No. 20060234311, each incorporated by reference in its entirety), Clausell-Tormos et al. (Chem. Biol., 2008, 15: -37- 427, incorporated by reference in its entirety), Love et al. (Nat. Biotechnol., 2006, 24: 703, incorporated by reference in its entirety), and Kelly et al. (Chem. Commun., 2007, 14: 1773, incorporated by reference in its entirety). Any method which localizes the antibody protein with the gene encoding the antibody, in a way in which they can both 5 be recovered while the linkage between them is maintained, is suitable. 2015201796 09 Apr 2015 2. Design of the Libraries

The antibody libraries of the invention are designed to reflect certain aspects of the preimmune repertoire as naturally created by the human immune system. Certain 10 libraries of the invention are based on rational design informed by the collection of

human V, D, and J genes, and other large databases of human heavy and light chain sequences (e.g., publicly known germline sequences; sequences from Jackson et al., J. Immunol Methods, 2007, 324: 26, incorporated by reference in its entirety; sequences from Lee et al., Immunogenetics, 2006, 57: 917, incorporated by reference in its 15 entirety; and sequences compiled for rearranged VK and νλ - see Appendices A and B filed herewith). Additional information may be found, for example, in Scaviner et al., Exp. Clin. Immunogene!, 1999, 16: 234; Tomlinson etal., J. Mol. Biol., 1992, 227: 799; and Matsuda et al., J. Exp. Med., 1998, 188: 2151 each incorporated by reference in its entirety. In certain embodiments of the invention, cassettes representing the possible V, 20 D, and J diversity found in the human repertoire, as well as junctional diversity (i.e., N1 and N2), are synthesized de novo as single or double-stranded DNA oligonucleotides. In certain embodiments of the invention, oligonucleotide cassettes encoding CDR sequences are introduced into yeast along with one or more acceptor vectors containing heavy or light chain chassis sequences. No primer-based PCR amplification or 25 template-directed cloning steps from mammalian cDNA or mRNA are employed. Through standard homologous recombination, the recipient yeast recombines the cassettes (e.g., CDR3s) with the acceptor vector(s) containing the chassis sequence(s) and constant regions, to create a properly ordered synthetic, full-length human heavy chain and/or light chain immunoglobulin library that can be genetically propagated, 30 expressed, displayed, and screened. One of ordinary skill in the art will readily recognize that the chassis contained in the acceptor vector can be designed so as to produce constructs other than full-length human heavy chains and/or light chains. For example, in certain embodiments of the invention, the chassis may be designed to -38- encode portions of a polypeptide encoding an antibody fragment or subunit of an antibody fragment, so that a sequence encoding an antibody fragment, or subunit thereof, is produced when the oligonucleotide cassette containing the CDR is recombined with the acceptor vector. 2015201796 09 Apr 2015 5 In certain embodiments, the invention provides a synthetic, preimmune human antibody repertoire comprising about 107 to about 1020 antibody members, wherein the repertoire comprises: (a) selected human antibody heavy chain chassis (i.e., amino acids 1 to 94 of the heavy chain variable region, using Rabat’s definition); 10 (b) a CDRH3 repertoire, designed based on the human IGHD and IGHJ germline sequences, the CDRH3 repertoire comprising the following: (i) optionally, one or more tail regions; (ii) one or more N1 regions, comprising about 0 to about 10 amino acids selected from the group consisting of fewer than 20 of the amino acid types 15 preferentially encoded by the action of terminal deoxynucleotidyl transferase (TdT) and functionally expressed by human B cells; (iii) one or DH segments, based on one or more selected IGHD segments, and one or more N- or C-terminal truncations thereof; (iv) one or more N2 regions, comprising about 0 to about 10 amino acids 20 selected from the group consisting of fewer than 20 of the amino acids preferentially encoded by the activity of TdT and functionally expressed by human B cells; and (v) one or more H3-JH segments, based on one or more IGHJ segments, and one or more N-terminal truncations thereof (e.g., down to XXWG); 25 (c) one or more selected human antibody kappa and/or lambda light chain chassis; and (d) a CDRL3 repertoire designed based on the human IGLV and IGLJ germline sequences, wherein “L” may be a kappa or lambda light chain.

The heavy chain chassis may be any sequence with homology to Rabat residues 30 1 to 94 of an immunoglobulin heavy chain variable domain. Non-limiting examples of heavy chain chassis are included in the Examples, and one of ordinary skill in the art will readily recognize that the principles presented therein, and throughout the specification, may be used to derive additional heavy chain chassis. -39-

As described above, the heavy chain chassis region is followed, optionally, by a “tail” region. The tail region comprises zero, one, or more amino acids that may or may not be selected on the basis of comparing naturally occurring heavy chain sequences. 2015201796 09 Apr 2015

For example, in certain embodiments of the invention, heavy chain sequences available 5 in the art may be compared, and the residues occurring most frequently in the tail position in the naturally occurring sequences included in the library (e.g., to produce sequences that most closely resemble human sequences). In other embodiments, amino acids that are used less frequently may be used. In still other embodiments, amino acids selected from any group of amino acids may be used. In certain embodiments of the 10 invention, the length of the tail is zero (no residue) or one (e.g., G/D/E) amino acid. For the purposes of clarity, and without being bound by theory, in the naturally occurring human repertoire, the first 2/3 of the codon encoding the tail residue is provided by the FRM3 region of the VH gene. The amino acid at this position in naturally occurring heavy chain sequences may thus be considered to be partially encoded by the IGHV 15 gene (2/3) and partially encoded by the CDRH3 (1/3). However, for the purposes of clearly illustrating certain aspects of the invention, the entire codon encoding the tail residue (and, therefore, the amino acid derived from it) is described herein as being part of the CDRH3 sequence.

As described above, there are two peptide segments derived from nucleotides 20 which are added by TdT in the naturally occurring human antibody repertoire. These segments are designated N1 and N2 (referred to herein as N1 and N2 segments, domains, regions or sequences). In certain embodiments of the invention, N1 and N2 are about 0, 1, 2, or 3 amino acids in length. Without being bound by theory, it is thought that these lengths most closely mimic the N1 and N2 lengths found in the human 25 repertoire (see Figure 2). In other embodiments of the invention, N1 and N2 may be about 4, 5, 6, 7, 8, 9, or 10 amino acids in length. Similarly, the composition of the amino acid residues utilized to produce the N1 and N2 segments may also vary. In certain embodiments of the invention, the amino acids used to produce N1 and N2 segments may be selected from amongst the eight most frequently occurring amino acids 30 in the N1 and N2 domains of the human repertoire (e.g., G, R, S, P, L, A, V, and T). In other embodiments of the invention, the amino acids used to produce the N1 and N2 segments may be selected from the group consisting of fewer than about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 of the amino acids preferentially encoded -40- by the activity of TdT and functionally expressed by human B cells. Alternatively, N1 and N2 may comprise amino acids selected from any group of amino acids. It is not required that N1 and N2 be of a similar length or composition, and independent variation of the length and composition of N1 and N2 is one method by which additional diversity 5 may be introduced into the library. 2015201796 09 Apr 2015

The DH segments of the libraries are based on the peptides encoded by the naturally occurring IGHD gene repertoire, with progressive deletion of residues at the bland C-termini. IGHD genes may be read in multiple reading frames, and peptides representing these reading frames, and their N- and C-terminal deletions are also 10 included in the libraries of the invention. In certain embodiments of the invention, DH segments as short as three amino acid residues may be included in the libraries. In other embodiments of the invention, DH segments as short as about 1, 2, 4, 5, 6, 7, or 8 amino acids may be included in the libraries.

The H3-JH segments of the libraries are based on the peptides encoded by the 15 naturally occurring IGHJ gene repertoire, with progressive deletion of residues at the N-terminus. The N-terminal portion of the IGHJ segment that makes up part of the CDRH3 is referred to herein as H3-JH. In certain embodiments of the invention, the H3-JH segment may be represented by progressive N-terminal deletions of one or more H3-JH residues, down to two H3-JH residues. In other embodiments of the invention, 20 the H3-JH segments of the library may contain N-terminal deletions (or no deletions) down to about 6, 5, 4, 3, 2, 1, or 0 H3-JH residues.

The light chain chassis of the libraries may be any sequence with homology to Kabat residues 1 to 88 of naturally occurring light chain (k or λ) sequences. In certain embodiments of the invention, the light chain chassis of the invention are synthesized in 25 combinatorial fashion, utilizing VL and JL segments, to produce one or more libraries of light chain sequences with diversity in the chassis and CDR3 sequences. In other embodiments of the invention, the light chain CDR3 sequences are synthesized using degenerate oligonucleotides or trinucleotides and recombined with the light chain chassis and light chain constant region, to form full-length light chains. 30 The instant invention also provides methods for producing and using such libraries, as well as libraries comprising one or more immunoglobulin domains or antibody fragments. Design and synthesis of each component of the claimed antibody libraries is provided in more detail below. -41 - 2.1. Design of the Antibody Library Chassis Sequences 2015201796 09 Apr 2015

One step in building certain libraries of the invention is the selection of chassis sequences, which are based on naturally occurring variable domain sequences (e.g., 5 IGHV and IGLV). This selection can be done arbitrarily, or by the selection of chassis that meet certain criteria. For example, the Kabat database, an electronic database containing non-redundant rearranged antibody sequences, can be queried for those heavy and light chain germline sequences that are most frequently represented. The BLAST search algorithm, or more specialized tools such as SoDA (Volpe et al., Bioinformatics, 10 2006, 22: 438-44, incorporated by reference in its entirety), can be used to compare rearranged antibody sequences with germline sequences, using the V BASE2 database (Retter et al., Nucleic Acids Res., 2005, 33: D671-D674), or similar collections of human V, D, and J genes, to identify the germline families that are most frequently used to generate functional antibodies. 15 Several criteria can be utilized for the selection of chassis for inclusion in the libraries of the invention. For example, sequences that are known (or have been determined) to express poorly in yeast, or other organisms used in the invention (e.g., bacteria, mammalian cells, fungi, or plants) can be excluded from the libraries. Chassis may also be chosen based on their representation in the peripheral blood of humans. In 20 certain embodiments of the invention, it may be desirable to select chassis that correspond to germline sequences that are highly represented in the peripheral blood of humans. In other embodiments, it may be desirable to select chassis that correspond to germline sequences that are less frequently represented, for example, to increase the canonical diversity of the library. Therefore, chassis may be selected to produce 25 libraries that represent the largest and most structurally diverse group of functional human antibodies. In other embodiments of the invention, less diverse chassis may be utilized, for example, if it is desirable to produce a smaller, more focused library with less chassis variability and greater CDR variability. In some embodiments of the invention, chassis may be selected based on both their expression in a cell of the 30 invention (e.g., a yeast cell) and the diversity of canonical structures represented by the selected sequences. One may therefore produce a library with a diversity of canonical structures that express well in a cell of the invention. -42- 2.1.1. Design of the Heavy Chain Chassis Sequences 2015201796 09 Apr 2015

In certain embodiments of the invention, the antibody library comprises variable heavy domains and variable light domains, or portions thereof. Each of these domains is built from certain components, which will be more fully described in the examples 5 provided herein. In certain embodiments, the libraries described herein may be used to isolate fully human antibodies that can be used as diagnostics and/or therapeutics. Without being bound by theory, antibodies with sequences most similar or identical to those most frequently found in peripheral blood (for example, in humans) may be less likely to be immunogenic when administered as therapeutic agents. 10 Without being bound by theory, and for the purposes of illustrating certain embodiments of the invention, the VH domains of the library may be considered to comprise three primary components: (1) a VH “chassis”, which includes amino acids 1 to 94 (using Kabat numbering), (2) the CDRH3, which is defined herein to include the Kabat CDRH3 proper (positions 95-102), and (3) the FRM4 region, including amino 15 acids 103 to 113 (Kabat numbering). The overall VH structure may therefore be depicted schematically (not to scale) as: (1) ... (94) (95) ... (102) (103) ...(113) 20 VH Chassis CDRH3 FRM4

The selection and design of VH chassis sequences based on the human IGHV germline repertoire will become more apparent upon review of the examples provided herein. In certain embodiments of the invention, the VH chassis sequences selected for 25 use in the library may correspond to all functionally expressed human IGHV germline sequences. Alternatively, IGHV germline sequences may be selected for representation in a library according to one or more criteria. For example, in certain embodiments of the invention, the selected IGHV germline sequences may be among those that are most highly represented among antibody molecules isolated from the peripheral blood of 30 healthy adults, children, or fetuses.

In certain embodiments, it may be desirable to base the design of the VH chassis on the utilization of IGHV germline sequences in adults, children, or fetuses with a disease, for example, an autoimmune disease. Without being bound by theory, it is -43 - possible that analysis of germline sequence usage in the antibody molecules isolated from the peripheral blood of individuals with autoimmune disease may provide information useful for the design of antibodies recognizing human antigens. 2015201796 09 Apr 2015

In some embodiments, the selection of IGHV germline sequences for 5 representation in a library of the invention may be based on their frequency of occurrence in the peripheral blood. For the purposes of illustration, four IGHV1 germline sequences (IGHV1-2, IGHV1-18, IGHV1-46, and IGHV1-69) comprise about 80% of the IGHV1 family repertoire in peripheral blood. Thus, the specific IGHV1 germline sequences selected for representation in the library may include those that are 10 most frequently occurring and that cumulatively comprise at least about 80% of the IGHV1 family repertoire found in peripheral blood. An analogous approach can be used to select specific IGHV germline sequences from any other IGHV family (i.e., IGHV1, IGHV2, IGHV3, IGHV4, IGHV5, IGHV6, and IGHV7). The specific germline sequences chosen for representation of a particular IGHV family in a library of the 15 invention may therefore comprise at least about 100%, 99%, 98%, 97%, 96% 95%, 94%, 93%, 92%, 91% 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% of the particular IGHV family member repertoire found in peripheral blood.

In some embodiments, the selected IGHV germline sequences may be chosen to 20 maximize the structural diversity of the VH chassis library. Structural diversity may be evaluated by, for example, comparing the lengths, compositions, and canonical structures of CDRH1 and CDRH2 in the IGHV germline sequences. In human IGHV sequences, the CDRH1 (Kabat definition) may have a length of 5, 6 or 7 amino acids, while CDRH2 (Kabat definition) may have length of 16, 17, 18 or 19 amino acids. The 25 amino acid compositions of the IGHV germline sequences and, in particular, the CDR domains, may be evaluated by sequence alignments, as presented in the Examples. Canonical structure may be assigned, for example, according to the methods described by Chothia et al., J. Mol. Biol., 1992,227: 799, incorporated by reference in its entirety.

In certain embodiments of the invention, it may be advantageous to design VH 30 chassis based on IGHV germline sequences that may maximize the probability of isolating an antibody with particular characteristics. For example, without being bound by theory, in some embodiments it may be advantageous to restrict the IGHV germline sequences to include only those germline sequences that are utilized in antibodies -44- undergoing clinical development, or antibodies that have been approved as therapeutics. On the other hand, in some embodiments, it may be advantageous to produce libraries containing VH chassis that are not represented amongst clinically utilized antibodies. Such libraries may be capable of yielding antibodies with novel properties that are 5 advantageous over those obtained with the use of “typical” IGHV germline sequences, or enabling studies of the structures and properties of “atypical” IGHV germline sequences or canonical structures. 2015201796 09 Apr 2015

One of ordinary skill in the art will readily recognize that a variety of other criteria can be used to select IGHV germline sequences for representation in a library of 10 the invention. Any of the criteria described herein may also be combined with any other criteria. Further exemplary criteria include the ability to be expressed at sufficient levels in certain cell culture systems, solubility in particular antibody formats (e.g., whole immunoglobulins and antibody fragments), and the thermodynamic stability of the individual domains, whole immunoglobulins, or antibody fragments. The methods of 15 the invention may be applied to select any IGHV germline sequence that has utility in an antibody library of the instant invention.

In certain embodiments of the invention, the VH chassis of the libraries may comprise from about Kabat residue 1 to about Kabat residue 94 of one or more of the following IGHV germline sequences: IGHV1-2, IGHV1-3, IGHV1-8, IGHV1-18, 20 IGHV 1-24, IGHV1-45, IGHV1-46, IGHV1-58, IGHV1-69, IGHV2-5, IGHV2-26, IGHV2-70, IGHV3-7, IGHV3-9, IGHV3-11, IGHV3-13, IGHV3-15, IGHV3-20, IGHV3-21, IGHV3-23, IGHV3-30, IGHV3-33, IGHV3-43, IGHV3-48, IGHV3-49, IGHV3-53, IGHV3-64, IGHV3-66, IGHV3-72, IGHV3-73, IGHV3-74, IGHV4-4, IGHV4-28, IGHV4-31, IGHV4-34, IGHV4-39, IGHV4-59, IGHV4-61, IGHV4-B, 25 IGHV5-51, IGHV6-1, and IGHV7-4-1. In some embodiments of the invention, a library may contain one or more of these sequences, one or more allelic variants of these sequences, or encode an amino acid sequence at least about 99.9%, 99.5%, 99%, 98.5%, 98%, 97.5%, 97%, 96.5%, 96%, 95.5%, 95%, 94.5%, 94%, 93.5%, 93%, 92.5%, 92%, 91.5%, 91%, 90.5%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 30 77.5%, 75%, 73.5%, 70%, 65%, 60%, 55%, or 50% identical to one or more of these sequences.

In other embodiments, the VH chassis of the libraries may comprise from about Kabat residue 1 to about Kabat residue 94 of the following IGHV germline sequences: -45 - IGHVl-2, IGHV1-18, IGHV1-46, IGHV1-69, IGHV3-7, IGHV3-15, IGHV3-23, IGHV3-30, IGHV3-33, IGHV3-48, IGHV4-31, IGHV4-34, IGHV4-39, IGHV4-59, IGHV4-61, IGHV4-B, and IGHV5-51. In some embodiments of the invention, a library may contain one or more of these sequences, one or more allelic variants of these 5 sequences, or encode an amino acid sequence at least about 99.9%, 99.5%, 99%, 98.5%, 98%, 97.5%, 97%, 96.5%, 96%, 95.5%, 95%, 94.5%, 94%, 93.5%, 93%, 92.5%, 92%, 91.5%, 91%, 90.5%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 77.5%, 75%, 73.5%, 70%, 65%, 60%, 55%, or 50% identical to one or more of these sequences. The amino acid sequences of these chassis are presented in Table 5. 2015201796 09 Apr 2015 10 2.1.1.1. Heavy Chain Chassis Variants

While the selection of the VH chassis with sequences based on the IGHV germline sequences is expected to support a large diversity of CDRH3 sequences, further diversity in the VH chassis may be generated by altering the amino acid residues 15 comprising the CDRH1 and/or CDRH2 regions of each chassis selected for inclusion in the library (see Example 2).

In certain embodiments of the invention, the alterations or mutations in the amino acid residues comprising the CDRH1 and CDRH2 regions, or other regions, of the IGHV germline sequences are made after analyzing the sequence identity within data 20 sets of rearranged human heavy chain sequences that have been classified according to the identity of the original IGHV germline sequence from which the rearranged sequences are derived. For example, from a set of rearranged antibody sequences, the IGHV germline sequence of each antibody is determined, and the rearranged sequences are classified according to the IGHV germline sequence. This determination is made on 25 the basis of sequence identity.

Next, the occurrence of any of the 20 amino acid residues at each position in these sequences is determined. In certain embodiments of the invention, one may be particularly interested in the occurrence of different amino acid residues at the positions within CDRH1 and CDRH2, for example if increasing the diversity of the antigen-30 binding portion of the VH chassis is desired. In other embodiments of the invention, it may be desirable to evaluate the occurrence of different amino acid residues in the framework regions. Without being bound by theory, alterations in the framework regions may impact antigen binding by altering the spatial orientation of the CDRs. -46-

After the occurrence of amino acids at each position of interest has been identified, alterations may be made in the VH chassis sequence, according to certain criteria. In some embodiments, the objective may be to produce additional VH chassis with sequence variability that mimics the variability observed in the heavy chain 5 domains of rearranged human antibody sequences (derived from respective IGHV 2015201796 09 Apr 2015 germline sequences) as closely as possible, thereby potentially obtaining sequences that are most human in nature (i.e., sequences that most closely mimic the composition and length of human sequences). In this case, one may synthesize additional VH chassis sequences that include mutations naturally found at a particular position and include one 10 or more of these VH chassis sequences in a library of the invention, for example, at a frequency that mimics the frequency found in nature. In another embodiment of the invention, one may wish to include VH chassis that represent only mutations that most frequently occur at a given position in rearranged human antibody sequences. For example, rather than mimicking the human variability precisely, as described above, and 15 with reference to exemplary Tables 6 and 7, one may choose to include only top 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, amino acid residues that most frequently occur at each position. For the purposes of illustration, and with reference to Table 6, if one wished to include the top four most frequently occurring amino acid residues at position 31 of the VH1-69 sequence, then position 31 in the VH1-69 20 sequence would be varied to include S, N, T, and R. Without being bound by theory, it is thought that the introduction of diversity by mimicking the naturally occurring composition of the rearranged heavy chain sequences is likely to produce antibodies that are most human in composition. However, the libraries of the invention are not limited to heavy chain sequences that are diversified by this method, and any criteria can be 25 used to introduce diversity into the heavy chain chassis, including random or rational mutagenesis. For example, in certain embodiments of the invention, it may be preferable to substitute neutral and/or smaller amino acid residues for those residues that occur in the IGHV germline sequence. Without being bound by theory, neutral and/or smaller amino acid residues may provide a more flexible and less sterically hindered 30 context for the display of a diversity of CDR sequences.

Example 2 illustrates the application of this method to heavy chains derived from a particular IGHV germline. One of ordinary skill in the art will readily recognize that this method can be applied to any germline sequence, and can be used to generate at -47- least about 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21,22, 23,24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1000, ΙΟ4, ΙΟ5, 106, or more variants of each heavy chain chassis. 2015201796 09 Apr 2015 5 2.1.2. Design of the Light Chain Chassis Sequences

The light chain chassis of the invention may be based on kappa and/or lambda light chain sequences. The principles underlying the selection of light chain variable (IGLV) germline sequences for representation in the library are analogous to those employed for the selection of the heavy chain sequences (described above and in 10 Examples 1 and 2). Similarly, the methods used to introduce variability into the selected heavy chain chassis may also be used to introduce variability into the light chain chassis.

Without being bound by theory, and for the purposes of illustrating certain embodiments of the invention, the VL domains of the library may be considered to comprise three primary components: (1) a VL “chassis”, which includes amino acids 1 15 to 88 (using Kabat numbering), (2) the VLCDR3, which is defined herein to include the Kabat CDRL3 proper (positions 89-97), and (3) the FRM4 region, including amino acids 98 to 107 (Kabat numbering). The overall VL structure may therefore be depicted schematically (not to scale) as: 20 (1)...(88) (89)...(97) (98)...(107) VL Chassis CDRL3 FRM4

In certain embodiments of the invention, the VL chassis of the libraries include 25 one or more chassis based on IGKV germline sequences. In certain embodiments of the invention, the VL chassis of the libraries may comprise from about Kabat residue 1 to about Kabat residue 88 of one or more of the following IGKV germline sequences: IGKV 1-05, IGKV 1-06, IGKV1-08, IGKV1-09, IGKV1-12, IGKV1-13, IGKV1-16, IGKV1-17, IGKV 1-27, IGKV1-33, IGKV1-37, IGKV1-39, IGKV1D-16, IGKV1D-17, 30 IGKV1D-43, IGKV1D-8, IGKV2-24, IGKV2-28, IGKV2-29, IGKV2-30, IGKV2-40, IGKV2D-26, IGKV2D-29, IGKV2D-30, IGKV3-11, IGKV3-15, IGKV3-20, IGKV3D-07, IGKV3D-11, IGKV3D-20, IGKV4-1, IGKV5-2, IGKV6-21, and IGKV6D-41. In some embodiments of the invention, a library may contain one or more of these -48- sequences, one or more allelic variants of these sequences, or encode an amino acid sequence at least about 99.9%, 99.5%, 99%, 98.5%, 98%, 97.5%, 97%, 96.5%, 96%, 95.5%, 95%, 94.5%, 94%, 93.5%, 93%, 92.5%, 92%, 91.5%, 91%, 90.5%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 77.5%, 75%, 73.5%, 70%, 65%, 2015201796 09 Apr 2015 5 60%, 55%, or 50% identical to one or more of these sequences.

In other embodiments, the VL chassis of the libraries may comprise from about Kabat residue 1 to about Kabat residue 88 of the following IGKV germline sequences: IGKV1-05, IGKV1-12, IGKV1-27, IGKV1-33, IGKV1-39, IGKV2-28, IGKV3-11, IGKV3-15, IGKV3-20, and IGKV4-1. In some embodiments of the invention, a library 10 may contain one or more of these sequences, one or more allelic variants of these sequences, or encode an amino acid sequence at least about 99.9%, 99.5%, 99%, 98.5%, 98%, 97.5%, 97%, 96.5%, 96%, 95.5%, 95%, 94.5%, 94%, 93.5%, 93%, 92.5%, 92%, 91.5%, 91%, 90.5%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 77.5%, 75%, 73.5%, 70%, 65%, 60%, 55%, or 50% identical to one or more of these 15 sequences. The amino acid sequences of these chassis are presented in Table 11.

In certain embodiments of the invention, the VL chassis of the libraries include one or more chassis based on IGXV germline sequences. In certain embodiments of the invention, the VL chassis of the libraries may comprise from about Kabat residue 1 to about Kabat residue 88 of one or more of the following IGXV germline sequences: 20 IGXV3-1, IGXV3-21, IGXV2-14, IGXVl-40, IGXV3-19, IGXV1-51, IGXVl-44, IGXV6-57, IGXV2-8, IGXV3-25, IGXV2-23, IGXV3-10, IGXV4-69, IGXVl-47, IGXV2-11, IGXV7-43, IGXV7-46, IGXV5-45, IGXV4-60, IGXV10-54, IGXV8-61, IGXV3-9, IGXVl-36, IGXV2-18, IGXV3-16, IGXV3-27, IGXV4-3, IGXV5-39, IGXV9-49, and IGXV3-12. In some embodiments of the invention, a library may contain one or more of 25 these sequences, one or more allelic variants of these sequences, or encode an amino acid sequence at least about 99.9%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% identical to one or more of these sequences.

In other embodiments, the VL chassis of the libraries may comprise from about 30 Kabat residue 1 to about Kabat residue 88 of the following IGXV germline sequences: IGXV3-1, IGXV3-21, IGXV2-14, IGXVl-40, IGXV3-19, IGXV1-51, IGXVl-44, IGXV6-57, IGLV4-69, IG/.V7-43, and IGLV5-45. In some embodiments of the invention, a library may contain one or more of these sequences, one or more allelic variants of these -49- sequences, or encode an amino acid sequence at least about 99.9%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% identical to one or more of these sequences. The amino acid sequences of these chassis are presented in Table 14. 2015201796 09 Apr 2015 5 2.2. Design of the Antibody Library CDRH3 Components

It is known in the art that diversity in the CDR3 region of the heavy chain is sufficient for most antibody specificities (Xu and Davis, Immunity, 2000, 13: 27-45, incorporated by reference in its entirety) and that existing successful libraries have been 10 created using CDRH3 as the major source of diversification (Hoogenboom et al„ J. Mol. Biol., 1992,227:381; Lee etal., J. Mol. Biol., 2004, 340: 1073 each of which is incorporated by reference in its entirety). It is also known that both the DH region and the N1/N2 regions contribute to the CDRH3 functional diversity (Schroeder et al„ J. Immunol., 2005, 174: 7773 and Mathis et ah, Eur J Immunol., 1995, 25: 3115, each of 15 which is incorporated by reference in its entirety). For the purposes of the present invention, the CDHR3 region of naturally occurring human antibodies can be divided into five segments: (1) the tail segment, (2) the N1 segment, (3) the DH segment, (4) the N2 segment, and (5) the JH segment. As exemplified below, the tail, N1 and N2 segments may or may not be present. 20 In certain embodiments of the invention, the method for selecting amino acid sequences for the synthetic CDRH3 libraries includes a frequency analysis and the generation of the corresponding variability profiles of existing rearranged antibody sequences. In this process, which is described in more detail in the Examples section, the frequency of occurrence of a particular amino acid residue at a particular position 25 within rearranged CDRH3s (or any other heavy or light chain region) is determined.

Amino acids that are used more frequently in nature may then be chosen for inclusion in a library of the invention. 2.2.1. Design and Selection of the DH Segment Repertoire 30 In certain embodiments of the invention, the libraries contain CDRH3 regions comprising one or more segments designed based on the IGHD gene germline repertoire. In some embodiments of the invention, DH segments selected for inclusion in the library are selected and designed based on the most frequent usage of human -50- IGHD genes, and progressive N-terminal and C-terminal deletions thereof, to mimic the in vivo processing of the IGHD gene segments. In some embodiments of the invention, the DH segments of the library are about 3 to about 10 amino acids in length. In some embodiments of the invention, the DH segments of the library are about 0, 1, 2, 3, 4, 5, 2015201796 09 Apr 2015 5 6, 7, 8, 9, or 10 amino acids in length, or a combination thereof. In certain embodiments, the libraries of the invention may contain DH segments with a wide distribution of lengths (e.g., about 0 to about 10 amino acids). In other embodiments, the length distribution of the DH may be restricted (e.g., about 1 to about 5 amino acids, about 3 amino acids, about 3 and about 5 amino acids, and so on). In certain 10 embodiments of the library, the shortest DH segments may be about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

In certain embodiments of the invention, libraries may contain DH segments representative of any reading frame of any IGHD germline sequence. In certain embodiments of the invention, the DH segments selected for inclusion in a library 15 include one or more of the following IGHD sequences, or their derivatives (i.e., any reading frame and any degree of N-terminal and C-terminal truncation): IGHD3-10, IGHD3-22, IGHD6-19, IGHD6-13, IGHD3-3, IGHD2-2, IGHD4-17, IGHD1-26, IGHD5-5 / 5-18, IGHD2-15, IGHD6-6, IGHD3-9, IGHD5-12, IGHD5-24, IGHD2-21, IGHD3-16, IGHD4-23, IGHD1-1, IGHD1-7, IGHD4-4/4-11, IGHD1-20, IGHD7-27, 20 IGHD2-8, and IGHD6-25. In some embodiments of the invention, a library may contain one or more of these sequences, allelic variants thereof, or encode an amino acid sequence at least about 99.9%, 99.5%, 99%, 98.5%, 98%, 97.5%, 97%, 96.5%, 96%, 95.5%, 95%, 94.5%, 94%, 93.5%, 93%, 92.5%, 92%, 91.5%, 91%, 90.5%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 77.5%, 75%, 73.5%, 70%, 65%, 25 60%, 55%, or 50% identical to one or more of these sequences.

For the purposes of illustration, progressive N-terminal and C-terminal deletions of IGHD3-10, reading frame 1, are enumerated in the Table 1. N-terminal and C-terminal deletions of other IGHD sequences and reading frames are also encompassed by the invention, and one of ordinary skill in the art can readily determine these 30 sequences using, for example, the non-limiting exemplary data presented in Table 16. and/or the methods outlined above. Table 18 (Example 5) enumerates certain DH segments used in certain embodiments of the invention. -51 - 10 15 25 2015201796 09 Apr 2015

Table 1: Example of Progressive N- and C-terminal Deletions of Reading Frame 1 for Gene IGHD3-10, Yielding DH Segments DH SEQ ID NO: DH SEQ ID NO: VLLWFGELL LWFGEL VLLWFGEL LWFGE VLLWFGE LWFG VLLLWFG LWF VLLWF WFGELL VLLW WFGEL VLL WFGE LLWFGELL WFG LLWFGEL FGELL LLWFGE FGEL LLWFG FGE LLWF GELL LLW GEL LWFGELL ELL

In certain embodiments of the invention, the DH segments selected for inclusion in a library include one or more of the following IGHD sequences, or their derivatives (/. e., any reading frame and any degree N-terminal and C-terminal truncation): IGHD3-10, IGHD3-22, IGHD6-19, IGHD6-13, IGHD3-03, IGHD2-02, IGHD4-17, IGHD1-26, IGHD5-5/5-18, and IGHD2-15. In some embodiments of the invention, a library may contain one or more of these sequences, allelic variants thereof, or encode an amino acid sequence at least about 99.9%, 99.5%, 99%, 98.5%, 98%, 97.5%, 97%, 96.5%, 96%, 95.5%, 95%, 94.5%, 94%, 93.5%, 93%, 92.5%, 92%, 91.5%, 91%, 90.5%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 77.5%, 75%, 73.5%, 70%, 65%, 60%, 55%, or 50% identical to one or more of these sequences.

In certain embodiments of the invention, the DH segments selected for inclusion in a library include one or more of the following IGHD sequences, wherein the notation “_x” denotes the reading frame of the gene, or their derivatives (i.e., any degree of N-terminal or C-terminal truncation): IGHD1-261, IGHD1-26 3, IGHD2-2 2, IGHD2-2 3, IGHD2-15 2, IGHD3-3_3, IGHD3-10 1, IGHD3-10 2, IGHD3-10 3, IGHD3-22 2, IGHD4-17 2, IGHD5-5_3, IGHD6-13 1, IGHD6-13 2, IGHD6-19 1, and IGHD6-19 2. In some embodiments of the invention, a library may contain one or more of these sequences, allelic variants thereof, or encode an amino acid sequence at least about 99.9%, 99.5%, 99%, 98.5%, 98%, 97.5%, 97%, 96.5%, 96%, 95.5%, 95%, 94.5%, 94%, 93.5%, 93%, 92.5%, 92%, 91.5%, 91%, 90.5%, 90%, 89%, 88%, 87%, 86%, -52- 85%, 84%, 83%, 82%, 81%, 80%, 77.5%, 75%, 73.5%, 70%, 65%, 60%, 55%, or 50% identical to one or more of these sequences. 2015201796 09 Apr 2015 5

In certain embodiments of the invention, the libraries are designed to reflect a pre-determined length distribution of N- and C-terminal deleted IGHD segments. For example, in certain embodiments of the library, the DH segments of the library may be designed to mimic the natural length distribution of DH segments found in the human repertoire. For example, the relative occurrence of different IGHD segments in rearranged human antibody heavy chain domains from Lee et al. (Immunogenetics, 2006, 57: 917, incorporated by reference in its entirety). Table 2 shows the relative occurrence of the top 68% of IGHD segments from Lee et al.

Table 2. Relative Occurrence of Top 68% of IGHD Gene Usage from Lee etal. IGHD Reading Frame Sequence (Parent) SEQ ID NO: Relative Occurrence IGHD3-10 1 VLLWFGELL 4.3% IGHD3-10 2 YYYGSGSYYN 8.4% IGHD3-10 3 ITMVRGVII 4.0% IGHD3-22 2 YYYDSSGYYY 15.6% IGHD6-19 1 GYSSGWY 7.4% IGHD6-19 2 GIAVAG 6.0% IGHD6-13 1 GYSSSWY 8.4% IGHD6-13 2 GIAAAG 5.3% IGHD3-3 3 ITIFGVVII 7.4% IGHD2-2 2 GYCSSTSCYT 5.2% IGHD2-2 3 DIWVPAAM 4.1% IGHD4-17 2 DYGDY 6.8% IGHD1-26 1 GIVGATT 2.9% IGHD1-26 3 YSGSYY 4.3% IGHD5-5 3 GYSYGY 4.3% IGHD2-15 2 GYCSGGSCYS 5.6%

In certain embodiments, these relative occurrences may be used to design a 15 library with DH prevalence that is similar to the IGHD usage found in peripheral blood. In other embodiments of the invention, it may be preferable to bias the library toward longer or shorter DH segments, or DH segments of a particular composition. In other embodiments, it may be desirable to use all DH segments selected for the library in equal proportion. 20 In certain embodiments of the invention, the most commonly used reading- frames of the ten most frequently occurring IGHD sequences are utilized, and progressive N-terminal and C-terminal deletions of these sequences are made, thus providing a total of 278 non-redundant DH segments that are used to create a CDRH3 -53 - repertoire of the instant invention (Table 18). In some embodiments of the invention, the methods described above can be applied to produce libraries comprising the top 1, 2, 3,4,5,6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21,22, 23,24, or 25 expressed IGHD sequences, and progressive N-terminal and C-terminal deletions 5 thereof. As with all other components of the library, while the DH segments may be selected from among those that are commonly expressed, it is also within the scope of the invention to select these gene segments based on the fact that they are less commonly expressed. This may be advantageous, for example, in obtaining antibodies toward self-antigens or in further expanding the diversity of the library. Alternatively, 2015201796 09 Apr 2015 10 DH segments can be used to add compositional diversity in a manner that is strictly relative to their occurrence in actual human heavy chain sequences.

In certain embodiments of the invention, the progressive deletion of IGHD genes containing disulfide loop encoding segments may be limited, so as to leave the loop intact and to avoid the presence of unpaired cysteine residues. In other embodiments of 15 the invention, the presence of the loop can be ignored and the progressive deletion of the IGHD gene segments can occur as for any other segments, regardless of the presence of unpaired cysteine residues. In still other embodiments of the invention, the cysteine residues can be mutated to any other amino acid. 20 2.2.2. Design and Selection of the H3-JH Segment Repertoire

There are six IGHJ (joining) segments, IGHJ1, IGHJ2, IGHJ3, IGHJ4, IGHJ5, and IGHJ6. The amino acid sequences of the parent segments and the progressive N-terminal deletions are presented in Table 20 (Example 5). Similar to the N- and C-terminal deletions that the IGHD genes undergo, natural variation is introduced into the 25 IGHJ genes by N-terminal “nibbling”, or progressive deletion, of one or more codons by exonuclease activity.

The H3-JH segment refers to the portion of the IGHJ segment that is part of CDRH3. In certain embodiments of the invention, the H3-JH segment of a library comprises one or more of the following sequences: AEYFQH (SEQ ID NO:_),

30 EYFQH (SEQ ID NO:_), YFQH (SEQ ID NO:_), FQH (SEQ ID NO:_), QH

(SEQ ID NO:_), H (SEQ ID NO:_), YWYFDL (SEQ ID NO:_), WYFDL

(SEQ ID NO:_), YFDL (SEQ ID NO:_), FDL (SEQ ID NO:_), DL (SEQ ID NO:_), L (SEQ ID NO:_), AFDV (SEQ ID NO:_), FDV (SEQ ID NO:_), -54-

DV (SEQ ID NO:_), V (SEQ ID NO:_), YFDY (SEQ ID NO:_), FDY (SEQ 2015201796 09 Apr 2015 ID NO:_), DY (SEQ ID NO:_), Y (SEQ ID NO:_), NWFDS (SEQ ID NO:

_), WFDS (SEQ ID NO:_), FDS (SEQ ID NO:_), DS (SEQ ID NO:_), S (SEQ ID NO:_), YYYYYGMDV (SEQ ID NO:_), YYYYGMDV (SEQ ID NO:

5 _), YYYGMDV (SEQ ID NO:__), YYGMDV (SEQ ID NO:_), YGMDV (SEQ

ID NO:_), GMDV (SEQ ID NO:_), MDV (SEQ ID NO:_), and DV (SEQ ID NO:_). In some embodiments of the invention, a library may contain one or more of these sequences, allelic variations thereof, or encode an amino acid sequence at least about 99.9%, 99.5%, 99%, 98.5%, 98%, 97.5%, 97%, 96.5%, 96%, 95.5%, 95%, 94.5%, 10 94%, 93.5%, 93%, 92.5%, 92%, 91.5%, 91%, 90.5%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 77.5%, 75%, 73.5%, 70%, 65%, 60%, 60%, 55%, or 50% identical to one or more of these sequences.

In other embodiments of the invention, the H3-JH segment may comprise about 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or more amino acids. For example, the H3-JH segment of 15 JH14 (Table 20) has a length of three residues, while non-deleted JH6 has an H3-JH segment length of nine residues. The FRM4-JH region of the IGHJ segment begins with

the sequence WG(Q/R)G (SEQ ID NO:_) and corresponds to the portion of the IGHJ segment that makes up part of framework 4. In certain embodiments of the invention, as enumerated in Table 20, there are 28 H3-JH segments that are included in a library. In 20 certain other embodiments, libraries may be produced by utilizing about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21,22, 23,24, 25,26, 27, 28, 29, or 30 of the IGHJ segments enumerated above or in Table 20. 2.2.3. Design and Selection of the N1 and N2 Segment Repertoires 25 Terminal deoxynucleotidyl transferase (TdT) is a highly conserved enzyme from vertebrates that catalyzes the attachment of 5' triphosphates to the 3' hydroxyl group of single- or double-stranded DNA. Hence, the enzyme acts as a template-independent polymerase (Koiwai eta/., Nucleic Acids Res., 1986, 14: 5777; Basu eta/., Biochem. Biophys. Res. Comm., 1983, 111: 1105, each incorporated by reference in its entirety). 30 In vivo, TdT is responsible for the addition of nucleotides to the V-D and D-J junctions of antibody heavy chains (Alt and Baltimore, PNAS, 1982, 79: 4118; Collins et a/., J. Immunol., 2004, 172: 340, each incorporated by reference in its entirety). Specifically, -55 -

TdT is responsible for creating the N1 and N2 (non-templated) segments that flank the D (diversity) region. 2015201796 17 Mar 2017

In certain embodiments of the invention, the length and composition of the N1 and N2 segments are designed rationally, according to statistical biases in amino acid usage found in naturally occurring N1 and N2 segments in human antibodies. One embodiment of a library produced via this method is described in Example 5. According to data compiled from human databases (Jackson et al., J. Immunol Methods, 2007, 324: 26, incorporated by reference in its entirety), there are an average of 3.02 amino acid insertions for N1 and 2.4 amino acid insertions for N2, not taking into account insertions of two nucleotides or less (Figure 2). In certain embodiments of the invention, N1 and N2 segments are restricted to lengths of zero to three amino acids. In other embodiments of the invention, N1 and N2 may be restricted to lengths of less than about 4, 5, 6, 7, 8, 9, or 10 amino acids.

In some embodiments of the invention, the composition of these sequences may be chosen according to the frequency of occurrence of particular amino acids in the N1 and N2 sequences of natural human antibodies (for examples of this analysis, see, Tables 21 to 23, in Example 5). In certain embodiments of the invention, the eight most commonly occurring amino acids in these regions (i.e., G, R, S, P, L, A, T, and V) are used to design the synthetic N1 and N2 segments. In other embodiments of the invention about the most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 most commonly occurring amino acids may be used in the design of the synthetic N1 and N2 segments. In still other embodiments, all 20 amino acids may be used in these segments. Finally, while it is possible to base the designed composition of the N1 and N2 segments of the invention on the composition of naturally occurring N1 and N2 segments, this is not a requirement. The N1 and N2 segments may comprise amino acids selected from any group of amino acids, or designed according to other criteria considered for the design of a library of the invention. A person of ordinary skill in the art would readily recognize that the criteria used to design any portion of a library of the invention may vary depending on the application of the particular library. It is an aspect of the invention that it may be possible to produce a functional library through the use of N1 and N2 segments selected from any group of amino acids, no N1 or N2 segments, or the use of N1 and N2 segments with compositions other than those described herein. -56-

One important difference between the libraries of the current invention and other libraries known in the art is the consideration of the composition of naturally occurring duplet and triplet amino acid sequences during the design of the library. Table 23 shows the top twenty-five naturally occurring duplets in the N1 and N2 regions. Many of these 2015201796 09 Apr 2015 5 can be represented by the general formula (G/P)(G/R/S/P/L/A/V/T) (SEQ ID NO:_) or (R/S/L/A/V/T)(G/P) (SEQ ID NO:_). In certain embodiments of the invention, the synthetic N1 and N2 regions may comprise all of these duplets. In other embodiments, the library may comprise the top 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 most common naturally occurring N1 and/or N2 duplets. In 10 other embodiments of the invention, the libraries may include duplets that are less frequently occurring (i.e., outside of the top 25). The composition of these additional duplets or triplets could readily be determined, given the methods taught herein.

Finally, the data from the naturally occurring triplet N1 and N2 regions demonstrates that the naturally occurring N1 and N2 triplet sequences can often be 15 represented by the formulas (G)(G)(G/R/S/P/L/A/V/T) (SEQ ID NO:_), (G)(R/S/P/L/A/V/T)(G) (SEQ ID NO:_), or (R/S/P/L/A/V/T)(G)(G) (SEQ ID NO: _). In certain embodiments of the invention, the library may comprise the top 2, 3, 4, 5,6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21,22, 23,24, or 25 most commonly occurring N1 and/or N2 triplets. In other embodiments of the invention, the 20 libraries may include triplets that are less frequently occurring (i.e., outside of the top 25). The composition of these additional duplets or triplets could readily be determined, given the methods taught herein.

In certain embodiments of the invention, there are about 59 total N1 segments and about 59 total N2 segments used to create a library of CDRH3s. In other 25 embodiments of the invention, the number of N1 segments, N2 segments, or both is increased to about 141 (see, for example, Example 5). In other embodiments of the invention, one may select a total of about 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 1000, 104, or more 30 N1 and/or N2 segments for inclusion in a library of the invention.

One of ordinary skill in the art will readily recognize that, given the teachings of the instant specification, it is well within the realm of normal experimentation to extend the analysis detailed herein, for example, to generate additional rankings of naturally -57- occurring duplet and triplet (or higher order) N regions that extend beyond those presented herein (e.g., using sequence alignment, the SoDA algorithm, and any database of human sequences (Volpe et al., Bioinformatics, 2006, 22: 438-44, incorporated by reference in its entirety). An ordinarily skilled artisan would also recognize that, based 5 on the information taught herein, it is now possible to produce libraries that are more diverse or less diverse (i.e., more focused) by varying the number of distinct amino acid sequences used in the N1 pool and/or N2 pool. 2015201796 09 Apr 2015

As described above, many alternative embodiments are envisioned, in which the compositions and lengths of the N1 and N2 segments vary from those presented in the 10 Examples herein. In some embodiments, sub-stoichiometric synthesis of trinucleotides may be used for the synthesis of N1 andN2 segments. Sub-stoichiometric synthesis with trinucleotides is described in Knappik et al. (U.S. Patent No. 6,300,064, incorporated by reference in its entirety). The use of sub-stoichiometric synthesis would enable synthesis with consideration of the length variation in the N1 and N2 sequences. 15 In addition to the embodiments described above, a model of the activity of TdT may also be used to determine the composition of the N1 and N2 sequences in a library of the invention. For example, it has been proposed that the probability of incorporating a particular nucleotide base (A, C, G, T) on a polynucleotide, by the activity of TdT, is dependent on the type of base and the base that occurs on the strand directly preceding 20 the base to be added. Jackson et al., (J. Immunol. Methods, 2007, 324: 26, incorporated by reference in its entirety) have constructed a Markov model describing this process. In certain embodiments of the invention, this model may be used to determine the composition of the N1 and/or N2 segments used in libraries of the invention. Alternatively, the parameters presented in Jackson et al. could be further refined to 25 produce sequences that more closely mimic human sequences. 2.2.4. Design of a CDRH3 Library Using the Nl, DH, N2, and H3-JH Segments

The CDRH3 libraries of the invention comprise an initial amino acid (in certain exemplary embodiments, G, D, E) or lack thereof (designated herein as position 95), 30 followed by the N1, DH, N2, and H3-JH segments. Thus, in certain embodiments of the invention, the overall design of the CDRH3 libraries can be represented by the following formula: [G/D/E/-] - [N1]-[DH]-[N2]-[H3-JH], -58 -

While the compositions of each portion of a CDRH3 of a library of the invention are more fully described above, the composition of the tail presented above (G/D/E/-) is non-limiting, and that any amino acid (or no amino acid) can be used in this position. Thus, certain embodiments of the invention may be represented by the following 5 formula: 2015201796 09 Apr 2015 [X]-[N1]-[DH]-[N2]-[H3-JH], wherein [X] is any amino acid residue or no residue.

In certain embodiments of the invention, a synthetic CDRH3 repertoire is combined with selected VH chassis sequences and heavy chain constant regions, via 10 homologous recombination. Therefore, in certain embodiments of the invention, it may be necessary to include DNA sequences flanking the 5’ and 3’ ends of the synthetic CDRH3 libraries, to facilitate homologous recombination between the synthetic CDRH3 libraries and vectors containing the selected chassis and constant regions. In certain embodiments, the vectors also contain a sequence encoding at least a portion of the non-15 nibbled region of the IGHJ gene (i.e., FRM4-JH). Thus, a polynucleotide encoding an N-terminal sequence (e.g., CA(K/R/T)) may be added to the synthetic CDRH3 sequences, wherein the N-terminal polynucleotide is homologous with FRM3 of the chassis, while a polynucleotide encoding a C-terminal sequence (e.g., WG(Q/R)G) may be added to the synthetic CDRH3, wherein the C-terminal polynucleotide is homologous 20 with FRM4-JH. Although the sequence WG(Q/R)G is presented in this exemplary embodiment, additional amino acids, C-terminal to this sequence in FRM4-JH may also be included in the polynucleotide encoding the C-terminal sequence. The purpose of the polynucleotides encoding the N-terminal and C-terminal sequences, in this case, is to facilitate homologous recombination, and one of ordinary skill in the art would 25 recognize that these sequences may be longer or shorter than depicted below.

Accordingly, in certain embodiments of the invention, the overall design of the CDRH3 repertoire, including the sequences required to facilitate homologous recombination with the selected chassis, can be represented by the following formula (regions homologous with vector underlined): 30 CAIR/K/T1-rX1-rN11-IDH1-rN21-IH3-JH1-rWGtO/R)G1.

In other embodiments of the invention, the CDRH3 repertoire can be represented by the following formula, which excludes the T residue presented in the schematic above: -59- CArR/Kl-rXl-rNll-rDHl-rN21-rH3-JHl-rWG(0/R^)Gl. 2015201796 09 Apr 2015

References describing collections of V, D, and J genes include Scaviner et al., Exp. Clin, Immunogene!, 1999, 16: 243 and Ruiz etal., Exp. Clin. Immunogenet, 1999, 16: 173, each incorporated by reference in its entirety. 5 2.2.5. CDRH3 Length Distributions

As described throughout this application, in addition to accounting for the composition of naturally occurring CDRH3 segments, the instant invention also takes into account the length distribution of naturally occurring CDRH3 segments. Surveys 10 by Zemlin et al. (JMB, 2003, 334: 733, incorporated by reference in its entirety) and Lee et al. (Immunogenetics, 2006, 57: 917, incorporated by reference in its entirety) provide analyses of the naturally occurring CDRH3 lengths. These data show that about 95% of naturally occurring CDRH3 sequences have a length from about 7 to about 23 amino acids. In certain embodiments, the instant invention provides rationally designed 15 antibody libraries with CDRH3 segments which directly mimic the size distribution of naturally occurring CDRH3 sequences. In certain embodiments of the invention, the length of the CDRH3s may be about 2 to about 30, about 3 to about 35, about 7 to about 23, about 3 to about 28, about 5 to about 28, about 5 to about 26, about 5 to about 24, about 7 to about 24, about 7 to about 22, about 8 to about 19, about 9 to about 22, about 20 9 to about 20, about 10 to about 18, about 11 to about 20, about 11 to about 18, about 13 to about 18, or about 13 to about 16 residues in length.

In certain embodiments of the invention, the length distribution of a CDRH3 library of the invention may be defined based on the percentage of sequences within a certain length range. For example, in certain embodiments of the invention, CDRH3s 25 with a length of about 10 to about 18 amino acid residues comprise about 84% to about 94% of the sequences of a the library. In some embodiments, sequences within this length range comprise about 89% of the sequences of a library.

In other embodiments of the invention, CDRH3s with a length of about 11 to about 17 amino acid residues comprise about 74% to about 84% of the sequences of a 30 library. In some embodiments, sequences within this length range comprise about 79% of the sequences of a library.

In still other embodiments of the invention, CDRH3s with a length of about 12 to about 16 residues comprise about 57% to about 67% of the sequences of a library. In -60- some embodiments, sequences within this length range comprise about 62% of the sequences of a library. 2015201796 09 Apr 2015

In certain embodiments of the invention, CDRH3s with a length of about 13 to about 15 residues comprise about 35% to about 45% of the sequences of a library. In 5 some embodiments, sequences within this length range comprise about 40% of the sequences of a library. 2.3. Design of the Antibody Library CDRL3 Components

The CDRL3 libraries of the invention can be generated by one of several 10 approaches. The actual version of the CDRL3 library made and used in a particular embodiment of the invention will depend on objectives for the use of the library. More than one CDRL3 library may be used in a particular embodiment; for example, a library containing CDRH3 diversity, with kappa and lambda light chains is within the scope of the invention. 15 In certain embodiments of the invention, a CDRL3 library is a VKCDR3 (kappa) library and/or a VXCDR3 (lambda) library. The CDRL3 libraries described herein differ significantly from CDRL3 libraries in the art. First, they consider length variation that is consistent with what is observed in actual human sequences. Second, they take into consideration the fact that a significant portion of the CDRL3 is encoded by the IGLV 20 gene. Third, the patterns of amino acid variation within the IGLV gene-encoded CDRL3 portions are not stochastic and are selected based on depending on the identity of the IGLV gene. Taken together, the second and third distinctions mean that CDRL3 libraries that faithfully mimic observed patterns in human sequences cannot use a generic design that is independent of the chassis sequences in FRM1 to FRM3. Fourth, 25 the contribution of JL to CDRL3 is also considered explicitly, and enumeration of each amino acid residue at the relevant positions is based on the compositions and natural variations of the JL genes themselves.

As indicated above, and throughout the application, a unique aspect of the design of the libraries of the invention is the germline or “chassis-based” aspect, which is meant 30 to preserve more of the integrity and variability of actual human sequences. This is in contrast to other codon-based synthesis or degenerate oligonucleotide synthesis approaches that have been described in the literature and that aim to produce “one-size-fits-all” (e.g., consensus) libraries (e.g.„ Knappik, et al., J Mol Biol, 2000, 296: 57; -61 -

Akamatsu et al., J Immunol, 1993, 151:4651, each incorporated by reference in its entirety). 2015201796 09 Apr 2015

In certain embodiments of the invention, patterns of occurrence of particular amino acids at defined positions within VL sequences are determined by analyzing data 5 available in public or other databases, for example, the NCBI database (see, for example, GI numbers in Appendices A and B filed herewith). In certain embodiments of the invention, these sequences are compared on the basis of identity and assigned to families on the basis of the germline genes from which they are derived. The amino acid composition at each position of the sequence, in each germline family, may then be 10 determined. This process is illustrated in the Examples provided herein. 2.3.1. Minimalist VKCDR3 Libraries

In certain embodiments of the invention, the light chain CDR3 library is a VKCDR3 library. Certain embodiments of the invention may use only the most 15 common VKCDR3 length, nine residues; this length occurs in a dominant proportion (greater than about 70%) of human VKCDR3 sequences. In human VKCDR3 sequences of length nine, positions 89-95 are encoded by the IGKV gene and positions 96-97 are encoded by the IGKJ gene. Analysis of human kappa light chain sequences indicates that there are not strong biases in the usage of the IGKJ genes. Therefore, in 20 certain embodiments of the invention, each of the five the IGKJ genes can be represented in equal proportions to create a combinatorial library of (M VK chassis) x (5 JK genes), or a library of size Mx 5. However, in other embodiments of the invention, it may be desirable to bias IGKJ gene representation, for example to restrict the size of the library or to weight the library toward IGKJ genes known to have particular properties. 25 As described in Example 6.1, examination of the first amino acid encoded by the IGKJ gene (position 96) indicated that the seven most common residues found at this position are L, Y, R, W, F, P, and I. These residues cumulatively account for about 85% of the residues found in position 96 in naturally occurring kappa light chain sequences.

In certain embodiments of the invention, the amino acid residue at position 96 may be 30 one of these seven residues. In other embodiments of the invention, the amino acid at this position may be chosen from amongst any of the other 13 amino acid residues. In still other embodiments of the invention, the amino acid residue at position 96 may be chosen from amongst the top 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, -62- or 20 amino acids that occur at position 96, or even residues that never occur at position 96. Similarly, the occurrence of the amino acids selected to occupy position 96 may be equivalent or weighted. In certain embodiments of the invention, it may be desirable to include each of the amino acids selected for inclusion in position 96 at equivalent 5 amounts. In other embodiments of the invention, it may be desirable to bias the composition of position 96 to include particular residues more or less frequently than others. For example, as presented in Example 6.1, arginine occurs at position 96 most frequently when the IGKJ1 germline sequence is used. Therefore, in certain embodiments of the invention, it may be desirable to bias amino acid usage at position 10 96 according to the origin of the IGKJ germline sequence(s) and/or the IGKV germline 2015201796 09 Apr 2015 sequence(s) selected for representation in a library.

Therefore, in certain embodiments of the invention, a minimalist VKCDR3 library may be represented by one or more of the following amino acid sequences: 15 [VK_Chassis]-[L3-VK]-[F/L/I/R/W/Y/P]-[JK*] [ VK_Chassis] - [L3-VK] - [X] - [ JK*]

In these schematic exemplary sequences, VKChassis represents any VK chassis 20 selected for inclusion in a library of the invention (e.g., see Table 11). Specifically, VK Chassis comprises about Kabat residues 1 to 88 of a selected IGKV sequence. L3-VK represents the portion of the VKCDR3 encoded by the chosen IGKV gene (in this embodiment, Kabat residues 89-95). F, L, I, R, W, Y, and P are the seven most commonly occurring amino acids at position 96 of VKCDR3s with length nine, X is any 25 amino acid, and JK* is an IGKJ amino acid sequence without the N-terminal residue (i.e., the N-terminal residue is substituted with F, L, I, R, W, Y, P, or X). Thus, in one possible embodiment of the minimalist VKCDR3 library, 70 members could be produced by utilizing 10 VK chassis, each paired with its respective L3-VK, 7 amino acids at position 96 (i.e., X), and one JK* sequence. Another embodiment of the library 30 may have 350 members, produced by combining 10 VK chassis, each paired with its respective L3-VK, with 7 amino acids at position 96, and all 5 JK* genes. Still another embodiment of the library may have 1,125 members, produced by combining 15 VK chassis, each paired with its respective H3-JK, with 15 amino acids at position 96 and all -63 - 5 JK* genes, and so on. A person of ordinary skill in the art will readily recognize that many other combinations are possible. Moreover, while it is believed that maintaining the pairing between the VK chassis and the L3-VK results in libraries that are more similar to human kappa light chain sequences in composition, the L3-VK regions may 5 also be combinatorially varied with different VK chassis regions, to create additional diversity. 2015201796 09 Apr 2015 2.3.2. VKCDR3 Libraries of About 105 Complexity

While the dominant length of VKCDR3 sequences in humans is about nine 10 amino acids, other lengths appear at measurable frequencies that cumulatively approach almost about 30% of VKCDR3 sequences. In particular, VKCDR3 of lengths 8 and 10 represent about 8.5% and about 16%, respectively, of VKCDR3 lengths in representative samples (Example 6.2; Figure 3). Thus, more complex VKCDR3 libraries may include CDR lengths of 8, 10, and 11 amino acids. Such libraries could 15 account for a greater percentage of the length distribution observed in collections of human VKCDR3 sequences, or even introduce VKCDR3 lengths that do not occur frequently in human VKCDR3 sequences (e.g., less than eight residues or greater than 11 residues).

The inclusion of a diversity of kappa light chain length variations in a library of 20 the invention also enables one to include sequence variability that occurs outside of the amino acid at the VK-JK junction (i.e., position 96, described above). In certain embodiments of the invention, the patterns of sequence variation within the VK, and/or JK segments can be determined by aligning collections of sequences derived from particular germline sequences. In certain embodiments of the invention, the frequency 25 of occurrence of amino acid residues within VKCDR3 can be determined by sequence alignments (e.g., see Example 6.2 and Table 30). In some embodiments of the invention, this frequency of occurrence may be used to introduce variability into the VK Chassis, L3-VK and/or JK segments that are used to synthesize the VKCDR3 libraries. In certain embodiments of the invention, the top 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 30 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids that occur at any particular position in a naturally occurring repertoire may be included at that position in a VKCDR3 library of the invention. In certain embodiments of the invention, the percent occurrence of any amino acid at any particular position within the VKCDR3 or a VK light chain may be -64- about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In certain embodiments of the invention, the percent occurrence of any amino acid at any position within a VKCDR3 or kappa light chain library of the invention may be within at 5 least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 140%, 160%, 180%, or 200% of the percent occurrence of any amino acid at any position within a naturally occurring VKCDR3 or kappa light chain domain. 2015201796 09 Apr 2015

In some embodiments of the invention, a VKCDR3 library may be synthesized 10 using degenerate oligonucleotides (see Table 31 for IUPAC base symbol definitions). In some embodiments of the invention, the limits of oligonucleotide synthesis and the genetic code may require the inclusion of more or fewer amino acids at a particular position in the VKCDR3 sequences. An illustrative embodiment of this approach is provided in Example 6.2. 15 2.3.3. More Complex VKCDR3 Libraries 20 30

The limitations inherent in using the genetic code and degenerate oligonucleotide synthesis may, in some cases, require the inclusion of more or fewer amino acids at a particular position within VKCDR3 (e.g., Example 6.2, Table 32), in comparison to those amino acids found at that position in nature. This limitation can be overcome through the use of a codon-based synthesis approach (Vimekas et al. Nucleic Acids Res., 1994, 22: 5600, incorporated by reference in its entirety), which enables precise synthesis of oligonucleotides encoding particular amino acids and a finer degree of control over the proportion of any particular amino acid incorporated at any position. Example 6.3 describes this approach in greater detail.

In some embodiments of the invention, a codon-based synthesis approach may be used to vary the percent occurrence of any amino acid at any particular position within the VKCDR3 or kappa light chain. In certain embodiments, the percent occurrence of any amino acid at any position in a VKCDR3 or kappa light chain sequence of the library may be about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments of the invention, the percent occurrence of any amino acid at any position may be about 1%, 2%, 3%, or 4%. In certain -65- embodiments of the invention, the percent occurrence of any amino acid at any position within a VKCDR3 or kappa light chain library of the invention may be within at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 140%, 160%, 180%, or 200% of the percent occurrence 5 of any amino acid at any position within a naturally occurring VKCDR3 or kappa light chain domain. 2015201796 09 Apr 2015

In certain embodiments of the invention, the VKCDR3 (and any other sequence used in the library, regardless of whether or not it is part of VKCDR3) may be altered to remove undesirable amino acid motifs. For example, peptide sequences with the pattern 10 N-X-(S or T)-Z, where X and Z are different from P, will undergo post-translational modification (N-linked glycosylation) in a number of expression systems, including yeast and mammalian cells. In certain embodiments of the invention, the introduction of N residues at certain positions may be avoided, so as to avoid the introduction of N-linked glycosylation sites. In some embodiments of the invention, these modifications 15 may not be necessary, depending on the organism used to express the library and the culture conditions. However, even in the event that the organism used to express libraries with potential N-linked glycosylation sites is incapable of N-linked glycosylation (e.g., bacteria), it may still be desirable to avoid N-X-(S/T) sequences, as the antibodies isolated from such libraries may be expressed in different systems (e.g., 20 yeast, mammalian cells) later (e.g., toward clinical development), and the presence of carbohydrate moieties in the variable domains, and the CDRs in particular, may lead to unwanted modifications of activity.

In certain embodiments of the invention, it may be preferable to create the individual sub-libraries of different lengths (e.g., one or more of lengths 5, 6, 7, 8, 9, 10, 25 11, or more) separately, and then mix the sub-libraries in proportions that reflect the length distribution of VKCDR3 in human sequences; for example, in ratios approximating the 1:9:2 distribution that occurs in natural VKCDR3 sequences of lengths 8, 9, and 10 (see Figure 3). In other embodiments, it may be desirable to mix these sub-libraries at ratios that are different from the distribution of lengths in natural 30 VKCDR3 sequences, for example, to produce more focused libraries or libraries with particular properties. 2.3.4. VACDR3 Libraries -66-

The principles used to design the minimalist VXCDR3 libraries of the invention are similar to those enumerated above, for the VKCDR3 libraries, and are explained in more detail in the Examples. One difference between the VXCDR3 libraries of the invention and the VKCDR3 libraries of the invention is that, unlike the IGKV genes, the 5 contribution of the IGVX genes to CDRL3 (i.e., L3-VX) is not constrained to a fixed number of amino acid residues. Therefore, while the combination of the VK (including L3-VK) and JK segments, with inclusion of position 96, yields CDRL3 with a length of only 9 residues, length variation may be obtained within a VXCDR3 library even when only the νλ (including L3-VX) and Ιλ segments are considered. 2015201796 09 Apr 2015 10 As for the VKCDR3 sequences, additional variability may be introduced into the VXCDR3 sequences via the same methods outlined above, namely determining the frequency of occurrence of particular residues within VXCDR3 sequences and synthesizing the oligonucleotides encoding the desired compositions via degenerate oligonucleotide synthesis or trinucleotides-based synthesis. 15 2.4. Synthetic Antibody Libraries

In certain embodiments of the invention, both the heavy and light chain chassis sequences and the heavy and light chain CDR3 sequences are synthetic. The polynucleotide sequences of the instant invention can be synthesized by various 20 methods. For example, sequences can be synthesized by split pool DNA synthesis as described in Feldhaus et al., Nucleic Acids Research, 2000, 28: 534; Omstein et al., Biopolymers, 1978, 17: 2341; and Brenner and Lemer, PNAS, 1992, 87: 6378 (each of which is incorporated by reference in its entirety).

In some embodiments of the invention, cassettes representing the possible V, D, 25 and J diversity found in the human repertoire, as well as junctional diversity, are

synthesized de novo either as double-stranded DNA oligonucleotides, single-stranded DNA oligonucleotides representative of the coding strand, or single-stranded DNA oligonucleotides representative of the non-coding strand. These sequences can then be introduced into a host cell along with an acceptor vector containing a chassis sequence 30 and, in some cases a portion of FRM4 and a constant region. No primer-based PCR amplification from mammalian cDNA or mRNA or template-directed cloning steps from mammalian cDNA or mRNA need be employed. -67- 2.5. Construction of Libraries by Yeast Homologous Recombination 2015201796 09 Apr 2015

In certain embodiments, the present invention exploits the inherent ability of yeast cells to facilitate homologous recombination at high efficiency. The mechanism of homologous recombination in yeast and its applications are briefly described below. 5 As an illustrative embodiment, homologous recombination can be carried out in, for example, Saccharomyces cerevisiae, which has genetic machinery designed to carry out homologous recombination with high efficiency. Exemplary S. cerevisiae strains include EM93, CEN.PK2, RM11-la, YJM789, and BJ5465. This mechanism is believed to have evolved for the purpose of chromosomal repair, and is also called “gap 10 repair” or “gap filling”. By exploiting this mechanism, mutations can be introduced into specific loci of the yeast genome. For example, a vector carrying a mutant gene can contain two sequence segments that are homologous to the 5' and 3' open reading frame (ORF) sequences of a gene that is intended to be interrupted or mutated. The vector may also encode a positive selection marker, such as a nutritional enzyme allele (e.g., ETRA3) 15 and/or an antibiotic resistant marker (e.g., Geneticin / G418), flanked by the two homologous DNA segments. Other selection markers and antibiotic resistance markers are known to one of ordinary skill in the art. In some embodiments of the invention, this vector (e.g., a plasmid) is linearized and transformed into the yeast cells. Through homologous recombination between the plasmid and the yeast genome, at the two 20 homologous recombination sites, a reciprocal exchange of the DNA content occurs between the wild type gene in the yeast genome and the mutant gene (including the selection marker gene(s)) that is flanked by the two homologous sequence segments. By selecting for the one or more selection markers, the surviving yeast cells will be those cells in which the wild-type gene has been replaced by the mutant gene (Pearson et al., 25 Yeast, 1998, 14: 391, incorporated by reference in its entirety). This mechanism has been used to make systematic mutations in all 6,000 yeast genes, or open reading frames (ORFs), for functional genomics studies. Because the exchange is reciprocal, a similar approach has also been used successfully to clone yeast genomic DNA fragments into a plasmid vector (Iwasaki et al., Gene, 1991, 109: 81, incorporated by reference in its 30 entirety).

By utilizing the endogenous homologous recombination machinery present in yeast, gene fragments or synthetic oligonucleotides can also be cloned into a plasmid vector without a ligation step. In this application of homologous recombination, a target -68- gene fragment (i.e., the fragment to be inserted into a plasmid vector, e.g., a CDR3) is obtained (e.g., by oligonucleotides synthesis, PCR amplification, restriction digestion out of another vector, etc.). DNA sequences that are homologous to selected regions of the plasmid vector are added to the 5' and 3’ ends of the target gene fragment. These 5 homologous regions may be fully synthetic, or added via PCR amplification of a target gene fragment with primers that incorporate the homologous sequences. The plasmid vector may include a positive selection marker, such as a nutritional enzyme allele (e.g., URA3), or an antibiotic resistance marker (e.g., Geneticin / G418). The plasmid vector is then linearized by a unique restriction cut located in-between the regions of sequence 10 homology shared with the target gene fragment, thereby creating an artificial gap at the cleavage site. The linearized plasmid vector and the target gene fragment flanked by sequences homologous to the plasmid vector are co-transformed into a yeast host strain. The yeast is then able to recognize the two stretches of sequence homology between the vector and target gene fragment and facilitate a reciprocal exchange of DNA content 15 through homologous recombination at the gap. As a consequence, the target gene fragment is inserted into the vector without ligation. 2015201796 09 Apr 2015

The method described above has also been demonstrated to work when the target gene fragments are in the form of single stranded DNA, for example, as a circular M13 phage derived form, or as single stranded oligonucleotides (Simon and Moore, Mol. Cell 20 Biol., 1987, 7: 2329; Ivanov etal., Genetics, 1996, 142: 693; and DeMarini etal., 2001, 30: 520., each incorporated by reference in its entirety). Thus, the form of the target that can be recombined into the gapped vector can be double stranded or single stranded, and derived from chemical synthesis, PCR, restriction digestion, or other methods.

Several factors may influence the efficiency of homologous recombination in 25 yeast. For example, the efficiency of the gap repair is correlated with the length of the homologous sequences flanking both the linearized vector and the target gene. In certain embodiments, about 20 or more base pairs may be used for the length of the homologous sequence, and about 80 base pairs may give a near-optimized result (Hua et al., Plasmid, 1997, 38: 91; Raymond et al., Genome Res., 2002, 12: 190, each 30 incorporated by reference in its entirety). In certain embodiments of the invention, at least about 5, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 187, 190, or 200 homologous base pairs may be used to facilitate -69- recombination. In other embodiments, between about 20 and about 40 base pairs are utilized. In addition, the reciprocal exchange between the vector and gene fragment is strictly sequence-dependent, i.e. it does not cause a frame shift. Therefore, gap-repair cloning assures the insertion of gene fragments with both high efficiency and precision. 2015201796 09 Apr 2015 5 The high efficiency makes it possible to clone two, three, or more targeted gene fragments simultaneously into the same vector in one transformation attempt (Raymond et al., Biotechniques, 1999, 26: 134, incorporated by reference in its entirety).

Moreover, the nature of precision sequence conservation through homologous recombination makes it possible to clone selected genes or gene fragments into 10 expression or fusion vectors for direct functional examination (El-Deiry et al., Nature Genetics, 1992, 1: 4549; Ishioka et al., PNAS, 1997, 94: 2449, each incorporated by reference in its entirety).

Libraries of gene fragments have also been constructed in yeast using homologous recombination. For example, a human brain cDNA library was constructed 15 as a two-hybrid fusion library in vector pJG4-5 (Guidotti and Zervos, Yeast, 1999, 15: 715, incorporated by reference in its entirety). It has also been reported that a total of 6,000 pairs of PCR primers were used for amplification of 6,000 known yeast ORFs for a study of yeast genomic protein interactions (Hudson et al., Genome Res., 1997, 7: 1169, incorporated by reference in its entirety). In 2000, Uetz et al. conducted a 20 comprehensive analysis-of protein-protein interactions in Saccharomyces cerevisiae (Uetz et al., Nature, 2000, 403: 623, incorporated by reference in its entirety). The protein-protein interaction map of the budding yeast was studied by using a comprehensive system to examine two-hybrid interactions in all possible combinations between the yeast proteins (Ito et al., PNAS, 2000, 97: 1143, incorporated by reference 25 in its entirety), and the genomic protein linkage map of Vaccinia virus was studied using this system (McCraith et al., PNAS, 2000, 97: 4879, incorporated by reference in its entirety).

In certain embodiments of the invention, a synthetic CDR3 (heavy or light chain) may be joined by homologous recombination with a vector encoding a heavy or light 30 chain chassis, a portion of FRM4, and a constant region, to form a full-length heavy or light chain. In certain embodiments of the invention, the homologous recombination is performed directly in yeast cells. In some embodiments, the method comprises: (a) transforming into yeast cells: -70- (i) a linearized vector encoding a heavy or light chain chassis, a portion of FRM4, and a constant region, wherein the site of linearization is between the end of FRM3 of the chassis and the beginning of the constant region; and 2015201796 09 Apr 2015 5 (ii) a library of CDR3 insert nucleotide sequences that are linear and double stranded, wherein each of the CDR3 insert sequences comprises a nucleotide sequence encoding CDR3 and 5'- and 3'-flanking sequences that are sufficiently homologous to the termini of the vector of (i) at the site of linearization to enable homologous recombination to occur 10 between the vector and the library of CDR3 insert sequences; and (b) allowing homologous recombination to occur between the vector and the CDR3 insert sequences in the transformed yeast cells, such that the CDR3 insert sequences are incorporated into the vector, to produce a vector encoding full-length heavy chain or light chain. 15 As specified above, the CDR3 inserts may have a 5’ flanking sequence and a 3' flanking sequence that are homologous to the termini of the linearized vector. When the CDR3 inserts and the linearized vectors are introduced into a host cell, for example, a yeast cell, the "gap" (the linearization site) created by linearization of the vector is filled by the CDR3 fragment insert through recombination of the homologous sequences at the 20 5' and 3' termini of these two linear double-stranded DNAs (i.e., the vector and the insert). Through this event of homologous recombination, libraries of circular vectors encoding full-length heavy or light chains comprising variable CDR3 inserts is generated. Particular instances of these methods are presented in the Examples. Subsequent analysis may be carried out to determine the efficiency of 25 homologous recombination that results in correct insertion of the CDR3 sequences into the vectors. For example, PCR amplification of the CDR3 inserts directly from selected yeast clones may reveal how many clones are recombinant. In certain embodiments, libraries with minimum of about 90% recombinant clones are utilized. In certain other embodiments libraries with a minimum of about 1%, 5% 10%, 15%, 20%, 25%, 30%, 30 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% recombinant clones are utilized. The same PCR amplification of selected clones may also reveal the insert size. -71 -

To verify the sequence diversity of the inserts in the selected clones, a PCR amplification product with the correct size of insert may be “fingerprinted” with restriction enzymes known to cut or not cut within the amplified region. From a gel electrophoresis pattern, it may be determined whether the clones analyzed are of the 5 same identity or of the distinct or diversified identity. The PCR products may also be sequenced directly to reveal the identity of inserts and the fidelity of the cloning procedure, and to prove the independence and diversity of the clones. Figure 1 depicts a schematic of recombination between a fragment (e.g., CDR3) and a vector (e.g., comprising a chassis, portion of FRM4, and constant region) for the construction of a 10 library. 2015201796 09 Apr 2015 2.6. Expression and Screening Systems

Libraries of polynucleotides generated by any of the techniques described herein, or other suitable techniques, can be expressed and screened to identify antibodies having 15 desired structure and/or activity. Expression of the antibodies can be carried out, for example, using cell-free extracts (and e.g., ribosome display), phage display, prokaryotic cells (e.g., bacterial display), or eukaryotic cells (e.g., yeast display). In certain embodiments of the invention, the antibody libraries are expressed in yeast.

In other embodiments, the polynucleotides are engineered to serve as templates 20 that can be expressed in a cell-free extract. Vectors and extracts as described, for example in U.S. Patent Nos. 5,324,637; 5,492,817; 5,665,563, (each incorporated by reference in its entirety) can be used and many are commercially available. Ribosome display and other cell-free techniques for linking a polynucleotide (/. e., a genotype) to a polypeptide (i.e., a phenotype) can be used, e.g., Profusion™ (see, e.g., U.S. Patent Nos. 25 6,348,315; 6,261,804; 6,258,558; and 6,214,553, each incorporated by reference in its entirety).

Alternatively, the polynucleotides of the invention can be expressed in an E. coli expression system, such as that described by Pluckthun and Skerra. (Meth. Enzymol., 1989, 178: 476; Biotechnology, 1991, 9: 273, each incorporated by reference in its 30 entirety). The mutant proteins can be expressed for secretion in the medium and/or in the cytoplasm of the bacteria, as described by Better and Horwitz, Meth. Enzymol., 1989, 178: 476, incorporated by reference in its entirety. In some embodiments, the single domains encoding VH and VL are each attached to the 3’ end of a sequence -72- encoding a signal sequence, such as the ompA, phoA or pelB signal sequence (Lei et al., J. Bacteriol., 1987, 169: 4379, incorporated by reference in its entirety). These gene fusions are assembled in a dicistronic construct, so that they can be expressed from a single vector, and secreted into the periplasmic space of E. coli where they will refold 5 and can be recovered in active form. (Skerra et al., Biotechnology, 1991, 9: 273, 2015201796 09 Apr 2015 incorporated by reference in its entirety). For example, antibody heavy chain genes can be concurrently expressed with antibody light chain genes to produce antibodies or antibody fragments.

In other embodiments of the invention, the antibody sequences are expressed on 10 the membrane surface of a prokaryote, e.g., E. coli, using a secretion signal and lipidation moiety as described, e.g., in US20040072740; US20030100023; and US20030036092 (each incorporated by reference in its entirety).

Higher eukaryotic cells, such as mammalian cells, for example myeloma cells (e.g., NS/0 cells), hybridoma cells, Chinese hamster ovary (CHO), and human 15 embryonic kidney (HEK) cells, can also be used for expression of the antibodies of the invention. Typically, antibodies expressed in mammalian cells are designed to be secreted into the culture medium, or expressed on the surface of the cell. The antibody or antibody fragments can be produced, for example, as intact antibody molecules or as individual VH and VL fragments, Fab fragments, single domains, or as single chains 20 (scFv) (Huston et al., PNAS, 1988, 85: 5879, incorporated by reference in its entirety).

Alternatively, antibodies can be expressed and screened by anchored periplasmic expression (APEx 2-hybrid surface display), as described, for example, in Jeong et al., PNAS, 2007, 104: 8247 (incorporated by reference in its entirety) or by other anchoring methods as described, for example, in Mazor et al., Nature Biotechnology, 2007, 25: 563 25 (incorporated by reference in its entirety).

In other embodiments of the invention, antibodies can be selected using mammalian cell display (Ho et al., PNAS, 2006, 103: 9637, incorporated by reference in its entirety).

The screening of the antibodies derived from the libraries of the invention can be 30 carried out by any appropriate means. For example, binding activity can be evaluated by standard immunoassay and/or affinity chromatography. Screening of the antibodies of the invention for catalytic function, e.g., proteolytic function can be accomplished using a standard assays, e.g., the hemoglobin plaque assay as described in U.S. Patent No. -73- 5,798,208 (incorporated by reference in its entirety). Determining the ability of candidate antibodies to bind therapeutic targets can be assayed in vitro using, e.g., a BIACORE™ instrument, which measures binding rates of an antibody to a given target or antigen based on surface plasmon resonance. In vivo assays can be conducted using 5 any of a number of animal models and then subsequently tested, as appropriate, in humans. Cell-based biological assays are also contemplated. 2015201796 09 Apr 2015

One aspect of the instant invention is the speed at which the antibodies of the library can be expressed and screened. In certain embodiments of the invention, the antibody library can be expressed in yeast, which have a doubling time of less than 10 about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In some embodiments, the doubling times are about 1 to about 3 hours, about 2 to about 4, about 3 to about 8 hours, about 3 to about 24, about 5 to about 24, about 4 to about 6 about 5 to about 22, about 6 to about 8, about 7 to about 22, about 8 to about 10 hours, about 7 to about 20, about 9 to about 20, about 9 to about 18, about 11 to about 18, about 15 11 to about 16, about 13 to about 16, about 16 to about 20, or about 20 to about 30 hours. In certain embodiments of the invention, the antibody library is expressed in yeast with a doubling time of about 16 to about 20 hours, about 8 to about 16 hours, or about 4 to about 8 hours. Thus, the antibody library of the instant invention can be expressed and screened in a matter of hours, as compared to previously known 20 techniques which take several days to express and screen antibody libraries. A limiting step in the throughput of such screening processes in mammalian cells is simply the time required to iteratively regrow populations of isolated cells, which, in some cases, have doubling times greater than the doubling times of the yeast used in the current invention.

In certain embodiments of the invention, the composition of a library may be 25 defined after one or more enrichment steps (for example by screening for antigen binding, or other properties). For example, a library with a composition comprising about x% sequences or libraries of the invention may be enriched to contain about 2x%, 3x%, 4x%, 5x%, 6x%, 7x%, 8x%, 9x%, 10x%, 20x%, 25x%, 40x%, 50x%, 60x% 75x%, 80x%, 90x%, 95x%,or 99x% sequences or libraries of the invention, after one or 30 more screening steps. In other embodiments of the invention, the sequences or libraries of the invention may be enriched about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1,000-fold, or more, relative to their occurrence prior to the one or more enrichment steps. In certain embodiments of the invention, a library -74- may contain at least a certain number of a particular type of sequence(s), such as CDRH3s, CDRL3s, heavy chains, light chains, or whole antibodies (e.g., at least about ΙΟ3, ΙΟ4, ΙΟ5, ΙΟ6, ΙΟ7, ΙΟ8, ΙΟ9, ΙΟ10, ΙΟ11, ΙΟ12, ΙΟ13, ΙΟ14, ΙΟ15, ΙΟ16, ΙΟ17, ΙΟ18, 1019, or 102°). In certain embodiments, these sequences may be enriched during one or more 5 enrichment steps, to provide libraries comprising at least about ΙΟ2, ΙΟ3, ΙΟ4, ΙΟ5, 106, ΙΟ7, ΙΟ8, ΙΟ9, ΙΟ10, ΙΟ11, ΙΟ12, ΙΟ13, ΙΟ14, ΙΟ15, ΙΟ16, ΙΟ17, 1018, or 1019 of the respective sequence(s). 2015201796 09 Apr 2015 2.7. Mutagenesis Approaches for Affinity Maturation 10 As described above, antibody leads can be identified through a selection process that involves screening the antibodies of a library of the invention for binding to one or more antigens, or for a biological activity. The coding sequences of these antibody leads may be further mutagenized in vitro or in vivo to generate secondary libraries with diversity introduced in the context of the initial antibody leads. The mutagenized 15 antibody leads can then be further screened for binding to target antigens or biological activity, in vitro or in vivo, following procedures similar to those used for the selection of the initial antibody lead from the primary library. Such mutagenesis and selection of primary antibody leads effectively mimics the affinity maturation process naturally occurring in a mammal that produces antibodies with progressive increases in the 20 affinity to an antigen. In one embodiment of the invention, only the CDRH3 region is mutagenized. In another embodiment of the invention, the whole variable region is mutagenized. In other embodiments of the invention one or more of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, andICDRL3 may be mutagenized. In some embodiments of the invention, “light chain shuffling” may be used as part of the affinity maturation 25 protocol. In certain embodiments, this may involve pairing one or more heavy chains with a number of light chains, to select light chains that enhance the affinity and/or biological activity of an antibody. In certain embodiments of the invention, the number of light chains to which the one or more heavy chains can be paired is at least about 2, 5, 10, 100, 1000, 104, 105, ΙΟ6, ΙΟ7, ΙΟ8, 109, or 1010. In certain embodiments of the 30 invention, these light chains are encoded by plasmids. In other embodiments of the invention, the light chains may be integrated into the genome of the host cell.

The coding sequences of the antibody leads may be mutagenized by a wide variety of methods. Examples of methods of mutagenesis include, but are not limited to -75 - site-directed mutagenesis, error-prone PCR mutagenesis, cassette mutagenesis, and random PCR mutagenesis. Alternatively, oligonucleotides encoding regions with the desired mutations can be synthesized and introduced into the sequence to be mutagenized, for example, via recombination or ligation. 2015201796 09 Apr 2015 5 Site-directed mutagenesis or point mutagenesis may be used to gradually change the CDR sequences in specific regions. This may be accomplished by using oligonucleotide-directed mutagenesis or PCR. For example, a short sequence of an antibody lead may be replaced with a synthetically mutagenized oligonucleotide in either the heavy chain or light chain region, or both. The method may not be efficient 10 for mutagenizing large numbers of CDR sequences, but may be used for fine tuning of a particular lead to achieve higher affinity toward a specific target protein.

Cassette mutagenesis may also be used to mutagenize the CDR sequences in specific regions. In a typical cassette mutagenesis, a sequence block, or a region, of a single template is replaced by a completely or partially randomized sequence. However, 15 the maximum information content that can be obtained may be statistically limited by the number of random sequences of the oligonucleotides. Similar to point mutagenesis, this method may also be used for fine tuning of a particular lead to achieve higher affinity towards a specific target protein.

Error-prone PCR, or "poison" PCR, may be used to mutagenize the CDR 20 sequences by following protocols described in Caldwell and Joyce, PCR Methods and Applications, 1992, 2: 28; Leung et al., Technique, 1989, 1:11; Shafikhani etal., Biotechniques, 1997, 23: 304; and Stemmer etal., PNAS, 1994, 91: 10747 (each of which is incorporated by reference in its entirety).

Conditions for error prone PCR may include (a) high concentrations of Mn2+

25 (e.g., about 0.4 to about 0.6 mM) that efficiently induces malfunction of Taq DNA polymerase; and (b) a disproportionally high concentration of one nucleotide substrate (e.g., dGTP) in the PCR reaction that causes incorrect incorporation of this high concentration substrate into the template and produces mutations. Additionally, other factors such as, the number of PCR cycles, the species of DNA polymerase used, and the 30 length of the template, may affect the rate of misincorporation of "wrong" nucleotides into the PCR product. Commercially available kits may be utilized for the mutagenesis of the selected antibody library, such as the "Diversity PCR random mutagenesis kit" (CLONTECH™). -76-

The primer pairs used in PCR-based mutagenesis may, in certain embodiments, include regions matched with the homologous recombination sites in the expression vectors. This design allows facile re-introduction of the PCR products back into the heavy or light chain chassis vectors, after mutagenesis, via homologous recombination. 2015201796 09 Apr 2015 5 Other PCR-based mutagenesis methods can also be used, alone or in conjunction with the error prone PCR described above. For example, the PCR amplified CDR segments may be digested with DNase to create nicks in the double stranded DNA.

These nicks can be expanded into gaps by other exonucleases such as Bal 31. The gaps may then be filled by random sequences by using DNA Klenow polymerase at a low 10 concentration of regular substrates dGTP, dATP, dTTP, and dCTP with one substrate (e.g., dGTP) at a disproportionately high concentration. This fill-in reaction should produce high frequency mutations in the filled gap regions. These method of DNase digestion may be used in conjunction with error prone PCR to create a high frequency of mutations in the desired CDR segments. 15 The CDR or antibody segments amplified from the primary antibody leads may also be mutagenized in vivo by exploiting the inherent ability of mutation in pre-B cells. The Ig genes in pre-B cells are specifically susceptible to a high-rate of mutation. The Ig promoter and enhancer facilitate such high rate mutations in a pre-B cell environment while the pre-B cells proliferate. Accordingly, CDR gene segments may be cloned into 20 a mammalian expression vector that contains a human Ig enhancer and promoter. This construct may be introduced into a pre-B cell line, such as 38B9, which allows the mutation of the VH and VL gene segments naturally in the pre-B cells (Liu and Van Ness, Mol. Immunol., 1999, 36: 461, incorporated by reference in its entirety). The mutagenized CDR segments can be amplified from the cultured pre-B cell line and re-25 introduced back into the chassis-containing vector(s) via, for example, homologous recombination.

In some embodiments, a CDR “hit” isolated from screening the library can be resynthesized, using degenerate codons or trinucleotides, and re-cloned into the heavy or light chain vector using gap repair. 30 3. Library Sampling

In certain embodiments of the invention, a library of the invention comprises a designed, non-random repertoire wherein the theoretical diversity of particular -77- components of the library (for example, CDRH3), but not necessarily all components or the entire library, can be over-sampled in a physical realization of the library, at a level where there is a certain degree of statistical confidence (e.g., 95%) that any given member of the theoretical library is present in the physical realization of the library at 5 least at a certain frequency (e.g., at least once, twice, three times, four times, five times, 2015201796 09 Apr 2015 or more) in the library.

In a library, it is generally assumed that the number of copies of a given clone obeys a Poisson probability distribution (see Feller, W. An Introduction to Probability Theory and Its Applications, 1968, Wiley New York, incorporated by reference in its 10 entirety). The probability of a Poisson random number being zero, corresponding to the probability of missing a given component member in an instance of a library (see below), is e~N, where N is the average of the random number. For example, if there are 106 possible theoretical members of a library and a physical realization of the library has 107 members, with an equal probability of each member of the theoretical library being 15 sampled, then the average number of times that each member occurs in the physical realization of the library is 107/106 =10, and the probability that the number of copies of a given member is zero is e v = e 10 = 0.000045; or a 99.9955% chance that there is at least one copy of any of the 106 theoretical members in this 10X oversampled library.

For a 2.3X oversampled library one is 90% confident that a given component is present. 20 For a 3X oversampled library one is 95% confident that a given component is present. For a 4.6X oversampled library one is 99% confident a given clone is present, and so on.

Therefore, if M is the maximum number of theoretical library members that can be feasibly physically realized, then M!3 is the maximum theoretical repertoire size for which one can be 95% confident that any given member of the theoretical library will be 25 sampled. It is important to note that there is a difference between a 95% chance that a given member is represented and a 95% chance that every possible member is represented. In certain embodiments, the instant invention provides a rationally designed library with diversity so that any given member is 95% likely to be represented in a physical realization of the library. In other embodiments of the invention, the library 30 is designed so that any given member is at least about 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 99.9% likely to be represented in a physical realization of the library. For a review, see, -78- e.g., Firth and Patrick, Biomol. Eng., 2005, 22: 105, and Patrick et al., Protein Engineering, 2003, 16: 451, each of which is incorporated by reference in its entirety. 2015201796 09 Apr 2015

In certain embodiments of the invention, a library may have a theoretical total diversity of X unique members and the physical realization of the theoretical total 5 diversity may contain at least about IX, 2X, 3X, 4X, 5X, 6X, 7X, 8X 9X, 10X, or more members. In some embodiments, the physical realization of the theoretical total diversity may contain about IX to about 2X, about 2X to about 3X, about 3X to about 4X, about 4X to about 5X, about 5X to about 6X members. In other embodiments, the physical realization of the theoretical total diversity may contain about IX to about 3X, 10 or about 3X to about 5X total members.

An assumption underlying all directed evolution experiments is that the amount of molecular diversity theoretically possible is enormous compared with the ability to synthesize it, physically realize it, and screen it. The likelihood of finding a variant with improved properties in a given library is maximized when that library is maximally 15 diverse. Patrick et al. used simple statistics to derive a series of equations and computer algorithms for estimating the number of unique sequence variants in libraries constructed by randomized oligonucleotide mutagenesis, error-prone PCR and in vitro recombination. They have written a suite of programs for calculating library statistics, such as GLUE, GLUE-IT, PEDEL, PEDEL-AA, and DRIVeR. These programs are 20 described, with instructions on how to access them, in Patrick et al., Protein

Engineering, 2003, 16: 451and Firth et al., Nucleic Acids Res., 2008, 36: W281 (each of which is incorporated by reference in its entirety).

It is possible to construct a physical realization of a library in which some components of the theoretical diversity (such as CDRH3) are oversampled, while other 25 aspects (VH/VL pairings) are not. For example, consider a library in which 108 CDRH3 segments are designed to be present in a single VH chassis, and then paired with 105 VL genes to produce 1013 (= 108 * 105) possible full heterodimeric antibodies. If a physical realization of this library is constructed with a diversity of 109 transformant clones, then the CDRH3 diversity is oversampled ten-fold (= 109/108), however the possible VH/VL 30 pairings are undersampled by 10'4 (= 109/1013). In this example, on average, each CDRH3 is paired only with 10 samples of the VL from the possible 105 partners. In certain embodiments of the invention, it is the CDRH3 diversity that is preferably oversampled. -79- 3.1. Other Variants of the Polynucleotide Sequences of the Invention 2015201796 09 Apr 2015

In certain embodiments, the invention relates to a polynucleotide that hybridizes with a polynucleotide taught herein, or that hybridizes with the complement of a 5 polynucleotide taught herein. For example, an isolated polynucleotide that remains hybridized after hybridization and washing under low, medium, or high stringency conditions to a polynucleotide taught herein or the complement of a polynucleotide taught herein is encompassed by the present invention.

Exemplary low stringency conditions include hybridization with a buffer solution 10 of about 30% to about 35% formamide, about 1 M NaCl, about 1% SDS (sodium dodecyl sulphate) at about 37°C, and a wash in about IX to about 2X SSC (20X SSC=3.0 M NaCl/0.3 M trisodium citrate) at about 50°C to about 55°C.

Exemplary moderate stringency conditions include hybridization in about 40% to about 45% formamide, about 1 M NaCl, about 1% SDS at about 37°C, and a wash in 15 about 0.5X to about IX SSC at abut 55°C to about 60°C.

Exemplary high stringency conditions include hybridization in about 50% formamide, about 1 M NaCl, about 1% SDS at about 37°C, and a wash in about 0.1X SSC at about 60° C to about 65° C.

Optionally, wash buffers may comprise about 0.1% to about 1% SDS. 20 The duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. 3.2. Sub-Libraries and Larger Libraries Comprising the Libraries or Sub-Libraries of the Invention 25 As described throughout the application, the libraries of the current invention are distinguished, in certain embodiments, by their human-like sequence composition and length, and the ability to generate a physical realization of the library which contains all members of (or, in some cases, even oversamples) a particular component of the library. Libraries comprising combinations of the libraries described herein (e.g., CDRH3 and 30 CDRL3 libraries) are encompassed by the invention. Sub-libraries comprising portions of the libraries described herein are also encompassed by the invention (e.g., a CDRH3 library in a particular heavy chain chassis or a sub-set of the CDRH3 libraries). One of ordinary skill in the art will readily recognize that each of the libraries described herein -80- has several components (e.g., CDRH3, VH, CDRL3, VL, etc.), and that the diversity of these components can be varied to produce sub-libraries that fall within the scope of the invention. 2015201796 09 Apr 2015

Moreover, libraries containing one of the libraries or sub-libraries of the 5 invention also fall within the scope of the invention. For example, in certain embodiments of the invention, one or more libraries or sub-libraries of the invention may be contained within a larger library, which may include sequences derived by other means, for example, non-human or human sequence derived by stochastic or semistochastic synthesis. In certain embodiments of the invention, at least about 1% of the 10 sequences in a polynucleotide library may be those of the invention (e.g., CDRH3 sequences, CDRL3 sequences, VH sequences, VL sequences), regardless of the composition of the other 99% of sequences. In other embodiments of the invention, at least about 0.001%, 0.01%, 0.1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91,%, 92%, 93%, 94%, 15 95%, 96%, 97%, 98% or 99% of the sequences in any polynucleotide library may be those of the invention, regardless of the composition of the other sequences. In some embodiments, the sequences of the invention may comprise about 0.001% to about 1%, about 1% to about 2%, about 2% to about 5%, about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 20 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99% of the sequences in any polynucleotide library, regardless of 25 the composition of the other sequences. Thus, libraries more diverse than one or more libraries or sub-libraries of the invention, but yet still comprising one or more libraries or sub-libraries of the invention, in an amount in which the one or more libraries or sublibraries of the invention can be effectively screened and from which sequences encoded by the one or more libraries or sub-libraries of the invention can be isolated, also fall 30 within the scope of the invention. 3.3. Alternative Scaffolds -81 -

In certain embodiments of the invention, the amino acid products of a library of the invention (e.g., a CDRH3 or CDRL3) may be displayed on an alternative scaffold. Several of these scaffolds have been shown to yield molecules with specificities and affinities that rival those of antibodies. Exemplary alternative scaffolds include those 5 derived from fibronectin (e.g., AdNectin), the β-sandwich (e.g., iMab), lipocalin (e.g., Anticalin), EETI-II/AGRP, BPTI/LACI-D1/ITI-D2 (e.g., Kunitz domain), thioredoxin (e.g., peptide aptamer), protein A (e.g., Affibody), ankyrin repeats (e.g., DARPin), γΒ-crystallin/ubiquitin (e.g., Affilin), CTLD3 (e.g., Tetranectin), and (LDLR-A module^ 2015201796 09 Apr 2015 (e.g., Avimers). Additional information on alternative scaffolds are provided in Binz et 10 al., Nat. Biotechnol., 2005 23: 1257 and Skerra, Current Opin. in Biotech., 2007 18: 295-304, each of which is incorporated by reference in its entirety. 4. Other Embodiments of the Invention

In certain embodiments, the invention comprises a synthetic preimmune human 15 antibody CDRH3 library comprising 107 to 108 polynucleotide sequences representative of the sequence diversity and length diversity found in known heavy chain CDR3 sequences.

In other embodiments, the invention comprises a synthetic preimmune human antibody CDRH3 library comprising polynucleotide sequences encoding CDRH3 20 represented by the following formula: [G/D/E/-] [N1 ] [DH] [N2] [H3-JH], wherein [G/D/E/-] is zero to one amino acids in length, [Nl] is zero to three amino acids, [DH] is three to ten amino acids in length, [N2] is zero to three amino acids in length, and [H3-JH] is two to nine amino acids in length. 25 In certain embodiments of the invention, [G/D/E/-] is represented by an amino acid sequence selected from the group consisting of: G, D, E, and nothing.

In some embodiments of the invention, [Nl] is represented by an amino acid sequence selected from the group consisting of: G, R, S, P, L, A, V, T, (G/P)(G/R/S/P/L/A/V/T), (R/S/L/A/V/T)(G/P), GG(G/R/S/P/L/A/V/T), 30 G(R/S/P/L/A/V/T)G, (R/S/P/L/A/V/T)GG, and nothing.

In certain embodiments of the invention, [N2] is represented by an amino acid sequence selected from the group consisting of: G, R, S, P, L, A, V, T, -82- (G/P)(G/R/S/P/L/A/V/T), (R/S/L/A/V/T)(G/P), GG(G/R/S/P/L/A/V/T), G(R/S/P/L/A/V/T)G, (R/S/P/L/A/V/T)GG, and nothing. 2015201796 09 Apr 2015

In some embodiments of the invention, [DH] comprises a sequence selected from the group consisting of: IGHD3-10 reading frame 1, IGHD3-10 reading frame 2, 5 IGHD3-10 reading frame 3, IGHD3-22 reading frame 2, IGHD6-19 reading frame 1, IGHD6-19 reading frame 2, IGHD6-13 reading frame 1, IGHD6-13 reading frame 2, IGHD3-03 reading frame 3, IGHD2-02 reading frame 2, IGHD2-02 reading frame 3, IGHD4-17 reading frame 2, IGHD1-26 reading frame 1, IGHD1-26 reading frame 3, IGHD5-5/5-18 reading frame 3, IGHD2-15 reading frame 2, and all possible N-terminal 10 and C-terminal truncations of the above-identified IGHDs down to three amino acids.

In certain embodiments of the invention, [H3-JH] comprises a sequence selected from the group consisting of: AEYFQH, EYFQH, YFQH, FQH, QH, YWYFDL, WYFDL, YFDL, FDL, DL, AFDV, FDV, DV, YFDY, FDY, DY, NWFDS, WFDS, FDS, DS, YYYYYGMDV, YYYYGMDV, YYYGMDV, YYGMDV, YGMDV, 15 GMDV, MDV, and DV.

In some embodiments of the invention, the sequences represented by [G/D/E/-][Nl][ext-DH][N2][H3-JH] comprise a sequence of about 3 to about 26 amino acids in length.

In certain embodiments of the invention, the sequences represented by 20 [G/D/E/-][Nl][ext-DH][N2][H3-JH] comprise a sequence of about 7 to about 23 amino acids in length.

In some embodiments of the invention, the library comprises about 107 to about 1010 sequences.

In certain embodiments of the invention, the library comprises about 107 25 sequences.

In some embodiments of the invention, the polynucleotide sequences of the libraries further comprise a 5’ polynucleotide sequence encoding a framework 3 (FRM3) region on the corresponding N-terminal end of the library sequence, wherein the FRM3 region comprises a sequence of about 1 to about 9 amino acid residues. 30 In certain embodiments of the invention , the FRM3 region comprises a sequence selected from the group consisting of CAR, CAK, and CAT.

In some embodiments of the invention, the polynucleotide sequences further comprise a 3 ’ polynucleotide sequence encoding a framework 4 (FRM4) region on the -83 - corresponding C-terminal end of the library sequence, wherein the FRM4 region comprises a sequence of about 1 to about 9 amino acid residues. 2015201796 09 Apr 2015

In certain embodiments of the invention, the library comprises a FRM4 region comprising a sequence selected from WGRG and WGQG. 5 In some embodiments of the invention, the polynucleotide sequences further comprise an FRM3 region coding for a corresponding polypeptide sequence comprising a sequence selected from the group consisting of CAR, CAK, and CAT; and an FRM4 region coding for a corresponding polypeptide sequence comprising a sequence selected from WGRG and WGQG. 10 In certain embodiments of the invention, the polynucleotide sequences further comprise 5 ’ and 3 ’ sequences which facilitate homologous recombination with a heavy chain chassis.

In some embodiments, the invention comprises a synthetic preimmune human antibody light chain library comprising polynucleotide sequences encoding human 15 antibody kappa light chains represented by the formula: [IGKV (1-95)] [F/L/I/R/W/Y] [JK].

In certain embodiments of the invention, [IGKV (1-95)] is selected from the group consisting of IGKV3-20 (1-95), IGKV1-39 (1-95), IGKV3-11 (1-95), IGKV3-15 (1-95), IGKV 1-05 (1-95), IGKV4-01 (1-95), IGKV2-28 (1-95), IGKV 1-33 (1-95), 20 IGKV 1-09 (1-95), IGKV1-12 (1-95), IGKV2-30 (1-95), IGKV1-27 (1-95), IGKV1-16 (1-95), and truncations of said group up to and including position 95 according to Kabat.

In some embodiments of the invention, [F/L/I/R/W/Y] is an amino acid selected from the group consisting of F, L, I, R, W, and Y.

In certain embodiments of the invention, [JK] comprises a sequence selected 25 from the group consisting of TFGQGTKVEIK and TFGGGT.

In some embodiments of the invention, the light chain library comprises a kappa light chain library.

In certain embodiments of the invention, the polynucleotide sequences further comprise 5 ’ and 3 ’ sequences which facilitate homologous recombination with a light 30 chain chassis.

In some embodiments, the invention comprises a method for producing a synthetic preimmune human antibody CDRH3 library comprising 107 to 108 polynucleotide sequences, said method comprising: -84- a) selecting the CDRH3 polynucleotide sequences encoded by the CDRH3 sequences, as follows: 2015201796 09 Apr 2015 {0 to 5 amino acids selected from the group consisting of fewer than ten of the amino acids preferentially encoded by terminal deoxynucleotidyl 5 transferase (TdT) and preferentially functionally expressed by human B cells}, followed by (all possible N or C-terminal truncations of IGHD alone and all possible combinations of N and C-terminal truncations}, followed by (0 to 5 amino acids selected from the group consisting of fewer 10 than ten of the amino acids preferentially encoded by TdT and preferentially functionally expressed by human B cells}, followed by (all possible N-terminal truncations of IGHJ, down to DXWG, wherein X is S, V, L, or Y}; and b) synthesizing the CDRH3 library described in a) by chemical synthesis, 15 wherein a synthetic preimmune human antibody CDRH3 library is produced.

In certain embodiments, the invention comprises a synthetic preimmune human antibody CDRH3 library comprising 107 to 1010 polynucleotide sequences representative of known human IGHD and IGHJ germline sequences encoding CDRH3, represented by the following formula: 20 {0 to 5 amino acids selected from the group consisting of fewer than ten of the amino acids preferentially encoded by terminal deoxynucleotidyl transferase (TdT) and preferentially functionally expressed by human B cells}, followed by (all possible N or C-terminal truncations of IGHD alone and all possible combinations of N and C-terminal truncations}, followed by 25 {0 to 5 amino acids selected from the group consisting of fewer than ten of the amino acids preferentially encoded by TdT and preferentially functionally expressed by human B cells}, followed by (all possible N-terminal truncations of IGHJ, down to DXWG, wherein X is S, V, L, or Y}. 30 In certain embodiments, the invention comprises a synthetic preimmune human antibody heavy chain variable domain library comprising 107 to 1010 polynucleotide sequences encoding human antibody heavy chain variable domains, said library comprising: -85 - a) an antibody heavy chain chassis, and 2015201796 09 Apr 2015 b) a CDRH3 repertoire designed based on the human IGHD and IGHJ germline sequences, as follows: {0 to 5 amino acids selected from the group consisting of fewer 5 than ten of the amino acids preferentially encoded by terminal deoxynucleotidyl transferase (TdT) and preferentially functionally expressed by human B cells}, followed by {all possible N or C-terminal truncations of IGHD alone and all possible combinations of N and C-terminal truncations}, followed by 10 {0 to 5 amino acids selected from the group consisting of fewer than ten of the amino acids preferentially encoded by TdT and preferentially functionally expressed by human B cells}, followed by {all possible N-terminal truncations of IGHJ, down to DXWG, wherein X is S, V, L, or Y}. 15 In some embodiments of the invention, the synthetic preimmune human antibody heavy chain variable domain library is expressed as a full length chain selected from the group consisting of an IgGl full length chain, an IgG2 full length chain, an IgG3 full length chain, and an IgG4 full length chain.

In certain embodiments of the invention, the human antibody heavy chain chassis 20 is selected from the group consisting of IGHV4-34, IGHV3-23, IGHV5-51, IGHV1-69, IGHV3-30, IGHV4-39, IGHV1-2, IGHV1-18, IGHV2-5, IGHV2-70, IGHV3-7, IGHV6-1, IGHV1-46, IGHV3-33, IGHV4-31, IGHV4-4, IGHV4-61, and IGHV3-15.

In some embodiments of the invention, the synthetic preimmune human antibody heavy chain variable domain library comprises 107 to 1010 polynucleotide sequences 25 encoding human antibody heavy chain variable domains, said library comprising: a) an antibody heavy chain chassis, and b) a synthetic preimmune human antibody CDRH3 library.

In some embodiments of the invention, the polynucleotide sequences are single-stranded coding polynucleotide sequences. 30 In certain embodiments of the invention, the polynucleotide sequences are single-stranded non-coding polynucleotide sequences.

In some embodiments of the invention, the polynucleotide sequences are double-stranded polynucleotide sequences. -86-

In certain embodiments, the invention comprises a population of replicable cells with a doubling time of four hours or less, in which a synthetic preimmune human antibody repertoire is expressed. 2015201796 09 Apr 2015

In some embodiments of the invention, the population of replicable cells are 5 yeast cells.

In certain embodiments, the invention comprises a method of generating a full-length antibody library comprising transforming a cell with a preimmune human antibody heavy chain variable domain library and a synthetic preimmune human antibody light chain library. 10 In some embodiments, the invention comprises a method of generating a full- length antibody library comprising transforming a cell with a preimmune human antibody heavy chain variable domain library and a synthetic preimmune human antibody light chain library.

In certain embodiments, the invention comprises a method of generating an 15 antibody library comprising synthesizing polynucleotide sequences by split-pool DNA synthesis.

In some embodiments of the invention, the polynucleotide sequences are selected from the group consisting of single-stranded coding polynucleotide sequences, single-stranded non-coding polynucleotide sequences, and double-stranded polynucleotide 20 sequences.

In certain embodiments, the invention comprises a synthetic full-length preimmune human antibody library comprising about 107 to about 1010 polynucleotide sequences representative of the sequence diversity and length diversity found in known heavy chain CDR3 sequences. 25 In certain embodiments, the invention comprises a method of selecting an antibody of interest from a human antibody library, comprising providing a synthetic preimmune human antibody CDRH3 library comprising a theoretical diversity of (N) polynucleotide sequences representative of the sequence diversity and length diversity found in known heavy chain CDR3 sequences, wherein the physical realization of that 30 diversity is an actual library of a size at least 3(N), thereby providing a 95% probability that a single antibody of interest is present in the library, and selecting an antibody of interest. -87-

In some embodiments of the invention, the theoretical diversity is about 107 to 2015201796 09 Apr 2015

Q about 10 polynucleotide sequences.

EXAMPLES 5 This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference.

In general, the practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, recombinant DNA 10 technology, PCR technology, immunology (especially, e.g., antibody technology),

expression systems (e.g., yeast expression, cell-free expression, phage display, ribosome display, and PROFUSION™), and any necessary cell culture that are within the skill of the art and are explained in the literature. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: Cold Spring Harbor Laboratory Press (1989); DNA Cloning, Vols. 15 1 and 2, (D.N. Glover, Ed. 1985); Oligonucleotide Synthesis (M.J. Gait, Ed. 1984); PCR

Handbook Current Protocols in Nucleic Acid Chemistry, Beaucage, Ed. John Wiley &amp; Sons (1999) (Editor); Oxford Handbook of Nucleic Acid Structure, Neidle, Ed., Oxford Univ Press (1999); PCR Protocols: A Guide to Methods and Applications, Innis et al., Academic Press (1990); PCR Essential Techniques: Essential Techniques, Burke, Ed., 20 John Wiley &amp; Son Ltd (1996); The PCR Technique: RT-PCR, Siebert, Ed., Eaton Pub. Co. (1998); Antibody Engineering Protocols (Methods in Molecular Biology), 510, Paul, S., Humana Pr (1996); Antibody Engineering: A Practical Approach (Practical Approach Series, 169), McCafferty, Ed., Irl Pr (1996); Antibodies: A Laboratory Manual, Harlow et al., C.S.H.L. Press, Pub. (1999); Current Protocols in Molecular 25 Biology, eds. Ausubel et al., John Wiley &amp; Sons (1992); Large-Scale Mammalian Cell Culture Technology, Lubiniecki, A., Ed., Marcel Dekker, Pub., (1990); Phage Display: A Laboratory Manual, C. Barbas (Ed.), CSHL Press, (2001); Antibody Phage Display, P O’Brien (Ed.), Humana Press (2001); Border et al., Nature Biotechnology, 1997, 15: 553; Border et al., Methods Enzymol., 2000, 328: 430; ribosome display as described by 30 Pluckthun et al. in U.S. Patent No. 6,348,315, and Profusion™ as described by Szostak et al. in U.S. Patent Nos. 6,258,558; 6,261,804; and 6,214,553; and bacterial periplasmic expression as described in US20040058403A1. Each of the references cited in this paragraph is incorporated by reference in its entirety. -88-

Further details regarding antibody sequence analysis using Kabat conventions and programs to screen aligned nucleotide and amino acid sequences may be found, e.g., in Johnson et al., Methods Mol. Biol., 2004, 248: 11; Johnson et al., Int. Immunol., 2015201796 09 Apr 2015 1998, 10: 1801; Johnson et al., Methods Mol. Biol., 1995, 51: 1; Wuet al., Proteins, 5 1993, 16: 1; and Martin, Proteins, 1996, 25: 130. Each of the references cited in this paragraph is incorporated by reference in its entirety.

Further details regarding antibody sequence analysis using Chothia conventions may be found, e.g., in Chothia et al., J. Mol. Biol., 1998, 278: 457; Morea et al.,

Biophys. Chem., 1997, 68: 9; Morea et al., J. Mol. Biol., 1998, 275: 269; Al-Lazikani et 10 al., J. Mol. Biol., 1997, 273: 927. Barre etal., Nat. Struct. Biol., 1994, 1: 915; Chothia et al., J. Mol. Biol., 1992,227: 799; Chothia et al., Nature, 1989, 342: 877; and Chothia et al., J. Mol. Biol., 1987, 196: 901. Further analysis of CDRH3 conformation may be found in Shirai et al., FEBS Lett., 1999, 455: 188 and Shirai et al., FEBS Lett., 1996, 399: 1. Further details regarding Chothia analysis are described, for example, in Chothia 15 et al., Cold Spring Harb. Symp. Quant Biol., 1987, 52: 399. Each of the references cited in this paragraph is incorporated by reference in its entirety.

Further details regarding CDR contact considerations are described, for example, in MacCallum et al., J. Mol. Biol., 1996, 262: 732, incorporated by reference in its entirety. 20 Further details regarding the antibody sequences and databases referred to herein are found, e.g., in Tomlinson et al., J. Mol. Biol., 1992, 227: 776, VBASE2 (Retter et al., Nucleic Acids Res., 2005, 33: D671); BLAST (www.ncbi.nlm.nih.gov/BLAST/); CDHIT (bioinformatics.ljcrf.edu/cd-hi/); EMBOSS (www.hgmp.mrc.ac.uk/Software/EMBOSS/); PHYLIP 25 (evolution.genetics.washington.edu/phylip.html); and FASTA (fasta.bioch.virginia.edu). Each of the references cited in this paragraph is incorporated by reference in its entirety.

Example 1: Design of an Exemplary VH Chassis Library

This example demonstrates the selection and design of exemplary, non-limiting 30 VH chassis sequences of the invention. VH chassis sequences were selected by examining collections of human IGHV germline sequences (Scaviner et al., Exp. Clin. Immunogenet., 1999, 16: 234; Tomlinson et al., J. Mol. Biol., 1992, 227: 799; Matsuda et al., J. Exp. Med., 1998, 188: 2151, each incorporated by reference in its entirety). As -89- discussed in the Detailed Description, as well as below, a variety of criteria can be used to select VH chassis sequences, from these data sources or others, for inclusion in the library. 2015201796 09 Apr 2015

Table 3 (adapted from information provided in Scaviner et al, Exp. Clin. 5 Immunogenet., 1999, 16: 234; Matsuda etal., J. Exp. Med., 1998, 188: 2151; and Wang et al. Immunol. Cell. Biol., 2008, 86: 111, each incorporated by reference in its entirety) lists the CDRH1 and CDRH2 length, the canonical structure and the estimated relative occurrence in peripheral blood, for the proteins encoded by each of the human IGHV germline sequences.

Table 3. IGHV Characteristics and Occurrence in Antibodies from Peripheral Blood IGHV Germline Length of CDRH1 Length of CDRH2 Canonical Structures1 Estimated Relative Occurrence in Peripheral Blood2 IGHV1-2 5 17 1-3 37 IGHV1-3 5 17 1-3 15 IGHV1-8 5 17 1-3 13 IGHV1-18 5 17 1-2 25 IGHV1-24 5 17 1-U 5 IGHV1-45 5 17 1-3 0 IGHV1-46 5 17 1-3 25 IGHV1-58 5 17 1-3 2 IGHV1-69 5 17 1-2 58 IGHV2-5 7 16 3-1 10 IGHV2-26 7 16 3-1 9 IGHV2-70 7 16 3-1 13 IGHV3-7 5 17 1-3 26 IGHV3-9 5 17 1-3 15 IGHV3-11 5 17 1-3 13 IGHV3-13 5 16 1-1 3 IGHV3-15 5 19 1-4 14 IGHV3-20 5 17 1-3 3 IGHV3-21 5 17 1-3 19 IGHV3-23 5 17 1-3 80 IGHV3-30 5 17 1-3 67 IGHV3-33 5 17 1-3 28 IGHV3-43 5 17 1-3 2 IGHV3-48 5 17 1-3 21 IGHV3-49 5 19 1-U 8 IGHV3-53 5 16 1-1 7 IGHV3-64 5 17 1-3 2 IGHV3-66 5 17 1-3 3 IGHV3-72 5 19 1-4 2 IGHV3-73 5 19 1-4 3 IGHV3-74 5 17 1-3 14 IGHV4-4 5 16 1-1 33 IGHV4-28 6 16 2-1 1 -90- IGHV4-31 7 16 3-1 25 IGHV4-34 5 16 1-1 125 IGHV4-39 7 16 3-1 63 IGHV4-59 5 16 1-1 51 IGHV4-61 7 16 3-1 23 IGHV4-B 6 16 2-1 7 IGHV5-51 5 17 1-2 52 IGHV6-1 7 18 3-5 26 IGHV7-4-1 5 17 1-2 8 'Adapted from Chothia et ah, J. Mol. Biol., 1992, 227: 799 2Adapted from Table SI of Wang et al., Immunol. Cell. Biol., 2008, 86: 111

In the currently exemplified library, 17 germline sequences were chosen for 5 representation in the VH chassis of the library (Table 4). As described in more detail below, these sequences were selected based on their relatively high representation in the peripheral blood of adults, with consideration given to the structural diversity of the chassis and the representation of particular germline sequences in antibodies used in the clinic. These 17 sequences account for about 76% of the total sample of heavy chain 10 sequences used to derive the results of Table 4. As outlined in the Detailed Description, these criteria are non-limiting, and one of ordinary skill in the art will readily recognize that a variety of other criteria can be used to select the VH chassis sequences, and that the invention is not limited to a library comprising the 17 VH chassis genes presented in Table 4. 2015201796 09 Apr 2015 15

Table 4. VH Chassis Selectee for Use in the Exemplary Library VH Chassis Relative Occurrence Length of CDRH1 Length of CDRH2 Comment VH1-2 37 5 17 Among highest usage for VH1 family VH1-18 25 5 17 Among highest usage for VH1 family VH1-46 25 5 17 Among highest usage for VH1 family VH1-69 58 5 17 Highest usage for VH1 family. The four chosen VH1 chassis represent about 80% of the VH1 repertoire. VH3-7 26 5 17 Among highest usage in VH3 family VH3-15 14 5 19 Not among highest usage, but it has unique structure (H2 of length 19). Highest occurrence among those with such structure. VH3-23 80 5 17 Highest usage in VH3 -91 - 2015201796 09 Apr 2015 family. VH3-30 67 5 17 Among highest usage in VH3 family VH3-33 28 5 17 Among highest usage in VH3 family VH3-48 21 5 17 Among highest usage in VH3 family. The six chosen VH3 chassis account for about 70% of the VH3 repertoire. VH4-31 25 7 16 Among highest usage in VH4 family VH4-34 125 5 16 Highest usage in VH4 family VH4-39 63 7 16 Among highest usage in VH4 family VH4-59 51 5 16 Among highest usage in VH4 family VH4-61 23 7 16 Among highest usage in VH4 family VH4-B 7 6 16 Not among highest usage in VH4 family, but has unique structure (H1 of length 6). The 6 chosen VH4 chassis account for close to 90% of the VH4 family repertoire VH5-51 52 5 17 High usage

In this particular embodiment of the library, VH chassis derived from sequences in the IGHV2, IGHV6 and IGHV7 germline families were not included. As described in the Detailed Description, this exemplification is not meant to be limiting, as, in some 5 embodiments, it may be desirable to include one or more of these families, particularly as clinical information on antibodies with similar sequences becomes available, to produce libraries with additional diversity that is potentially unexplored, or to study the properties and potential of these IGHV families in greater detail. The modular design of the library of the present invention readily permits the introduction of these, and other, 10 VH chassis sequences. The amino acid sequences of the VH chassis utilized in this particular embodiment of the library, which are derived from the IGHV germline sequences, are presented in Table 5. The details of the derivation procedures are presented below. 15 Table 5. Amino Acid Sequences for VH Chassis Selected for Inclusion in the

Exemplary Library _____

Chassis SEQ ID NO: FRM1 CDRH1 FRM2 CDRH2 FRM3 VH1-2 QVQLVQSG AEVKKPGA GYYMH WVRQAPG QGLEWMG WINPNSG GTNYAQK RVTMTRDTSI STAYMELSRL -92- 2015201796 09 Apr 2015

SVKVSCKA SGYTFT FQG RSDDTAVYYC AR VH1-18 QVQLVQSG SYGIS WVRQAPG WISAYNG RVTMTTDTST AEVKKPGA QGLEWMG NTNYAQK STAYMELRSL SVKVSCKA LQG RSDDTAVYYC SGYTFT AR VH1-46 QVQLVQSG SYYMH WVRQAPG IINPSGG RVTMTRDTST AEVKKPGA QGLEWMG STSYAQK STVYMELSSL SVKVSCKA FQG RSEDTAVYYC SGYTFT AR VH1-69 QVQLVQSG SYAIS WVRQAPG GIIPIFG RVTITADKST AEVKKPGS QGLEWMG TANYAQK STAYMELSSL SVKVSCKA FQG RSEDTAVYYC SGGTFS AR VH3-7 EVQLVESG SYWMS WVRQAPG NIKQDGS RFTISRDNAK GGLVQPGG KGLEWVA EKYYVDS NSLYLQMNSL SLRLSCAA VKG RAEDTAVYYC SGFTFS AR VH3-151 EVQLVESG NAWMS WVRQAPG RIKSKTD RFTISRDDSK GGLVKPGG KGLEWVG GGTTDYA NTLYLQMNSL SLRLSCAA APVKG RAEDTAVYYC SGFTFS AR VH3-23 EVQLLESG SYAMS WVRQAPG AISGSGG RFTISRDNSK GGLVQPGG KGLEWVS STYYADS NTLYLQMNSL SLRLSCAA VKG RAEDTAVYYC SGFTFS AK VH3-30 QVQLVESG SYGMH WVRQAPG VISYDGS RFTISRDNSK GGVVQPGR KGLEWVA NKYYADS NTLYLQMNSL SLRLSCAA VKG RAEDTAVYYC SGFTFS AR VH3-33 QVQLVESG SYGMH WVRQAPG VIWYDGS RFTISRDNSK GGVVQPGR KGLEWVA NKYYADS NTLYLQMNSL SLRLSCAA VKG RAEDTAVYYC SGFTFS AR VH3-48 EVQLVESG SYSMN WVRQAPG YISSSSS RFTISRDNAK GGLVQPGG KGLEWVS TIYYADS NSLYLQMNSL SLRLSCAA VKG RAEDTAVYYC SGFTFS AR VH4-31 QVQLQESG SGGYY WIRQHPG YIYYSGS RVTISVDTSK PGLVKPSQ WS KGLEWIG TYYNPSL NQFSLKLSSV TLSLTCTV KS TAADTAVYYC SGGSIS AR -93 - 2015201796 09 Apr 2015

VH4-342 QVQLQQWG GYYWS WIRQPPG EIDHSGS RVTISVDTSK AGLLKPSE KGLEWIG TNYNPSL NQFSLKLSSV TLSLTCAV KS TAADTAVYYC YGGSFS AR VH4-39 QLQLQESG SSSYY WIRQPPG SIYYSGS RVTISVDTSK PGLVKPSE WG KGLEWIG TYYNPSL NQFSLKLSSV TLSLTCTV KS TAADTAVYYC SGGSIS AR VH4-59 QVQLQESG SYYWS WIRQPPG YIYYSGS RVTISVDTSK PGLVKPSE KGLEWIG TNYNPSL NQFSLKLSSV TLSLTCTV KS TAADTAVYYC SGGSIS AR VH4-61 QVQLQESG SGSYY WIRQPPG YIYYSGS RVTISVDTSK PGLVKPSE ws KGLEWIG TNYNPSL NQFSLKLSSV TLSLTCTV KS TAADTAVYYC SGGSVS AR VH4-B QVQLQESG SGYYW WIRQPPG SIYHSGS RVTISVDTSK PGLVKPSE G KGLEWIG TYYNPSL NQFSLKLSSV TLSLTCAV KS TAADTAVYYC SGYSIS AR VH5-51 EVQLVQSG SYWIG WVRQMPG IIYPGDS QVTISADKSI AEVKKPGE KGLEWMG DTRYSPS STAYLQWSSL SLKISCKG FQG KASDTAVYYC SGYSFT AR ίο 'The original KT sequence in VH3-15 was mutated to RA (bold/underlined) and TT to AR (bold/underlined), in order to match other VH3 family members selected for inclusion in the library. The modification to RA was made so that no unique sequence stretches of up to about 20 amino acids are created. Without being bound by theory, this modification is expected to reduce the odds of introducing novel T-cell epitopes in the VH3-15-derived chassis sequence. The avoidance of T cell epitopes is an additional criterion that can be considered in the design of certain libraries of the invention. 2The original NHS motif in VH4-34 was mutated to DHS, in order to remove a possible N-linked glycosylation site in CDR-H2. In certain embodiments of the invention, for example, if the library is transformed into yeast, this may prevent unwanted N-linked glycosylation.

Table 5 provides the amino acid sequences of the seventeen chassis. In 15 nucleotide space, most of the corresponding germline nucleotide sequences include two additional nucleotides on the 3’ end (i.e., two-thirds of a codon). In most cases, those two nucleotides are GA. In many cases, nucleotides are added to the 3’ end of the IGHV-derived gene segment in vivo, prior to recombination with the IGHD gene segment. Any additional nucleotide would make the resulting codon encode one of the 20 following two amino acids: Asp (if the codon is GAC or GAT) or Glu (if the codon is -94- GAA or GAG). One, or both, of the two 3’-terminal nucleotides may also be deleted in the final rearranged heavy chain sequence. If only the A is deleted, the resulting amino acid is very frequently a G. If both nucleotides are deleted, this position is “empty,” but followed by a general V-D addition or an amino acid encoded by the IGHD gene. 2015201796 09 Apr 2015 5 Further details are presented in Example 5. This first position, after the CAR or CAK motif at the C-terminus of FRM3 (Table 5), is designated the “tail.” In the currently exemplified embodiment of the library, this residue may be G, D, E, or nothing. Thus, adding the tail to any chassis enumerated above (Table 5) can produce one of the following four schematic sequences, wherein the residue following the VH chassis is the 10 tail: (1) [VH_Chassis]-[G] (2) [VH_Chassis]-[D] (3) [VH_Chassis]-[E] (4) [VH_Chassis] 15

These structures can also be represented in the format: [ VH_Chassis]- [G/D/E/-], wherein the hyphen symbol (-) indicates an empty or null position.

Using the CDRH3 numbering system defined in the Definitions section, the 20 above sequences could be denoted to have amino acid 95 as G, D, or E, for instances (1), (2), and (3), respectively, while the sequence of instance 4 would have no position 95, and CDRH3 proper would begin at position 96 or 97.

In some embodiments of the invention, VH3-66, with canonical structure 1-1 (five residues in CDRH1 and 16 for CDRH2) may be included in the library. The 25 inclusion of VH3-66 may compensate for the removal of other chassis from the library, which may not express well in yeast under some conditions {e.g., VH4-34 and VH4-59).

Example 2: Desisn of VH Chassis Variants with Variation Within CDRH1 and CDRH2

30 This example demonstrates the introduction of further diversity into the VH chassis by creating mutations in the CDRH1 and CDRH2 regions of each chassis shown in Example 1. The following approach was used to select the positions and nature of the amino acid variation for each chassis: First, the sequence identity between rearranged human heavy chain antibody sequences was analyzed (Lee et al., Immunogenetics, 35 2006, 57: 917; Jackson et al., J. Immunol. Methods, 2007, 324: 26) and they were -95 - classified by the origin of their respective IGHV germline sequence. As an illustrative example, about 200 sequences in the data set exhibited greatest identity to the IGHV1-69 germline, indicating that they were likely to have been derived from IGHV 1-69. Next, the occurrence of amino acid residues at each position within the CDRH1 and 5 CDRH2 segments, in each germline family selected in Example 1 was determined. For VH1-69, these occurrences are illustrated in Tables 6 and 7. Second, neutral and/or smaller amino acid residues were favored, where possible, as replacements. Without being bound by theory, the rationale for the choice of these amino acid residues is the desire to provide a more flexible and less sterically hindered context for the display of a 10 diversity of CDR sequences.

Table 6. Occurrence of Amino Acid Residues at Each Position Within IGHV 1-69- derived CDRH1 Seq uences 31 32 33 34 35 S Y A 1 S A 1 0 129 0 0 C 0 1 0 0 2 D 0 5 1 0 0 E 0 0 0 0 0 F 0 9 1 8 0 G 0 0 24 0 3 H 2 11 0 0 4 1 2 0 0 159 1 K 3 0 0 0 0 L 0 10 2 5 0 M 1 0 0 0 0 N 21 2 2 0 27 P 0 0 1 0 0 Q 1 1 0 0 5 R 9 0 0 0 1 S 133 3 7 0 129 T 12 1 10 0 12 V 0 0 7 13 0 w 0 0 0 0 0 Y 0 142 1 0 1 2015201796 09 Apr 2015 -96- 2015201796 09 Apr 2015

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Tab e 7. Occurrence of Amino Acid Residues at Each Position Within IGHV l-69-derived CDRH2 Sequences 50 51 52 52A 53 54 55 56 57 58 59 60 61 62 63 64 65 G I I P I F G T A N Y A Q K F Q G A 0 0 7 0 2 0 4 3 132 0 0 178 0 0 0 0 0 C 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 D 1 0 0 0 0 0 11 0 1 21 0 0 0 2 0 0 12 E 2 0 0 0 0 0 4 0 0 2 0 1 1 4 0 2 0 F 0 1 0 1 7 119 0 0 0 0 0 0 0 0 180 0 0 G 135 0 1 0 0 0 155 0 3 1 0 0 0 0 0 0 173 H 0 0 0 0 1 0 0 0 0 4 4 0 3 0 0 4 0 1 0 166 159 0 132 2 0 34 0 2 1 0 0 0 0 0 0 K 1 0 0 0 0 0 0 4 1 5 0 0 2 156 0 3 0 L 0 1 2 0 16 37 0 1 0 0 0 0 0 0 3 2 0 M 0 6 2 0 9 1 0 3 1 0 0 0 0 0 0 0 0 N 0 0 1 0 2 0 5 0 0 132 1 0 0 8 0 0 0 P 0 2 0 181 1 3 0 0 15 0 0 3 6 0 0 0 0 Q 0 0 0 0 0 0 1 0 1 0 0 0 173 2 0 164 0 R 44 0 0 0 0 0 1 4 0 3 0 0 0 13 0 9 0 S 1 0 1 1 2 6 3 5 8 7 0 2 0 0 1 0 0 T 1 1 7 2 2 1 0 127 15 8 3 1 0 0 0 0 0 V 0 8 5 0 11 4 0 4 8 0 0 0 0 0 0 0 0 w 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 Y 0 0 0 0 0 11 1 0 0 0 176 0 0 0 1 1 0 5 -97-

The original germline sequence is provided in the second row of the tables, in bold font, beneath the residue number (Kabat system). The entries in the table indicate the number of times a given amino acid residue (first column) is observed at the 5 indicated CDRH1 (Table 6) or CDRH2 (Table 7) position. For example, at position 33 the amino acid type G (glycine) is observed 24 times in the set of IGHVl-69-based sequences that were examined. Thus, applying the criteria above, variants were constructed with N at position 31, L at position 32 (H can be charged, under some conditions), G and T at position 33, no variants at position 34 and N at position 35, 2015201796 09 Apr 2015 10 resulting in the following VH1-69 chassis CDRH1 single-amino acid variant sequences: NYAIS (SEQ ID NO ) SLAIS (SEQ ID NO ) SYGIS (SEQ ID NO ) SYTIS (SEQ ID NO ) SYAIN (SEQ ID NO )

Similarly, the analysis that produced Table 7 provided a basis for choosing the following single-amino acid variant sequences for VH1-69 chassis CDRH2s: SIIPIFGTANYAQKFQG (SEQ ID NO: _) 20 GIAPIFGTANYAQKFQG (SEQ ID NO: _) GIIPILGTANYAQKFQG (SEQ ID NO: _) GIIPIFGTASYAQKFQG (SEQ ID NO: _) A similar approach was used to design and construct variants of the other 25 selected chassis; the resulting CDRH1 and CDRH2 variants for each of the exemplary chassis are provided in Table 8. One of ordinary skill in the art will readily recognize that the methods described herein can be applied to create variants of other VH chassis and VL chassis. -98- 2015201796 09 Apr 2015

Table 8. VH Chassis Variants

Chassis CDRH1 SEQ ID NO: CDRH2 SEQ ID NO: Chassis CDRH1 SEQ ID NO: CDRH2 SEQ ID NO: 1-18.0 SYGIS WISAYNGNT 3-48.0 SYSMN YISSSSSTI NYAQKLQG YYADSVKG 1-18.1 NYGIS WISAYNGNT 3-48.11 NYSMN YISSSSSTI NYAQKLQG YYADSVKG 1-18.2 SNGIS WISAYNGNT 3-48.2 IYSMN YISSSSSTI NYAQKLQG YYADSVKG 1-18.3 SYAIS WISAYNGNT 3-48.3 SNSMN YISSSSSTI NYAQKLQG YYADSVKG 1-18.4 SYGIT WISAYNGNT 3-48.4 SYEMN YISSSSSTI NYAQKLQG YYADSVKG 1-18.5 SYGIH WISAYNGNT 3-48.5 SYNMN YISSSSSTI NYAQKLQG YYADSVKG 1-18.6 SYGIS SISAYNGNT 3-48.6 SYSMT YISSSSSTI NYAQKLQG YYADSVKG 1-18.7 SYGIS WISTYNGNT 3-48.7 SYSMN TISSSSSTI NYAQKLQG YYADSVKG 1-18.8 SYGIS WISPYNGNT 3-48.8 SYSMN YISGSSSTI NYAQKLQG YYADSVKG 1-18.9 SYGIS WISAYNGNT 3-48.9 SYSMN YISSSSSTI YYAQKLQG LYADSVKG

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1-2.0 GYYMH WINPNSGGT NYAQKFQG 3-7.0 SYWMS NIKQDGSEK YYVDSVKG 1-2.1 DYYMH WINPNSGGT NYAQKFQG 3-7.1 TYWMS NIKQDGSEK YYVDSVKG 1-2.2 RYYMH WINPNSGGT NYAQKFQG 3-7.2 NYWMS NIKQDGSEK YYVDSVKG 1-2.3 GSYMH WINPNSGGT NYAQKFQG 3-7.3 SSWMS NIKQDGSEK YYVDSVKG 1-2.4 GYSMH WINPNSGGT NYAQKFQG 3-7.4 SYGMS NIKQDGSEK YYVDSVKG 1-2.5 GYYMQ WINPNSGGT NYAQKFQG 3-7.5 SYWMT NIKQDGSEK YYVDSVKG 1-2.6 GYYMH SINPNSGGT NYAQKFQG 3-7.6 SYWMS SIKQDGSEK YYVDSVKG 1-2.7 GYYMH WINPSSGGT NYAQKFQG 3-7.7 SYWMS NINQDGSEK YYVDSVKG 1-2.8 GYYMH WINPNSGGT KYAQKFQG 3-7.8 SYWMS NIKSDGSEK YYVDSVKG 1-2.9 GYYMH WINPNSGGT SYAQKFQG 3-7.9 SYWMS NIKQDGSEK QYVDSVKG 1-46.0 SYYMH IINPSGGST SYAQKFQG 4-31.0 SGGYYWS YIYYSGSTY YNPSLKS - 100- 2015201796 09 Apr 2015

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1-46.1 NYYMH IINPSGGST SYAQKFQG 4-31.1 SGSYYWS YIYYSGSTY YNPSLKS 1-46.2 SSYMH IINPSGGST SYAQKFQG 4-31.2 SGTYYWS YIYYSGSTY YNPSLKS 1-46.3 SYSMH IINPSGGST SYAQKFQG 4-31.3 SGGTYWS YIYYSGSTY YNPSLKS 1-46.4 SYYIH IINPSGGST SYAQKFQG 4-31.4 SGGYSWS YIYYSGSTY YNPSLKS 1-46.5 SYYMV IINPSGGST SYAQKFQG 4-31.5 SGGYYWS SIYYSGSTY YNPSLKS 1-46.6 SYYMS IINPSGGST SYAQKFQG 4-31.6 SGGYYWS NIYYSGSTY YNPSLKS 1-46.7 SYYMH VINPSGGST SYAQKFQG 4-31.7 SGGYYWS YIYYSGNTY YNPSLKS 1-46.8 SYYMH IINPGGGST SYAQKFQG 4-31.8 SGGYYWS YIYYSGSTS YNPSLKS 1-46.9 SYYMH IINPSGGST TYAQKFQG 4-31.9 SGGYYWS YIYYSGSTV YNPSLKS 1-69.0 SYAIS GIIPIFGTA NYAQKFQG 4-34.0 GYYWS ElDHSGSTN YNPSLKS 1-69.1 NYAIS GIIPIFGTA NYAQKFQG 4-34.1 DYYWS ElDHSGSTN YNPSLKS - 101 - 2015201796 09 Apr 2015

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1-69.2 SLA IS GIIPIFGTA NYAQKFQG 4-34.2 GYYWT ElDHSGSTN YNPSLKS 1-69.3 SYGIS GIIPIFGTA NYAQKFQG 4-34.3 GYYWS DIDHSGSTN YNPSLKS 1-69.4 SYTIS GIIPIFGTA NYAQKFQG 4-34.4 GYYWS EISHSGSTN YNPSLKS 1-69.5 SYAIN GIIPIFGTA NYAQKFQG 4-34.5 GYYWS EIDQSGSTN YNPSLKS 1-69.6 SYAIS SIIPIFGTA NYAQKFQG 4-34.6 GYYWS ElDHGGSTN YNPSLKS 1-69.7 SYAIS GIAPIFGTA NYAQKFQG 4-34.7 GYYWS EIDHSGNTN YNPSLKS 1-69.8 SYAIS GIIPILGTA NYAQKFQG 4-34.8 GYYWS ElDHSGSTS YNPSLKS 1-69.9 SYAIS GIIPIFGTA SYAQKFQG 4-34.9 GYYWS ElDHSGSTD YNPSLKS 3-15.0 NAWMS RIKSKTDGG TTDYAAPVK G 4-39.0 SSSYYWG SIYYSGSTY YNPSLKS 3-15.1 KAWMS RIKSKTDGG TTDYAAPVK G 4-39.1 TSSYYWG SIYYSGSTY YNPSLKS 3-15.2 DAWMS RIKSKTDGG 4-39.2 SNSYYWG SIYYSGSTY - 102- 2015201796 09 Apr 2015

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TTDYAAPVK G YNPSLKS 3-15.3 NALMS RIKSKTDGG TTDYAAPVK G 4-39.3 SSDYYWG SIYYSGSTY YNPSLKS 3-15.4 NAAMS RIKSKTDGG TTDYAAPVK G 4-39.4 SSNYYWG SIYYSGSTY YNPSLKS 3-15.5 NAWMN RIKSKTDGG TTDYAAPVK G 4-39.5 SSRYYWG SIYYSGSTY YNPSLKS 3-15.6 NAWMS SIKSKTDGG TTDYAAPVK G 4-39.6 SSSYAWG SIYYSGSTY YNPSLKS 3-15.7 NAWMS RIKSTTDGG TTDYAAPVK G 4-39.7 SSSYYWG NIYYSGSTY YNPSLKS 3-15.8 NAWMS RIKSKADGG TTDYAAPVK G 4-39.8 SSSYYWG SISYSGSTY YNPSLKS 3-15.9 NAWMS RIKSKTDGG TTGYAAPVK G 4-39.9 SSSYYWG SIYYSGSTS YNPSLKS 3-23.0 SYAMS AISGSGGST YYADSVKG 4-59.0 SYYWS YIYYSGSTN YNPSLKS - 103 - 2015201796 09 Apr 2015

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3-23.1 NY AMS AISGSGGST YYADSVKG 4-59.1 TYYWS YIYYSGSTN YNPSLKS 3-23.2 TYAMS AISGSGGST YYADSVKG 4-59.2 SSYWS YIYYSGSTN YNPSLKS 3-23.3 SSAMS AISGSGGST YYADSVKG 4-59.3 SYSWS YIYYSGSTN YNPSLKS 3-23.4 SYAMS GISGSGGST YYADSVKG 4-59.4 SYYWS FIYYSGSTN YNPSLKS 3-23.5 SYAMS SISGSGGST YYADSVKG 4-59.5 SYYWS HIYYSGSTN YNPSLKS 3-23.6 SYAMS TISGSGGST YYADSVKG 4-59.6 SYYWS SIYYSGSTN YNPSLKS 3-23.7 SYAMS VI SGSGGST YYADSVKG 4-59.7 SYYWS YIYSSGSTN YNPSLKS 3-23.8 SYAMS AISASGGST YYADSVKG 4-59.8 SYYWS YIYYSGSTD YNPSLKS 3-23.9 SYAMS AISGSGGST SYADSVKG 4-59.9 SYYWS YIYYSGSTT YNPSLKS 3-30.0 SYGMH VISYDGSNK YYADSVKG 4-61.0 SGSYYWS YIYYSGSTN YNPSLKS 3-30.1 NYGMH VISYDGSNK YYADSVKG 4-61.1 SGGYYWS YIYYSGSTN YNPSLKS - 104- 2015201796 09 Apr 2015

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3-30.2 SYAMH VISYDGSNK YYADSVKG 4-61.2 SGNYYWS YIYYSGSTN YNPSLKS 3-30.3 SYGFH VISYDGSNK YYADSVKG 4-61.3 SGSSYWS YIYYSGSTN YNPSLKS 3-30.4 SYGMH FISYDGSNK YYADSVKG 4-61.4 SGSYSWS YIYYSGSTN YNPSLKS 3-30.5 SYGMH LISYDGSNK YYADSVKG 4-61.5 SGSYYWT YIYYSGSTN YNPSLKS 3-30.6 SYGMH VISSDGSNK YYADSVKG 4-61.6 SGSYYWS RIYYSGSTN YNPSLKS 3-30.7 SYGMH VISYDGNNK YYADSVKG 4-61.7 SGSYYWS SIYYSGSTN YNPSLKS 3-30.8 SYGMH VISYDGSIK YYADSVKG 4-61.8 SGSYYWS YIYTSGSTN YNPSLKS 3-30.9 SYGMH VISYDGSNQ YYADSVKG 4-61.9 SGSYYWS YIYYSGSTS YNPSLKS 3-33.0 SYGMH VIWYDGSNK YYADSVKG 4-B.O SGYYWG SIYHSGSTY YNPSLKS 3-33.1 TYGMH VIWYDGSNK YYADSVKG 4-B.1 SAYYWG SIYHSGSTY YNPSLKS 3-33.2 NYGMH VIWYDGSNK YYADSVKG 4-B.2 SGSYWG SIYHSGSTY YNPSLKS - 105 - 2015201796 09 Apr 2015

Attorney Docket No.: ADS-011.25

3-33.3 SSGMH VIWYDGSNK YYADSVKG 4-B.3 SGYNWG SIYHSGSTY YNPSLKS 3-33.4 SYAMH VIWYDGSNK YYADSVKG 4-B.4 SGYYWA SIYHSGSTY YNPSLKS 3-33.5 SYGMN VIWYDGSNK YYADSVKG 4-B.5 SGYYWG TIYHSGSTY YNPSLKS 3-33.6 SYGMH LIWYDGSNK YYADSVKG 4-B.6 SGYYWG SSYHSGSTY YNPSLKS 3-33.7 SYGMH FIWYDGSNK YYADSVKG 4-B.7 SGYYWG SIYHSGNTY YNPSLKS 3-33.8 SYGMH VIWYDGSNK SYADSVKG 4-B.8 SGYYWG SIYHSGSTN YNPSLKS 3-33.9 SYGMH VIWYDGSNK GYADSVKG 4-B.9 SGYYWG SIYHSGSTG YNPSLKS 5-51.0 SYWIG IIYPGDSDT RYSPSFQG 5-51.1 TYWIG IIYPGDSDT RYSPSFQG 5-51.2 NYWIG IIYPGDSDT RYSPSFQG 5-51.3 SNWIG IIYPGDSDT RYSPSFQG - 106- 2015201796 09 Apr 2015

Attorney Docket No.: ADS-011.25

5-51.4 SYYIG IIYPGDSDT RYSPSFQG 5-51.5 SYWIS IIYPGDSDT RYSPSFQG 5-51.6 SYWIG SIYPGDSDT RYSPSFQG 5-51.7 SYWIG IIYPADSDT RYSPSFQG 5-51.8 SYWIG IIYPGDSST RYSPSFQG 5-51.9 SYWIG IIYPGDSDT TYSPSFQG 1

Contains an N-linked glycosylation site w lich can be removed, if desired, as described herein. - 107-

As specified in the Detailed Description, other criteria can be used to select which amino acids are to be altered and the identity of the resulting altered sequence. This is true for any heavy chain chassis sequence, or any other sequence of the 5 invention. The approach outlined above is meant for illustrative purposes and is nonlimiting. 2015201796 09 Apr 2015

Example 3: Desisn of an Exemplary VK Chassis Library 10 This example describes the design of an exemplary VK chassis library. One of ordinary skill in the art will recognize that similar principles may be used to design a νλ library, or a library containing both VK and νλ chassis. Design of a νλ chassis library is presented in Example 4.

As was previously demonstrated in Example 1, for IGHV germline sequences, 15 the sequence characteristics and occurrence of human IGKV germline sequences in antibodies from peripheral blood were analyzed. The data are presented in Table 9.

Table 9. IGKV Gene Characteristics and Occurrence in Antibodies from Peripheral Blood _ IGKV Gene Alternative Names CDRL1 Length CDRL2 Length Canonical Structures1 Estimated Relative Occurrence in Peripheral Blood2 IGKV1-05 L12 11 7 2-1-(11) 69 IGKV1-06 L11 11 7 2-1-(1) 14 IGKV1-08 L9 11 7 2-1-(1) 9 IGKV1-09 L8 11 7 2-1-(1) 24 IGKV1-12 L5, L19 11 7 2-1-(1) 32 IGKV1-13 L4, L18 11 7 2-1-(1) 13 IGKV1-16 L1 11 7 2-1-(1) 15 IGKV1-17 A30 11 7 2-1-(1) 34 IGKV1-27 A20 11 7 2-1-(1) 27 IGKV1-33 08, 018 11 7 2-1-(1) 43 IGKV1-37 014, 04 11 7 2-1-(1) 3 IGKV1-39 02, 012 11 7 2-1-(1) 147 IGKV1D-16 L15 11 7 2-1-(1) 6 IGKV1D-17 L14 11 7 2-1-(1) 1 IGKV1D-43 L23 11 7 2-1-(1) 1 IGKV1D-8 L24 11 7 2-1-(1) 1 IGKV2-24 A23 16 7 4-1-(1) 8 IGKV2-28 A19, A3 16 7 4-1-(1) 62 IGKV2-29 A18 16 7 4-1-(1) 6 IGKV2-30 A17 16 7 4-1-(1) 30 IGKV2-40 01, 011 17 7 3-1-0) 3 IGKV2D-26 A8 16 7 4-1-(1) 0 -108- 2015201796 09 Apr 2015 IGKV2D-29 A2 16 7 4-1-(1) 20 IGKV2D-30 A1 16 7 4-1-(1) 4 IGKV3-11 L6 11 7 2-1-(1) 87 IGKV3-15 L2 11 7 2-1-(1) 53 IGKV3-20 A27 12 7 6-1-(1) 195 IGKV3D-07 L25 12 7 6-1-(1) 0 IGKV3D-11 L20 11 7 2-1-(11) 0 IGKV3D-20 A11 12 7 6-1-(1) 2 IGKV4-1 B3 17 7 3-1-(1) 83 IGKV5-2 B2 11 7 2-1-(1) 1 IGKV6-21 A10, A26 11 7 2-1-(1) 6 IGKV6D-41 A14 11 7 2-1-(1) 0

Adapted from Tomlinson et al. EMBO J., 1995, 14: 4628, incorporated by reference in its entirety. The number in parenthesis refers to canonical structures in CDRL3, if one assuming the most common length (see Example 5 for further detail about CDRL3). 2Estimated from sets of human VK sequences compiled from the NCBI database; full set of GI numbers provided in Appendix A.

The 14 most commonly occurring IGKV germline genes (bolded in column 6 of Table 9) account for just over 90% of the usage of the entire repertoire in peripheral 10 blood. From the analysis of Table 9, ten IGKV germline genes were selected for representation as chassis in the currently exemplified library (Table 10). All but VI-12 and VI-27 are among the top 10 most commonly occurring. IGKV germline genes VH2-30, which was tenth in terms of occurrence in peripheral blood, was not included in the currently exemplified embodiment of the library, in order to maintain the 15 proportion of chassis with short (i.e., 11 or 12 residues in length) CDRL1 sequences at about 80% in the final set of 10 chassis. Vl-12 was included in its place. Vl-17 was more similar to other members of the V1 family that were already selected ; therefore, VI-27 was included, instead of Vl-17. In other embodiments, the library could include 12 chassis (e.g., the ten of Table 10 plus Vl-17 and V2-30), or a different set of any “N” 20 chassis, chosen strictly by occurrence (Table 9) or any other criteria. The ten chosen VK chassis account for about 80% of the usage in the data set believed to be representative of the entire kappa light chain repertoire.

Table 10. VK Chassis Selected for i Jse in the Exemp ary Library Chassis CDR-L1 Length CDR-L2 Length Canonical Structures Estimated Relative Occurrence in Peripheral Blood VK1-5 11 7 2-1-(11) 69 VK1-12 11 7 2-1-(1) 32 VK1-27 11 7 2-1-(1) 27 VK1-33 11 7 2-1-(1) 43 VK1-39 11 7 2-1-(1) 147 VK2-28 16 7 4-1-(1) 62 - 109- 2015201796 09 Apr 2015 VK3-11 11 7 2-1-(1) 87 VK3-15 11 7 2-1-(1) 53 VK3-20 12 7 6-1-(1) 195 VK4-1 17 7 _3±m_ 83

The amino acid sequences of the selected VK chassis enumerated in Table 10 are provided in Table 11. 5 Table 11. Amino Acid Sequences for VK Chassis Selected for Inclusion in the Exemplary Library _____

Chassis FRM1 CDRL1 FRM2 CDRL2 FRM3 CDRL3 SEQID 1 NO: VK1-5 DIQMTQS RASQSI WYQQKP DASSLE GVPSRFSGSGSGT QYNSY PSTLSAS SSWLA GKAPKL S EFTLTISSLQPDD S VGDRVTI TC LIY FATYYC VK1-12 DIQMTQS RASQGI WYQQKP AASSLQ GVPSRFSGSGSGT QANSF PSSVSAS SSWLA GKAPKL S DFTLTISSLQPED P VGDRVTI TC LIY FATYYC VK1-27 DIQMTQS RASQGI WYQQKP AASTLQ GVPSRFSGSGSGT KYNSA PSSLSAS SNYLA GKVPKL S DFTLTISSLQPED P VGDRVTI TC LIY VATYYC VK1-33 DIQMTQS QASQDI WYQQKP DASNLE GVPSRFSGSGSGT QYDNL PSSLSAS SNYLN GKAPKL T DFTFTISSLQPED P VGDRVTI TC LIY IATYYC VK1-39 DIQMTQS RASQSI WYQQKP AASSLQ GVPSRFSGSGSGT QSYST PSSLSAS SSYLN GKAPKL S DFTLTISSLQPED P VGDRVTI TC LIY FATYYC VK2-28 DIVMTQS RSSQSL WYLQKP LGSNRA GVPDRFSGSGSGT QALQT PLSLPVT LHSNGY GQSPQL S DFTLKISRVEAED P PGEPASI SC NYLD LIY VGVYYC VK3-11 EIVLTQS RASQSV WYQQKP DASNRA GIPARFSGSGSGT QRSNW PATLSLS SSYLA GQAPRL T DFTLTISSLEPED P PGERATL SC LIY FAVYYC VK3-15 EIVMTQS RASQSV WYQQKP GASTRA GIPARFSGSGSGT QYNNW PATLSVS SSNLA GQAPRL T EFTLTISSLQSED P PGERATL SC LIY FAVYYC VK3-20 EIVLTQS RASQSV WYQQKP GASSRA GIPDRFSGSGSGT QYGSS PGTLSLS SSSYLA GQAPRL T DFTLTISRLEPED P PGERATL LIY FAVYYC - 110- 2015201796 09 Apr 2015

SC VK4-1 DIVMTQS PDSLAVS LGERATI NC KSSQSV LYSSNN KNYLA WYQQKP GQPPKL LIY WASTRE S GVPDRFSGSGSGT DFTLTISSLQAED VAVYYC QYYST P 1 Note that the portion of the IGKV gene contributing to VKCDR3 is not considered part of the chassis as described herein. The VK chassis is defined as Kabat residues 1 to 88 of the IGKV-encoded sequence, or from the start of FRM1 to the end of FRM3. The portion of the VKCDR3 sequence contributed 5 by the IGKV gene is referred to herein as the L3-VK region.

Example 4: Desien of an Exemplary VX Chassis Library

This example, describes the design of an exemplary νλ chassis library. As was 10 previously demonstrated in Examples 1-3, for the VH and VK chassis sequences, the sequence characteristics and occurrence of human IgAV germline-derived sequences in peripheral blood were analyzed. As with the assignment of other sequences set forth herein to germline families, assignment of νλ sequences to a germline family was performed via SoDA and VBASE2 (Volpe and Kepler, Bioinformatics, 2006, 22: 438; 15 Mollova et al., BMS Systems Biology, 2007, IS: P30, each incorporated by reference in its entirety). The data are presented in Table 12.

Table 12. IGAV Gene Characteristics and Occurrence in Peripheral Blood IGAV Gene Alternative Name Canonical Structures1 Contribution of IGVA Gene to CDRL3 Estimated Relative Occurrence in Peripheral Blood2 IGAV3-1 3R 11 -7(*) 8 11.5 IGAV3-21 3H 11-7 (*) 9 10.5 IGAV2-14 2A2 14-7 (A) 9 10.1 IGAV1-40 1E 14-7 (A) 9 7.7 IGAV3-19 3L 11 -7(*) 9 7.6 IGAV1-51 1B 13-7 (A) 9 7.4 IGAV 1-44 1C 13-7 (A) 9 7.0 IGAV6-57 6A 13-7(B) 7 6.1 IGAV2-8 2C 14-7 (A) 9 4.7 IGAV3-25 3M 11-7(*) 9 4.6 IGAV2-23 2B2 14-7 (A) 9 4.3 IGAV3-10 3P 11-7 (*) 9 3.4 IGAV4-69 4B 12-11 (*) 7 3.0 IGAV1-47 1G 13-7 (A) 9 2.9 IGAV2-11 2E 14-7 (A) 9 1.3 IGAV7-43 7A 14-7(B) 8 1.3 IGAV7-46 7B 14-7(B) 8 1.1 IGAV5-45 5C 14-11 (*) 8 1.0 IGAV4-60 4A 12-11 (*) 7 0.7 - Ill - IGAV10- 54 8A 14-7(B) 8 0.7 IGAV8-61 10A 13-7(C ) 9 0.7 IGAV3-9 3J 11-7 (*) 8 0.6 IGAV1-36 1A 13-7(A) 9 0.4 IGAV2-18 2D 14-7(A) 9 0.3 IGAV3-16 3A 11-7(*) 9 0.2 IGAV3-27 11-70 7 0.2 IGAV4-3 5A 14-110 8 0.2 IGAV5-39 4C 12-110 12 0.2 IGAV9-49 9A 12-120 12 0.2 IGAV3-12 3I 11-70 9 0.1 'Adapted from Williams et al. J. Mol. Biol. 1996: 264, 220-32. The (*) indicates that the canonical structure is entirely defined by the lengths of CDRs LI and L2. When distinct structures are possible for identical LI and L2 length combinations, the structure present in a given gene is set forth as A, B, or C. 5 2015201796 09 Apr 2015 2Estimated from a set of human VA sequences compiled from the NCBI database; full set of GI codes set forth in Appendix B.

To choose a subset of the sequences from Table 12 to serve as chassis, those 10 represented at less than 1% in peripheral blood (as extrapolated from analysis of published sequences corresponding to the GI codes provided in Appendix B) were first discarded. From the remaining 18 germline sequences, the top occurring genes for each unique canonical structure and contribution to CDRL3, as well as any germline gene represented at more than the 5% level, were chosen to constitute the exemplary νλ 15 chassis. The list of 11 such sequences is given in Table 13, below. These 11 sequences represent approximately 73% of the repertoire in the examined data set (Appendix B).

Table 13. Υλ Chassis Selected for Use in the Exemplary Library

Chassis CDRL1 Length CDRL2 Length Canonical Structure Relative Occurrence VA3-1 11 7 11-70 11.5 VA3-21 11 7 11-70 10.5 VA2-14 14 7 14-7(A) 10.1 VA1-40 14 7 14-7(A) 7.7 VA3-19 11 7 11-70 7.6 VA1-51 13 7 13-7(A) 7.4 VA1-44 13 7 13-7(A) 7.0 VA6-57 13 7 13-7(B) 6.1 VA4-69 12 11 12-110 3.0 VA7-43 14 7 14-7(B) 1.3 VA5-45 11 11 14-110 1.0 - 112-

The amino acid sequences of the selected Vk chassis enumerated in Table 13 are provided in Table 14, below.

Table 14. Amino Acid Sequences for νλ Chassis Selected for Inclusion in the 5 Exemplary Library _____

Chassis FRM1 CDRL1 FRM2 CDRL2 FRM3 CDRL32 νλ1-40 QSVLTQP PSVSGAP GQRVTIS C TGSSSN IGAGYD ---VH WYQQLP GTAPRL LIY GN---- SNRPS GVPDRFSGSRSG— TSASLAITGLQAEDE ADYYC QSYDSS LSG νλ1-44 QSVLTQP PSASGTP GQRVTIS C SGSSSN IGSNT----VN WYQQLP GTAPRL LIY SN---- NQRPS GVPDRFSGSRSG— TSASLAISGLQSEDE ADYYC AAWDDS LNG νλ1-51 QSVLTQP PSVSAAP GQRVTIS C SGSSSN IGNNY----VS WYQQLP GTAPRL LIY DN---- NRRPS GIPDRFSGSRSG— TSATLGITGLQTGDE ADYYC GTWDSS LSA νλ2-14 QSALTQP ASVSGSP GQSITIS C TGTSSD VGGYNY ---VS WYQQHP GRAPRL MIY EV---- SNRPS GVSNRFSGSRSG— NTASLTISGLQAEDE ADYYC SSYTSS STL νλ3-11 SYELTQP PSVSVSP GQTASIT C SGDKLG DRY--- ---AS WYQQRP GQSPVL VIY QD---- SRRPS GIPERFSGSNSG— NTATLTISGTQAMDE ADYYC QAWDSS TA- νλ3-19 SSELTQD PAVSVAL GQTVRIT C QGDSLR SYY — ---AS WYQQRP GQAPVL VIY GR---- NNRPS GIPDRFSGSSSG— NTASLTITGAQAEDE ADYYC NSRDSS GNH νλ3-21 SYVLTQP PSVSVAP GKTARIT C GGNNIG SRS--- ---VH WYQQRP GQAPVL VIY YD---- SDRPS GIPERFSGSNSG— NTATLTISRVEAGDE ADYYC QVWDSS SDH νλ4-69 QLVLTQS PSASASL GASVKLT C TLSSGH SSYA— ---IA WHQQQP ERGPRY LMR LNSDGS HSRGD GIPDRFSGSSSG— AERYLTISSLQSEDE ADYYC QTWGTG I-- νλ6-57 NFMLTQP HSVSESP GKTVTIS C TRSSGS IASNY----VQ WYQQRP GSSPTT VIY ED---- NQRPS GVPDRFSGSIDSSSN SASLTISGLRTEDEA DYYC QSYDSS N— νλ5-45 QAVLTQP ASLSASP GASASLT C TLRSGI NVGTYR ---IY WYQQRP GSPPQY LLR YRSDSD RQQGS GVPSRFSGSRDASAN AGILLISGLQSEDEA DYYC MIWHSS AS- νλ7-43 QTVVTQE PSLTVSP GGTVTLT C ASSTGA VTSGYY ---PN WFQQRP GQAPRA LIY ST---- SNRHS WTPARFSGSLLG-- GRAALTLSGVQPEDE AEYYC LLYYGG AQ- 2015201796 09 Apr 2015 - 113 - 1 The last amino acid in CDRL1 of the νλ3-1 chassis, S, differs from the corresponding one in the IGkV3-l germline gene, C. This was done to avoid having a potentially unpaired CYS (C) amino acid in the resulting synthetic light chain. 2015201796 09 Apr 2015 5 2 Note that, as for the VK chassis, the portion of the IGkV gene contributing to

VkCDR3 is not considered part of the chassis as described herein. The νλ chassis is defined as Kabat residues 1 to 88 of the IGLV-encoded sequence, or from the start of FRM1 to the end of FRM3. The portion of the VXCDR3 sequence contributed by the IGkV gene is referred to herein as the L3-Vk region. 10

Example 5: Design of a CDRH3 Library

This example describes the design of a CDHR3 library from its individual components. In nature, the CDRH3 sequence is derived from a complex process involving recombination of three different genes, termed IGHV, IGHD and IGHJ. In 15 addition to recombination, these genes may also undergo progressive nucleotide deletions: from the 3’ end of the IGHV gene, either end of the IGHD gene, and/or the 5’ end of the IGHJ gene. Non-templated nucleotide additions may also occur at the junctions between the V, D and J sequences. Non-templated additions at the V-D junction are referred to as “Nl”, and those at the D-J junction are referred to as “N2”. 20 The D gene segments may be read in three forward and, in some cases, three reverse reading frames.

In the design of the present exemplary library, the codon (nucleotide triplet) or single amino acid was designated as a fundamental unit, to maintain all sequences in the desired reading frame. Thus, all deletions or additions to the gene segments are carried 25 out via the addition or deletion of amino acids or codons, and not single nucleotides. According to the CDRH3 numbering system of this application, CDRH3 extends from amino acid number 95 (when present; see Example 1) to amino acid 102.

Example 5.1: Selection of the DH Segments 30 In this illustrative example, selection of DH gene segments for use in the library was performed according to principles similar to those used for the selection of the chassis sequences. First, an analysis of IGHD gene usage was performed, using data from Lee et al., Immunogenetics, 2006, 57: 917; Corbett et al., PNAS, 1982, 79: 4118; and Souto-Cameiro et al., J. Immunol., 2004, 172: 6790 (each incorporated by reference 35 in its entirety), with preference for representation in the library given to those IGHD genes most frequently observed in human sequences. Second, the degree of deletion on either end of the IGHD gene segments was estimated by comparison with known heavy - 114- chain sequences, using the SoDA algorithm (Volpe etal., Bioinformatics, 2006, 22: 438, incorporated by reference in its entirety) and sequence alignments. For the presently exemplified library, progressively deleted DH segments, as short as three amino acids, were included. As enumerated in the Detailed Description, other embodiments of the 5 invention comprise DH segments with deletions to a different length, for example, about 1, 2, 4, 5, 6, 7, 8, 9, or 10 amino acids. Table 15 shows the relative occurrence of IGHD gene usage in human antibody heavy chain sequences isolated mainly from peripheral blood B cells (list adapted from Lee et al., Immunogenetics, 2006, 57: 917, incorporated by reference in its entirety). 2015201796 09 Apr 2015 10

Table 15. Usage of IGHD Genes Based on Relative Occurrence in Peripheral Blood* IGHD Gene Estimated Relative Occurrence in Peripheral Blood3 IGHD3-10 117 IGHD3-22 111 IGHD6-19 95 IGHD6-13 93 IGHD3-3 82 IGHD2-2 63 IGHD4-17 61 IGHD1-26 51 IGHD5-5/5-181 49 IGHD2-15 47 IGHD6-6 38 IGHD3-9 32 IGHD5-12 29 IGHD5-24 29 IGHD2-21 28 IGHD3-16 18 IGHD4-23 13 IGHD1-1 9 IGHD1-7 9 IGHD4-4/4-112 7 IGHD1-20 6 IGHD7-27 6 IGHD2-8 4 IGHD6-25 3 Although c istinct genes in the genome, the nucleotic IGHD5-18 are 100% identical and thus indistinguishable in rearranged VH sequences. 2IGHD4-4 and IGHD4-11 are also 100% identical. 3Adapted from Lee et al. Immunogenetics, 2006, 57: 917, by merging the information for distinct alleles of the same IGHD gene. 15 * IGHD 1-14 may also be included in the libraries of the invention. - 115 -

The translations of the ten most commonly expressed IGHD gene sequences found in naturally occurring human antibodies, in three reading frames, are shown in Table 16. Those reading frames which occur most commonly in peripheral blood have been highlighted in gray. As in Table 15, data regarding IGHD sequence usage and 5 reading frame statistics were derived from Lee et al., 2006, and data regarding IGHD sequence reading frame usage were further complemented by data derived from Corbett et al., PNAS, 1982, 79: 4118 and Souto-Cameiro et al., J. Immunol, 2004, 172: 6790, each of which is incorporated by reference in its entirety.

10 Table 16. Translations of the Ten Most Common Naturally Occurring IGHD

Sequences, n Three Reading Frames (RF) IGHD RF 1 SEQ ID NO RF 2 SEQ ID NO RF 3 SEQ ID NO IGHD3-10 FGELL YYYGEGSYYN ITMVR.GVH IGHD3-22 VLLL###WLLL yyyd:::;gyyy ITMIVWIT IGHD6-19 SGWY G f AVAG V#QWLV IGHD6-13 GYSSSWY GIAAAG V#QQLV IGHD3-03 VLRFLEWLLY YYDFWSGYYT ITIFGWII IGHD2-02 WIL##YQLLC GYCSSTSCYT DIVVVi'AAM IGHD4-17 #LR#L DYGDY TTVT IGHD1-26 *: VGATT V#WELL YSGSYY IGHD5- 5/5-18 VDTAMVT WIQLWL GYEYGY IGHD2-15 RIL#WW#LLL GYCSGGSCYS DIWWAAT # represents a stop codon.

Reading frames highlighted in gray correspond to the most commonly used reading frames. 2015201796 09 Apr 2015 15 In the presently exemplified library, the top 10 IGHD genes most frequently used in heavy chain sequences occurring in peripheral blood were chosen for representation in the library. Other embodiments of the library could readily utilize more or fewer D genes. The amino acid sequences of the selected IGHD genes, including the most commonly used reading frames and the total number of variants after progressive N- and 20 C-terminal deletion to a minimum of three residues, are listed in Table 17. As depicted in Table 17, only the most commonly occurring alleles of certain IGHD genes were included in the illustrative library. This is, however, not required, and other embodiments of the invention may utilize IGHD reading frames that occur less frequently in the peripheral blood. 25

Table 17. D Genes Selected for use in the Exemplary Library IGHD Gene1 Amino Acid Sequence SEQ ID NO: Total Number of Variants2 -116- 2015201796 09 Apr 2015 IGHD1-26 1 GIVGATT 15 IGHD1-26 3 YSGSYY 10 IGHD2-2 2 GYCSSTSCYT 9a IGHD2-2 3 DIWVPAAM 28 IGHD2-15 2 GYCSGGSCYS 9 IGHD3-3 3 ITIFGWII 28 IGHD3-10 1 VLLWFGELL 28 IGHD3-10 2 YYYGSGSYYN 36 IGHD3-10 3 ITMVRGVII 28 IGHD3-22 2 YYYDSSGYYY 36 IGHD4-17 2 DYGDY 6 IGHD5-5 3 GYSYGY 10 IGHD6-13 1 GYSSSWY 15 IGHD6-13 2 GIAAAG 10 IGHD6-19 1 GYSSGWY 15 IGHD6-19 2 GIAVAG 10 ^he reac ing frame (RF) is specified as _RF after the name of the gene. 2 In most cases the total number of variants is given by (N-l) times (N-2) divided by two, where N is the total length in amino acids of the intact D segment. 3As detailed herein, the number of variants for segments containing a putative disulfide 5 bond (two C or Cys residues) is limited in this illustrative embodiment.

For each of the selected sequences of Table 17, variants were generated by systematic deletion from the N- and/or C-termini, until there were three amino acids remaining. For example, for the IGHD4-17 2 above, the full sequence DYGDY (SEQ

10 ID NO:_) may be used to generate the progressive deletion variants: DYGD (SEQ ID NO:_), YGDY (SEQ ID NO:_), DYG (SEQ ID NO:_), GDY (SEQ ID NO:_) and YGD (SEQ ID NO:_). In general, for any full-length sequence of size N, there will be a total of (N-l)*(N-2)/2 total variants, including the original full sequence. For the disulfide-loop-encoding segments, as exemplified by reading frame 2 of both 15 IGHD2-2 and IGHD2-15, (i.e., IGHD2-2 2 and IGH2-15 2), the progressive deletions were limited, so as to leave the loop intact i.e., only amino acids N-terminal to the first Cys, or C-terminal to the second Cys, were deleted in the respective DH segment variants. The foregoing strategy was used to avoid the presence of unpaired cysteine residues in the exemplified version of the library. However, as discussed in the Detailed 20 Description, other embodiments of the library may include unpaired cysteine residues, or the substitution of these cysteine residues with other amino acids. In the cases where the truncation of the IGHD gene is limited by the presence of the Cys residues, only 9 variants (including the original full sequence) were generated; e.g., for IGHD2-2 2, the

variants would be: GYCSSTSCYT (SEQ ID NO:_), GYCSSTSCY (SEQ ID 25 NO:_), YCSSTSCYT (SEQ ID NO:_), CSSTSCYT (SEQ ID NO:_), - 117-

GYCSSTSC (SEQ ID NO:_), YCSSTSCY (SEQ ID NO:_), CSSTSCY (SEQ ID 2015201796 09 Apr 2015 NO:_), YCSSTSC (SEQ ID NO:_) and CSSTSC (SEQ ID NO:_).

According to the criteria outlined above, 293 DH sequences were obtained from the selected IGHD gene segments, including the original IGHD gene segments. Certain 5 sequences are redundant. For example, it is possible to obtain the ΥΎΥ variant from either IGHD3-10 2 (full sequence YYYGSGSYYN (SEQ ID NO:_)), or in two different ways from IGHD3-22 2 (SEQ ID NO:_) (YYYDSSGYYY). When redundant sequences are removed, the number of unique DH segment sequences in this illustrative embodiment of the library is 278. These sequences are enumerated in Table 10 18.

Table 18. DH Gene Segments Used in the Presently Exemplified Library*

DH Segment Designation1 Peptide SEQ ID NO: DH Segment Designation Peptide SEQ ID NO: IGHD1-26 1-1 ATT IGHD3-10 2-20 YYGSG IGHD1-26 1-2 GAT IGHD3-10 2- 21 YYYGS IGHD1-26 1-3 GIV IGHD3-10 2-22 GSGSYY IGHD1-26 1-4 IVG IGHD3-10 2-23 SGSYYN IGHD1-26 1-5 VGA IGHD3-10 2-24 YGSGSY IGHD1-26 1-6 GATT IGHD3-10 2-25 YYGSGS IGHD1-26 1-7 GIVG IGHD3-10 2-26 YYYGSG IGHD1-26 1-8 IVGA IGHD3-10 2-27 GSGSYYN IGHD1-26 1-9 VGAT IGHD3-10 2-28 YGSGSYY IGHD1-26 1-10 GIVGA IGHD3-10 2-29 YYGSGSY IGHD1-26 1-11 IVGAT IGHD3-10 2-30 YYYGSGS IGHD1-26 1-12 VGATT IGHD3-10 2-31 YGSGSYYN IGHD1-26 1-13 GIVGAT IGHD3-10 2-32 YYGSGSYY IGHD1-26 1-14 IVGATT IGHD3-10 2-33 YYYGSGSY IGHD1-26 1-15 GIVGATT IGHD3-10 2-34 YYGSGSYYN IGHD1-26 3-1 YSG IGHD3-10 2-35 YYYGSGSYY IGHD1-26 3-2 YSGS IGHD3-10 2-36 YYYGSGSYYN - 118- 2015201796 09 Apr 2015

IGHD1-26 3-3 YSGSY IGHD3-10 3-1 GVI IGHD1-26 3-4 YSGSYY IGHD3-10 3-2 ITM IGHD2-02 2-1 CSSTSC IGHD3-10 3-3 MVR IGHD2-02 2-2 CSSTSCY IGHD3-10 3-4 RGV IGHD2-02 2-3 YCSSTSC IGHD3-10 3-5 TMV IGHD2-02 2-4 CSSTSCYT IGHD3-10 3-6 VII IGHD2-02 2-5 GYCSSTSC IGHD3-10 3-7 VRG IGHD2-02 2-6 YCSSTSCY IGHD3-10 3-8 GVI I IGHD2-02 2-7 GYCSSTSCY IGHD3-10 3-9 I TMV IGHD2-02 2-8 YCSSTSCYT IGHD3-10 310 MVRG IGHD2-02 2-9 GYCSSTSCYT IGHD3-10 311 RGV I IGHD2-02 3-1 AAM IGHD3-10 312 TMVR IGHD2-02 3-2 DIV IGHD3-10 313 VRGV IGHD2-02 3-3 IW IGHD3-10 314 I TMVR IGHD2-02 3-4 PAA IGHD3-10 315 MVRGV IGHD2-02 3-5 VPA IGHD3-10 316 RGV 11 IGHD2-02 3-6 VVP IGHD3-10 317 TMVRG IGHD2-02 3-7 VW IGHD3-10 318 VRGV I IGHD2-02 3-8 DIVV IGHD3-10 319 ITMVRG IGHD2-02 3-9 IWV IGHD3-10 320 MVRGVI IGHD2-02 3-10 PAAM IGHD3-10 321 TMVRGV IGHD2-02 3-11 VPAA IGHD3-10 322 VRGVII IGHD2-02 3-12 WPA IGHD3-10 323 ITMVRGV IGHD2-02 3-13 WVP IGHD3-10 324 MVRGVII IGHD2-02 3-14 DIWV IGHD3-10 325 TMVRGVI IGHD2-02 3-15 IVWP IGHD3-10 326 ITMVRGVI IGHD2-02 3-16 VPAAM IGHD3-10 327 TMVRGVII IGHD2-02 3-17 WPAA IGHD3-10 328 ITMVRGVII IGHD2-02 3-18 WVPA IGHD3-22 2-1 DSS IGHD2-02 3-19 DIVWP IGHD3-22 2-2 GYY IGHD2-02 3-20 IVWP A IGHD3-22 2-3 SGY IGHD2-02 3-21 WPAAM IGHD3-22 2-4 SSG IGHD2-02 3-22 WVPAA IGHD3-22 2-5 YDS IGHD2-02 3-23 DI WVPA IGHD3-22 2-6 YYD IGHD2-02 3-24 I WVPAA IGHD3-22 2-7 DSSG IGHD2-02 3-25 VWP AAM IGHD3-22 2-8 GYYY IGHD2-02 3-26 DIVWPAA IGHD3-22 2-9 SGYY - 119- 2015201796 09 Apr 2015 IGHD2-02 3-27 IWVPAAM IGHD3-22 210 SSGY IGHD2-02 3-28 DIWVPAAM IGHD3-22 211 YDSS IGHD2-15 2-1 CSGGSC IGHD3-22 212 YYDS IGHD2-15 2-2 CSGGSCY IGHD3-22 213 YYYD IGHD2-15 2-3 YCSGGSC IGHD3-22 214 DSSGY IGHD2-15 2-4 CSGGSCYS IGHD3-22 215 SGYYY IGHD2-15 2-5 GYCSGGSC IGHD3-22 216 SSGYY IGHD2-15 2-6 YCSGGSCY IGHD3-22 217 YDSSG IGHD2-15 2-7 GYCSGGSCY IGHD3-22 218 YYDSS IGHD2-15 2-8 YCSGGSCYS IGHD3-22 219 YYYDS IGHD2-15 2-9 GYCSGGSCYS IGHD3-22 220 DSSGYY IGHD3-03 3-1 FGV IGHD3-22 221 SSGYYY IGHD3-03 3-2 GW IGHD3-22 222 YDSSGY IGHD3-03 3-3 IFG IGHD3-22 223 YYDSSG IGHD3-03 3-4 ITI IGHD3-22 224 YYYDSS IGHD3-03 3-5 TIF IGHD3-22 225 DSSGYYY IGHD3-03 3-6 WI IGHD3-22 226 YDSSGYY IGHD3-03 3-7 FGW IGHD3-22 227 YYDSSGY IGHD3-03 3-8 GW I IGHD3-22 228 YYYDSSG IGHD3-03 3-9 I FGV IGHD3-22 229 YDSSGYYY IGHD3-03 3-10 ITI F IGHD3-22 230 YYDSSGYY IGHD3-03 3-11 TIFG IGHD3-22 231 YYYDSSGY IGHD3-03 3-12 WII IGHD3-22 232 YYDSSGYYY IGHD3-03 3-13 FGW I IGHD3-22 233 YYYDSSGYY IGHD3-03 3-14 GWII IGHD3-22 234 YYYDSSGYYY IGHD3-03 3-15 I FGW IGHD4-17 2-1 DYG IGHD3-03 3-16 ITIFG IGHD4-17 2-2 GDY IGHD3-03 3-17 TIFGV IGHD4-17 2-3 YGD IGHD3-03 3-18 FGWII IGHD4-17 2-4 DYGD IGHD3-03 3-19 IFGWI IGHD4-17 2-5 YGDY IGHD3-03 3-20 ITIFGV IGHD4-17 2-6 DYGDY IGHD3-03 3-21 TIFGW IGHD5-5 3-1 SYG IGHD3-03 3-22 I FGWII IGHD5-5 3-2 YGY - 120- 2015201796 09 Apr 2015

IGHD3-03 3-23 ITIFGW IGHD5-5 3-3 YSY IGHD3-03 3-24 TIFGVVI IGHD5-5 3-4 GYSY IGHD3-03 3-25 ITIFGWI IGHD5-5 3-5 SYGY IGHD3-03 3-26 TIFGVVII IGHD5-5 3-6 YSYG IGHD3-03 3-27 ITIFGWI I IGHD5-5 3-7 GYSYG IGHD3-10 1-1 ELL IGHD5-5 3-8 YSYGY IGHD3-10 1-2 FGE IGHD5-5 3-9 GYSYGY IGHD3-10 1-3 GEL IGHD6-13 1-1 SSS IGHD3-10 1-4 LLW IGHD6-13 1-2 SSW IGHD3-10 1-5 LWF IGHD6-13 1-3 SWY IGHD3-10 1-6 VLL IGHD6-13 1-4 SSSW IGHD3-10 1-7 WFG IGHD6-13 1-5 SSWY IGHD3-10 1-8 FGEL IGHD6-13 1-6 YSSS IGHD3-10 1-9 GELL IGHD6-13 1-7 GYSSS IGHD3-10 1-10 LLWF IGHD6-13 1-8 SSSWY IGHD3-10 1-11 LWFG IGHD6-13 1-9 YSSSW IGHD3-10 1-12 VLLW IGHD6-13 Ι-ΙΟ GYSSSW IGHD3-10 1-13 WFGE IGHD6-13 111 YSSSWY IGHD3-10 1-14 FGELL IGHD6-13 112 GYSSSWY IGHD3-10 1-15 LLWFG IGHD6-19 1-1 GWY IGHD3-10 1-16 LWFGE IGHD6-19 1-2 GYS IGHD3-10 1-17 VLLWF IGHD6-19 1-3 SGW IGHD3-10_1-18 WFGEL IGHD6-19_1-4 YSS IGHD3-10 1-19 LLWFGE IGHD6-19 1-5 GYSS IGHD3-10 1-20 LWFGEL IGHD6-19 1-6 SGWY IGHD3-10 1-21 VLLWFG IGHD6-19 1-7 SSGW IGHD3-10 1-22 WFGELL IGHD6-19 1-8 YSSG IGHD3-10 1-23 LLWFGEL IGHD6-19 1-9 GYSSG IGHD3-10 1-24 LWFGELL IGHD6-19 Ι-ΙΟ SSGWY IGHD3-10 1-25 VLLWFGE IGHD6-19 111 YSS GW IGHD3-10 1-26 LLWFGELL IGHD6-19 112 GY S S GW IGHD3-10 1-27 VLLWFGEL IGHD6-19 113 YSSGWY IGHD3-10 1-28 VLLWFGELL IGHD6-19 114 GYSSGWY IGHD3-10 2-1 GSG IGHD6-19 2-1 AVA IGHD3-10 2-2 GSY IGHD6-19 2-2 GIA IGHD3-10 2-3 SGS IGHD6-19 2-3 IAV IGHD3-10 2-4 SYY IGHD6-19 2-4 VAG IGHD3-10 2-5 YGS IGHD6-19 2-5 AVAG IGHD3-10 2-6 YYG IGHD6-19 2-6 GIAV IGHD3-10_2-7 YYN IGHD6-19_2-7 I AVA IGHD3-10 2-8 YYY IGHD6-19 2-8 GIAVA IGHD3-10 2-9 GSGS IGHD6-19 2-9 I AVAG IGHD3-10 2-10 GSYY IGHD6-19 210 GIAVAG IGHD3-10 2-11 SGSY IGHD6-13 2-1 AAA IGHD3-10 2-12 SYYN IGHD6-13 2-2 AAG - 121 -

IGHD3-10 2-13 YGSG IGHD6-13 2-3 IAA IGHD3-10 2-14 YYGS IGHD6-13 2-4 AAAG IGHD3-10 2-15 YYYG IGHD6-13 2-5 GIAA IGHD3-10 2-16 GSGSY IGHD6-13 2-6 I AAA IGHD3-10 2-17 GSYYN IGHD6-13 2-7 GIAAA IGHD3-10 2-18 SGSYY IGHD6-13 2-8 I AAAG IGHD3-10 2-19 YGSGS IGHD6-13 2-9 GIAAAG 'The sequence designation is formatted as follows: (IGHD Gene Name)_(Reading Frame)-(V ariant Number) * Note that the origin of certain variants is rendered somewhat arbitrary when redundant 5 segments are deleted from the library (i.e., certain segments may have their origins with more than one parent, including the one specified in the table).

Table 19 shows the length distribution of the 278 DH segments selected according to the methods described above. 2015201796 09 Apr 2015 10

Table 19 Length Distributions of DH Segments Selected for Inclusion in the Exemplary Library DH Size Number of Occurrences 3 78 4 64 5 50 6 38 7 27 8 20 9 12 10 4 15 As specified above, based on the CDRH3 numbering system defined in this application, IGHD-derived amino acids (i.e., DH segments) are numbered beginning with position 97, followed by positions 97A, 97B, etc. In the currently exemplified embodiment of the library, the shortest DH segment has three amino acids: 97, 97A and 97B, while the longest DH segment has 10 amino acids: 97, 97A, 97B, 97C, 97D, 97E, 20 97F, 97G, 97H and 971.

Example 5.2: Selection of the H3-JH Segments

There are six human germline IGHJ genes. During in vivo assembly of antibody genes, these segments are progressively deleted at their 5’ end. In this exemplary 25 embodiment of the library, IGHJ gene segments with no deletions, or with 1, 2, 3, 4, 5, 6, or 7 deletions (at the amino acid level), yielding JH segments as short as 13 amino - 122- acids, were included (Table 20). Other embodiments of the invention, in which the IGHJ gene segments are progressively deleted (at their 5’ / N-terminal end) to yield 15, 14, 12, or 11 amino acids are also contemplated.

Table 20. IGHJ Gene Segments Selected for use in the Exemplary Library

IGHJ Segment rH3-JH]-[FRM4]1 SEQ ID NO: H3-JH SEQ ID NO: JH1 parent or JH1 1 AEYFQHWGQGTLVTVSS AEYFQH JH1_2 EYFQHWGQGTLVTVSS EYFQH JH1_3 YFQHWGQGTLVTVSS YFQH JH1_4 FQHWGQGTLVTVS S FQH JH1_5 QHWGQGTLVTVS S QH JH2 parent or JH2 1 YWYFDLWGRGTLVTVS S YWYFDL JH2_2 WYFDLWGRGTLVTVSS WYFDL JH2_3 YFDLWGRGTLVTVSS YFDL JH2_4 FDLWGRGTLVTVSS FDL JH2_5 DLWGRGTLVTVSS DL JH3 parent or JH3 1 AFDVWGQGTMVTVSS AFDV JH3_2 FDVWGQGTMVTVSS FDV JH3_3 DVWGQGTMVTVS S DV JH4 parent or JH4 1 YFDYWGQGTLVTVSS YFDY JH4_2 FDYWGQGTLVTVS S FDY JH4_3 DYWGQGTLVTVS S DY JH5 parent or JH5 1 NWFDSWGQGTLVTVSS NWFDS JH5_2 WFDSWGQGTLVTVSS WFDS JH5_3 FDSWGQGTLVTVSS FDS JH5_4 DSWGQGTLVTVSS DS JH6 parent or JH6 1 YYYYYGMDVWGQGTTVTVSS YYYYYGMDV JH62 YYYYGMDVWGQGTTVTVSS YYYYGMDV JH63 YYYGMDVWGQGTTVTVSS YYYGMDV JH64 YYGMDVWGQGTTVTVSS YYGMDV JH65 YGMDVWGQGTTVTVSS YGMDV JH66 GMDVWGQGTTVTVSS GMDV JH6_7 MDVWGQGTTVTVS S MDV JH6_8 DVWGQGTTVTVS S DV 1H3-JH is defined as the portion of the IGHJ segment included within the Kabat definition of CDRH3; FRM4 is defined as the portion of the IGHJ segment encoding framework region four. 2015201796 09 Apr 2015 5 10 According to the CDRH3 numbering system of this application, the contribution of, for example, JH61 to CDRH3, would be designated by positions 99F, 99E, 99D, 99C, 99B, 99A, 100, 101 and 102 (Y, Y, Y, Y, Y, G, M, D and V, respectively). Similarly, - 123 - the JH4_3 sequence would contribute amino acid positions 101 and 102 (D and Y, respectively) to CDRH3. However, in all cases of the exemplified library, the JH segment will contribute amino acids 103 to 113 to the FRM4 region, in accordance with the standard Kabat numbering system for antibody variable regions (Kabat, op. cit. 2015201796 09 Apr 2015 5 1991). This may not be the case in other embodiments of the library.

Example 5.3: Selection of the N1 and N2 Segments

While the consideration of V-D-J recombination enhanced by mimicry of the naturally occurring process of progressive deletion (as exemplified above) can generate 10 enormous diversity, the diversity of the CDRH3 sequences in vivo is further amplified by non-templated addition of a varying number of nucleotides at the V-D junction and the D-J junction. N1 and N2 segments located at the V-D and D-J junctions, respectively, were identified in a sample containing about 2,700 antibody sequences (Jackson et al., J. 15 Immunol. Methods, 2007, 324: 26) also analyzed by the SoDA method of Volpe et al., Bioinformatics, 2006, 22: 438-44; (both Jackson et al., and Volpe et al., are incorporated by reference in their entireties). Examination of these sequences revealed patterns in the length and composition of N1 and N2. For the construction of the currently exemplified CDRH3 library, specific short amino acid sequences were derived from the above 20 analysis and used to generate a number of N1 and N2 segments that were incorporated into the CDRH3 design, using the synthetic scheme described herein.

As described in the Detailed Description, certain embodiments of the invention include N1 and N2 segments with rationally designed length and composition, informed by statistical biases in these parameters that are found by comparing naturally occurring 25 N1 and N2 segments in human antibodies. According to data compiled from human databases (see, e.g., Jackson et al., J. Immunol Methods, 2007, 324: 26, incorporated by reference in its entirety), there are an average of about 3.02 amino acid insertions for N1 and about 2.4 amino acid insertions for N2, not taking into account insertions of two nucleotides or less. Figure 2 shows the length distributions of the N1 and N2 regions in 30 human antibodies. In this exemplary embodiment of the invention, N1 and N2 were fixed to a length of 0, 1, 2, or 3 amino acids. The naturally occurring composition of these sequences in human antibodies was used as a guide for the inclusion of different amino acid residues. - 124-

The naturally occurring composition of single amino acid, two amino acids, and three amino acids N1 additions is defined in Table 21, and the naturally occurring composition of the corresponding N2 additions is defined in Table 22. The most frequently occurring duplets in the N1 and N2 set are compiled in Table 23. 2015201796 09 Apr 2015 5

Table 21. Composition of Naturally Occurring 1,2, and 3 Amino Acid N1 Additions*

Position 1 Number of Occurrences Position 2 Number of Occurrences Position 3 Number of Occurrences R 251 G 97 G 101 G 249 P 67 R 66 P 173 R 67 P 47 L 130 S 42 s 47 S 117 L 39 L 38 A 84 V 33 A 33 V 62 E 24 V 28 K 61 A 21 T 27 I 55 D 18 E 24 Q 51 I 18 D 22 T 51 T 18 K 18 D 50 K 16 F 14 E 49 Y 16 I 13 F 3 H 13 W 13 H 32 F 12 N 10 N 30 Q 11 Y 10 W 28 N 5 H 8 Y 21 W 5 Q 5 M 16 C 4 C 3 C 3 M 4 M 3 1546 530 530 * Defined as the sequence C-terminal to “CARX”, or equivalent, of VH, wherein “X” is the “tail” (e.g., D, E, G, or no amino acid residue). 10 - 125 - 2015201796 09 Apr 2015 5

Table 22. Composition of Naturally Occurring 1, 2, and 3 Amino Acid N2 Additions* Position 1 Number of Occurrences Position 2 Number of Occurrences Position 3 Number of Occurrences G 242 G 244 G 156 P 219 P 138 P 79 R 180 R 86 S 54 L 132 S 85 R 51 S 123 T 77 L 49 A 97 L 74 A 41 T 78 A 69 T 31 V 75 V 46 V 29 E 57 E 41 D 23 D 56 Y 38 E 23 F 54 D 36 W 23 H 54 K 30 Q 19 Q 53 F 29 F 17 I 49 W 27 Y 17 N 45 H 24 H 16 Y 40 I 23 I 11 K 35 Q 23 K 11 w 29 N 21 N 8 M 20 M 8 C 6 C 6 C 5 M 6 1644 1124 670 * Defined as the sequence C-terminal to the D segment but not encoded by IGHJ genes. - 126- 2015201796 09 Apr 2015

Table 23. Top Twenty-Five Naturally Occurring N1 and N2 Duplets Sequence Number of Occurrences Cumulative Frequency Individual Frequency GG 17 0.037 0.037 PG 15 0.070 0.033 RG 15 0.103 0.033 PP 13 0.132 0.029 GP 12 0.158 0.026 GL 11 0.182 0.024 PT 10 0.204 0.022 TG 10 0.226 0.022 G V 9 0.246 0.020 RR 9 0.266 0.020 SG 8 0.284 0.018 RP 7 0.299 0.015 IG 6 0.312 0.013 GS 6 0.325 0.013 SR 6 0.338 0.013 PA 6 0.352 0.013 LP 6 0.365 0.013 VG 6 0.378 0.013 KG 6 0.389 0.011 GW 5 0.400 0.011 FP 5 0.411 0.011 LG 5 0.422 0.011 RS 5 0.433 0.011 TP 5 0.444 0.011 EG 5 0.455 0.011

Example 5.3.1 Selection of the N1 Segments 5 Analysis of the identified N1 segments, located at the junction between V and D, revealed that the eight most frequently occurring amino acid residues were G, R, S, P, L, A, T and V (Table 21). The number of amino acid additions in the N1 segment was frequently none, one, two, or three (Figure 2). The addition of four or more amino acids was relatively rare. Therefore, in the currently exemplified embodiment of the library, 10 the N1 segments were designed to include zero, one, two or three amino acids.

However, in other embodiments, N1 segments of four, five, or more amino acids may also be utilized. G and P were always among the most commonly occurring amino acid residues in the N1 regions. Thus, in the present exemplary embodiment of the library, the N1 segments that are dipeptides are of the form GX, XG, PX, or XP, where X is any 15 of the eight most commonly occurring amino acids listed above. Due to the fact that G - 127- residues were observed more frequently than P residues, the tripeptide members of the exemplary N1 library have the form GXG, GGX, or XGG, where X is, again, one of the eight most frequently occurring amino acid residues listed above. The resulting set of N1 sequences used in the present exemplary embodiment of the library, include the 5 “zero” addition amounts to 59 sequences, which are listed in Table 24.

Table 24. N1 Sequences Selected for Inclusion in the Exemplary Library

Segment Type Sequences Number “Zero” (no addition) V segment joins directly to D segment 1 Monomers G, P, R, A, S, L, T, V 8 Dimers GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP 28 Trimers GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV 22 2015201796 09 Apr 2015

In accordance with the CDRH3 numbering system of the application, the 10 sequences enumerated in Table 24 contribute the following positions to CDRH3: the monomers contribute position 96, the dimers to 96 and 96A, and the trimers to 96, 96A and 96B. In alternative embodiments, where tetramers and longer segments could be included among the N1 sequences, the corresponding numbers would go on to include 96C, and so on. 15

Example 5.3.2 Selection of the N2 Segments

Similarly, analysis of the identified N2 segments, located at the junction between D and J, revealed that the eight most frequently occurring amino acid residues were also G, R, S, P, L, A, T and V (Table 22). The number of amino acid additions in the N2 20 segment was also frequently none, one, two, or three (Figure 2). For the design of the N2 segments in the exemplary library, an expanded set of sequences was utilized. Specifically, the sequences in Table 25 were used, in addition to the 59 sequences enumerated in Table 24, for N1. 25 Table 25. Extra Sequences in N2 Additions___

Segment Type Sequence Number New Number Total Monomers D, E, F, Η, I, K, M, Q, W, Y 10 18 - 128-

Dimers AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS 54 82 Trimers AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, WGT 18 40

The presently exemplified embodiment of the library, therefore, contains 141 total N2 sequences, including the “zero” state. One of ordinary skill in the art will readily recognize that these 141 sequences may also be used in the N1 region, and that 5 such embodiments are within the scope of the invention. In addition, the length and compositional diversity of the N1 and N2 sequences can be further increased by utilizing amino acids that occur less frequently than G, R, S, P, L, A, T and V, in the N1 and N2 regions of naturally occurring antibodies, and including N1 and N2 segments of four, five, or more amino acids in the library. Tables 21 to 23 and Figure 2 provides 10 information about the composition and length of the N1 and N2 sequences in naturally occurring antibodies that is useful for the design of additional N1 and N2 regions which mimic the natural composition and length. 2015201796 09 Apr 2015

In accordance with the CDRH3 numbering system of the application, N2 sequences will begin at position 98 (when present) and extend to 98A (dimers) and 98B 15 (trimers). Alternative embodiments may occupy positions 98C, 98D, and so on.

Example 5.4. A CDRH3 Library

When the “tail” (i.e., G/D/E/-) is considered, the CDRH3 in the exemplified library may be represented by the general formula: [G/D/E/-] - [N1]-[DH]-[N2]-[H3-JH]

In the currently exemplified, non-limiting, embodiment of the library, [G/D/E/-] represents each of the four possible terminal amino acid “tails”; N1 can be any of the 59 25 sequences in Table 24; DH can be any of the 278 sequences in Table 18; N2 can be any of the 141 sequences in Tables 24 and 25; and H3-JH can be any of the 28 H3-JH sequences in Table 20. The total theoretical diversity or repertoire size of this CDRH3 - 129- library is obtained by multiplying the variations at each of the components, i.e., 4 x 59 x 278 x 141 x 28 = 2.59 x 108. 2015201796 09 Apr 2015

However, as described in the previous examples, redundancies may be eliminated from the library. In the presently exemplified embodiment, the tail and N1 5 segments were combined, and redundancies were removed from the library. For example, considering the VH chassis, tail, and N1 regions, the sequence [VH_Chassis]-[G] may be obtained in two different ways: [VH_Chassis] + [G] + [nothing] or [VHChassis] + [nothing] + [G], Removal of redundant sequences resulted in a total of 212 unique [G/D/E/-]-[Nl] segments out of the 236 possible combinations (i.e., 4 tails x 10 59 Nl). Therefore, the actual diversity of the presently exemplified CDRH3 library is 212 x 278 x 141 x 28 = 2.11 x 108. Figure 23 depicts the frequency of occurrence of different CDRH3 lengths in this library, versus the preimmune repertoire of Lee et al.

Table 26 further illustrates specific exemplary sequences from the CDRH3 library described above, using the CDRH3 numbering system of the present application. 15 In instances where a position is not used, the hyphen symbol (-) is included in the table instead. -130- 2015201796 09 Apr 2015

Attorney Docket No.: ADS-011.25

Table 26. Examples of Designed CDRH3 Sequences According to the Library Exemplified in Examples 1 to 5 | [Tail] | [N1] | [DH] | [N2] j [H3- No. 1 |'~95.....f'96......96A | G | - 'ΤέΒ'ΤθΓ - I Y "97A" '"'T™ 97B 97C Y 97D 97E 97F \\\\\\\\\\\\\\\\\\\\\\\\\\ 97G.....97H WXXXXWWWWWXXXXWXX' No. 2 1 D | G - - I G Y C s G G s c Y No. 3 1 e | R - - 1 1 T 1 F G V - - - No. 4 1 | p P - I V L L W F G E L L No. 5 1 G 1 G s G | Y Y Y G S G S Y Y No. 6 1 ° | - - - | R G V 1 1 - - - - No. 7 I E | s G - | Y Y Y D s S G Y Y No. $ 1 s _ - 1 D Y G D Y _ _ _ _

G G

M

No. 9 No. 10

P G

W F G

P S

C S G G S C

A Y \xxx\x\\x\V\\x\\x\\x\\x\\x\\xv $ CDRH3 $ JH] 991B~ ~99A~ "99Γ Too'' ''ToT 102 id .encjth D V I 6 - - F Q H l 16 - - Y F D Y I 14 - - - - D L I 14 A E Y F Q H I 21 Y Y G M D V I 16 w Y F D L I 21 - - - F D I I 11 Y Y G M D V 13 N W F D P i 13 xxwwwwwwwwwwwwwwwwwxxxxxxxxxVxxxxxxxxxxxxxxxxx^ \x\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\xx\xx\xx\xx\xx\\\\\\\xx\xx\xxxxxxxxxxxxxxxxxx\x\xx\xx\\\\\\\\\\\wxxxxxxxxxxxxxw->''Ax\xx\xx\xx\xx\xxxxxxxxxxxxxxxxxxxxx\xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx\xx 131

Example 6: Design ofVKCDR3 Libraries 2015201796 09 Apr 2015

This example describes the design of a number of exemplary VKCDR3 libraries. As specified in the Detailed Description, the actual version(s) of the VKCDR3 library 5 made or used in particular embodiments of the invention will depend on the objectives for the use of the library. In this example the Rabat numbering system for light chain variable regions was used.

In order to facilitate examination of patterns of occurrence, human kappa light chain sequences were obtained from the publicly available NCBI database (Appendix 10 A). As for the heavy chain sequences (Example 2), each of the sequences obtained from the publicly available database was assigned to its closest germline gene, on the basis of sequence identity. The amino acid compositions at each position were then determined within each kappa light chain subset. 15 Example 6.1.: A Minimalist VKCDR3 Library

This example describes the design of a “minimalist” VKCDR3 library, wherein the VKCDR3 repertoire is restricted to a length of nine residues. Examination of the VKCDR3 lengths of human sequences shows that a dominant proportion (over 70%) has nine amino acids within the Rabat definition of CDRL3: positions 89 through 97. Thus, 20 the currently exemplified minimalist design considers only VRCDR3 of length nine. Examination of human kappa light chain sequences shows that there are not strong biases in the usage of IGRJ genes; there are five such IRJ genes in humans. Table 27 depicts IGRJ gene usage amongst three data sets, namely Juul et al. (Clin. Exp. Immunol., 1997, 109: 194, incorporated by reference in its entirety), Rlein and Zachau 25 (Eur. J. Immunol., 1993, 23: 3248, incorporated by reference in its entirety), and the kappa light chain data set provided in Appendix A (labeled LUA).

Table 27. IGRJ Gene Usage in Various Data Sets

Gene Klein Juul LUA IGKJ1 35.0% 29.0% 29.3% IGKJ2 25.0% 23.0% 24.1% IGKJ3 7.0% 8.0% 12.1% IGKJ4 26.0% 24.0% 26.5% IGKJ5 6.0% 18.0% 8.0% 30 - 132-

Thus, a simple combinatorial of “M” VK chassis and the 5 IGKJ genes would generate a library of size Μ x 5. In the Kabat numbering system, for VKCDR3 of length nine, amino acid number 96 is the first encoded by the IGKJ gene. Examination of the amino acid occupying this position in human sequences showed that the seven 5 most common residues are L, Y, R, W, F, P, and I, cumulatively accounting for about 85% of the residues found in position 96. The remaining 13 amino acids account for the other 15%. The occurrence of all 20 amino acids at position 96 is presented in Table 28. 2015201796 09 Apr 2015

Table 28. Occurrence of 20 Amino Acid Residues at Position 96 in Human VK Data Set

Type Number Percent Cumulative L 333 22.3 22.3 Y 235 15.8 38.1 R 222 14.9 52.9 W 157 10.5 63.5 F 148 9.9 73.4 1 96 6.4 79.8 P 90 6.0 85.9 Q 53 3.6 89.4 N 39 2.6 92.0 H 31 2.1 94.1 V 21 1.4 95.5 G 20 1.3 96.8 C 14 0.9 97.8 K 7 0.5 98.3 S 6 0.4 98.7 A 5 0.3 99.0 D 5 0.3 99.3 E 5 0.3 99.7 T 5 0.3 100.0 M 0 0.0 100.0

To determine the origins of the seven residues most commonly found in position 96, known human IGKJ amino acid sequences were examined (Table 29). 15 Table 29. Known Human IGKJ Amino Acid Sequences

Gene Sequence IGKJ1 WTFGQGTKVEIK IGKJ2 YTFGQGTKLEIK IGKJ3 FTFGPGTKVDIK IGKJ4 LTFGGGTKVEIK IGKJ5 ITFGQGTRLEIK

Without being bound by theory, five of the seven most commonly occurring amino acids found in position 96 of rearranged human sequences appear to originate - 133 - from the first amino acid encoded by each of the five human IGKJ genes, namely, W, Y, F, L, and I. 2015201796 09 Apr 2015

Less evident were the origins of the P and R residues. Without being bound by theory, most of the human IGKV gene nucleotide sequences end with the sequence CC, 5 which occurs after {i.e., 3’ to) the end of the last full codon (e.g., that encodes the C-terminal residue shown in Table 11). Therefore, regardless of which nucleotide is placed after this sequence {i.e., CCX, where X may be any nucleotide) the codon will encode a proline (P) residue. Thus, when the IGKJ gene undergoes progressive deletion (just as in the IGHJ of the heavy chain; see Example 5), the first full amino acid is lost 10 and, if no deletions have occurred in the IGKV gene, a P residue will result.

To determine the origin of the arginine residue at position 96, the origin of IGKJ genes in rearranged kappa light chain sequences containing R at position 96 were analyzed. The analysis indicated that R occurred most frequently at position 96 when the IGKJ gene was IGKJ1. The germline W (position 1; Table 29) for IGKJ1 is encoded 15 by TGG. Without being bound by theory, a single nucleotide change of T to C (yielding CGG) or A (yielding AGG) will, therefore, result in codons encoding Arg (R). A change to G (yielding GGG) results in a codon encoding Gly (G). R occurs about ten times more often at position 96 in human sequences than G (when the IGKJ gene is IGKJ1), and it is encoded by CGG more often than AGG. Therefore, without being 20 bound by theory, C may originate from one of the aforementioned two Cs at the end of IGKV gene. However, regardless of the mechanism(s) of occurrence, R and P are among the most frequently observed amino acid types at position 96, when the length of VKCDR3 is 9. Therefore, a minimalist VKCDR3 library may be represented by the following amino acid sequence: 25 [VK_ChassisHL3-VK]-[F/L/I/R/W/Y/P]-[TFGGGTKVEIK]

In this sequence, VK Chassis represents any selected VK chassis (for non-limiting examples, see Table 11), specifically Kabat residues 1 to 88 encoded by the IGKV gene. 30 L3-VK represents the portion of the VKCDR3 encoded by the chosen IGKV gene (in this embodiment, residues 89-95). F/L/I/R/W/Y/P represents any one of amino residues F, L, I, R, W, Y, or P. In this exemplary representation, IKJ4 (minus the first residue) has been depicted. Without being bound by theory, apart from IGKJ4 being among the -134- most commonly used IGKJ genes in humans, the GGG amino acid sequence is expected to lead to larger conformational flexibility than any of the alternative IGKJ genes, which contain a GXG amino acid sequence, where X is an amino acid other than G. In some embodiments, it may be advantageous to produce a minimalist pre-immune repertoire 5 with a higher degree of conformational flexibility. Considering the ten VK chassis depicted in Table 11, one implementation of the minimalist VKCDR3 library would have 70 members resulting from the combination of 10 VK chassis by 7 junction (position 96) options and one IGKJ-derived sequence (e.g., IGKJ4). Although this embodiment of the library has been depicted using IGKJ4, it is possible to design a 10 minimalist VKCDR3 library using one of the other four IGKJ sequences. For example, another embodiment of the library may have 350 members (10 VK chassis by 7 junctions by 5 IGKJ genes). 2015201796 09 Apr 2015

One of ordinary skill in the art will readily recognize that one or more minimalist VKCDR3 libraries may be constructed using any of the IGKJ genes. Using the notation 15 above, these minimalist VKCDR3 libraries may have sequences represented by, for example: 20 JK1. [VK_Chassis]-[L3-VK]-[F/L/I/R/W/Y/P]-[TFGQGTKVEIK]; JK2: [VKChassis]-11.3-VK]-[ F/E/I/R/W/Y/P] -| TFGQGTK I E IK ]; JK3: [VK Chassis]- [L3-VK]-| F/L/I/R/W/Y/P]-[TFGPGTKVDI K]; and JK5: [VK Chassis]- [L3-VK]-[F/L/I/R/W/Y7PHTFGQGTRLEIK].

Example 6.2: A VKCDR3 Library of About 10s Complexity

In this example, the nine residue VKCDR3 repertoire described in Example 6.1 25 is expanded to include VKCDR3 lengths of eight and ten residues. Moreover, while the previously enumerated VKCDR3 library included the VK chassis and portions of the IGKJ gene not contributing to VKCDR3, the presently exemplified version focuses only on residues comprising a portion of VKCDR3. This embodiment may be favored, for example, when recombination with a vector which already contains VK chassis 30 sequences and constant region sequences is desired.

While the dominant length of VKCDR3 sequences in humans is nine amino acids, other lengths appear at measurable rates that cumulatively approach almost 30% of kappa light chain sequences. In particular, VKCDR3 of lengths 8 and 10 represent, -135 - respectively, about 8.5% and about 16% of sequences in representative samples (Figure 3). Thus, a more complex VKCDR3 library includes CDR lengths of 8 to 10 amino acids; this library accounts for over 95% of the length distribution observed in typical collections of human VKCDR3 sequences. This library also enables the inclusion of 5 additional variation outside of the junction between the VK and JK genes. The present example describes such a library. The library comprises 10 sub-libraries, each designed around one of the 10 exemplary VK chassis depicted in Table 11. Clearly, the approach exemplified here can be generalized to consider M different chassis, where M may be less than or more than 10. 2015201796 09 Apr 2015 10 To characterize the variability within the polypeptide segment occupying Kabat positions 89 to 95, human kappa light chain sequence collections derived from each of the ten germline sequences of Example 3 were aligned and compared separately (i.e., within the germline group). This analysis enabled us to discern the patterns of sequence variation at each individual position in each kappa light chain sequence, grouped by 15 germline. The table below shows the results for sequences derived from IGKV1-39.

Table 30. Percent Occurrence of Amino Acid Types in IGKVl-39-Derived Sequences

Amino Acid P89 P90 P91 P92 P93 P94 P95 A 0 0 1 0 0 4 1 C 0 0 0 0 0 0 0 D 0 0 1 1 3 0 0 E 0 1 0 0 0 0 0 F 0 0 0 5 0 2 0 G 0 0 2 1 2 0 0 H 1 1 0 4 0 0 0 1 0 0 1 0 4 5 1 K 0 0 0 1 2 0 0 L 3 0 0 1 1 3 7 M 0 0 0 0 0 1 0 N 0 0 3 2 6 2 0 P 0 0 0 0 0 4 85 Q 96 97 0 0 0 0 0 R 0 0 0 0 5 0 2 S 0 0 80 4 65 6 3 T 0 0 9 0 10 65 1 V 0 0 0 0 0 1 1 w 0 0 0 0 0 0 0 Y 0 0 2 80 0 3 0

For example, at position 89, two amino acids, Q and L, account for about 99% of 20 the observed variability, and thus in the currently exemplified library (see below), only -136- Q and L were included in position 89. In larger libraries, of course, additional, less frequently occurring amino acid types (e.g., H), may also be included. 2015201796 09 Apr 2015

Similarly, at position 93 there is more variation, with amino acid types S, T, N, R and I being among the most frequently occurring. The currently exemplified library 5 thus aimed to include these five amino acids at position 93, although clearly others could be included in more diverse libraries. However, because this library was constructed via standard chemical oligonucleotide synthesis, one is bound by the limits of the genetic code, so that the actual amino acid set represented at position 93 of the exemplified library consists of S, T, N, R, P and H, with P and H replacing I (see exemplary 9 10 residue VKCDR3 in Table 32, below). This limitation may be overcome by using codon-based synthesis of oligonucleotides, as described in Example 6.3, below. A similar approach was followed at the other positions and for the other sequences: analysis of occurrences of amino acid type per position, choice from among most frequently occurring subset, followed by adjustment as dictated by the genetic code. 15 As indicated above, the library employs a practical and facile synthesis approach using standard oligonucleotide synthesis instrumentation and degenerate oligonucleotides. To facilitate description of the library, the IUPAC code for degenerate nucleotides, as given in Table 31, will be used. 20 Table 31. Degenerate Base Symbol Definition_ IUPAC Symbol Base Pair Composition A A (100%) C C (100%) G G (100%) T T(100%) R A (50%) G (50%) Y C (50%) T (50%) W A (50%) T (50%) S C (50%) G (50%) M A (50%) C (50%) K G (50%) T (50%) B C (33%) G (33%) T (33%) (*) D A (33%) G (33%) T (33%) H A (33%) C (33%) T (33%) V A (33%) C (33%) G (33%) N A (25%) C (25%) G (25%) T (25%) (*) 33% is short hand here for 1/3 (i.e., 33.3333 ... %)

Using the VK1-39 chassis with VKCDR3 of length nine as an example, the VKCDR3 library may be represented by the following four oligonucleotides (left - 137- column in Table 32), with the corresponding amino acids encoded at each position of CDRL3 (Kabat numbering) provided in the columns on the right.

Table 32. Exemplary Oligonucleotides Encoding a VK1-39 CDR3 Library

Oligonucleotide Sequence 89 90 91 92 93 94 95 95A 96 97 CWGSAAWCATHCMVTABTCCTTWCACT LQ EQ ST FSY HNPRST 1ST P - FY T CWGSAAWCATHCMVTABTCCTMTCACT LQ EQ ST FSY HNPRST 1ST P - IL T CWGSAAWCATHCMVTABTCCTWGGACT LQ EQ ST FSY HNPRST 1ST P - WR T CWGSAAWCATHCMVTABTCCTCBTACT LQ EQ ST FSY HNPRST 1ST P PLR - T 2015201796 09 Apr 2015 5

For example, the first codon (CWG) of the first nucleotide of Table 32, corresponding to Kabat position 89, represents 50% CTG and 50% CAG, which encode Leu (L) and Gin (Q), respectively. Thus, the expressed polypeptide would be expected to have L and Q each about 50% of the time. Similarly, for Kabat position 95A of the 10 fourth oligonucleotide, the codon CBT represents 1/3 each of CCT, CGT and CTT, corresponding in turn to 1/3 each of Pro (P), Leu (L) and Arg (R) upon translation. By multiplying the number of options available at each position of the peptide sequence, one can obtain the complexity, in peptide space, contributed by each oligonucleotide. For the VK1-39 example above, the numbers are 864 for the first three oligonucleotides 15 and 1,296 for the fourth oligonucleotide. Thus, the oligonucleotides encoding VK1-39 CDR3s of length nine contribute 3,888 members to the library. However, as shown in Table 32, sequences with L or R at position 95A (when position 96 is empty) are identical to those with L or R at position 96 (and 95A empty). Therefore, the 3,888 number overestimates the LR contribution and the actual number of unique members is 20 slightly lower, at 3,024. As depicted in Table 33, for the complete list of oligonucleotides that represent VKCDR3 of sizes 8, 9, and 10, for all 10 VK chassis, the overall complexity is about 1.3 x 105 or 1.2 x 105 unique sequences after correcting for over-counting of the LR contribution for the size 9 VKCDR3. - 138 - 2015201796 09 Apr 2015

Table 33. Degenerate Oligonucleotides Encoding an Exemplary VKCDR3 Library

Chassis c D R L3 Le ng th Junctio n Type (1) Degenerate Oligonucleotide SEQ ID NO: 89 90 91 92 93 94 95 95A 96 97 VK1-5 8 1 CASCASTMCVRTRSTT WCTWCACT HQ HQ SY DGHNRS AGST FY _ _ FY T VK1-5 8 2 CASCASTMCVRTRSTT WCMTCACT HQ HQ SY DGHNRS AGST FY _ _ IL T VK1-5 8 3 CASCASTMCVRTRSTT WCWGGACT HQ HQ SY DGHNRS AGST FY _ _ WR T VK1-5 8 4 CASCASTMCVRTRSTT WCYCTACT HQ HQ SY DGHNRS AGST FY PS _ _ T VK1-5 9 1 CASCASTMCVRTRSTT WCYCTTWCACT HQ HQ SY DGHNRS AGST FY PS _ FY T VK1-5 9 2 CASCASTMCVRTRSTT WCYCTMTCACT HQ HQ SY DGHNRS AGST FY PS _ IL T VK1-5 9 3 CASCASTMCVRTRSTT WCYCTWGGACT HQ HQ SY DGHNRS AGST FY PS _ WR T VK1-5 9 4 CASCASTMCVRTRSTT WCYCTYCTACT HQ HQ SY DGHNRS AGST FY PS PS _ T VK1-5 1 0 1 CASCASTMCVRTRSTT WCYCTCBTTWCACT HQ HQ SY DGHNRS AGST FY PS PLR FY T VK1-5 1 0 2 CASCASTMCVRTRSTT WCYCTCBTMTCACT HQ HQ SY DGHNRS AGST FY PS PLR IL T VK1-5 1 0 3 CASCASTMCVRTRSTT WCYCTCBTWGGACT HQ HQ SY DGHNRS AGST FY PS PLR WR T

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VK1-12 8 1 CASCASDCTRVCARTT TSTWCACT HQ HQ AST ADGNST NS FL _ _ FY T VK1-12 8 2 CASCASDCTRVCARTT TSMTCACT HQ HQ AST ADGNST NS FL _ _ IL T VK1-12 8 3 CASCASDCTRVCARTT TSWGGACT HQ HQ AST ADGNST NS FL _ _ WR T VK1-12 8 4 CASCASDCTRVCARTT TSCCTACT HQ HQ AST ADGNST NS FL P _ _ T VK1-12 9 1 CASCASDCTRVCARTT TSCCTTWCACT HQ HQ AST ADGNST NS FL P _ FY T VK1-12 9 2 CASCASDCTRVCARTT TSCCTMTCACT HQ HQ AST ADGNST NS FL P _ IL T VK1-12 9 3 CASCASDCTRVCARTT TSCCTWGGACT HQ HQ AST ADGNST NS FL P _ WR T VK1-12 9 4 CASCASDCTRVCARTT TSCCTCBTACT HQ HQ AST ADGNST NS FL P PLR _ T VK1-12 1 0 1 CASCASDCTRVCARTT TSCCTCBTTWCACT HQ HQ AST ADGNST NS FL P PLR FY T VK1-12 1 0 2 CASCASDCTRVCARTT TSCCTCBTMTCACT HQ HQ AST ADGNST NS FL P PLR IL T VK1-12 1 0 3 CASCASDCTRVCARTT TSCCTCBTWGGACT HQ HQ AST ADGNST NS FL P PLR WR T VK1-27 8 1 CASMAGTWCRRTASKG BATWCACT HQ KQ FY DGNS RST AGV _ _ FY T VK1-27 8 2 CASMAGTWCRRTASKG BAMTCACT HQ KQ FY DGNS RST AGV _ _ IL T VK1-27 8 3 CASMAGTWCRRTASKG BAWGGACT HQ KQ FY DGNS RST AGV _ _ WR T VK1-27 8 4 CASMAGTWCRRTASKG BACCTACT HQ KQ FY DGNS RST AGV P _ _ T VK1-27 9 1 CASMAGTWCRRTASKG BACCTTWCACT HQ KQ FY DGNS RST AGV P _ FY T VK1-27 9 2 CASMAGTWCRRTASKG HQ KQ FY DGNS RST AGV P - IL T - 140- 2015201796 09 Apr 2015

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BACCTMTCACT VK1-27 9 3 CASMAGTWCRRTASKG BACCTWGGACT HQ KQ FY DGNS RST AGV P _ WR T VK1-27 9 4 CASMAGTWCRRTASKG BACCTCBTACT HQ KQ FY DGNS RST AGV P PLR _ T VK1-27 1 0 1 CASMAGTWCRRTASKG BACCTCBTTWCACT HQ KQ FY DGNS RST AGV P PLR FY T VK1-27 1 0 2 CASMAGTWCRRTASKG BACCTCBTMTCACT HQ KQ FY DGNS RST AGV P PLR IL T VK1-27 1 0 3 CASMAGTWCRRTASKG BACCTCBTWGGACT HQ KQ FY DGNS RST AGV P PLR WR T VK1-33 8 1 CASCWTTMCRATRVCB WTTWCACT HQ HL SY DN ADGNS T DFH LVY _ _ FY T VK1-33 8 2 CASCWTTMCRATRVCB WTMTCACT HQ HL SY DN ADGNS T DFH LVY _ _ IL T VK1-33 8 3 CASCWTTMCRATRVCB WTWGGACT HQ HL SY DN ADGNS T DFH LVY _ _ WR T VK1-33 8 4 CASCWTTMCRATRVCB WTCCTACT HQ HL SY DN ADGNS T DFH LVY P _ _ T VK1-33 9 1 CASCWTTMCRATRVCB WTCCTTWCACT HQ HL SY DN ADGNS T DFH LVY P _ FY T VK1-33 9 2 CASCWTTMCRATRVCB WTCCTMTCACT HQ HL SY DN ADGNS T DFH LVY P _ IL T VK1-33 9 3 CASCWTTMCRATRVCB WTCCTWGGACT HQ HL SY DN ADGNS T DFH LVY P _ WR T VK1-33 9 4 CASCWTTMCRATRVCB WTCCTCBTACT HQ HL SY DN ADGNS T DFH LVY P PLR _ T VK1-33 1 0 1 CASCWTTMCRATRVCB WTCCTCBTTWCACT HQ HL SY DN ADGNS T DFH LVY P PLR FY T VK1-33 1 0 2 CASCWTTMCRATRVCB WTCCTCBTMTCACT HQ HL SY DN ADGNS T DFH LVY P PLR IL T VK1-33 1 0 3 CASCWTTMCRATRVCB WTCCTCBTWGGACT HQ HL SY DN ADGNS T DFH LVY P PLR WR T - 141 - 2015201796 09 Apr 2015

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VK1-39 8 1 CWGSAAWCATHCMVTA BTTWCACT LQ EQ ST FSY HNPRS T 1ST _ _ FY T VK1-39 8 2 CWGSAAWCATHCMVTA BTMTCACT LQ EQ ST FSY HNPRS T 1ST _ _ IL T VK1-39 8 3 CWGSAAWCATHCMVTA BTWGGACT LQ EQ ST FSY HNPRS T 1ST _ _ WR T VK1-39 8 4 CWGSAAWCATHCMVTA BTCCTACT LQ EQ ST FSY HNPRS T 1ST P _ _ T VK1-39 9 1 CWGSAAWCATHCMVTA BTCCTTWCACT LQ EQ ST FSY HNPRS T 1ST P _ FY T VK1-39 9 2 CWGSAAWCATHCMVTA BTCCTMTCACT LQ EQ ST FSY HNPRS T 1ST P _ IL T VK1-39 9 3 CWGSAAWCATHCMVTA BTCCTWGGACT LQ EQ ST FSY HNPRS T 1ST P _ WR T VK1-39 9 4 CWGSAAWCATHCMVTA BTCCTCBTACT LQ EQ ST FSY HNPRS T 1ST P PLR _ T VK1-39 1 0 1 CWGSAAWCATHCMVTA BTCCTCBTTWCACT LQ EQ ST FSY HNPRS T 1ST P PLR FY T VK1-39 1 0 2 CWGSAAWCATHCMVTA BTCCTCBTMTCACT LQ EQ ST FSY HNPRS T 1ST P PLR IL T VK1-39 1 0 3 CWGSAAWCATHCMVTA BTCCTCBTWGGACT LQ EQ ST FSY HNPRS T 1ST P PLR WR T VK3-11 8 1 CASCASAGWRGKRVCT SGTWCACT HQ HQ RS GRS ADGNS T sw _ _ FY T VK3-11 8 2 CASCASAGWRGKRVCT SGMTCACT HQ HQ RS GRS ADGNS T sw _ _ IL T VK3-11 8 3 CASCASAGWRGKRVCT SGWGGACT HQ HQ RS GRS ADGNS T sw _ _ WR T VK3-11 8 4 CASCASAGWRGKRVCT SGCCTACT HQ HQ RS GRS ADGNS T sw P _ _ T VK3-11 9 1 CASCASAGWRGKRVCT SGCCTTWCACT HQ HQ RS GRS ADGNS T sw P _ FY T VK3-11 9 2 CASCASAGWRGKRVCT HQ HQ RS GRS ADGNS sw P - IL T - 142- 2015201796 09 Apr 2015

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SGCCTMTCACT T VK3-11 9 3 CASCASAGWRGKRVCT SGCCTWGGACT HQ HQ RS GRS ADGNS T SW P _ WR T VK3-11 9 4 CASCASAGWRGKRVCT SGCCTCBTACT HQ HQ RS GRS ADGNS T SW P PER _ T VK3-11 1 0 1 CASCASAGWRGKRVCT SGCCTCBTTWCACT HQ HQ RS GRS ADGNS T SW P PER FY T VK3-11 1 0 2 CASCASAGWRGKRVCT SGCCTCBTMTCACT HQ HQ RS GRS ADGNS T SW P PER IE T VK3-11 1 0 3 CASCASAGWRGKRVCT SGCCTCBTWGGACT HQ HQ RS GRS ADGNS T SW P PER WR T VK3-15 8 1 CASCASTMCVRTRRKT GGTWCACT HQ HQ SY DGHNRS DEGKN RS W _ _ FY T VK3-15 8 2 CASCASTMCVRTRRKT GGMTCACT HQ HQ SY DGHNRS DEGKN RS W _ _ IE T VK3-15 8 3 CASCASTMCVRTRRKT GGWGGACT HQ HQ SY DGHNRS DEGKN RS W _ _ WR T VK3-15 8 4 CASCASTMCVRTRRKT GGCCTACT HQ HQ SY DGHNRS DEGKN RS W P _ _ T VK3-15 9 1 CASCASTMCVRTRRKT GGCCTTWCACT HQ HQ SY DGHNRS DEGKN RS W P _ FY T VK3-15 9 2 CASCASTMCVRTRRKT GGCCTMTCACT HQ HQ SY DGHNRS DEGKN RS W P _ IE T VK3-15 9 3 CASCASTMCVRTRRKT GGCCTWGGACT HQ HQ SY DGHNRS DEGKN RS W P _ WR T VK3-15 9 4 CASCASTMCVRTRRKT GGCCTCBTACT HQ HQ SY DGHNRS DEGKN RS W P PER _ T VK3-15 1 0 1 CASCASTMCVRTRRKT GGCCTCBTTWCACT HQ HQ SY DGHNRS DEGKN RS W P PER FY T VK3-15 1 0 2 CASCASTMCVRTRRKT GGCCTCBTMTCACT HQ HQ SY DGHNRS DEGKN RS W P PER IE T VK3-15 1 0 3 CASCASTMCVRTRRKT GGCCTCBTWGGACT HQ HQ SY DGHNRS DEGKN RS W P PER WR T - 143 - 2015201796 09 Apr 2015

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VK3-20 8 1 CASCASTWCGRTRVKK CATWCACT HQ HQ FY DG ADEGK NRST AS _ _ FY T VK3-20 8 2 CASCASTWCGRTRVKK CAMTCACT HQ HQ FY DG ADEGK NRST AS _ _ IL T VK3-20 8 3 CASCASTWCGRTRVKK CAWGGACT HQ HQ FY DG ADEGK NRST AS _ _ WR T VK3-20 8 4 CASCASTWCGRTRVKK CACCTACT HQ HQ FY DG ADEGK NRST AS P _ _ T VK3-20 9 1 CASCASTWCGRTRVKK CACCTTWCACT HQ HQ FY DG ADEGK NRST AS P _ FY T VK3-20 9 2 CASCASTWCGRTRVKK CACCTMTCACT HQ HQ FY DG ADEGK NRST AS P _ IL T VK3-20 9 3 CASCASTWCGRTRVKK CACCTWGGACT HQ HQ FY DG ADEGK NRST AS P _ WR T VK3-20 9 4 CASCASTWCGRTRVKK CACCTCBTACT HQ HQ FY DG ADEGK NRST AS P PLR _ T VK3-20 1 0 1 CASCASTWCGRTRVKK CACCTCBTTWCACT HQ HQ FY DG ADEGK NRST AS P PLR FY T VK3-20 1 0 2 CASCASTWCGRTRVKK CACCTCBTMTCACT HQ HQ FY DG ADEGK NRST AS P PLR IL T VK3-20 1 0 3 CASCASTWCGRTRVKK CACCTCBTWGGACT HQ HQ FY DG ADEGK NRST AS P PLR WR T VK2-28 8 1 ATGCASRBTCKTSASA BTTWCACT M HQ AGI STV LR DEHQ 1ST _ _ FY T VK2-28 8 2 ATGCASRBTCKTSASA BTMTCACT M HQ AGI STV LR DEHQ 1ST _ _ IL T VK2-28 8 3 ATGCASRBTCKTSASA BTWGGACT M HQ AGI STV LR DEHQ 1ST _ _ WR T VK2-28 8 4 ATGCASRBTCKTSASA BTCCTACT M HQ AGI STV LR DEHQ 1ST P _ _ T VK2-28 9 1 ATGCASRBTCKTSASA BTCCTTWCACT M HQ AGI STV LR DEHQ 1ST P _ FY T VK2-28 9 2 ATGCASRBTCKTSASA M HQ AGI LR DEHQ 1ST P - IL T - 144- 2015201796 09 Apr 2015

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BTCCTMTCACT STV VK2-28 9 3 ATGCASRBTCKTSASA BTCCTWGGACT M HQ AGI STV LR DEHQ 1ST P _ WR T VK2-28 9 4 ATGCASRBTCKTSASA BTCCTCBTACT M HQ AGI STV LR DEHQ 1ST P PLR _ T VK2-28 1 0 1 ATGCASRBTCKTSASA BTCCTCBTTWCACT M HQ AGI STV LR DEHQ 1ST P PLR FY T VK2-28 1 0 2 ATGCASRBTCKTSASA BTCCTCBTMTCACT M HQ AGI STV LR DEHQ 1ST P PLR IL T VK2-28 1 0 3 ATGCASRBTCKTSASA BTCCTCBTWGGACT M HQ AGI STV LR DEHQ 1ST P PLR WR T VK4-1 8 1 CASCASTWCTWCRVCA BTTWCACT HQ HQ FY FY ADGNS T 1ST _ _ FY T VK4-1 8 2 CASCASTWCTWCRVCA BTMTCACT HQ HQ FY FY ADGNS T 1ST _ _ IL T VK4-1 8 3 CASCASTWCTWCRVCA BTWGGACT HQ HQ FY FY ADGNS T 1ST _ _ WR T VK4-1 8 4 CASCASTWCTWCRVCA BTCCTACT HQ HQ FY FY ADGNS T 1ST P _ _ T VK4-1 9 1 CASCASTWCTWCRVCA BTCCTTWCACT HQ HQ FY FY ADGNS T 1ST P _ FY T VK4-1 9 2 CASCASTWCTWCRVCA BTCCTMTCACT HQ HQ FY FY ADGNS T 1ST P _ IL T VK4-1 9 3 CASCASTWCTWCRVCA BTCCTWGGACT HQ HQ FY FY ADGNS T 1ST P _ WR T VK4-1 9 4 CASCASTWCTWCRVCA BTCCTCBTACT HQ HQ FY FY ADGNS T 1ST P PLR _ T VK4-1 1 0 1 CASCASTWCTWCRVCA BTCCTCBTTWCACT HQ HQ FY FY ADGNS T 1ST P PLR FY T VK4-1 1 0 2 CASCASTWCTWCRVCA BTCCTCBTMTCACT HQ HQ FY FY ADGNS T 1ST P PLR IL T VK4-1 1 0 3 CASCASTWCTWCRVCA BTCCTCBTWGGACT HQ HQ FY FY ADGNS T 1ST P PLR WR T - 145 - 2015201796 09 Apr 2015

Attorney Docket No.: ADS-011.25

[Alternative for VK1-33] (2) VK1-33 8 1 CASCWATMCRATRVCB WTTWCACT HQ QL SY DN ADGNS T DFH LVY _ _ FY T VK1-33 8 2 CASCWATMCRATRVCB WTMTCACT HQ QL SY DN ADGNS T DFH LVY _ _ IL T VK1-33 8 3 CASCWATMCRATRVCB WTWGGACT HQ QL SY DN ADGNS T DFH LVY _ _ WR T VK1-33 8 4 CASCWATMCRATRVCB WTCCTACT HQ QL SY DN ADGNS T DFH LVY P _ _ T VK1-33 9 1 CASCWATMCRATRVCB WTCCTTWCACT HQ QL SY DN ADGNS T DFH LVY P _ FY T VK1-33 9 2 CASCWATMCRATRVCB WTCCTMTCACT HQ QL SY DN ADGNS T DFH LVY P _ IL T VK1-33 9 3 CASCWATMCRATRVCB WTCCTWGGACT HQ QL SY DN ADGNS T DFH LVY P _ WR T VK1-33 9 4 CASCWATMCRATRVCB WTCCTCBTACT HQ QL SY DN ADGNS T DFH LVY P PLR _ T VK1-33 1 0 1 CASCWATMCRATRVCB WTCCTCBTTWCACT HQ QL SY DN ADGNS T DFH LVY P PLR FY T VK1-33 1 0 2 CASCWATMCRATRVCB WTCCTCBTMTCACT HQ QL SY DN ADGNS T DFH LVY P PLR IL T VK1-33 1 0 3 CASCWATMCRATRVCB WTCCTCBTWGGACT HQ QL SY DN ADGNS T DFH LVY P PLR WR T L, type 3 as RW, and type 4 has a deletion (1) Junction type 1 has position 96 as FY, type 2 as (2) Two embodiments are shown for the VK1-33 library.

In one embodiment, the second codon was CWT. In another embodiment, it was CWA or CWG. - 146-

Example 6.3: More Complex VKCDR3 Libraries 2015201796 09 Apr 2015

This example demonstrates how a more faithful representation of amino acid 5 variation at each position may be obtained by using a codon-based synthesis approach (Vimekas et al. Nucleic Acids Res., 1994, 22: 5600). This synthetic scheme also allows for finer control of the proportions of particular amino acids included at a position. For example, as described above for the VK1-39 sequences, position 89 was designed as 50% Q and 50% L; however, as Table 30 shows, Q is used much more frequently than 10 L. The more complex VKCDR3 libraries of the present example account for the different relative occurrence of Q and L, for example, 90% Q and 10% L. Such control is better exercised within codon-based synthetic schemes, especially when multiple amino acid types are considered.

This example also describes an implementation of a codon-based synthetic 15 scheme, using the ten VK chassis described in Table 11. Similar approaches, of course, can be implemented with more or fewer such chassis. As indicated in the Detailed Description, a unique aspect of the design of the present libraries, as well as those of the preceding examples, is the germline or chassis-based aspect, which is meant to preserve more of the integrity and variation of actual human kappa light chain sequences. This is 20 in contrast to other codon-based synthesis or degenerate oligonucleotide synthesis approaches that have been described in the literature and that aim to produce “one-size-fits-all” (e.g., consensus) kappa light chain libraries (e.g.,, Knappik, et al., J Mol Biol, 2000, 296: 57; Akamatsu et al., J Immunol, 1993, 151: 4651).

With reference to Table 30, obtained for VK1-39, one can thus design the length 25 nine VKCDR3 library of Table 34. Here, for practical reasons, the proportions at each position are denoted in multiples of five percentage points. As better synthetic schemes are developed, finer resolution may be obtained - for example to resolutions of one, two, three, or four percent. 30 Table 34. Amino Acid Composition (%) at Each VKCDR3 Position for VK1-39 Library

With CDR Length of Nine Residues Amino Acid 89 90 91 92 93 94 95 96 (*) 97 (*) A 5 5 D 5 5 E 5 5 - 147- F 5 10 G 5 5 5 5 H 5 5 5 5 1 5 5 K 5 L 10 5 10 20 M N 0 0 5 0 5 P 5 85 5 Q 85 90 5 R 5 5 10 S 80 5 60 5 5 T 10 10 65 90 V 5 w 15 Y 5 75 5 15 Number Different 3 3 4 6 8 8 3 11 3 (*) The composition of positions 96 and 97, determined largely by junction and IGKJ diversity, could be the same for length 9 VK CDR3 of all chassis.

The library of Table 34 would have 1.37 x 106 unique polypeptide sequences, 2015201796 09 Apr 2015 5 calculated by multiplying together the numbers in the bottom row of the table.

The underlined 0 entries for Asn (N) at certain positions represent regions where the possibility of having N-linked glycosylation sites in the VKCDR3 has been minimized or eliminated. Peptide sequences with the pattern N-X-(S or T)-Z, where X and Z are different from P, may undergo post-translational modification in a number of 10 expression systems, including yeast and mammalian cells. Moreover, the nature of such modification depends on the specific cell type and, even for a given cell type, on culture conditions. N-linked glycosylation may be disadvantageous when it occurs in a region of the antibody molecule likely to be involved in antigen binding (e.g., a CDR), as the function of the antibody may then be influenced by factors that may be difficult to 15 control. For example, considering position 91 above, one can observe that position 92 is never P. Position 94 is not P in 95% of the cases. However, position 93 is S or T in 75 % (65 + 10) of the cases. Thus, allowing N at position 91 would generate the undesirable motif N-X-(T/S)-Z (with both X and Z distinct from P), and a zero occurrence has therefore been implemented, even though N is observed with some 20 frequency in actual human sequences (see Table 30). A similar argument applies for N at positions 92 and 94. It should be appreciated, however, that if the antibody library were to be expressed in a system incapable of N-linked glycosylation, such as bacteria, or under culture conditions in which N-linked glycosylation did not occur, this -148- consideration may not apply. However, even in the event that the organism used to express libraries with potential N-linked glycosylation sites is incapable of N-linked glycosylation (e.g., bacteria), it may still be desirable to avoid N-X-(S/T) sequences, as the antibodies isolated from such libraries may be expressed in different systems (e.g., 2015201796 09 Apr 2015 5 yeast, mammalian cells) later (e.g., toward clinical development), and the presence of carbohydrate moieties in the variable domains, and the CDRs in particular, may lead to unwanted modifications of activity. These embodiments are also included within the scope of the invention. To our knowledge, VKCDR3 libraries known in the art have not considered this effect, and thus a proportion of their members may have the undesirable 10 qualities mentioned above.

We also designed additional sub-libraries, related to the library outlined in Table 34, for VKCDR3 of lengths 8 and 10. In these embodiments, the compositions at positions 89 to 94 and 97 remain the same as those depicted in Table 34. Additional diversity, introduced at positions 95 and 95A, the latter being defined for VKCDR3 of 15 length 10 only, are illustrated in Table 35.

Table 35. Amino Acid Composition (%) for VK1-39 Libraries of Lengths 8 and 10

Amino Acid Position 95- Length 8 (*) Position 95-Length 10 (**) Position 95A-Length 10 A D E F 5 G 5 H 1 10 5 K L 20 10 10 M N P 25 85 60 Q R 10 5 10 S 5 5 T 5 V 5 w 10 Y 10 Number Different 9 3 8 (*) Position 96 is c eleted in VKCDR3 of size 8. (**) This is the same composition as in VKCDR3 of size 9. - 149-

The total number of unique members in the VK1-39 library of length 8, thus, can be obtained as before, and is 3.73 x 105 (or, 3χ3χ4χ6χ8χ8χ9χ3). Similarly, the complexity of the VK1-39 library of length 10 would be 10.9 x 106 (or 8 times that of 5 the library of size 9, as there is additional 8-fold variation at the insertion position 95 A). Thus, there would be a total of 12.7 x 106 unique members in the overall VK1-39 library, as obtained by summing the number of unique members for each of the specified lengths. In certain embodiments of the invention, it may be preferable to create the individual sub-libraries of lengths 8, 9 and 10 separately, and then mix the sub-libraries 10 in proportions that reflect the length distribution of VKCDR3 in human sequences; for example, in ratios approximating the 1:9:2 distribution that occurs in natural VKCDR3 sequences (see Figure 3). The present invention provides the compositions and methods for one of ordinary skill synthesizing VKCDR3 libraries corresponding to other VK chassis. 2015201796 09 Apr 2015 15

Example 7: A Minimalist VXCDR3 Library

This example describes the design of a minimalist VkCDR3 library. The principles used in designing this library (or more complex νλ libraries) are similar to 20 those used to design the VKCDR3 libraries. However, unlike the VK genes, the contribution of the ^λν segment to CDRL3 is not constrained to a fixed number of amino acids. Therefore, length variation may be obtained in a minimalist VXCDR3 library even when only considering combinations between Vk chassis and 1λ sequences.

Examination of the VλCDR3 lengths of human sequences shows that lengths of 25 9 to 12 account for almost about 95% of sequences, and lengths of 8 to 12 account for about 97% of sequences (Figure 4). Table 36 shows the usage (percent occurrence) of the six known IGXJ genes in the rearranged human lambda light chain sequences compiled from the NCBI database (see Appendix B), and Table 37 shows the sequences encoded by the genes. 30 - 150-

Table 36. IGXJ Gene Usage in the Lambda Light Chain Sequences Compiled from the NCBI Database (see Appendix B)

GeneAllele LUA ύλ1 01 20.2% JX2 01 42.2% ύλ3 02 36.2% ύλθ 01 0.6% JX7 01 0.9% 2015201796 09 Apr 2015 5

Table 37. Observed Human IGXJ Amino Acid Sequences

Gene Sequence ΙΟλϋ1-01 YVFGTGTKVTVL ΙΟλϋ2-01 WFGGGTKLTVL ΙΘλύ 3-01 WVFGGGTKLTVL Ιΰλϋ3-02 WFGGGTKLTVL IGAJ6-01 NVFGSGTKVTVL IGAJ7-01 AVFGGGTQLTVL IGAJ7-02 AVFGGGTQLTAL IGXJ3-01 and IGXJ7-02 are not represented among the sequences that were analyzed; therefore, they were not included in Table 36. As illustrated in Table 36, 10 IGXJ1-01, IGXJ2-01, and IGXJ3-02 are over-represented in their usage, and have thus been bolded in Table 37. In some embodiments of the invention, for example, only these three over-represented sequences may be utilized. In other embodiments of the invention, one may use all six segments, any 1, 2, 3, 4, or 5 of the 6 segments, or any combination thereof may be utilized. 15 As shown in Table 14, the portion of CDRL3 contributed by the IGXV gene segment is 7, 8, or 9 amino acids. The remainder of CDRL3 and FRM4 are derived from the IGXJ sequences (Table 37). The IGXJ sequences contribute either one or two amino acids to CDRL3. If two amino acids are contributed by IGXJ, the contribution is from the N-terminal two residues of the IGXJ segment: YV (IGXJ1-01), W (IGXJ2-01), 20 WV (IGXJ3-01), W (IGXJ3-02), or AV (IGXJ7-01 and IGXJ7-02). If one amino acid is -151 - contributed from IGXJ, it is a V residue, which is formed after the deletion of the N-terminal residue of a IGXJ segment. 2015201796 09 Apr 2015

In this non-limiting exemplary embodiment of the invention, the FRM4 segment was fixed as FGGGTKLTVL, corresponding to IGXJ2-01 and IG/J3-02. 5 Seven of the 11 selected chassis (VX1-40, VX3-19, VX3-21, VX6-57, VX1-44, VX1-51, and VX4-69) have an additional two nucleotides following the last full codon. In four of those seven cases, analysis of the data set provided in Appendix B showed that the addition of a single nucleotide (i.e. without being limited by theory, via the activity of TdT) lead to a further increase in CDRL3 length. This effect can be 10 considered by introducing variants for the L3-VX sequences contributed by these four IGXV sequences (Table 38). -152-

Attorney Docket No.: ADS-011.25 2015201796 09 Apr 2015

Table 38. Variants with an additional residue in CDRL3

Name Locus FRM1 CDR1 FRM2 CDR2 FRM3 CDR3 / L3-W. 1E+ IGVkl- 40+ QSVLTQPPSVSGAPG QRVTISC TGSSSNIGAG YD---VH WYQQLPGTAP KLLI YGN---- SNRPS GVPDRFSGSKSG-- TSASLAITGLQAEDEADYYC QSYDSSL SGS 3L+ IGVk3- 19+ SSELTQDPAVSVALG QTVRITC QGDSLRSYY------AS WYQQKPGQAP VLVI YGK---- NNRPS GIPDRFSGSSSG--NTASLTITGAQAEDEADYYC NSRDSSG NHH/Q 3H+ IGVk3-21 + SYVLTQPPSVSVAPG KTARITC GGNNIGSKS- -----VH WYQQKPGQAP VLVI YYD---- SDRPS GIPERFSGSNSG— NTATLTISRVEAGDEADYYC QVWDSSS DHP 6A+ IGVk6- 57+ NFMLTQPHSVSESPG KTVTISC TRSSGSIASN Y----VQ WYQQRPGSSP TTVI YED---- NQRPS GVPDRFSGSIDSSSNSASLT ISGLKTEDEADYYC QSYDSSN H/Q— (+) sequences are derived from their parents by the addition of an amino acid at the end of the respective CDR3 (bold underlined). H/Q can be introduced in a single sequence by use of the degenerate codon CAW or similar. - 153 -

Thus, the final set of chassis in the currently exemplified embodiment of the invention is 15: eleven contributed by the chassis in Table 14 and an additional four contributed by the chassis of Table 38. The corresponding L3-VA domains of the 15 chassis contribute 5 from 7 to 10 amino acids to CDRL3. When considering the amino acids contributed by the IGXJ sequences, the total variation in the length of CDRL3 is 8 to 12 amino acids, approximating the distribution in Figure 4. Thus, in this exemplary embodiment of the invention, the minimalist νλ library may be represented by the following: 15 Chassis x 5 IG?J-derived segments = 75 sequences. Here, the 15 chassis are νλ1-40, νλ1-44, 2015201796 09 Apr 2015 10 νλ1-51, νλ2-14, νλ3-1*, νλ3-19, νλ3-21, νλ4-69, νλ6-57, νλ5-45, νλ7-43, νλΐ- 40+, νλ3-19+, νλ3-21+, and νλ6-57+. The 5 IGXJ-derived segments are YVFGGGTKLTVL (IG/J1), VVFGGGTKLTVL (IGXJ2), WVFGGGTKLTVL (IGXJ3), AVFGGGTKLTVL (IGXJ), and -VFGGGTKLTVL (from any of the preceding sequences). 15

Example 8: Matching to “Reference” Antibodies CDRH3 sequences of human antibodies of interest that are known in the art, (e.g., antibodies that have been used in the clinic) have close counterparts in the 20 designed library of the invention. A set of fifteen CDRH3 sequences from clinically relevant antibodies is presented in Table 39.

Table 39. CDRH3 Sequences of Reference Antibodies

Antibody Name Target Origin Status CDHR3 sequence SEQ ID NO: CAB1 TNF-oc Phage display -human library FDA Approved AKVSYLSTASSLDY CAB2 EGFR Transgenic mouse FDA Approved VRDRVTGAFDI CAB3 IL-12/IL-23 Phage display -human library Phase III KTHGSHDN CAB4 lnterleukin- 1-3 Transgenic mouse Phase III ARDLRTGPFDY CAB5 RANKL Transgenic mouse Phase III AKDPGTTVIMSWFDP - 154- 2015201796 09 Apr 2015

CAB6 IL-12/IL-23 Transgenic mouse Phase III ARRRPGQGYFDF CAB7 TNF-ot Transgenic mouse Phase III ARDRGASAGGNYYYYGMDV CAB8 CTLA4 Transgenic mouse Phase III ARDPRGATLYYYYYGMDV CAB9 CD20 Transgenic mouse Phase III AKDIQYGNYYYGMDV CAB 10 CD4 Transgenic mouse Phase III ARVINWFDP CAB11 CTLA4 Transgenic mouse Phase III ARTGWLGPFDY CAB 12 IGF1-R Transgenic mouse Phase II AKDLGWSDSYYYYYGMDV CAB 13 EGFR Transgenic mouse Phase II ARDGITMVRGVMKDYFDY CAB 14 EGFR Phage display -human library Phase II ARVSIFGVGTFDY CAB 15 BLyS Phage display -human library Phase II ARSRDLLLFPHHALSP

Each of the above sequences was compared to each of the members of the library of Example 5, and the member, or members, with the same length and fewest number of amino acid mismatches was, or were, recorded. The results are summarized in Table 40, 5 below. For most of the cases, matches with 80% identity or better were found in the exemplified CDRH3 library. To the extent that the specificity and binding affinity of each of these antibodies is influenced by their CDRH3 sequence, without being bound by theory, one or more of these library members could have measurable affinity to the relevant targets. 10

Table 40. Match of Reference Antibody CDRH3 to Designed Library

Antibody Name Number of Mismatches (*) Length % Identity of Best Match CAB1 5 14 64% CAB2 2 11 82% CAB3 4 8 50% CAB4 2 11 82% CAB5 3 15 80% CAB6 3 12 75% CAB7 2 20 90% CAB8 0 19 100% CAB9 3 15 80% CAB 10 1 9 89% -155 - CAB11 1 11 91% CAB 12 2 18 89% CAB 13 2 18 89% CAB 14 1 13 92% CAB 15 7 16 56% (*) For the best-matching sequence(s) in li arary

Given that a physical realization of a library with about 108 distinct members could, in practice, contain every single member, then such sequences with close percent 5 identity to antibodies of interest would be present in the physical realization of the library. This example also highlights one of many distinctions of the libraries of the current invention over those of the art; namely, that the members of the libraries of the invention may be precisely enumerated. In contrast, CDRH3 libraries known in the art cannot be explicitly enumerated in the manner described herein. For example, many 10 libraries known in the art (e.g., Hoet et al., Nat. Biotechnol., 2005, 23: 344; Griffiths et al., EMBO J., 1994, 13: 3245; Griffiths etal., EMBO J., 1993, 12: 725; Marks etal., J. Mol. Biol., 1991, 222: 581, each incorporated by reference in its entirety) are derived by cloning of natural human CDRH3 sequences and their exact composition is not characterized, which precludes enumeration. 2015201796 09 Apr 2015 15 Synthetic libraries produced by other (e.g., random or semi-random / biased) methods (Knappik, et al., J Mol Biol, 2000, 296: 57, incorporated by reference in its entirety) tend to have very large numbers of unique members. Thus, while matches to a given input sequence (for example, at 80% or greater) may exist in a theoretical representation of such libraries, the probability of synthesizing and then producing a 20 physical realization of the theoretical library that contains such a sequence and then selecting an antibody corresponding to such a match, in practice, may be remotely small. For example, a CDRH3 of length 19 in the Knappik library may have over 1019 distinct sequences. In a practical realization of such a library a tenth or so of the sequences may have length 19 and the largest total library may have in the order of 1010 to 1012 25 transformants; thus, the probability of a given pre-defined member being present, in practice, is effectively zero (less than one in ten million). Other libraries (e.g., Enzelberger et al. W02008053275 and Ladner US20060257937, each incorporated by reference in its entirety) suffer from at least one of the limitations described throughout this application. 30 Thus, for example, considering antibody CAB14, there are seven members of the designed library of Example 5 that differ at just one amino acid position from the - 156- sequence of the CDRH3 of CAB14 (given in Table 39). Since the total length of this CDRH3 sequence is 13, the percent of identical amino acids is 12/13 or about 92% for each of these 7 sequences of the library of the invention. It can be estimated that the probability of obtaining such a match (or better) in the library of Knappik et al. is about 5 1.4 x 10'9; it would be lower still, about 5.5 x 10~10, in a library with equal amino acid 2015201796 09 Apr 2015 proportions (/. e., completely random). Therefore, in a physical realization of the library with about 1010 transformants of which about a tenth may have length 13, there may be one or two instances of these best matches. However, with longer sequences such as CAB 12, the probability of having members in the Knappik library with about 89% or 10 better matching are under about 10~15, so that the expected number of instances in a physical realization of the library is essentially zero. To the extent that sequences of interest resemble actual human CDRH3 sequences, there will be close matches in the library of Example 5, which was designed to mimic human sequences. Thus, one of the many relative advantages of the present library, versus those in the art, becomes more 15 apparent as the length of the CDRH3 increases.

Example 9: Split Pool Synthesis of Oligonucleotides Encoding the DH, N2, and 113-JH Sesments 20 This example outlines the procedures used to synthesize the oligonucleotides used to construct the exemplary libraries of the invention. Custom Primer Support ™ 200 dT40S resin (GE Healthcare) was used to synthesize the oligonucleotides, using a loading of about 39 pmol/g of resin. Columns (diameter = 30 pm) and frits were purchased from Biosearch Technologies, Inc. A column bed volume of 30 pL was used 25 in the synthesis, with 120 nmol of resin loaded in each column. A mixture of dichloromethane (DCM) and methanol (MeOH), at a ratio of 400/122 (v/v) was used to load the resin. Oligonucleotides were synthesized using a Dr. Oligo ® 192 oligonucleotide synthesizer and standard phosphorothioate chemistry.

The split pool procedure for the synthesis of the [DH]-[N2]-[H3-JH]

30 oligonucleotides was performed as follows: First, oligonucleotide leader sequences, containing a randomly chosen 10 nucleotide sequence (ATGCACAGTT; SEQ ID NO:_), a BsrDI recognition site (GCAATG), and a two base “overlap sequence” (TG, AC, AG, CT, or GA) were synthesized. The purpose of each of these segments is explained below. After synthesis of this 18 nucleotide sequence, the DH segments were - 157- synthesized; approximately 1 g of resin (with the 18 nucleotide segment still conjugated) was suspended in 20 mL of DCM/MeOH. About 60 pL of the resulting slurry (120 nmol) was distributed inside each of 278 oligonucleotide synthesis columns. These 278 columns were used to synthesize the 278 DH segments of Table 18, 3’ to the 18 5 nucleotide segment described above. After synthesis, the 278 DH segments were pooled as follows: the resin and frits were pushed out of the columns and collected inside a 20 mL syringe barrel (without plunger). Each column was then washed with 0.5 mL MeOH, to remove any residual resin that was adsorbed to the walls of the column. The resin in the syringe barrel was washed three times with MeOH, using a low porosity 10 glass filter to retain the resin. The resin was then dried and weighed. 2015201796 09 Apr 2015

The pooled resin (about 1.36 g) containing the 278 DH segments was subsequently suspended in about 17 mL of DCM/MeOH, and about 60 pL of the resulting slurry was distributed inside each of two sets of 141 columns. The 141 N2 segments enumerated in Tables 24 and 25 were then synthesized, in duplicate (282 total 15 columns), 3’ to the 278 DH segments synthesized in the first step. The resin from the 282 columns was then pooled, washed, and dried, as described above.

The pooled resin obtained from the N2 synthesis (about 1.35 g) was suspended in about 17 mL of DCM/MeOH, and about 60 pL of the resulting slurry was distributed inside each of 280 columns, representing 28 H3-JH segments synthesized ten times 20 each. A portion (described more fully below) of each of the 28 IGHJ segments, including H3-JH of Table 20 were then synthesized, 3’ to the N2 segments, in ten of the columns. Final oligonucleotides were cleaved and deprotected by exposure to gaseous ammonia (85°C, 2 h, 60 psi).

Split pool synthesis was used to synthesize the exemplary CDRH3 library. 25 However, it is appreciated that recent advances in oligonucleotide synthesis, which enable the synthesis of longer oligonucleotides at higher fidelity and the production of the oligonucleotides of the library by synthetic procedures that involve splitting, but not pooling, may be used in alternative embodiments of the invention. The split pool synthesis described herein is, therefore, one possible means of obtaining the 30 oligonucleotides of the library, but is not limiting. One other possible means of synthesizing the oligonucleotides described in this application is the use of trinucleotides. This may be expected to increase the fidelity of the synthesis, since frame shift mutants would be reduced or eliminated. -158-

Example 10: Construction of the CDRH3 and Heavy Chain Libraries 2015201796 09 Apr 2015

This example outlines the procedures used to create exemplary CDRH3 and heavy chain libraries of the invention. A two step process was used to create the 5 CDRH3 library. The first step involved the assembly of a set of vectors encoding the tail and N1 segments, and the second step involved utilizing the split pool nucleic acid synthesis procedures outlined in Example 9 to create oligonucleotides encoding the DH, N2, and H3-JH segments. The chemically synthesized oligonucleotides were then ligated into the vectors, to yield CDRH3 residues 95-102, based on the numbering 10 system described herein. This CDRH3 library was subsequently amplified by PCR and recombined into a plurality of vectors containing the heavy chain chassis variants described in Examples 1 and 2. CDRH1 and CDRH2 variants were produced by QuikChange ® Mutagenesis (Stratagene ™), using the oligonucleotides encoding the ten heavy chain chassis of Example 1 as a template. In addition to the heavy chain chassis, 15 the plurality of vectors contained the heavy chain constant regions (i.e., CHI, CH2, and CH3) from IgGl, so that a full-length heavy chain was formed upon recombination of the CDRH3 with the vector containing the heavy chain chassis and constant regions. In this exemplary embodiment, the recombination to produce the full-length heavy chains and the expression of the full-length heavy chains were both performed in S. cerevisiae. 20 To generate full-length, heterodimeric IgGs, comprising a heavy chain and a light chain, a light chain protein was also expressed in the yeast cell. The light chain library used in this embodiment was the kappa light chain library, wherein the VKCDR3s were synthesized using degenerate oligonucleotides (see Example 6.2). Due to the shorter length of the oligonucleotides encoding the light chain library (in 25 comparison to those encoding the heavy chain library), the light chain CDR3 oligonucleotides could be synthesized de novo, using standard procedures for oligonucleotide synthesis, without the need for assembly from sub-components (as in the heavy chain CDR3 synthesis). One or more light chains can be expressed in each yeast cell which expresses a particular heavy chain clone from a library of the invention. 30 One or more light chains have been successfully expressed from both episomal (e.g., plasmid) vectors and from integrated sites in the yeast genome.

Below are provided further details on the assembly of the individual components for the synthesis of a CDRH3 library of the invention, and the subsequent combination - 159- of the exemplary CDRH3 library with the vectors containing the chassis and constant regions. In this particular exemplary embodiment of the invention, the steps involved in the process may be generally characterized as (i) synthesis of 424 vectors encoding the tail andNl regions; (ii) ligation of oligonucleotides encoding the [DH]-[N2]-[H3-JH] 2015201796 09 Apr 2015 5 segments into these 424 vectors; (iii) PCR amplification of the CDRH3 sequences from the vectors produced in these ligations; and (iv) homologous recombination of these PCR-amplified CDRH3 domains into the yeast expression vectors containing the chassis and constant regions. 10 Example 10.1: Synthesis of Vectors Encoding the Tail and N1 Regions

This example demonstrates the synthesis of 424 vectors encoding the tail and N1 regions of CDRH3. In this exemplary embodiment of the invention, the tail was restricted to G, D, E, or nothing, and the N1 region was restricted to one of the 59 sequences shown in Table 24. As described throughout the specification, many other 15 embodiments are possible.

In the first step of the process, a single “base vector” (pJM204, a pUC-derived cloning vector) was constructed, which contained (i) a nucleic acid sequence encoding two amino acids that are common to the C-terminal portion of all 28 IGHJ segments (SS), and (ii) a nucleic acid sequence encoding a portion of the CHI constant region 20 from IgGl. Thus, the base vector contains an insert encoding a sequence that can be depicted as: [SS]-[CH1~], wherein SS is a common portion of the C-terminus of the 28 IGHJ segments and CH1~ is a portion of the CHI constant region from IgGl, namely: 25 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLG (SEQ ID NO:_).

Next, 424 different oligonucleotides were cloned into the base vector, upstream (i.e., 5’) from the region encoding the [SS]-[CH1~], These 424 oligonucleotides were synthesized by standard methods and each encoded a C-terminal portion of one of the 17 30 heavy chain chassis enumerated in Table 5, plus one of four exemplary tail segments (G/D/E/-), and one of 59 exemplary N1 segments (Table 24). These 424 oligonucleotides, therefore, encode a plurality of sequences that may be represented by: [~FRM3]-[G/D/E/-]-[Nl], - 160- wherein ~FRM3 represents a C-terminal portion of a FRM3 region from one of the 17 heavy chain chassis of Table 5, G/D/E/- represents G, D, E, or nothing, and N1 represents one of the 59 N1 sequences enumerated in Table 24. As described throughout the specification, the invention is not limited to the chassis exemplified in 5 Table 5, their CDRH1 and CDRH2 variants (Table 8), the four exemplary tail options used in this example, or the 59 N1 segments presented in Table 24. 2015201796 09 Apr 2015

The oligonucleotide sequences represented by the sequences above were synthesized in two groups: one group containing a ~FRM3 region identical to the corresponding region on 16 of the 17 the heavy chain chassis enumerated in Table 5,

10 and another group containing a -FRM3 region that is identical to the corresponding region on VH3-15. In the former group, an oligonucleotide encoding DTAVYYCAR

(SEQ ID NO:_) was used for -FRM3. During subsequent PCR amplification, the V residue of VH5-51 was altered to an M, to correspond to the VH5-51 germline sequence. In the latter group (that with a sequence common to VH3-15), a larger 15 oligonucleotide, encoding the sequence

AISGS GGS T Y Y AD S VKGRFTISRDN SKNTL YLQMN SLRAEDT AVYY C AK (SEQ ID NO:_) was used for ~FRM3. Each of the two oligonucleotides encoding the ~FRM3 regions were paired with oligonucleotides encoding one of the four tail regions (G/D/E/-) and one of the 59 N1 segments, yielding a total of 236 possible combinations 20 for each -FRM3 (i.e., 1 x 4 x 59), or a total of 472 possible combinations when both ~FRM3 sequences are considered. However, 48 of these combinations are redundant and only a single representation of these sequences was used in the currently exemplified CDRH3 library, yielding 424 unique oligonucleotides encoding [-FRM3]-[G/D/E/-]-[Nl] sequences. 25 After the oligonucleotides encoding the [~FRM3]-[G/D/E/-]-[Nl] and [SS]- [CH1~] segments were cloned into the vector, as described above, additional sequences were added to the vector to facilitate the subsequent insertion of the oligonucleotides encoding the [DH]-[N2]-[H3-JH] fragments synthesized during the split pool synthesis. These additional sequences comprise a polynucleotide encoding a selectable marker 30 protein, flanked on each side by a recognition site for a type II restriction enzyme, for example: [Type IIRS 1]-[selectable marker protein]-[Type II RS 2]. -161 -

In this exemplary embodiment, the selectable marker protein is ccdB and the type II restriction enzyme recognition sites are specific for BsrDI and Bbsl. In certain strains of E. coli, the ccdB protein is toxic, thereby preventing the growth of these bacteria when the gene is present. 2015201796 09 Apr 2015 5 An example of the 5 ’ end of one of the 212 vectors with a -FRM3 region based on the VH3-23 chassis, D tail residue and an N1 segment of length zero is presented below: VH3-23

A I S GSG G S T Y · GCTATTAG TGGTAGTGGT GGTAGCACAT CGATAATC ACCATCACCA CCATCGTGTA 10 15 1C1 ]. VH3-23

• YAD SVK GRFT ISR DNS KNTL YLQ MNS ACTACGCAGA CTCCGTGAAG GGCCGGTTCA CCATCTCCAG AGACAATTCC AAGAACACGC TGTATCTGCA AATGAACAGC

TGATGCGTCT GAGGCACTTC CCGGCCAAGT GGTAGAGGTC TCTGTTAAGG TTCTTGTGCG ACATAGACGT TTACTTGTCG

20 VH3-23 ccdB

BsrDI

LRAEDTAVYYCAK

25 1121 CTGAGAGCCG AGGACACGGC GGTGTACTAC TGCGCCAAGG ACCATTGCGC TTAGCCTAGG TTATATTCCC CAGAACATCA

GACTCTCGGC TCCTGTGCCG CCACATGATG ACGCGGTTCC TGGTAACGCG AATCGGATCC AATATAAGGG GTCTTGTAGT

An example of one of the 212 vectors with a -FRM3 region based on one of the other 16 chassis, with a D residue as the tail and an N1 segment of length zero is 30 presented below:

Framework. 3 35 40 45 50 55 60 65 9 61.

D T A VYYC AR GACACGGCG GTGTACTACT GCGCCAGAGA

CTGTGCCGC CACATGATGA CGCGGTCTCT

ccdB

BsrDI

1041 CCATTGCGCT TAGCCTAGGT TATATTCCCC AGAACATCAG GTTAATGGCG TTTTTGATGT CATTTTCGCG GTGGCTGAGA

GGTAACGCGA ATCGGATCCA ATATAAGGGG TCTTGTAGTC CAATTACCGC AAAAACTACA GTAAAAGCGC CACCGACTCT

ccdB

1121 TCAGCCACTT CTTCCCCGAT AACGGAAACC GGCACACTGG CCATATCGGT GGTCATCATG CGCCAGCTTT CATCCCCGAT

AGTCGGTGAA GAAGGGGCTA TTGCCTTTGG CCGTGTGACC GGTATAGCCA CCAGTAGTAC GCGGTCGAAA GTAGGGGCTA

ccdB

1201 ATGCACCACC GGGTAAAGTT CACGGGAGAC TTTATCTGAC AGCAGACGTG CACTGGCCAG GGGGATCACC ATCCGTCGCC TACGTGGTGG CCCATTTCAA GTGCCCTCTG AAATAGACTG TCGTCTGCAC GTGACCGGTC CCCCTAGTGG TAGGCAGCGG

ccdB

1281 CGGGCGTGTC AATAATATCA CTCTGTACAT CCACAAACAG ACGATAACGG CTCTCTCTTT TATAGGTGTA AACCTTAAAC GCCCGCACAG TTATTATAGT GAGACATGTA GGTGTTTGTC TGCTATTGCC GAGAGAGAAA ATATCCACAT TTGGAATTTG

ccdB

1361 TGCATTTCAC CAGCCCCTGT TCTCGTCAGC AAAAGAGCCG TTCATTTCAA TAAACCGGGC GACCTCAGCC ATCCCTTCCT

ACGTAAAGTG GTCGGGGACA AGAGCAGTCG TTTTCTCGGC AAGTAAAGTT ATTTGGCCCG CTGGAGTCGG TAGGGAAGGA

ccdB

1441 GATTTTCCGC TTTCCAGCGT TCGGCACGCA GACGACGGGC TTCATTCTGC ATGGTTGTGC TTACCAGACC GGAGATATTG

CTAAAAGGCG AAAGGTCGCA AGCCGTGCGT CTGCTGCCCG AAGTAAGACG TACCAACACG AATGGTCTGG CCTCTATAAC

ccdB

1521 ACATCATATA TGCCTTGAGC AACTGATAGC TGTCGCTGTC AACTGTCACT GTAATACGCT GCTTCATAGC ATACCTCTTT

TGTAGTATAT ACGGAACTCG TTGACTATCG ACAGCGACAG TTGACAGTGA CATTATGCGA CGAAGTATCG TATGGAGAAA

ccdB

1601 TTGACATACT TCGGGTATAC ATATCAGTAT ATATTCTTAT ACCGCAAAAA TCAGCGCGCA AATATGCATA CTGTTATCTG 162 2015201796 09 Apr 2015

AACTGTATGA AGCCCATATG TATAGTCATA TATAAGAATA TGGCGTTTTT AGTCGCGCGT TTATACGTAT GACAATAGAC CCdB CHI 5 10 15

Bbsl

A STK GPS VFPL APS 1681 GCTTTTAGTA AGCCGCCTAG GTCATCAGAA GACAACTCAG CTAGCACCAA GGGCCCATCG GTCTTTCCCC TGGCACCCTC CGAAAATCAT TCGGCGGATC CAGTAGTCTT CTGTTGAGTC GATCGTGGTT CCCGGGTAGC CAGAAAGGGG ACCGTGGGAG CHI •SKS TSGG T A A LGC LVKD YFP EPV TVSW 1761 CTCCAAGAGC ACCTCTGGGG GCACAGCGGC CCTGGGCTGC CTGGTCAAGG ACTACTTCCC CGAACCGGTG ACGGTGTCGT GAGGTTCTCG TGGAGACCCC CGTGTCGCCG GGACCCGACG GACCAGTTCC TGATGAAGGG GCTTGGCCAC TGCCACAGCA CHI • NSG ALT SGVH TFP A V L QSSG L 1841 GGAACTCAGG CGCCCTGACC AGCGGCGTGC ACACCTTCCC GGCTGTCCTA CAGTCCTCAG GACTC CCTTGAGTCC GCGGGACTGG TCGCCGCACG TGTGGAAGGG CCGACAGGAT GTCAGGAGTC CTGAG 20 All 424 vectors were sequence verified. A schematic diagram of the content of the 424 vectors, before and after cloning of the [DH]-[N2]-[H3-JH] fragment is presented in Figure 5. Below is an exemplary sequence from one of the 424 vectors containing a FRM3 region from VH3-23. 25 30 35 40 45 50 55 60 65 70 primer EMK135 VH3-23

A I S GSG GST YYAD SVK GRF 561 GCTATTA GTGGTAGTGG TGGTAGCACA TACTACGCAG ACTCCGTGAA GGGCCGGTTC CGATAAT CACCATCACC ACCATCGTGT ATGATGCGTC TGAGGCACTT CCCGGCCAAG VH3-23

TISR DNS KNT LYLQ MNS LRA EDTA VYY

641 ACCATCTCCA GAGACAATTC CAAGAACACG CTGTATCTGC AAATGAACAG CCTGAGAGCC GAGGACACGG CGGTGTACTA TGGTAGAGGT CTCTGTTAAG GTTCTTGTGC GACATAGACG TTTACTTGTC GGACTCTCGG CTCCTGTGCC GCCACATGAT VH3-23 D J1 JH6

Nl_9 N2

•CAR DAGG YYY GSG SYYN AAA YYY YYGM '/'21 CTGCGCCAAG GACGCCGGAG GATATTATTA TGGGTCAGGA AGCTATTACA ACGCTGCGGC TTACTACTAC TATTATGGCA GACGCGGTTC CTGCGGCCTC CTATAATAAT ACCCAGTCCT TCGATAATGT TGCGACGCCG AATGATGATG ATAATACCGT JH6

J1 CHI

Nhel

• DVW GQG TTVT VSS AST KGPS VFP LAP

801 TGGACGTGTG GGGACAAGGT ACAACAGTCA CCGTCTCCTC AGCTAGCACC AAGGGCCCAT CGGTCTTTCC CCTGGCACCC

ACCTGCACAC CCCTGTTCCA TGTTGTCAGT GGCAGAGGAG TCGATCGTGG TTCCCGGGTA GCCAGAAAGG GGACCGTGGG

CHI

SSKS TSG GTA ALGC LVK DYF PEPV TVS

631 TCCTCCAAGA GCACCTCTGG GGGCACAGCG GCCCTGGGCT GCCTGGTCAA GGACTACTTC CCCGAACCGG TGACGGTGTC

AGGAGGTTCT CGTGGAGACC CCCGTGTCGC CGGGACCCGA CGGACCAGTT CCTGATGAAG GGGCTTGGCC ACTGCCACAG EK137 CHI Primer

CHI

•WNS GALT SGV HTF PAVL QSS GLY SLSS

961 GTGGAACTCA GGCGCCCTGA CCAGCGGCGT GCACACCTTC CCGGCTGTCC TACAGTCCTC AGGACTCTAC TCCCTCAGCA

CACCTTGAGT CCGCGGGACT GGTCGCCGCA CGTGTGGAAG GGCCGACAGG ATGTCAGGAG TCCTGAGATG AGGGAGTCGT

CHI

• VVT VPS SSLG 1041 GCGTGGTGAC CGTGCCCTCC AGCAGCTTGG GC

CGCACCACTG GCACGGGAGG TCGTCGAACC CG 163

Example 10.2: Cloning of the Oligonucleotides Encoding the DH, N2, H3-JH Segments into the Vectors Containing the Tail and N1 Segments 2015201796 09 Apr 2015

This example describes the cloning of the oligonucleotides encoding the [D]- [N2]-[H3-JH] segments (made via split pool synthesis; Example 9) into the 424 vectors 5 produced in Example 10.1. To summarize, the [DH]-[N2]-[H3-JH] oligonucleotides produced via split pool synthesis were amplified by PCR, to produce double-stranded oligonucleotides, to introduce restriction sites that would create overhangs complementary to those on the vectors (i.e., BsrDI and Bbsl), and to complete the 3’ portion of the IGHJ segments that was not synthesized in the split pool synthesis. The

10 amplified oligonucleotides were then digested with the restriction enzymes BsrDI (cleaves adjacent to the DH segment) and Bbsl (cleaves near the end of the JH segment).

The cleaved oligonucleotides were then purified and ligated into the 424 vectors which had previously been digested with BsrDI and Bbsl. After ligation, the reactions were purified, ethanol precipitated, and resolubilized. 15 This process for one of the [DH]-[N2]-[H3-JH] oligonucleotides synthesized in

the split pool synthesis is illustrated below. The following oligonucleotide (SEQ ID NO:_) is one of the oligonucleotides synthesized during the split pool synthesis: 1 ATGCACAGTTGCAAT GTGT ATTACT AT GGAT CT GGTT CTT ACT AT AAT GT 50 51 GGGCGGAT ATT ATTACT ACTAT GGT AT GGACGT AT GGGGGCAAGGGACC 99 20

The first 10 nucleotides (ATGCACAGTT; SEQ ID NO:_) represent a portion of a random sequence that is increased to 20 base pairs in the PCR amplification step, below. This portion of the sequence increases the efficiency of BsrDI digestion and facilitates the downstream purification of the oligonucleotides. 25 Nucleotides 11-16 (underlined) represent the BsrDI recognition site. The two base overlap sequence that follows this site (in this example TG; bold) was synthesized to be complementary to the two base overhang created by digesting certain of the 424 vectors with BsrDI (i.e., depending on the composition of the tail / N1 region of the particular vector). Other oligonucleotides contain different two-base overhangs, as 30 described below.

The two base overlap is followed by the DH gene segment (nucleotides 19-48), in this example, by a 30 bp sequence (TATTACTATGGATCTGGTTCTTACTATAAT, SEQ ID NO:_) which encodes the ten residue DH segment YYYGSGSYYN (i.e., IGHD3-10 2 of Table 17; SEQ ID NO:_). - 164- 2015201796 09 Apr 2015

The region of the oligonucleotide encoding the DH segment is followed, in this example, by a nine base region (GTGGGCGGA; bold; nucleotides 49-57), encoding the N2 segment (in this case VGG; Table 24).

The remainder of this exemplary oligonucleotide represents the portion of the JH 5 segment that is synthesized during the split pool synthesis (T ATT ATT ACT AC T AT GGT AT GG AC GT AT GGGGGC AAGGG AC C; SEQ ID NO:

_; nucleotides 58-99; underlined), encoding the sequence YYYYYGMDVWGQGT (Table 20; SEQ ID NO:_). The balance of the IGHJ segment is added during the subsequent PCR amplification described below. 10 After the split pool-synthesized oligonucleotides were cleaved from the resin and deprotected, they served as a template for a PCR reaction which added an additional randomly chosen 10 nucleotides (e.g., GACGAGCTTC; SEQ ID NO:_) to the 5’ end and the rest of the IGHJ segment plus the Bbsl restriction site to the 3’ end. These additions facilitate the cloning of the [DH]-[N2]-[JH] oligonucleotides into the 424 15 vectors. As described above (Example 9), the last round of the split pool synthesis involves 280 columns: 10 columns for each of the oligonucleotides encoding one of 28 H3-JH segments. The oligonucleotide products obtained from these 280 columns are pooled according to the identity of their H3-JH segments, for a total of 28 pools. Each of these 28 pools is then amplified in five separate PCR reactions, using five forward 20 primers that each encode a different two base overlap (preceding the DH segment; see above) and one reverse primer that has a sequence corresponding to the familial origin of the H3-JH segment being amplified. The sequences of these 11 primers are provided below: 25 30

Forward primers AC GACGAGCTT CAAT GCACAGTT GC AAT G AC AG GACGAGCTT CAAT GCACAGTT GCAATGAG CT GACGAGCTT CAAT GCACAGTT GCAATGCT GA GACGAGCTT CAAT GCACAGTT GCAATGGA TG GACGAGCTT CAAT GCACAGTT GCAATGTG (SEQ ID NO:_) (SEQ ID NO:_) (SEQ ID NO:_) (SEQ ID NO:_) (SEQ ID NO:_)

Reverse Primers

DHl TGCATCAGTGCGACTAACGGAAGACTC TGAGGAGACGGTGACCAAGGTGCCCTGGCCCCA (SEQ ID NO:_)

35 J H2 TGCATCAGTGCGACTAACGGAAGACTC TGAGGAGACAGTGACCAAGGTGCCACGGCCCCA (SEQ ID NO:_)

DH3 T GCAT CAGTGCGACTAACGGAAGACTCTGAAGAGACGGT GACCATTGTCCCTTGGCCCCA (SEQ ID NO:_)

DH4 T GCAT CAGTGCGACTAACGGAAGACTCTGAGGAGACGGT GACCAAGGTTCCTTGGCCCCA - 165 - (SEQ ID NO:_) 2015201796 09 Apr 2015

DH5 T GCAT CAGTGCGACTAACGGAAGACT CTGAGGAGACGGT GACCAAGGTTCCCTGGCCCCA (SEQ ID NO:_)

DH6 T GCAT CAGTGCGACTAACGGAAGACT CTGAGGAGACGGT GACCGT GGT CCCTT GCCCCCA 5 (SEQ ID NO:_)

Amplifications were performed using Taq polymerase, under standard conditions. The oligonucleotides were amplified for eight cycles, to maintain the representation of sequences of different lengths. Melting of the strands was performed 10 at 95°C for 30 seconds, with annealing at 58°C and a 15 second extension time at 72°C.

Using the exemplary split-pool derived oligonucleotide enumerated above as an example, the PCR amplification was performed using the TG primer and the JH6 primer, where the annealing portion of the primers has been underlined: 15 TG GACGAGCTTCAATGCACAGTTGCAATGTG (SEQ ID NO:_)

DH6 TGCATCAGTGCGACTAACGGAAGACTCTGAGGAGACGGTGACCGTGGTCCCTTGCCCCCA (SEQ ID NO:_) 20 The portion of the TG primer that is 5’ to the annealing portion includes the random 10 base pairs described above. The portion of the JH6 primer that is 5’ to the annealing portion includes the balance of the JH6 segment and the Bbsl restriction site. The following PCR product (SEQ ID NO:_) is formed in the reaction (added sequences underlined):

25 GACGAGCTTCATGCACAGTTGCAATGTGTATTACTATGGATCTGGTTCTTACTATAATGTGGGCGGATATTAT TACTACTATGGTAT GGACGTAT GGGGGCAAGGGACCACGGT CACCGT CTCCT CAGAGT CTT CCGTTAGT CGCA CTGATGCAG

The PCR products from each reaction were then combined into five pools, based 30 on the forward primer that was used in the reaction, creating sets of sequences yielding the same two-base overhang after BsrDI digestion. The five pools of PCR products were then digested with BsRDI and Bbsl (100 pg of PCR product; 1 mL reaction volume; 200 U Bbsl; 100 U BsrDI; 2h; 37°C; NEB Buffer 2). The digested oligonucleotides were extracted twice with phenol/chloroform, ethanol precipitated, air 35 dried briefly and resolubilized in 300 pL of TE buffer by sitting overnight at 4°C.

Each of the 424 vectors described in the preceding sections was then digested with BsrDI and Bbsl, each vector yielding a two base overhang that was complimentary to one of those contained in one of the five pools of PCR products. Thus, one of the five pools of restriction digested PCR products are ligated into each of the 424 vectors, - 166- depending on their compatible ends, for a total of 424 ligations. 2015201796 09 Apr 2015

Example 10.3: PCR Amplification of the CDRH3 from the 424 Vectors

This example describes the PCR amplification of the CDRH3 regions from the 424 5 vectors described above. As set forth above, the 424 vectors represent two sets: one for the VH3-23 family, with FRM3 ending in CAK (212 vectors) and one for the other 16 chassis, with FRM3 ending in CAR (212 vectors). The CDRH3s in the VH3-23-based vectors were amplified using a reverse primer (EK137; see Table 41) recognizing a portion of the CHI region of the plasmid and the VH3-23-specific primer EK135 (see 10 Table 41). Amplification of the CDRH3s from the 212 vectors with FRM3 ending in CAR was performed using the same reverse primer (EK137) and each of five FRM3-specific primers shown in Table 41 (EK139, EK140, EK141, EK143, and EK144). Therefore, 212 VH3-23 amplifications and 212 x 5 FRM3 PCR reactions were performed, for a total of 1,272 reactions. An additional PCR reaction amplified the 15 CDRH3 from the 212 VH3-23-based vectors, using the EK 133 forward primer, to allow the amplicons to be cloned into the other 5 VH3 family member chassis while making the last three amino acids of these chassis CAK instead of the original CAR (VH3-23*). The primers used in each reaction are shown in Table 41. 20 Table 41. Primers Used for Amplification of CDRH3 Sequences

Primer No. Compatible Chassis Primer Sequence SEQ ID NO EK135 VH3-23 CACATACTACGCAGACTCCGTG EK133 VH3-48; VH3-7; VH3-15; VH3-30; VH3-33; VH3-23* CAAATGAACAGCCTGAGAGCCGAGGACACGGCGGTGTACTACTG EK139 VH4-B; VH4-31; VH4-34; VH4-39; VH4-59; AAGCTGAGTTCTGTGACCGCCGCAGACACGGCGGTGTACTACTG -167-

VH4-61 EK140 VH1-46; VH1-69 GAGCTGAGCAGCCTGAGATCTGAGGACACGGCGGTGTACTACTG EK141 VH1-2 GAGCTGAGCAGGCTGAGATCTGACGACACGGCGGTGTACTACTG EK143 VH5-51 CAGTGGAGCAGCCTGAAGGCCTCGGACACGGCGATGTACTACTG EK144 VH1-18 GAGCTGAGGAGCCTGAGATCTGACGACACGGCGGTGTACTACTG EK137 CH1 Rev. Primer GTAGGACAGCCGGGAAGG

Example 10.4: Homologous Recombination of PCR-Amplified CDRH3 Regions Into Heavy Chain Chassis 2015201796 09 Apr 2015 5 After amplification, reaction products were pooled according to the respective VH chassis that they would ultimately be cloned into. Table 42 enumerates these pools, with the PCR primers used to obtain the CDRH3 sequences in each pool provided in the last two columns.

Table 42. PCR Primers Used to Amplify CDRH3 Regions from 424 Vectors

Pool # (Arbitrary) HC Chassis Target 5’ Primer 3’ Primer 1 1-46 EK140 EK137 1-69 EK140 EK137 2 1-2 EK141 EK137 3 1-18 EK144 EK137 4 4-B EK139 EK137 4-31 EK139 EK137 4-342 EK139 EK137 4-39 EK139 EK137 4-59 EK139 EK137 4-61 EK139 EK137 5 5-51 EK143 EK137 6 3-151 EK133 EK137 3-7 EK133 EK137 3-33 EK133 EK137 3-33 EK133 EK137 3-48 EK133 EK137 7 3-23 EMK135 EK137 8 3-23* EK133 EK137 -168- 2015201796 09 Apr 2015

Allowed the amplicons to be cloned into the other 5 VH3 family member chassis {i.e., other than VH3-23), while making the last three amino acids of these chassis CAK instead of the original CAR. 5 1 As described in Table 5, the original KT sequence in VH3-15 was mutated to RA, and the original TT to AR. 2 As described in Table 5, the potential site for N-linked glycosylation was removed from CDRH2 of this chassis. 10

After pooling of the amplified CDRH3 regions, according to the process outlined above, the heavy chain chassis expression vectors were pooled according to their origin and cut, to create a “gap” for homologous recombination with the amplified CDRH3s. Figure 6 shows a schematic structure of a heavy chain vector, prior to recombination 15 with a CDRH3. In this exemplary embodiment of the invention, there were a total of 152 vectors encoding heavy chain chassis and IgGl constant regions, but no CDRH3. These 152 vectors represent 17 individual variable heavy chain gene families (Table 5; Examples 1 and 2). Fifteen of the families were represented by the heavy chain chassis sequences described in Table 5 and the CDRH1/H2 variants described in Table 8 (i.e., 20 150 vectors). VH 3-30 differs from VH3-33 by a single amino acid; thus VH3-30 was included in the VH3-33 pool of variants. The 4-34 VH family member was kept separate from all others and, in this exemplary embodiment, no variants of it were included in the library. Thus, a total of 16 pools, representing 17 heavy chain chassis, were generated from the 152 vectors. 25 The vector pools were digested with the restriction enzyme Sfil, which cuts at two sites in the vector that are located between the end of the FRM3 of the variable domain and the start of the CHI. 30 35 40 45 50

SOI

VH3-48 SVK GRFT ISR DNA KNSL YLQ MNS LRAE-CTCTGTGAAG GGCCGATTCA CCATCTCCAG AGACAATGCC AAGAACTCAC TGTATCTGCA AATGAACAGC CTGAGAGCTG GAGACACTTC CCGGCTAAGT GGTAGAGGTC TCTGTTACGG TTCTTGAGTG ACATAGACGT TTACTTGTCG GACTCTCGAC Constant DTAVYYCAR VH3-4

Sfil

Sfil

VTVSS common to all J

D T A VYY CAR V T 88: AGGACACGGC GGTGTACTAC TGCGCCAGAG GCCAATAGGG CCAACTATAA CAGGGGTACC CCGGCCAATA AGGCCGTCAC TCCTGTGCCG CCACATGATG ACGCGGTCTC CGGTTATCCC GGTTGATATT GTCCCCATGG GGCCGGTTAT TCCGGCAGTG VTVSS common to all J hIgGlml7,1

Nhel VSS ASTK GPS VFP LAPS SKS TSG GTA CGTCTCCTCA GCTAGCACCA AGGGCCCATC GGTCTTCCCC CTGGCACCCT CCTCCAAGAG CACCTCTGGG GGCACAGCGG GCAGAGGAGT CGATCGTGGT TCCCGGGTAG CCAGAAGGGG GACCGTGGGA GGAGGTTCTC GTGGAGACCC CCGTGTCGCC

The gapped vector pools were then mixed with the appropriate {i.e., compatible) - 169- pool of CDRH3 amplicons, generated as described above, at a 50:1 insert to vector ratio. The mixture was then transformed into electrocompetent yeast (S. cerevisiae), which already contained plasmids or integrated genes comprising a VK light chain library (described below). The degree of library diversity was determined by plating a dilution 5 of the electroporated cells on a selectable agar plate. In this exemplified embodiment of the invention, the agar plate lacked tryptophan and the yeast lacked the ability to endogenously synthesize tryptophan. This deficiency was remedied by the inclusion of the TRP marker on the heavy chain chassis plasmid, so that any yeast receiving the plasmid and recombining it with a CDRH3 insert would grow. The electroporated cells 10 were then outgrown approximately 100-fold, in liquid media lacking tryptophan. Aliquots of the library were frozen in 50% glycerol and stored at -80°C. Each transformant obtained at this stage represents a clone that can express a full IgG molecule. A schematic diagram of a CDRH3 integrated into a heavy chain vector and the accompanying sequence are provided in Figure 5. 2015201796 09 Apr 2015 15 A heavy chain library pool was then produced, based on the approximate representation of the heavy chain family members as depicted in Table 43.

Table 43. Occurrence of Heavy Chain Chassis in Data Sets Used to Design Library, Expected (Designed) Library, and Actual (Observed) Library

Chassis Relative Occurrence in Data Sets (1) Expected (2) Observed (3) VH1-2 5.1 6.0 6.4 VH1-18 3.4 3.7 3.8 VH1-46 3.4 5.2 4.7 VH1-69 8.0 8.0 10.7 VH3-7 3.6 6.1 4.5 VH3-15 1.9 6.9 3.6 VH3-23 11.0 13.2 17.1 VH3-33/30 13.1 12.5 6.6 VH3-48 2.9 6.3 7.5 VH4-31 3.4 2.5 4.3 VH4-34 17.2 7.0 4.7 VH4-39 8.7 3.9 3.0 VH4-59 7.0 7.8 9.2 VH4-61 3.2 1.9 2.4 VH4-B 1.0 1.4 0.8 VH5-51 7.2 7.7 10.5 (1) As detailed in Example 1, these 17 sequences account for a Dout 76% of the entire sample of human VH sequences used to represent the human repertoire. (2) Based on pooling of sub-libraries of each chassis type. - 170- (3) Usage in 531 sequences from library; cf. Figure 20. 2015201796 09 Apr 2015 5 Example 10.5: K94R Mutation in VH3-23 and R94K Mutation in VH3-33, VH3-30, VH3-7, and VH3-48

This example describes the mutation of position 94 in VH3-23, VH3-33, VH3-30, VH3-7, and VH3-48. In VH3-23, the amino acid at this position was mutated from K to R. In VH3-33, VH3-30, VH3-7, and VH3-48, this amino acid was mutated from R 10 to K. In VH3-32, this position was mutated from K to R. The purpose of making these mutations was to enhance the diversity of CDRH3 presentation in the library. For example, in naturally occurring VH3-23 sequences, about 90% have K at position 94, while about 10% have position R. By making these changes the diversity of the CDRH3 presentation is increased, as is the overall diversity of the library.

15 Amplification was performed using the 424 vectors as a template. For the K94R mutation, the vectors containing the sequence DTAVYYCAK (VH3-23) were amplified with a PCR primer that changed the K to a R and added 5’ tail for homologous recombination with the VH3-48, VH3-33, VH-30, and VH3-7. The “T” base in 3-48 does not change the amino acid encoded and thus the same primer with a T::C mismatch 20 still allows homologous recombination into the 3-48 chassis.

Furthermore, the amplification products from the 424 vectors (produced as described above) containing the DTAVYYCAR sequence can be homologously recombined into the VH3-23 (CAR) vector, changing R to K in this framework and thus further increasing the diversity of CDRH3 presentation in this chassis. 25 240 294

VH3-48 (240) TCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCGGTGTACTACTGCGCCAGA

30 VH3-33/30(240) TCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGTGTACTACTGCGCCAGA

VH3-7 (240) TCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGTGTACTACTGCGCCAGA

VH3-23 (240) TCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGTGTACTACTGCGCCAAG 35

Example 11: VK Library Construction

This example describes the construction of a VK library of the invention. The - 171 - exemplary VK library described herein corresponds to the VKCDR3 library of about 105 complexity, described in Example 6.2. As described in Example 6, and throughout the application, other VK libraries are within the scope of the invention, as are νλ libraries. 2015201796 09 Apr 2015

Ten VK chassis were synthesized (Table 11), which did not contain VKCDR3, 5 but instead had two Sfil restriction sites in the place of VKCDR3, as for the heavy chain vectors. The kappa constant region followed the Sfil restriction sites. Figure 8 shows a schematic structure of a light chain vector, prior to recombination with a CDRL3.

Ten VKCDR oligonucleotide libraries were then synthesized, as described in Example 6.2, using degenerate oligonucleotides (Table 33). The oligonucleotides were 10 then PCR amplified, as separate pools, to make them double stranded and to add additional nucleotides required for efficient homologous recombination with the gapped (by Sfil) vector containing the VK chassis and constant region sequences. The VKCDR3 pools in this embodiment of the invention represented lengths 8, 9, and 10 amino acids, which were mixed post-PCR at a ratio 1:8:1. The pools were then cloned 15 into the respective Sfil gapped VK chassis via homologous recombination, as described for the CDRH3 regions, set forth above. A schematic diagram of a CDRL3 integrated into a light chain vector and the accompanying sequence are provided in Figure 9. A kappa light chain library pool was then produced, based on the approximate representation of the VK family members found in the circulating pool of B cells. The 20 10 kappa variable regions used and the relative frequency in the final library pool are shown in Table 44.

Table 44. Occurrence of VK Chassis in Data Sets Used to Design Library, Expected (Designed) Library, and Actual (Observed) Library Chassis Relative Occurrence in Data Sets (1) Expected (2) Observed (3) VK1-5 8.6 7.1 5.8 VK1-12 4.0 3.6 3.5 VK1-27 3.3 3.6 8.1 VK1-33 5.3 7.1 3.5 VK1-39 18.5 21.4 17.4 VK2-28 7.7 7.1 5.8 VK3-11 10.9 10.7 20.9 VK3-15 6.6 7.1 4.7 VK3-20 24.5 21.4 18.6 VK4-1 10.4 10.7 11.6 25 - 172- (1) As indicated in Example 3, these 10 chassis account for about 80% of the occurrences in the entire data set of VK sequences examined. 2015201796 09 Apr 2015 (2) Rounded off ratios from the data in column 2, then normalized for actual experimental set up. The relative rounded ratios are 6 for VK1-39 and VK3-20, 3 5 for VK3-11 and VK4-1, 2 for VK-15, VK1-33, VK2-28 and VK3-15, and 1 for VK1-12 and VK1-27. (3) Chassis usage in set of 86 sequences obtained from library; see also Figure 22. 10 Example 12: Characterization of Exemplary Libraries

This example shows the characteristics of exemplary libraries of the invention, constructed according to the methods described herein.

Example 12.1. Characterization of the Heavy Chains 15 To characterize the product of the split pool synthesis, ten of the 424 vectors containing the [Tail]-[N1]-[DH]-[N2]-[H3-JH] product were selected at random and transformed into E. coli. The split pool product had a theoretical diversity of about 1.1 x 106 (i.e., 278 x 141 x 28). Ninety-six colonies were selected from the transformation and forward and reverse sequences were generated for each clone. Of the 96 sequencing 20 reactions, 90 yielded sequences from which the CDRH3 region could be identified, and about 70% of these sequences matched a designed sequence in the libraiy. The length distribution of the sequenced CDRH3 segments from the ten vectors, as compared to the theoretical distribution (based on design), is provided in Figure 10. The length distribution of the individual DH, N2, and H3-JH segments obtained from the ten 25 vectors are shown in Figures 11-13.

Once the length distribution of the CDRH3 components of the library that were contained in the vector matched design were verified, the CDRH3 domains and heavy chain family representation in yeast that had been transformed according to the process described in Example 10.4 were characterized. Over 500 single-pass sequences were 30 obtained. Of these, 531 yielded enough sequence information to identify the heavy chain chassis and 291 yielded enough sequence information to characterize the CDRH3. These CDRH3 domains have been integrated with the heavy chain chassis and constant region, according to the homologous recombination processes described herein. The length distribution of the CDRH3 domains from 291 sequences, compared to the 35 theoretical length distribution, is shown in Figure 14. The mean theoretical length was 14.4 ± 4 amino acids, while the average observed length was 14.3 ± 3 amino acids. The - 173 - observed length of each portion of the CDRH3, as compared to theoretical, is presented in Figures 15-18. Figure 19 depicts the familial origin of the JH segments identified in the 291 sequences, and Figure 20 shows the representation of 16 of the chassis of the library. The VH3-15 chassis was not represented amongst these sequences. This was 5 corrected later by introducing yeast transformants containing the VH3-15 chassis, with CDRH3 diversity, into the library at the desired composition. 2015201796 09 Apr 2015

Example 12.2. Characterization of the Light Chains

The length distribution of the CDRL3 components, from the VKCDR3 library 10 described in Example 6.2, were determined after yeast transformation via the methods described in Example 10.4. A comparison of the CDRL3 length from 86 sequences of the library to the human sequences and designed sequences is provided in Figure 21. Figure 22 shows the representation of the light chain chassis from amongst the 86 sequences selected from the library. About 91% of the CDRL3 sequences were exact 15 matches to the design, and about 9% differed by a single amino acid.

Example 13: Characterization of the Composition of the Desisned CDRH3 Libraries

This example presents data on the composition of the CDRH3 domains of exemplary libraries, and a comparison to other libraries of the art. More specifically, 20 this example presents an analysis of the occurrence of the 400 possible amino acid pairs (20 amino acids x 20 amino acids) occurring in the CDRH3 domains of the libraries. The prevalence of these pairs is computed by examination of the nearest neighbor (i -i+1; designated IP1), next nearest neighbor (i - i+2; designated IP2), and next-next nearest neighbor (i - i+3; designated IP3) of the i residue in CDRH3. Libraries 25 previously known in the art (e.g., Knappik et al, J. Mol. Biol., 2000, 296: 57; Sidhu et al, J. Mol. Biol., 2004, 338: 299; and Lee et al., J. Mol. Biol. 2004, 340: 1073, each of which is incorporated by reference in its entirety) have only considered the occurrence of the 20 amino acids at individual positions within CDRH3, while maintaining the same composition across the center of CDRH3, and not the pair-wise occurrences 30 considered herein. In fact, according to Sidhu et al. (J. Mol. Biol., 2004, 338: 299, incorporated by reference in its entirety), “[i]n CDR-H3, there was some bias towards certain residue types, but all 20 natural amino acid residues occurred to a significant extent, and there was very little position-specific bias within the central portion of the - 174- loop”. Thus, the present invention represents the first recognition that, surprisingly, a position-specific bias does exist within the central portion of the CDRH3 loop, when the occurrences of amino acid pairs recited above are considered. This example shows that the libraries described herein more faithfully reproduce the occurrence of these pairs as 5 found in human sequences, in comparison to other libraries of the art. The composition of the libraries described herein may thus be considered more “human” than other libraries of the art. 2015201796 09 Apr 2015

To examine the pair-wise composition of CDRH3 domains, a portion of CDRH3 beginning at position 95 was chosen. For the purposes of comparison with data 10 presented in Knappik et al. and Lee et al., the last five residues in each of the analyzed CDRH3s were ignored. Thus, for the purposes of this analysis, both members of the pair i - i + X (X=l to 3) must fall within the region starting at position 95 and ending at (but including) the sixth residue from the C-terminus of the CDRH3. The analyzed portion is termed the “central loop” (see Definitions). 15 To estimate pair distributions in representative libraries of the invention, a sampling approach was used. A number of sequences were generated by choosing randomly and, in turn, one of the 424 tail plus N1 segments, one of the 278 DH segments, one of the 141 N2 segments and one of the 28 JH segments (the latter truncated to include only the 95 to 102 Kabat CDRH3). The process was repeated 20 10,000 times to generate a sample of 10,000 sequences. By choosing a different seed for the random number generation, an independent sample of another 10,000 sequences was also generated and the results for pair distributions were observed to be nearly the same. For the calculations presented herein, a third and much larger sample of 50,000 sequences was used. A similar approach was used for the alternative library 25 embodiment (Nl-141), whereby the first segment was selected from 1068 tail+N 1 segments (resulting after eliminating redundant sequences from 2 times 4 times 141 or 1128 possible combinations).

The pair-wise composition of Knappik et al. was determined based on the percent occurrences presented in Figure 7a of Knappik et al. (p.71). The relevant data 30 are reproduced below, in Table 45.

Table 45. Composition of CDRH3 positions 95-100s (corresponding to positions 95-99B of the libraries of the current invention) of CDRH3 of Knappik et al. (from Figure - 175 - 2015201796 09 Apr 2015 7a of Knappik et al.) Amino Acid Planned (%) Found (%) A 4.1 3.0 C 1.0 1.0 D 4.1 4.2 E 4.1 2.3 F 4.1 4.9 G 15.0 10.8 H 4.1 4.6 1 4.1 4.5 K 4.1 2.9 L 4.1 6.6 M 4.1 3.3 N 4.1 4.5 P 4.1 4.8 Q 4.1 2.9 R 4.1 4.1 S 4.1 5.6 T 4.1 4.5 V 4.1 3.7 w 4.1 2.0 Y 15.0 19.8

The pair-wise composition of Lee et al. was determined based on the libraries depicted in Table 5 of Lee et al., where the positions corresponding to those CDRH3 5 regions analyzed from the current invention and from Knappik et al. are composed of an “XYZ” codon in Lee et al. The XYZ codon of Lee et al. is a degenerate codon with the following base compositions: position 1 (X): 19% A, 17% C, 38% G, and 26% T; position 2 (Y): 34% A, 18% C, 31% G, and 17% T; and 10 position 3 (Z): 24% G and 76% T.

When the approximately 2% of codons encoding stop codons are excluded (these do not occur in functionally expressed human CDRH3 sequences), and the percentages are renormalized to 100%, the following amino acid representation can be deduced from the composition of the XYZ codon of Lee et al. (Table 46). 15

Table 46. Composition of CDRH3 of Lee et al., Based on the Composition of the Degenerate XYZ Codon. - 176-

Type Percent Type Percent A 6.99% M 0.79% C 6.26% N 5.02% D 10.03% P 3.13% E 3.17% Q 1.42% F 3.43% R 6.83% G 12.04% S 9.35% H 4.49% T 3.49% 1 2.51% V 6.60% K 1.58% w 1.98% L 4.04% Y 6.86%

The occurrences of each of the 400 amino acid pairs, in each of the IP1, IP2, and IP3 configurations, can be computed for Knappik et al. and Lee et al. by multiplying 5 together the individual amino acid compositions. For example, for Knappik et al, the occurrence of YS pairs in the library is calculated by multiplying 15% by 4.1%, to yield 6.1%; note that the occurrence of SY pairs would be the same. Similarly, for the XYZ codon-based libraries of Lee et al., the occurrence of YS pairs would be 6.86% (Y) multiplied by 9.35% (S), to give 6.4%; the same, again, for SY. 2015201796 09 Apr 2015 10 For the human CDRH3 sequences, the calculation is performed by ignoring the last five amino acids in the Kabat definition. By ignoring the C-terminal 5 amino acids of the human CDRH3, these sequences may be compared to those of Lee et al, based on the XYZ codons. While Lee et al. also present libraries with “NNK” and “NNS” codons, the pair-wise compositions of these libraries are even further away from human 15 CDRH3 pair-wise composition. The XYZ codon was designed by Lee et al. to replicate, to some extent, the individual amino acid type biases observed in CDRH3.

An identical approach was used for the libraries of the invention, after using the methods described above to produce sample sequences. While it is possible to perform these calculations with all sequences in the library, independent random samples of 20 10,000 to 20,000 members gave indistinguishable results. The numbers reported herein were thus generated from samples of 50,000 members.

Three tables were generated for IP1, IP2 and IP3, respectively (Tables 47, 48, and 49). Out of the 400 pairs, a selection from amongst the 20 most frequently occurring is included in the tables. The sample of about 1,000 human sequences (Lee et 25 al., 2006) is denoted as “Preimmune,” a sample of about 2,500 sequences (Jackson et al., 2007) is denoted as “Humabs,” and the more affinity matured subset of the latter, - 177- which excludes all of the Preimmune set, is denoted as “Matured.” Synthetic libraries in the art are denoted as HuCAL (Knappik, et al., 2000) and XYZ (Lee et al., e 2004). Two representative libraries of the invention are included: LUA-59 includes 59 N1 segments, 278 DH segments, 141 N2 segments, and 28 H3-JH segments (see Examples, 5 above). LUA-141 includes 141 N1 segments, 278 DH segments, 141 N2 segments, and 28 H3-JH segments (see Examples, above). Redundancies created by combination of the N1 and tail sequences were removed from the dataset in each respective library. In certain embodiments, the invention may be defined based on the percent occurrence of any of the 400 amino acid pairs, particularly those in Tables 47-49. In certain 10 embodiments, the invention may be defined based on at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more of these pairs. In certain embodiments of the invention, the percent occurrence of certain pairs of amino acids may fall within ranges indicated by “LUA-“ (lower boundary) and “LUA+” (higher boundary), in the following tables. In some embodiments of the invention, the lower boundary for the 15 percent occurrence of any amino acid pairs may be about 0.1, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, and 5. In some 2015201796 09 Apr 2015 embodiments of the invention, the higher boundary for the percent occurrence of any amino acid pairs may be about 0.1, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7, 7.25, 7.5, 20 7.75, and 8. According to the present invention, any of the lower boundaries recited may be combined with any of the higher boundaries recited, to establish ranges, and vice-versa. -178- 2015201796 09 Apr 2015

Table 47. Percent Occurrence of i - i+1 (IP1) Amino Acid Pairs in Human Sequences, Exemplary Libraries of the Invention, and the Libraries

Pairs Preimmune Humabs Matured LUA- 59 LUA- 141 HuCAL XYZ LUA- LUA+ Range HuCAL XYZ YY 5.87 4.44 3.27 5.83 5.93 2.25 0.47 2.50 6.50 4.00 0 0 SG 3.54 3.41 3.26 3.90 3.72 0.61 1.13 2.50 4.50 2.00 0 0 SS 3.35 2.65 2.26 2.82 3.08 0.16 0.88 2.00 4.00 2.00 0 0 GS 2.59 2.37 2.20 3.82 3.52 0.61 1.13 1.50 4.00 2.50 0 0 GY 2.55 2.34 2.12 3.15 2.56 2.25 0.83 2.00 3.50 1.50 1 0 GG 2.19 2.28 2.41 6.78 3.51 2.25 1.45 2.00 7.00 5.00 1 0 YS 1.45 1.30 1.23 1.40 1.52 0.61 0.64 0.75 2.00 1.25 0 0 YG 1.35 1.21 1.10 1.64 1.69 2.25 0.83 0.75 2.00 1.25 0 1 SY 1.31 1.07 0.90 1.65 1.77 0.61 0.64 0.75 2.00 1.25 0 0 YD 1.67 1.40 1.17 0.88 0.90 0.61 0.69 0.75 2.25 1.50 0 0 DS 1.53 1.31 1.16 1.20 1.46 0.16 0.94 0.75 2.00 1.25 0 1 DY 1.40 1.23 1.11 0.34 0.48 0.61 0.69 0.25 2.00 1.75 1 1 VV 1.37 0.94 0.64 2.30 2.30 0.16 0.44 0.50 2.50 2.00 0 0 GD 1.20 1.21 1.25 0.49 0.44 0.61 1.21 0.25 1.75 1.50 1 1 AA 1.16 0.93 0.75 1.27 1.46 0.16 0.49 0.60 1.50 0.90 0 0 RG 1.08 1.26 1.38 1.69 1.38 0.61 0.82 1.00 2.00 1.00 0 0 VA 0.91 0.66 0.46 0.36 0.35 0.16 0.46 0.25 1.00 0.75 0 1 GV 0.84 0.89 0.95 2.87 2.16 0.61 0.79 0.80 3.00 2.20 0 0 CS 0.82 0.55 0.38 0.79 0.80 0.04 0.59 0.50 1.00 0.50 0 1 GR 0.74 0.90 1.00 1.01 0.79 0.61 0.82 0.70 1.25 0.55 0 1 5 The pairs in bold comprise about 19% to about 24% of occurrences (among the possible 400 pairs) for the Preimmune (Lee, et al., 2006), Humabs (Jackson, et al„ 2007) and matured (Jackson minus Lee) sets. They account for about 27% to about 31% of the occurrences in the LUA libraries, but only about 12% in the HuCAL library and about 8% in the “XYZ” library. This is a reflection of the fact that pair-wise biases do exist in the human and LUA libraries, but not in the others. The last 2 columns indicate whether the corresponding pair-wise compositions fall within the LUA- and LUA+ boundaries: 0 if outside, 1 if within.

Attorney Docket No.: ADS-011.25 - 179- 2015201796 09 Apr 2015

Table 48. Percent Occurrence of i - i+2 (IP2) Amino Acid Pairs in Human Sequences, Exemplary Libraries of the Invention, and the Libraries of Knappik et al. and Lee et al.

Pairs Preimmune Humabs Matured LUA- 59 LUA- 141 HuCAL XYZ LUA- LUA+ Range HuCAL XYZ YY 3.57 2.59 1.78 2.99 3.11 2.25 0.47 2.5 4.5 2 0 0 GY 3.34 2.91 2.56 4.96 3.78 2.25 0.83 2.5 5.5 3 0 0 SY 2.94 2.41 2.01 3.03 3.42 0.61 0.64 2 4 2 0 0 YS 2.88 2.34 1.95 3.24 3.32 0.61 0.64 1.75 3.75 2 0 0 SG 2.60 2.29 2.05 2.84 2.96 0.61 1.13 2 3.5 1.5 0 0 SS 2.27 2.01 1.84 2.30 2.50 0.16 0.88 1.5 3 1.5 0 0 GS 2.16 2.12 2.10 2.96 2.32 0.61 1.13 1.5 3 1.5 0 0 GG 1.92 2.25 2.44 6.23 3.68 2.25 1.45 1.5 7 5.5 1 0 YG 1.17 1.14 1.15 1.39 1.47 2.25 0.83 1 2 1 0 0 DS 2.03 1.67 1.40 1.21 1.48 0.16 0.94 1 2.5 1.5 0 0 YD 1.71 1.39 1.11 0.89 0.92 0.61 0.69 0.75 1.75 1 0 0 VG 1.35 1.17 1.01 1.75 1.54 0.61 0.79 1 2 1 0 0 DY 1.06 1.02 0.99 0.23 0.40 0.61 0.69 0.2 1.2 1 1 1 WG 1.06 0.76 0.53 0.85 0.91 0.61 0.24 0.75 1.25 0.5 0 0 RY 0.98 1.00 0.96 0.70 0.91 0.61 0.47 0.6 1 0.4 1 0 GC 0.97 0.75 0.64 0.94 0.81 0.15 0.75 0.5 1 0.5 0 1 DG 0.95 1.05 1.08 1.78 1.05 0.61 1.21 0.75 2 1.25 0 1 GD 0.94 0.88 0.86 0.47 0.36 0.61 1.21 0.25 1 0.75 1 0 VV 0.94 0.59 0.35 0.95 0.90 0.16 0.44 0.5 1 0.5 0 0 AA 0.90 0.73 0.59 0.72 0.74 0.16 0.49 0.5 1 0.5 0 0 5 The pairs in bold comprise about 18% to about 23% of occurrences (among the possible 400 pairs) for the Preimmune (Lee, et al., 2006), Humabs (Jackson, et al., 2007) and matured (Jackson minus Lee) sets. They account for about 27% to about 30% of the occurrences in the LUA libraries, but only about 12% in the HuCAL library and about 8% in the “XYZ” library. Because of the nature of the construction of the central loops in the HuCAL and XYZ libraries, these numbers are the same for the IP1, IP2, and IP3 pairs. The last 2 columns indicate whether the corresponding pair-wise compositions fall within the LUA- and LUA+ boundaries: 0 if outside, 1 if within.

Attorney Docket No.: ADS-011.25 10 - 180- 2015201796 09 Apr 2015

Table 49. Percent Occurrence of i - i+3 (IP3) Amino Acid Pairs in Human Sequences, Exemplary Libraries of the Invention, and the Libraries of Knappik et al. and Lee et al.

Pairs Preimmune Humabs Matured LUA- 59 LUA- 141 HuCAL XYZ LUA- LUA+ Range HuCAL XYZ GY 3.55 2.85 2.32 5.80 4.42 2.25 0.83 2.5 6.5 4 0 0 SY 3.38 3.01 2.67 3.78 4.21 0.61 0.64 1 5 4 0 0 YS 3.18 2.56 2.05 3.20 3.33 0.61 0.64 2 4 2 0 0 SS 2.26 1.74 1.37 1.81 2.18 0.16 0.88 1 3 2 0 0 GS 2.23 2.13 2.00 4.60 3.33 0.61 1.13 2 5 3 0 0 YG 2.14 1.65 1.35 2.69 2.79 2.25 0.83 1.5 3 1.5 1 0 YY 1.86 1.48 1.12 1.18 1.27 2.25 0.47 0.75 2 1.25 0 0 GG 1.60 1.87 2.11 4.73 2.84 2.25 1.45 1.5 5 3.5 1 0 SG 0.90 1.04 1.12 0.93 1.25 0.61 1.13 0.75 1.5 0.75 0 1 DG 2.01 1.94 1.84 2.51 2.03 0.61 1.21 1.5 3 1.5 0 0 DS 1.48 1.31 1.22 0.41 0.55 0.16 0.94 0.25 1.5 1.25 0 1 VA 1.18 0.83 0.55 1.48 1.46 0.16 0.46 0.5 2 1.5 0 0 AG 1.13 1.09 1.03 0.97 1.04 0.61 0.84 0.9 2 1.1 0 0 TY 1.05 0.90 0.76 1.01 1.16 0.61 0.24 0.75 1.75 1 0 0 PY 1.02 0.88 0.79 1.23 0.86 0.61 0.21 0.75 1.75 1 0 0 RS 1.02 0.88 0.77 0.38 0.55 0.16 0.64 0.25 1.25 1 0 1 RY 1.02 1.12 1.14 0.68 0.88 0.61 0.47 0.65 1.25 0.6 0 0 LY 1.01 0.88 0.75 0.69 0.76 0.61 0.28 0.65 1.25 0.6 0 0 DY 0.93 0.84 0.77 0.72 0.95 0.61 0.69 0.7 1.3 0.6 0 0 GC 0.90 0.62 0.48 0.86 0.68 0.15 0.75 0.5 1 0.5 0 1 5 The pairs in bold make up about 16 to about 21% of the occurrences (among the possible 400 pairs) for the Preimmune (Lee, et al., 2006), Humabs (Jackson, et al., 2007) and matured (Jackson minus Lee) sets. They account for 26 to 29% of the occurrences in the LUA libraris, but only about 12% in the HuCAL library and about 8% for the “XYZ” library. Because of the nature of the construction of the central loops in the HuCAL and XYZ libraries, these numbers are the same for the IP1, IP2, and IP3 pairs. The last 2 columns indicate whether the corresponding pair-wise compositions fall within the LUA- and LUA+ boundaries: 0 if outside, 1 if within.

Attorney Docket No.: ADS-011.25 10 - 181 -

The analysis provided in this example demonstrates that the composition of the libraries of the present invention more closely mimics the composition of human sequences than other libraries known in the art. Synthetic libraries of the art do not intrinsically reproduce the composition of the “central loop” portion actual human 5 CDRH3 sequences at the level of pair percentages. The libraries of the invention have a more complex pair-wise composition that closely reproduces that observed in actual human CDRH3 sequences. The exact degree of this reproduction versus a target set of actual human CDRH3 sequences may be optimized, for example, by varying the compositions of the segments used to design the CDRH3 libraries. Moreover, it is also 10 possible to utilize these metrics to computationally design libraries that exactly mimic the pair-wise compositional prevalence found in human sequences. 2015201796 09 Apr 2015

Example 14: Information Content of Exemplary Libraries

One way to quantify the observation that certain libraries, or collection of 15 sequences, may be intrinsically more complex or “less random” than others is to apply information theory (Shannon, Bell Sys. Tech. J., 1984, 27: 379; Martin et al., Bioinformatics, 2005, 21: 4116; Weiss et al., J. Theor. Biol., 2000, 206: 379, each incorporated by reference in its entirety). For example, a metric can be devised to quantify the fact that a position with a fixed amino acid represents less “randomness” 20 than a position where all 20 amino acids may occur with equal probability. Intermediate situations should lead, in turn, to intermediate values of such a metric. According to information theory this metric can be represented by the formula:

Here,/' is the normalized frequency of occurrence of i, which may be an amino acid type 25 (in which case N would be equal to 20). When all f are zero except for one, the value of I is zero. In any other case the value of I would be smaller, i.e., negative, and the lowest value is achieved when all f values are the same and equal to N. For the amino acid case, N is 20, and the resulting value of I would be -4.322. Because I is defined with base 2 logarithms, the units of I are bits. 30 The I value for the HuCAL and XYZ libraries at the single position level may be derived from Tables 45 and 46, respectively, and are equal to -4.08 and -4.06. The corresponding single residue frequency occurrences in the non-limiting exemplary -182- libraries of the invention and the sets of human sequences previously introduced, taken within the “central loop” as defined above, are provided in Table 50.

Table 50. Amino Acid Type Frequencies in Central Loop

Type Preimmune Humabs Matured LUA-59 LUA-141 A 5.46 5.51 5.39 5.71 6.06 C 1.88 1.46 1.22 1.33 1.34 D 7.70 7.51 7.38 4.76 5.23 E 2.40 2.90 3.28 3.99 4.68 F 2.29 2.60 2.81 1.76 2.17 G 14.86 15.42 15.82 24.90 18.85 H 1.46 1.79 2.01 0.20 0.67 1 3.71 3.26 2.99 3.99 4.34 K 1.06 1.27 1.44 0.21 0.67 L 4.48 4.84 5.16 4.12 4.54 M 1.18 1.03 0.93 0.94 1.03 N 1.81 2.43 2.84 0.41 0.65 P 4.12 4.10 4.13 5.68 3.96 Q 1.60 1.77 1.95 0.21 0.68 R 5.05 5.90 6.41 3.35 4.11 S 12.61 11.83 11.37 11.18 12.77 T 4.59 5.11 5.47 4.36 4.95 V 6.21 5.55 5.12 8.13 7.67 w 2.79 2.91 3.07 1.57 1.98 Y 14.74 12.81 11.24 13.20 13.63 2015201796 09 Apr 2015

The information content of these sets, computed by the formula given above, would then be -3.88, -3.93, -3.96, -3.56, and -3.75, for the preimmune, human, matured, LUA-59 and LUA-141 sets, respectively. As the frequencies deviate more from completely uniform (5% for each of the 20), then numbers tend to be larger, or less negative. 10 The identical approach can be used to analyze pair compositions, or frequencies, by calculating the sum in the formula above over the 20x20 or 400 values of the frequencies for each of the pairs. It can be shown that any pair frequency made up of the simple product of two singleton frequency sets is equal to the sum of the individual singleton I values. If the two singleton frequency sets are the same or approximately so, 15 this means that I (independent pairs) = 2 * I (singles). It is thus possible to define a special case of the mutual information, MI, for a general set of pair frequencies as MI (pair) = I(pair) - 2 * I (singles) to measure the amount of information gained by the structure of the pair frequencies themselves (compare to the standard definitions in Martin et al., 2005, for example, after considering that I (X) = -H(X) in their notation). 20 When there is no such structure, the value of MI is simply zero. -183-

Values of MI computed from the pair distributions discussed above (over the entire set of 400 values) are given in Table 51.

Table 51. Mutual Information Within Central Loop of CDRH3

Library or Set i - i+1 i - i+2 i - i+3 Preimmune 0.226 0.192 0.163 Humabs 0.153 0.128 0.111 Matured 0.124 0.107 0.100 LUA-59 0.422 0.327 0.278 LUA-141 0.376 0.305 0.277 Hu CAL 0.000 0.000 0.000 XYZ 0.000 0.000 0.000 2015201796 09 Apr 2015 5

It is notable that the MI values decrease within sets of human sequences as those sequences undergo further somatic mutation, a process that over many independent sequences is essentially random. It is also worth noting that the MI values decrease as the pairs being considered sit further and further apart, and this is the case for both sets 10 of human sequences, and exemplary libraries of the invention. In both cases, as the two amino acids in a pair become further separated the odds of their straddling an actual segment (V, D, J plus V-D or D-J insertions) increase, and their pair frequencies become closer to a simple product of singleton frequencies. - 184- 2015201796 09 Apr 2015

Attorney Docket No.: ADS-011.25

Table 52 contains sequence information on certain immunoglobulin gene segments cited in the application. These sequences are nonlimiting, and it is recognized that allelic variants exist and encompassed by the present invention. Accordingly, the methods present herein can be utilized with mutants of these sequences.

Table 52. Sequence Information for Certain Immunoglobulin Gene Segments Cited Herein

SEQ ID NO: Sequence Peptide or Nucleotide Sequence Observations IGHV1-3 Q V Q L VQS G A E V K KP G AS V KVS C KAS G YT FT S YAM H WVRQ APGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSAST AYMELSSLRSEDTAVYYCAR IGHV1-8 v1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQ ATGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRNTSIS TAYMELSSLRSEDTAVYYCAR IGHV1-8 v2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQ ATGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRDTSIS TAYMELSSLRSEDTAVYYCAR N to D mutation avoids NTS potential glycosylation site in the original germline sequence (v1 above). XTS, where X is not N, and NTZ, where Z is not S or T are also options. NPS is yet another option that is much less likely to be N-linked glycosylated. IGHV1-24 QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQ APGKGLEWMGGFDPEDGETIYAQKFQGRVTMTEDTSTDT AYMELSSLRSEDTAVYYCAT IGHV1-45 QMQLVQSGAEVKKTGSSVKVSCKASGYTFTYRYLHWVRQ APGQALEWMGWITPFNGNTNYAQKFQDRVTITRDRSMST AYMELSSLRSEDTAMYYCAR - 185- 2015201796 09 Apr 2015

Attorney Docket No.: ADS-011.25

IGHV1-58 QMQLVQSGPEVKKPGTSVKVSCKASGFTFTSSAVQWVRQ ARGQRLEWIGWIWGSGNTNYAQKFQERVTITRDMSTSTA YMELSSLRSEDTAVYYCAA IGHV2-5 QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQ PPGKALEWLALIYWDDDKRYSPSLKSRLTITKDTSKNQVVL TMTNMD PVDTATYYCAH R IGHV2-26 QVTLKESGPVLVKPTETLTLTCTVSGFSLSNARMGVSWIRQ PPGKALEWLAHIFSNDEKSYSTSLKSRLTISKDTSKSQWLT MTNMDPVDTATYYCARI IGHV2-70 v1 RVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMCVSWIRQ PPGKALEWLARIDWDDDKYYSTSLKTRLTISKDTSKNQVVL TMTNMDPVDTATYYCARI IGHV2-70 v2 RVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQ PPGKALEWLARIDWDDDKYYSTSLKTRLTISKDTSKNQWL TMTNMDPVDTATYYCARI C to G mutation avoids unpaired Cys in v1 above. G was chosen by analogy to other germline sequences, but other amino acid types, R, S, T, as non-limiting examples, are possible. IGHV3-9 EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQ APGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSL YLQMNSLRAEDTALYYCAKD IGHV3-11 QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQ APGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAR IGHV3-13 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMHWVRQ ATGKGLEWVSAIGTAGDTYYPGSVKGRFTISRENAKNSLYL QMNSLRAGDTAVYYCAR IGHV3-20 EVQLVESGGGWRPGGSLRLSCAASGFTFDDYGMSWVR QAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKN SLYLQMNSLRAEDTALYHCAR IGHV3-21 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQ APGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCAR -186- 2015201796 09 Apr 2015

Attorney Docket No.: ADS-011.25 IGHV3-43 EVQLVESGGWVQPGGSLRLSCAASGFTFDDYTMHWVRQ APGKGLEWVSLISWDGGSTYYADSVKGRFTISRDNSKNSL YLQMNSLRTEDTALYYCAKD IGHV3-49 EVQLVESGGGLVQPGRSLRLSCTASGFTFGDYAMSWVRQ APGKGLEWVGFIRSKAYGGTTEYAASVKGRFTISRDDSKSI AYLQMNSLKTEDTAVYYCTR IGHV3-53 EVQLVESGGGLIQPGGSLRLSCAASGFTVSSNYMSWVRQ APGKGLEWVSVIYSGGSTYYADSVKGRFTISRDNSKNTLYL QMNSLRAEDTAVYYCAR IGHV3-64 EVQLVESGGGLVQPGGSLRLSCSASGFTFSSYAMHWVRQ APGKGLEYVSAISSNGGSTYYADSVKGRFTISRDNSKNTLY LQMSSLRAEDTAVYYCVK IGHV3-66 EVQLVESGGGLVQPGGSLRLSCAASGFTVSSNYMSWVRQ APGKGLEWVSVIYSGGSTYYADSVKGRFTISRDNSKNTLYL QMNSLRAEDTAVYYCAR IGHV3-72 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMDWVRQ APGKGLEWVGRTRNKANSYTTEYAASVKGRFTISRDDSKN SLYLQMNSLKTEDTAVYYCAR IGHV3-73 EVQLVESGGGLVQPGGSLKLSCAASGFTFSGSAMHWVRQ ASGKGLEWVGRIRSKANSYATAYAASVKGRFTISRDDSKN TAYLQMNSLKTEDTAVYYCTR IGHV3-74 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMHWVR QAPGKGLVWVSRINSDGSSTSYADSVKGRFTISRDNAKNT LYLQMNSLRAEDTAVYYCAR IGHV4-4v1 QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVR QPPGKGLEWIGEIYHSGSTNYNPSLKSRVTISVDKSKNQFS LKLSSVTAADTAVYYCAR Contains CDRH1 with size 6 (Kabat definition); canonical structure H1-2. Sequence corresponds to allele *02 of IGHV4-4. IGHV4-4v2 QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQP AGKGLEWIGRIYTSGSTNYNPSLKSRVTMSVDTSKNQFSL KLSSVTAADTAVYYCAR Contains CDRH1 with size 5 (Kabat definition); canonical structure H1-1. Sequence corresponds to allele *07 of - 187- 2015201796 09 Apr 2015

Attorney Docket No.: ADS-011.25

IGHV4-4 IGHV4-28 QVQLQESGPGLVKPSDTLSLTCAVSGYSISSSNWWGWIR QPPGKGLEWIGYIYYSGSTYYNPSLKSRVTMSVDTSKNQF SLKLSSVTAVDTAVYYCAR IGHV6-1 QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIR QSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKN QFSLQLNSVTPEDTAVYYCAR IGHV7-4-1 QVQLVQSGSELKKPGASVKVSCKASGYTFTSYAMNWVRQ APGQGLEWMGWINTNTGNPTYAQGFTGRFVFSLDTSVST AYLQISSLKAEDTAVYYCAR IGKV1-06 AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKP GKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCLQDYNYP IGKV1-08 v1 AIRMTQSPSSFSASTGDRVTITCRASQGISSYLAWYQQKP GKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISCLQSE DFATYYCQQYYSYP IGKV1-08 v2 AIRMTQSPSSFSASTGDRVTITCRASQGISSYLAWYQQKP GKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQSE DFATYYCQQYYSYP C to S mutation avoids unpaired Cys. in v1 above. S was chosen by analogy to other germline sequences, but amino acid types, N, R, S, as non-limiting examples, are also possible IGKV1-09 DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPG KAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDF ATYYCQQLNSYP IGKV1-13 AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPG KAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDF ATYYCQQFNSYP IGKV1-16 DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWFQQKP GKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQYNSYP IGKV1-17 DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKP - 188- 2015201796 09 Apr 2015

Attorney Docket No.: ADS-011.25

GKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPE DFATYYCLQHNSYP IGKV1-37 v1 DIQLTQSPSSLSASVGDRVTITCRVSQGISSYLNWYRQKPG KVPKLLIYSASNLQSGVPSRFSGSGSGTDFTLTISSLQPED VATYYGQRTYNAP IGKV1-37 v2 DIQLTQSPSSLSASVGDRVTITCRVSQGISSYLNWYRQKPG KVPKLLIYSASNLQSGVPSRFSGSGSGTDFTLTISSLQPED VATYY C Q RTY NAP Restores conserved Cys, missing in v1 above, just prior to CDRL3. IGKV1D-16 DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKP EKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQYNSYP IGKV1D-17 NIQMTQSPSAMSASVGDRVTITCRARQGISNYLAWFQQKP GKVPKHLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPE DFATYYCLQHNSYP IGKV1D-43 AIRMTQSPFSLSASVGDRVTITCWASQGISSYLAWYQQKP AKAPKLFIYYASSLQSGVPSRFSGSGSGTDYTLTISSLQPE DFATYYCQQYYSTP IGKV1D-8 v1 VIWMTQSPSLLSASTGDRVTISCRMSQGISSYLAWYQQKP GKAPELLIYAASTLQSGVPSRFSGSGSGTDFTLTISCLQSE DFATYYCQQYYSFP IGKV1D-8 v2 VIWMTQSPSLLSASTGDRVTISCRMSQGISSYLAWYQQKP GKAPELLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQSE DFATYYCQQYYSFP C to S mutation avoids unpaired Cys. in v1 above. S was chosen by analogy to other germline sequences, but amino acid types, N, R, S, as non-limiting examples, are also possible IGKV2-24 DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWL QQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISR VEAEDVGVYYCMQATQFP IGKV2-29 DIVMTQTFLSLSVTRQQPASISCKSSQSLLHSDGVTYLYWY LQRPQQSPQLLTYEVSSRFSGVPDRFSGSGSGTDFTLKIS RVEAEDVGVYYCMQGTHLP IGKV2-30 DVVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLNW - 189- 2015201796 09 Apr 2015

Attorney Docket No.: ADS-011.25 FQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKI SRVEAEDVGVYYCMQGTHWP IGKV2-40 DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSDDGNTYLDW YLQKPGQSPQLLIYTLSYRASGVPDRFSGSGSGTDFTLKIS RVEAEDVGVYYCMQRIEFP IGKV2D-26 EIVMTQTPLSLSITPGEQASMSCRSSQSLLHSDGYTYLYWF LQKARPVSTLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISR VEAEDFGVYYCMQDAQD IGKV2D-29 DIVMTQTPLSLSVTPGQPASISCKSSQSLLHSDGKTYLYWY LQKPGQPPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISR VEAEDVGVYYCMQSIQLP IGKV2D-30 DWMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLNW FQQRPGQSPRRLIYKVSNWDSGVPDRFSGSGSGTDFTLKI SRVEAEDVGVYYCMQGTHWP IGKV3D-07 EIVMTQSPATLSLSPGERATLSCRASQSVSSSYLSWYQQK PGQAPRLLIYGASTRATGIPARFSGSGSGTDFTLTISSLQPE DFAVYYCQQDYNLP IGKV3D-11 EIVLTQSPATLSLSPGERATLSCRASQGVSSYLAWYQQKP GQAPRLLIYDASNRATGIPARFSGSGPGTDFTLTISSLEPED FAVYYCQQRSNWH IGKV3D-20 EIVLTQSPATLSLSPGERATLSCGASQSVSSSYLAWYQQK PGLAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPE DFAVYYCQQYGSSP IGKV5-2 v1 ETTLTQSPAFMSATPGDKVNISCKASQDIDDDMNWYQQKP GEAAIFIIQEATTLVPGIPPRFSGSGYGTDFTLTINNIESEDA AYYFCLQHDNFP IGKV5-2 v2 ETTLTQSPAFMSATPGDKVTISCKASQDIDDDMNWYQQKP GEAAIFIIQEATTLVPGIPPRFSGSGYGTDFTLTINNIESEDA AYYFCLQHDNFP N to D mutation avoids NIS potential glycosylation site in v1 above. XIS, where X is not N, and NIZ, where Z is not S or T are also options. NPS is yet another option that is much less likely to be N-linked glycosylated. - 190- 2015201796 09 Apr 2015

Attorney Docket No.: ADS-011.25

IGKV6-21 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSLHWYQQKPD QSPKLLIKYASQSFSGVPSRFSGSGSGTDFTLTINSLEAED AATYYCHQSSSLP IGKV6D-21 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSLHWYQQKPD QSPKLLIKYASQSFSGVPSRFSGSGSGTDFTLTINSLEAED AATYYCHQSSSLP IGKV7-3 DIVLTQSPASLAVSPGQRATITCRASESVSFLGINLIHWYQQ KPGQPPKLLIYQASNKDTGVPARFSGSGSGTDFTLTINPVE ANDTANYYCLQSKNFP IGAV1-36 QSVLTQPPSVSEAPRQRVTISCSGSSSNIGNNAVNWYQQL PGKAPKLLIYYDDLLPSGVSDRFSGSKSGTSASLAISGLQS E D EADYY CAAWD DS LN G IGAV1-47 QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQL PGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRS E D EADYY CAAWD DS LSG IGAV10-54 QAGLTQPPSVSKGLRQTATLTCTGNSNNVGNQGAAWLQQ HQGHPPKLLSYRNNNRPSGISERLSASRSGNTASLTITGLQ PEDEADYYCSAWDSSLSA IGAV2-11 v1 QSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQ QHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNTASLTISGL QAE D EADYY CCSYAGSYTF IGAV2-11 v2 QSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQ QHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNTASLTISGL QAE D EADYY CSSYAGSYTF C to S mutation avoids unpaired Cys in v1 above. S was chosen by analogy to other germline sequences, but other amino acid types, such as Q, G, A, L, as nonlimiting examples, are also possible IGAV2-18 QSALTQPPSVSGSPGQSVTISCTGTSSDVGSYNRVSWYQ QPPGTAPKLMIYEVSNRPSGVPDRFSGSKSGNTASLTISGL QAEDEADYYCSLYTSSSTF IGAV2-23 v1 QSALTQPASVSGSPGQSITISCTGTSSDVGSYNLVSWYQQ - 191 - 2015201796 09 Apr 2015

Attorney Docket No.: ADS-011.25

HPGKAPKLMIYEGSKRPSGVSNRFSGSKSGNTASLTISGL QAE D EADYYCCSYAGSSTL IGAV2-23 v2 QSALTQPASVSGSPGQSITISCTGTSSDVGSYNLVSWYQQ HPGKAPKLMIYEGSKRPSGVSNRFSGSKSGNTASLTISGL QAE D EADYYCSSYAGSSTL C to S mutation avoids unpaired Cys in v1 above. S was chosen by analogy to other germline sequences, but other amino acid types, such as Q, G, A, L, as nonlimiting examples, are also possible IGAV2-8 QSALTQPPSASGSPGQSVTISCTGTSSDVGGYNYVSWYQ QHPGKAPKLMIYEVSKRPSGVPDRFSGSKSGNTASLTVSG LQAEDEADYYCSSYAGSNNF IGAV3-10 SYELTQPPSVSVSPGQTARITCSGDALPKKYAYWYQQKSG QAPVLVIYEDSKRPSGIPERFSGSSSGTMATLTISGAQVED EADYYCYSTDSSGNH IGAV3-12 SYELTQPHSVSVATAQMARITCGGNNIGSKAVHWYQQKP GQDPVLVIYSDSNRPSGIPERFSGSNPGNTTTLTISRIEAGD EADYYCQVWDSSSDH IGAV3-16 SYELTQPPSVSVSLGQMARITCSGEALPKKYAYWYQQKPG QFPVLVIYKDSERPSGIPERFSGSSSGTIVTLTISGVQAEDE ADYYCLSADSSGTY IGAV3-25 SYELMQPPSVSVSPGQTARITCSGDALPKQYAYWYQQKP GQAPVLVIYKDSERPSGIPERFSGSSSGTTVTLTISGVQAE DEADYYCQSADSSGTY IGAV3-27 SYELTQPSSVSVSPGQTARITCSGDVLAKKYARWFQQKPG QAPVLVIYKDSERPSGIPERFSGSSSGTTVTLTISGAQVEDE ADYYCYSAADNN IGAV3-9 SYELTQPLSVSVALGQTARITCGGNNIGSKNVHWYQQKPG QAPVLVIYRDSNRPSGIPERFSGSNSGNTATLTISRAQAGD EADYYCQVWDSSTA IGAV4-3 LPVLTQPPSASALLGASIKLTCTLSSEHSTYTIEWYQQRPG RSPQYIMKVKSDGSHSKGDGIPDRFMGSSSGADRYLTFSN LQSDDEAEYHCGESHTIDGQVG - 192- 2015201796 09 Apr 2015

Attorney Docket No.: ADS-011.25

IGAV4-60 QPVLTQSSSASASLGSSVKLTCTLSSGHSSYIIAWHQQQP GKAPRYLMKLEGSGSYNKGSGVPDRFSGSSSGADRYLTIS NLQLEDEADYYCETWDSNT IGAV5-39 QPVLTQPTSLSASPGASARFTCTLRSGINVGTYRIYWYQQK PGSLPRYLLRYKSDSDKQQGSGVPSRFSGSKDASTNAGLL LISGLQSEDEADYYCAIWYSSTS IGAV7-46 QAWTQEPSLTVSPGGTVTLTCGSSTGAVTSGHYPYWFQ QKPGQAPRTLIYDTSNKHSWTPARFSGSLLGGKAALTLSG AQPEDEAEYYCLLSYSGAR IGAV8-61 QTVVTQEPSFSVSPGGTVTLTCGLSSGSVSTSYYPSWYQ QTPGQAPRTLIYSTNTRSSGVPDRFSGSILGNKAALTITGA QADDESDYYCVLYMGSGI IGAV9-49 QPVLTQPPSASASLGASVTLTCTLSSGYSNYKVDWYQQRP GKGPRFVMRVGTGGIVGSKGDGIPDRFSVLGSGLNRYLTI KNIQEEDESDYHCGADHGSGSNFV IGHD1-1 GGTACAACTGGAACGAC See (1) below. IGHD1-14 GGTATAACCGGAACCAC IGHD1-20 GGTATAACTGGAACGAC IGHD1-7 G GTAT AACT G GAACT AC IGHD2-21 v1 AGCAT ATT GT GGTGGT GATTGCT ATT CC IGHD2-21 v2 AGCAT ATT GT GGTGGT GACTGCT ATT CC Common allelic variant encoding a different amino acid sequence, compared to v1, in 2 of 3 forward reading frames. IGHD2-8 AGGATATTGTACTAATGGTGTATGCTATACC IGHD3-16 GT ATT AT GATT AC GTTT GGGGGAGTTATGCTTATACC IGHD3-9 GT ATT ACG AT ATTTT G ACT G GTT ATT ATAAC IGHD4-23 TGACTACGGTGGTAACTCC IGHD4-4/4-11 T GACTACAGTAACTAC IGHD5-12 GTGGATATAGTGGCTACGATTAC IGHD5-24 GT AGAGAT G GCT ACAATT AC - 193 - 2015201796 09 Apr 2015

Attorney Docket No.: ADS-011.25

IGHD6-25 GGGTATAGCAGCGGCTAC IGHD6-6 G AGTATAGCAGCTCGT CC IGHD7-27 CTAACTGGGGA (1) Each of the IGHD nucleotide sequences can be read in three (3) forward reading frames, and, possibly, in 3 reverse reading frames. For example, the nucleotide sequence given for IGHD1-1, depending on how it inserts in full V-DJ rearrangement, may encode the full peptide sequences: GTTGT, VQLER and YNWND in the forward direction, and WPVV, SFQLY and RSSCT in the reverse direction. Each of these sequences, in turn, could generate progressively deleted segments as explained in the Examples to produce suitable components for libraries 5 of the invention. -194-

Example 15: Selection of Antibodies from the Library 2015201796 09 Apr 2015

In this example, the selection of antibodies from a library of the invention (described in Examples 9-11 and other Examples) is demonstrated. These selections demonstrate that the libraries of the invention encode antibody proteins capable of 5 binding to antigens. In one selection, antibodies specific for “Antigen X”, a protein antigen, were isolated from the library using the methods described herein. Figure 24 shows binding curves for six clones specifically binding Antigen X, and their Kd values. This selection was performed using yeast with the heavy chain on a plasmid vector and the kappa light chain library integrated into the genome of the yeast. 10 In a separate selection, antibodies specific for a model antigen, hen egg white lysozyme (HEL) were isolated. Figure 25 shows the binding curves for 10 clones specifically binding HEL; each gave a Kd >500nM. This selection was performed using yeast with the heavy chain on a plasmid vector and the kappa light chain library on a plasmid vector. The sequences of the heavy and light chains were determined for clones 15 isolated from the library and it was demonstrated that multiple clones were present. A portion of the FRM3s (underlined) and the entire CDRH3s from four clones are shown below (Table 53 and Table 54, the latter using the numbering system of the invention).

Table 53. Sequences of CDRH3, and a Portion of FRM3, from Four HEL Binders Seq Name SEQ ID NO: FRM3 and CDRH3 Tail N1 DH N2 H3-JH CR080362 AKGPSVPAARAEYFQH G PS VPA AR AEYFQH CR080363 AREGGLGYYYREWYFDL E GGL GYYY RE WYFDL CR080372 akpdygaeyfqh - P DYG - AEYFQH EK080902 akeivvpsaeyfqh E - IW PS AEYFQH 20 - 195-

Attorney Docket No.: ADS-011.25 2015201796 09 Apr 2015

| Cjones sJTailjJ CR080362 CR080363 CR080372 EK080902

G E

P G P

S G

V G D I

P Y Y Y

A Y G V

Table 54. Sequences of CDRH3 from Four HEL Binders in Numbering System of the Invention, According to the Numbering System of the Invention A R .(N2J...............I............ [H3-JH] § CDRH3 Length 98A 98B si 99E 99 D 99 C 99 B 99A 99 100 101 102 | R - I - A E Y F Q H | 14 E _ W Y F D L 1 15 j; A E Y F Q H 1 10 s A E Y F Q H | 12 196

The heavy chain chassis isolated were VH3-23.0 (for EK080902 and CR080363), VH3- 23.6 (for CR080362), and VH3-23.4 (for CR080372). These variants are defined in Table 8 of Example 2. Each of the four heavy chain CDRH3 sequences matched a designed sequence from the exemplified library. The CDRL3 sequence of one of the 5 clones (ED080902) was also determined, and is shown below, with the surrounding FRM regions underlined: 2015201796 09 Apr 2015 CDRL3: YYCOESFHIPYTFGGG.

In this case, the CDRL3 matched the design of a degenerate VK1-39 oligonucleotide sequence in row 49 of Table 33. The relevant portion of this table is reproduced below, 10 with the amino acids occupying each position of the isolated CDRL3 bolded and underlined:

mssis CDR Length Junction type Degenerate Oligonucleotide SEQ ID 89 90 91 92 93 94 95 96 ! 97 0-39 9 1 CWGSAAWCATH CMVTABTCCTT WCACT LQ EQ ST FSY HNPRST 1ST P FY T 15 - 197-

EQUIVALENTS 2015201796 09 Apr 2015

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the 5 following claims. - 198- 2015201796 09 Apr 2015 APPENDIX A Numbers of Kappa Light Chains Used to Derive the VK Libraries 23868 2385488 16923194 58222611 70798854 98956311 32779 2385490 16923202 58222613 70798856 98956323 32810 2385492 16923208 58222615 70798858 98956325 33059 2385494 17226623 58222617 70798860 98956327 33144 2385495 17226631 58222619 70798862 98956337 33156 2385497 17226635 58222621 70798866 98956341 33170 2597932 17226639 58222623 70798868 98956343 33173 2597935 17226643 58222625 70798872 98956349 33183 2597937 17226645 58222627 70798874 98956355 33185 2597943 17226655 58222629 70798878 98956357 33189 2597946 17381491 58222631 70798880 98956365 33191 2597948 17385013 58222633 70798882 98956375 33195 2597950 17385015 58222635 70798884 98956379 33200 2597952 17385017 58222637 70798886 98956381 33202 2599531 17385019 58222639 70798888 98956383 33221 2599533 17385021 58222641 70798890 98956400 33227 2599535 17483729 58222643 70798892 98956404 33230 2599545 18025561 58222645 70798894 98956406 33233 2625059 18025563 58222647 70798896 98956414 33237 2632152 18025573 58222649 70798898 98956418 33268 2654047 18025575 58222651 70798900 98956422 33288 2654051 18025577 58222653 70798902 98956426 33290 2654055 18025579 58222655 70798904 98956428 33294 2773084 18025581 58222657 70798906 98956430 33296 2920359 18025583 58222659 70798914 98956432 33298 2995674 18025585 58222661 70798916 98956436 33300 2995676 18025587 58222663 70798918 98956440 33302 2995678 18025589 58222665 70798920 99022977 33304 2995680 18025591 58222667 70798922 99022979 33324 2995682 18025593 58222669 70798926 99022981 33330 2995688 18025595 58222671 70798928 99022983 33415 2995690 18025597 58222673 70798930 99022985 33416 3023134 18025599 58222675 70798934 99022987 33417 3023136 18025603 58222677 70798936 99022989 33418 3023138 18025605 58222679 70798940 99022991 33421 3023140 18025607 58222681 70798942 99022993 33422 3023142 18025611 58222683 70798946 99022995 33423 3023144 18025613 58222685 70798948 99022997 33424 3023146 18025617 58222687 70798950 99022999 33426 3023148 18025621 58222689 70798952 99023002 33647 3251385 18025623 58222691 70798954 99023004 33649 3251387 18025627 58222693 70798956 99023006 33655 3251389 18025629 58222695 71058688 99023008 33657 3251391 18025635 58222697 71058704 99023010 33659 3251744 18025639 58222699 71058712 99023012 33665 3251749 18025641 58222701 71058717 99023474 33669 3251983 18025645 58222703 71058719 99023476 33679 3251985 18025651 58222705 71058721 99023478 33683 3288824 18025653 58222707 71058723 99023480 33685 3378165 18025655 58222709 71058725 99023482 33756 3378177 18025657 58222711 71058727 99023484 - 199- 2015201796 09 Apr 2015 34022 3378183 18025659 36657 3451194 18025661 37860 3603382 18025665 37909 3603384 18025667 38361 3603386 18025669 38362 3603388 18025677 38363 3603390 18025679 38367 3603392 18025681 38436 3603394 18025683 38438 3603396 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70798782 70798784 70798786 70798788 70798792 70798794 70798796 70798798 70798800 70798802 70798804 70798806 70798808 70798810 70798812 70798814 70798816 70798818 70798820 70798824 70798826 70798828 70798830 70798832 70798834 70798836 70798838 70798840 70798842 94035284 94035289 94035298 94035300 94035312 94469910 94469912 94469914 94469922 94469924 94469926 95007504 95007510 95007512 95007514 95007516 95007518 95007520 95007522 95007524 95007526 95007528 95007530 95007532 95007534 95007536 95007538 95007540 95007542 95007544 95101759 95101761 95101767 95101769 95101777 98956195 98956209 98956219 98956223 98956232 98956244 98956249 98956255 98956261 98956263 98956271 98956277 98956279 98956281 98956285 98956289 98956291 98956293 157903220 158055245 158055254 158055268 158055282 158055285 158055288 158058441 158731523 158731524 158731525 158731526 158731527 158731528 158731529 158731530 158731531 158731532 158731533 158731534 158731536 158731538 158731539 158731540 158731541 158731542 158731545 158731546 158731547 158731548 158731550 158731551 158731552 158731553 158731554 158731555 158731556 158731557 158731558 158731559 158731560 158731561 158731562 158731563 158731564 158731565 158731566 158731567 158731568 158731569 158744132 158744140 158744148 -212 2015201796 09 Apr 2015 2345029 15859220 2345031 15986229 2345033 16508167 2385484 16554974 2385486 16923186 58222601 70798844 58222603 70798846 58222605 70798848 58222607 70798850 58222609 70798852 98956299 158744156 98956301 158744164 98956303 158746355 98956305 158746363 98956307 158746371 -213- 2015201796 09 Apr 2015 APPENDIX B GI Numbers of Lambda Light Chains Used to Derive the Υλ Libraries 31454 3142529 4566076 9968397 51103608 77379760 32808 3142531 4566078 9968401 51103612 77379824 32812 3142533 4566082 9968403 51103614 77379826 33335 3142535 4566084 9968405 51103616 77379828 33368 3142537 4566086 9968409 51490956 77379830 33383 3142539 4566088 9968411 54781261 77379832 33387 3142541 4566090 9968413 61815560 77379834 33412 3142543 4566092 9968415 62720404 77379836 33429 3142545 4566094 9968417 62720406 77379838 33431 3142547 4566096 9968419 62720408 77379840 33433 3142549 4566098 9968421 62720412 77379842 33703 3142553 4566101 9968423 62860947 77379846 33711 3142556 4566105 9968425 62860950 77379848 37918 3142558 4732059 9968427 62860967 77379850 37920 3142562 4761253 9968429 62860969 77379855 37922 3142564 4761255 9968433 62860971 77379857 37923 3142566 4761257 9968435 62860973 77379859 38359 3142569 4761259 9968437 62860975 77379861 38360 3142573 4761261 9968439 62860977 77379863 38364 3142577 4761263 10636511 62860979 77379865 38365 3142579 4761265 10636514 62860985 77379867 38366 3142581 4761267 10636518 62861006 77379869 38368 3142583 4761269 10636521 62861008 77379871 186078 3142585 4761271 10636527 62861010 77379875 186080 3142587 4761273 11992185 62861047 77379877 186082 3142589 4761277 11992187 62999489 77379879 186084 3142591 4761279 11992189 62999497 77379882 186086 3142593 4927957 11992191 62999501 77379884 186088 3142595 5019504 11992195 62999509 77379886 186090 3142597 5019506 11992197 70888031 77379888 186092 3142599 5019516 11992199 70888035 77379890 186094 3142601 5019518 11992201 70888037 77379894 186096 3142603 5019520 12666922 70888041 77379896 186097 3142612 5019528 12666924 70888043 77379900 186111 3142614 5019530 12666926 70888045 77379908 186162 3142616 5019532 12666928 70888047 77379910 186164 3142618 5019534 12666930 70888049 77379912 186168 3142620 5019536 12666932 70888051 77379916 186170 3142649 5174362 12666934 70888053 77379918 186172 3142651 5174364 12666936 70888055 80975584 186175 3142653 5174366 12666938 70888057 80975588 298556 3142656 5174378 12666940 70888059 80975598 405223 3142658 5524086 12666942 70888061 80975622 405227 3142660 5524106 12666944 70888063 80975628 409040 3142662 5524108 12666946 70888065 80975632 409041 3142668 5524118 12666948 70888067 80975636 409043 3142670 5524122 12666952 70888069 81020028 433485 3142672 5524132 12666954 70888071 81020064 434041 3142674 5578817 12666956 70888073 86438995 434045 3142676 5578819 12666958 70888075 86439001 439514 3142678 5578823 12666960 70888077 86439005 -214- 439516 3142680 441251 3142684 460854 3153359 460856 3153361 460860 3153365 465157 3153366 465167 3153368 465171 3153374 465175 3153376 469249 3335577 483911 3335579 487824 3335585 487825 3335587 487828 3335591 493153 3388046 506426 3388048 506428 3388050 515765 3388054 532599 3388056 532600 3388058 532603 3388060 560845 3388062 575230 3388064 575238 3388066 575242 3388070 685021 3388072 773591 3388074 871362 3388080 987068 3747019 987076 3821077 998390 3821078 998394 3821079 1055278 3821080 1070329 3821081 1070341 3821082 1070349 3821083 1143195 3821084 1200068 3821086 1235776 3821087 1235778 3821089 1235780 3821090 1235782 3821091 1255606 3821092 1255610 3821093 1255611 3821094 1255613 3821095 1552313 3821096 1561599 3821097 1770407 4103646 1864134 4103648 1864140 4103650 1864142 4103652 1864144 4103654 5578825 12666962 5578827 12830380 5578829 12830382 5578831 12830384 5578833 13276707 5911837 13877276 6492194 14279402 6492196 14279404 6492206 14279406 6492208 17226627 6492210 17226649 6492212 18307305 6643078 18307307 6643082 18307309 6643086 18307311 6643088 18307313 6643090 18307315 6643098 18307317 6643104 18307319 6643106 18307321 6643114 18307329 6643118 21311290 6643120 21311292 6643124 21669150 6643126 21669152 6643128 21669154 6643136 21669156 6643138 21669158 6643154 21669160 6643156 21669162 6643158 21669164 6643162 21669166 6643168 21669172 6643170 21669174 6643172 21669176 6643176 21669178 6643178 21669180 6643180 21669182 6643182 21669184 6643184 21669186 6643186 21669188 6643188 21669190 6643192 21669192 6643196 21669194 6643198 21669196 6643200 21669198 6643202 21669200 6643204 21669204 6643210 21669206 6643214 21669210 6643218 21669212 6643220 21669214 6643224 21669218 2015201796 09 Apr 2015 70888079 86439015 70888081 86439017 70888083 86439087 70888085 86439089 70888087 86439091 70888089 86439093 70888091 86439095 70888093 86439097 70888095 86439099 70888097 86439101 70888099 86439105 70888103 86439127 70888105 86439133 70888109 86439137 70888111 86439139 70888113 86439141 70888115 90994749 70888117 95007506 70888121 95007546 70888123 95007548 70888125 95007550 70888127 95007552 70888129 95007554 70888133 95007556 70888137 95007558 70888139 95007560 70888141 95007562 70888143 95007564 70888147 95007566 70888149 95007570 70888151 95007572 70888155 95007576 70888157 95007578 70888159 109240683 70888161 109240697 70888163 109240743 70888165 109240749 70888167 109240754 70888169 109240756 70888171 109240758 70888173 116795127 70888179 116795192 70888181 146336934 70888183 156632919 70888185 156632943 70888187 156632945 70888193 156632975 70888195 156633095 70888197 156633103 70888199 156633141 70888201 156633153 70888204 156633155 70888206 156633159 -215 2015201796 09 Apr 2015 2078365 2654039 2654043 2865485 3023094 3023096 3023098 3023100 3023102 3023104 3023106 3023108 3023110 3023112 3023114 3023116 3023118 3023120 3023122 3023126 3023130 3023132 3091153 3091155 3091157 3091159 3091161 3091163 3091165 3091167 3091169 3091171 3091173 3091175 3091177 3091179 3091181 3091183 3091185 3091187 3091191 3091193 3091195 3091197 3091201 3091203 3091205 3091207 3091209 3091213 3093861 3093863 3093865 4103656 4103658 4103660 4103672 4324023 4324025 4324029 4324031 4324037 4324039 4324043 4324047 4324055 4324057 4324061 4324063 4324067 4324069 4324073 4324075 4324077 4324085 4324087 4324089 4324091 4324093 4324097 4324103 4324107 4324111 4324113 4324115 4324117 4324123 4324125 4324127 4324139 4324145 4324151 4324155 4324157 4324159 4324163 4324169 4324175 4324177 4324181 4324187 4324189 4324193 4324197 4324199 4324205 6643226 6643230 6643232 6643238 6643240 6643242 6643244 6643248 6643250 6643254 6643256 6643258 6643268 6643272 6643274 6643276 6643278 6643280 6643282 6643286 6643290 6643292 6643294 6643296 6643302 6643304 6643308 6643314 6643318 6643328 6643344 6643352 6643354 6643358 6643360 6643362 6643366 6643368 6643374 6643376 6643378 6643382 6643386 6643390 6643392 6643402 6643416 6643418 6643424 6643428 6643436 6643446 6643448 21669220 21669222 21669224 21669226 21669228 21669230 21669232 21669234 21669236 21669238 21669240 21669242 21669244 21669248 21669252 21669254 21669256 21669260 21669262 21669264 21669266 21669268 21669270 21669272 21669274 21669276 21669278 21669280 21669288 21998780 21998782 21998784 21998786 21998792 21998794 21998800 21998802 21998804 23194484 23194488 23194492 23194496 23343556 24474079 27369031 27369033 27369035 27369037 27369045 27369047 27369051 27369053 27369058 70888208 70888210 70888212 70888216 70888218 70888220 70888222 70888224 70888228 70888230 70888232 70888234 70888236 70888238 70888240 70888242 70888244 70888246 70888248 70888250 70888252 70888254 70888258 70888260 70888262 70888264 70888266 70888268 70888270 70888272 70888274 70888276 70888278 70888280 70888282 70888284 70888286 70888288 70888290 70888292 70888294 70888296 70888304 70888306 71482628 71482632 77378177 77378188 77378257 77378266 77378268 77378270 77378273 156633171 156633179 156633199 156633203 156633209 156633211 156633225 156633229 156633237 156633241 156633245 156633253 156633255 156633267 156633283 157093725 170684323 170684325 170684329 170684331 170684333 170684335 170684339 170684341 170684345 170684349 170684351 170684355 170684363 170684365 170684369 170684371 170684373 170684375 170684379 170684381 170684385 170684387 170684389 170684397 170684405 170684407 170684409 170684411 170684417 170684419 170684423 170684425 170684427 170684429 170684431 170684433 170684439 -216 2015201796 09 Apr 2015 3093867 3093869 3093871 3093873 3093875 3093877 3093879 3093881 3093883 3093885 3093887 3093889 3093891 3093895 3093903 3142451 3142453 3142455 3142457 3142459 3142461 3142465 3142467 3142471 3142475 3142477 3142479 3142481 3142483 3142485 3142487 3142489 3142491 3142493 3142495 3142497 3142499 3142503 3142505 3142507 3142509 3142511 3142515 3142517 3142519 3142521 3142527 4324207 4324209 4324211 4324213 4324215 4324221 4324223 4324229 4324231 4324245 4324247 4324249 4324251 4324255 4324257 4324261 4324263 4324265 4324271 4324273 4324275 4324283 4324285 4468355 4468367 4468369 4468371 4565964 4565966 4565996 4566007 4566009 4566016 4566021 4566023 4566025 4566029 4566045 4566049 4566051 4566053 4566055 4566057 4566059 4566061 4566065 4566074 6643450 6643452 6643456 6643470 6643474 6643478 6643484 6643488 6643492 6643500 6643512 6643514 6643528 6643534 6643558 6643560 6643562 6643564 6643572 6643574 6643580 6643582 6643584 6643586 6643588 6643592 6643596 6643598 6643600 6643602 6643604 6643606 6643614 6643628 6643630 6649891 6649893 8920222 8920226 9864840 9968383 9968385 9968387 9968389 9968391 9968393 9968395 27369060 27369064 27369068 27369075 27369082 27369084 27369088 27818828 28394695 28394699 28394703 28394707 28394711 28394715 28848877 28848881 28848885 29342115 33304654 40647151 47271301 47271303 47271319 47271321 47271323 47271325 50199320 50199322 50199328 50199330 50199338 50199340 50871689 51103426 51103428 51103430 51103434 51103436 51103572 51103574 51103576 51103588 51103590 51103592 51103600 51103602 51103606 77378277 77378280 77378282 77378284 77378286 77378288 77378291 77378293 77378298 77378300 77378303 77378305 77378307 77378309 77378312 77378316 77378318 77378320 77378322 77378377 77378379 77378381 77378383 77378385 77378387 77378389 77378392 77378394 77378396 77378398 77378400 77378402 77379590 77379620 77379622 77379624 77379632 77379642 77379644 77379646 77379675 77379677 77379726 77379728 77379730 77379738 77379740 170684443 170684449 170684451 170684453 170684461 170684469 170684473 170684489 170684495 170684497 170684499 170684501 170684507 170684513 170684515 170684517 170684527 170684531 170684535 170684537 170684539 170684541 170684545 170684549 170684553 170684555 170684557 170684561 170684565 170684567 170684569 170684571 170684583 170684589 170684591 170684593 170684597 170684599 170684601 170684603 170684607 170684609 170684613 170684617 170684619 -217

Claims (12)

1. WHAT IS CLAIMED IS:

   1. A library of synthetic polynucleotides, wherein said polynucleotides encode a plurality of antibody proteins comprising: (i) at least 106 unique antibody CDRH3 amino acid sequences comprising: (a) an N1 amino acid sequence selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV, D, E, F, Η, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, WGT; (b) a human CDRH3 DH amino acid sequence, N- and C-terminal truncations thereof, or a sequence of at least about 80% identity to any of them; (c) an N2 amino acid sequence selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV, D, E, F, H, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, WGT; and (d) a human CDRH3 H3-JH amino acid sequence or N-terminal truncations thereof; and (ii) a VKCDR3 amino acid sequence comprising about I to about 10 amino acids found at Kabat positions 89, 90, 91, 92, 93, 94, 95, 95 A, 96, and 97, in selected VKCDR3 amino acid sequences derived from a particular IGKV or IGKJ germline sequence.
2. 2. The library of claim 1, wherein the VKCDR3 amino acid sequence comprises one or more of the amino acid sequences listed in Table 33 or a sequence at least about 80% identical to any of them.
3. 3. An antibody isolated from the library of claim 1 or 2.
4. 4. The library of any one of claims 1 or 2, wherein the antibody proteins are expressed in a heterodimeric form.
5. 5. The library of claim 4, wherein the antibody proteins are expressed as antibody fragments.
6. 6. The library of claim 5, wherein the antibody fragments are selected from the group consisting of Fab, Fab', F(ab')2, Fv fragments, diabodies, linear antibodies, and single-chain antibodies.
7. 7. A library of synthetic polynucleotides, wherein said polynucleotides encode a plurality of antibody proteins comprising: (i) at least 106 unique antibody CDRH3 amino acid sequences comprising: (a) an N1 amino acid sequence selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV, D, E, F, Η, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, WGT; (b) a human CDRH3 DH amino acid sequence, N- and C-terminal truncations thereof, or a sequence of at least about 80% identity to any of them; (c) an N2 amino acid sequence selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV, D, E, F, H, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, YD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, WGT; and (d) a human CDRH3 H3-JH amino acid sequence or N-terminal truncations thereof; and (ii) a VKCDR3 amino acid sequence of at least about 80% identity to a amino acid sequence represented by the following formula: [VR_Chassis]-[L3-VR]-[X]-[JR*], wherein: (a) VK Chassis is an amino acid sequence selected from the group consisting of about Rabat amino acid 1 to about Rabat amino acid 88 encoded by IGRV1 -05, IGRV1-06, IGRV 1-08, IGRV 1-09, IGRV1-12, IGRV1-13, IGRV1- 16, IGRV1-17, IGRV 1-27, IGRV1-33, IGRV1-37, IGRV 1-39, IGRV1D-16, IGRV1D-17, IGRV1D-43, IGRV1D-8, IGRV2-24, IGRV2-28, IGRV2-29, IGRV2- 30, IGRV2-40, IGRV2D-26, IGRV2D-29, IGRV2D-30, IGRV3-11, IGRV3-15, IGRV3-20, IGRV3D-07, IGRV3D-11, IGRV3D-20, IGRV4-1, IGRV5-2, IGRV6- 21, and IGRV6D-41, or a sequence of at least about 80% identity to any of them; (b) L3-VR is the portion of the VRCDR3 encoded by the IGRV gene segment; and (c) X is any amino acid residue; and (d) JR* is an amino acid sequence selected from the group consisting of amino acid sequences encoded by IGJR1, IGJR2, IGJR3 , IGJR4, and IG JR5 , wherein the first residue of each IGJR amino acid sequence is not present.
8. 8. An antibody isolated from the library of claim 7.
9. 9. A method of preparing a synthetic polynucleotide library comprising providing and assembling the polynucleotide sequences of claims 1 or 7.
10. 10. A method of isolating one or more host cells expressing one or more antibodies, the method comprising: (i) expressing the antibodies of any one of claims 1 or 7 in one or more host cells, (ii) contacting the host cells with one or more antigens; and (iii) isolating one or more host cells having antibodies that bind to the one or more antigens.
11. 11. The method of claim 10, further comprising isolating one or more antibodies from the one or more host cells.
12. 12. The method of claims 10 or 11, further comprising the step of isolating one or more polynucleotide sequences encoding one or more antibodies from the one or more host cells.