NZ738496B2 - Cys80 conjugated immunoglobulins - Google Patents

Abstract

Provided herein are methods for generating conjugated immunoglobulins, the method comprising: decapping a cysteine at amino acid position 80 ("Cys80") in a light chain variable region of an immunoglobulin, wherein the immunoglobulin comprises a heavy chain variable region and the light chain variable region; and conjugating a thiol-reactive compound to the Cys80, wherein the thiol-reactive compound comprises a thiol-reactive group. Antigen-binding molecules and methods for generating the same, immunoglobulins as well as nucleic acid molecules encoding the immunoglobulins and host cells comprising the nucleic acid molecules, conjugated immunoglobulins, and light chain variable regions for use in a conjugated immunoglobulin are also provided.

Description

CYSSO CONJUGATED GLOBULINS CROSS REFERENCE TO RELATED ATIONS This application claims priority to US. Provisional Application No. 62/182,020, ?led June 19, 2015, the disclosure of which is hereby incorporated by reference in its entirety.

SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, d on June 16, 2016, is named 104018.000953_SL.txt and is 1 bytes in size.

TECHNICAL FIELD ed herein are CysSO conjugated immunoglobulins and methods of creating the same.

BACKGROUND The utility of monoclonal antibodies extends from basic research to therapeutic and stic applications. The ability to conjugate antibodies to functional agents extends their functionality even further. The manufacture of conjugated antibodies usually involves conjugation of a linker, drug, or other functional agent to reactive lysine or cysteine residues on the heavy (HC) and light (LC) chains of a monoclonal antibody (mAb). Lysine conjugation is typically mediated by succinimide (NHS)-based or isothiocyanate—based chemistry. Given the number of exposed lysines on the surface of an dy, amine—based conjugation ches result in multiple s being modi?ed, though not all lysine es are modi?ed to the same extent. Therefore, the ?nal product is a heterogeneous mixture of mAbs with a distribution of drug-to-antibody (DAR) ratios.

Most cysteines within an antibody are involved in either inter- or intra—chain disul?de bonds. Conjugation to cysteines thus es at least partial reduction of the antibody.

Like lysine-based conjugation, cysteine-based conjugation results in a heterogeneous mixture of conjugated dies differing in drug load and conjugation site. Each species of conjugated antibody may have distinct properties, which in turn could lead to wide variation of in viva pharmacokinetic ties. Additionally, such heterogeneity can present challenges in manufacturing of the conjugated antibody.

Disclosed herein are methods for generating a conjugated immunoglobulin, the methods comprising: decapping a cysteine at amino acid position 80 ("CysSO") in a light chain variable region of an immunoglobulin derived from rabbit, wherein the immunoglobulin comprises a heavy chain variable region and the light chain variable region; and conjugating a thiol-reactive compound to the Cys80, wherein the thiol-reactive compound ses a thiol- reactive group.

Also provided are methods for generating an antigen-binding molecule, the methods comprising incubating a ?rst conjugated immunoglobulin with a second conjugated globulin to generate the antigen-binding molecule, wherein: the ?rst conjugated immunoglobulin ses a ?rst heavy chain le region and a ?rst light chain variable region, the ?rst light chain variable region having a cysteine at position 80 ("Cys801") wherein Cys801 is ated to a ?rst thiol-reactive compound comprising a ?rst thiol-reactive group, and the second conjugated immunoglobulin comprises a second heavy chain variable region and a second light chain variable region, the second light chain variable region having a cysteine at position 80 ("Cy5802") wherein Cy5802 is conjugated to a second thiol-reactive compound comprising a second thiol-reactive group.

Immunoglobulins comprising a heavy chain le region and a light chain variable region, the light chain le region having a cysteine at position 80 ("Cys80") and an amino acid other than Phe, Lys, or Cys at position 83 are also ed herein, as are nucleic acid molecules encoding the immunoglobulins and host cells comprising the nucleic acid molecules.

Further provided are conjugated immunoglobulins comprising the disclosed immunoglobulins, wherein the cysteine at position 80 is conjugated to a thiol-reactive compound, the thiol-reactive compound comprising a reactive group.

Also disclosed herein are methods of treating cancer in a subject comprising administering to the subject a pharmaceutically effective amount of a ated mesothelin globulin, wherein the conjugated mesothelin immunoglobulin comprises: any of the disclosed mesothelin immunoglobulins, and a thiol-reactive compound comprising a thiol- ve group, a linker, and a functional agent.

Provided are antigen-binding molecules comprising: a first conjugated immunoglobulin comprising a first heavy chain le region and a first light chain variable , the first light chain variable region having a cysteine at position 80 ("Cys801"), wherein Cys801 is conjugated to a first thiol-reactive compound comprising a first thiol-reactive group, and a second conjugated immunoglobulin comprising a second heavy chain variable region and a second light chain variable , the second light chain variable region having a cysteine at position 80 ("Cys802") wherein Cys802 is conjugated to a second thiol-reactive compound sing a second thiol-reactive group.

Light chain variable regions for use in a conjugated globulin, the light chain variable region having a cysteine at amino acid position 80 ("Cys80") and an amino acid residue other than Phe, Lys, or Cys at amino acid position 83, wherein the Cys80 is unpaired are also disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS The summary, as well as the following detailed description, is further understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosed methods, conjugated immunoglobulins, antigen-binding molecules, immunoglobulins, and light chain le regions, there are shown in the drawings exemplary ments; however, the methods, conjugated immunoglobulins, antigen-binding molecules, immunoglobulins, and light chain variable regions are not limited to the specific ments disclosed. In the drawings: represents an alignment of rabbit and human light chain sequences. (A) Alignment of the germline V? sequences from rabbit S2, X02336) and human (IGKV1- , Z00001). The bold residue indicates Cys80 (according to either Kabat or Chothia numbering) in the rabbit sequence. (B) Alignment of the germline C? sequences from rabbit (IGKC1, K01360) and human (IGKC, J00241). The bold residue indicates Cys171 (EU numbering) in the rabbit sequence. represents structural models of (A) rabbit mAb, showing the Cys80- Cys171 disulfide bond, (B) human mAb, and (C) -human chimeric mAb showing the unpaired Cys80. rates an alignment of rabbit ne V? families. The residue at position 80 is indicated by the arrow. illustrates an exemplary mass spectrometry analysis of xi155D5 light chain when (A) reduced using harsh conditions (20 mM DTT, 60°C, 5 minutes) and (B) reduced using mild conditions (100 uM DTT, 22°C, 30 minutes). illustrates an exemplary SE-HPLC analysis of the stability of xi155D5.

(A) Stability of xi155D5 stored at -80°C. (B) Stability of xi155D5 stored at 37°C for 1 week.

Only a very slight increase in formation of ates or degradation products was observed. Y axis, mAU, x axis, retention time (minutes). ents an ary decapping experiment showing that the cysteine capping Cys80 can be removed by mild reducing conditions r containing 5 mM cysteine for 16 hours followed by washing with a ne—free ontaining buffer for 60 hours; all incubations carried out at 4°C). The mass of xi155D5 before (A) and after (B) decapping was 145,464 and 145,221 Da, respectively. The difference (243 Da) corresponding approximately to two free cysteines. represents an exemplary conjugation experiment showing that an uncapped Cys80 can be conjugated to maleimide-PEG2-biotin. (A) The light chain of reduced xi155D5 (predicted mass of 23,399 Da) had a mass of 23,384 Da, and (B) after incubation with maleimide—PEGZ-biotin, 94% of the product showed a mass increase by 526 Da (23,910 Da).

Asterisks denote non-light chain peaks. represents rabbit 155D5 VK and VH sequences aligned with the most homologous human germline variable domains. Framework region (FWR) and complementary determining region (CDR) based on Kabat numbering are ?ed above the sequences. CDRs based on Chothia numbering are underlined. The inal half of Kabat CDR2 is not considered a CDR by Chothia numbering and is italicized. rates an exemplary standard protein A puri?cation of zu155D5-1. zu155D5-1 was found capped (A), as evidenced by the change of mass after decapping (B) by 233 Da, approximately corresponding to two capping cysteines. illustrates exemplary structural models of chimerized xi155D5.

Modelling to ine CysSO proximity was conducted as in The residues ing between 5 and zu155D5-1 were highlighted and the distance to Cys80 was measured for each. (A) Residues Vall 1, Ala14, Gly17, Thr18, (B) Lys63; (C) Thr76, Gly77, Val78, Ala83, and (D) Glu103 and Leu104, are within 11 A of Cys80, except for Lys63 (18 A). These residues were changed back to the rabbit amino acids in the presence of Cys80. illustrates exemplary humanized mAbs of 155D5, 1E4, 166B3, and 33011 light chain sequences. represents (A-D) ?ow cytometry screening of sera from immunized s and (E) ELISA screening of sera from immunized animals. (A-D) Cells were incubated with post—immunization bleed sera at the indicated dilutions (A: post bleed, rabbit 1 serum diluted in 111000; B: post bleed, rabbit 1 serum diluted in 1:5000; C: post bleed, rabbit 2 serum diluted in 1:1000; and D: post bleed, rabbit 2 serum diluted in ). Signal from cells transiently expressing human MSLN ) and that from MSLN-negative cells (-MSLN) are shown. (E) ELISA plates were coated with 1 ug/mL of human MSLN at 4°C overnight and blocked using 1% BSA in PBS with 0.01% Tween (PBST) for 2 hours at room temperature.

After blocking buffer was removed, serial diluted samples of pre- and mmunization bleeds were added to wells. The plate was incubated for 2 hours at room temperature and then washed three times with PBST. HRP-conjugated goat anti-rabbit antibody was added in blocking buffer and incubated for 1 hour. The plate was washed three times and TMB substrate was added. The reaction was stopped and absorbance was measured at 450 nm. represents an ary protein peak of deconvoluted mass spectrometry analysis before and after ation of maleimido-PEGZ-auristatin F (AuF) to Cys80. illustrates (A) MSLN-AuF Cys80 conjugated mAbs cytotoxicity against MSLN-negative A431 cells and (B) MSLN-AuF Cys80 conjugated mAbs cytotoxicity against MSLN-positive A431-MSLN cells. represents the average tumor volumes among different treatment groups. represents (A) the average A431-MSLN (left ?ank) tumor volumes among different treatment groups and (B) the average tumor volumes among different treatment groups for A431 (right ?ank). ents an SDS-PAGE analysis of an ary Xi155D5-800CW conjugated antibody. (A) mwm, molecular weight marker; lane 1, Xi155D5, unconjugated, non- d; lane 2, xi155D5, x1155D5-800CW, non-reduced; lane 3, blank, lane 4, unconjugated Xi155D5, reduced; lane 5, xi155D5, xi155D5-800CW, reduced. All lanes contain 5 pg protein, Coomassie—stained. (B) Same gel as in A, imaged on IVIS system. Results indicate that IRDye 800CW is conjugated only on the light chain of 5. ELISA analysis of 5-800CW indicates that full binding to CA9 is retained (data not shown).

WO 05618 2016/038041 illustrates the tumor-specific localization of an exemplary DyeIR 800CW-conjugated antibody (xi155D5-800CW). Human colo205 (A-D) and HT-29 (E-H) cells were grafted into nude mice, which were later injected with xi155D5-800CW. Fluorescent signal (orange-red) was monitored at s times including 0 hr (A and E), 4 hr (B and F), 24 hr (C and G) and 72 hr (D and H) (shown only 0-72 hours and right ?ank). Approximate location of kidney (K) and tumor (T) is shown. illustrates an exemplary puri?cation of a xi155D5/xil2 bivalent/bispecific antigen-binding molecule. Gel-?ltration chromatography graph showing the peak corresponding to the xilSSDS/xil2 bivalent/bispecific antigen-binding le (referred to as ") (A). Fraction’s molecular size was analyzed by SDS-PAGE (B). The fractions containing the xi155D5/xil2 bivalent/bispecific n-binding le (biFab) were pooled and the mass was determined by mass spectrometry (C). illustrates the bispecificity of an exemplary xi155D5/xi12 bivalent/bispeciflc antigen-binding le (biFab). Biotinylated human CA9 was captured on streptavidin Octet biosensor tips. Compounds were added as indicated by the first arrow ("compound") and then allowed to bind. Subsequently, soluble human TEM-l (second arrow; "TEM-l") was added and its binding to the captured CA9/compound complexes was measured.

The se shift (double arrow), indicating capturing of the soluble TEM—l, was observed only with xi155D5/xi12 bivalent/bispeciflc antigen-binding molecule (biFab). illustrates an SDS-PAGE analysis of xi330l l-l6) Cys80 conjugated mAbs and xil-55—2-A[3(1—16) CysSO conjugated mAbs. Shown are the products before (B) and after (A) conjugation of mAb-DBCO with peptide ABC-16). MW, molecular weight marker. LC, light chain. illustrates an exemplary mass spectrometry analysis of xi1—55-2 and xi330ll parental light chain (LC) (A and B, tively), xil-55—2-DBCO Cys80 conjugated LC and xi330l l-DBCO Cys80 conjugated LC (C and D, respectively), and xi12-A[3(1-16) Cys80 conjugated LC and xi330l l-l6) CysSO conjugated LC (E and F, respectively). illustrates an exemplary scheme for generating n binding molecules. Immunoglobulins derived from rabbit can be digested with papain to generate Fabs.

A first Fab can be incubated with maleimido—DBCO and a second Fab can be incubated with maleimido—azide. The ?rst and second Fabs can be combined to form a bi-specific-Fab-Fab binding molecule. SPAAC = strain promoted alkyne—azide conjugation.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS The disclosed methods, conjugated immunoglobulins, antigen-binding molecules, immunoglobulins, and light chain variable regions may be understood more readily by reference to the following detailed description taken in connection with the anying ?gures, which form a part of this sure. It is to be understood that the disclosed methods, conjugated immunoglobulins, antigen-binding molecules, globulins, and light chain variable regions are not d to the c embodiments described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed methods, conjugated immunoglobulins, antigen-binding molecules, immunoglobulins, and light chain variable regions.

Unless speci?cally stated otherwise, any ption as to a possible mechanism or mode of action or reason for improvement is meant to be illustrative only, and the disclosed s, conjugated immunoglobulins, n-binding les, immunoglobulins, and light chain variable regions are not to be constrained by the correctness or incorrectness of any such suggested mechanism or mode of action or reason for improvement.

Throughout this text, the descriptions refer to conjugated immunoglobulins, antigen-binding molecules, immunoglobulins, and light chain variable regions and methods of generating the same. Where the disclosure describes or claims a e or ment associated with a conjugated immunoglobulin, antigen-binding molecule, immunoglobulin, or light chain variable region, such a feature or embodiment is equally applicable to the methods of generating the same. Likewise, where the disclosure describes or claims a feature or embodiment associated with a method of generating a conjugated immunoglobulin, antigen- binding molecule, immunoglobulin, or light chain variable region, such a feature or embodiment is y applicable to the conjugated immunoglobulin, antigen-binding molecule, immunoglobulin, or light chain variable region.

Reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise. When a range of values is expressed, another ment includes from the one particular value and/or to the other particular value. Further, reference to values stated in ranges include each and every value within that range. All ranges are inclusive and combinable.

When values are expressed as imations, by use of the antecedent ," it will be understood that the particular value forrm another embodiment.

It is to be appreciated that certain features of the disclosed methods, conjugated immunoglobulins, antigen-binding molecules, immunoglobulins, and light chain variable s which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment, Conversely, various features of the disclosed methods, conjugated immunoglobulins, antigen-binding molecules, immunoglobulins, and light chain variable regions that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination.

As used herein, the singular forms "a," "an," and "the" include the plural.

The term "comprising" is intended to include examples encompassed by the terms "consisting essentially of" and "consisting of"; similarly, the term "consisting essentially of" is intended to include examples encompassed by the term "consisting of." Various terms relating to aspects of the description are used throughout the cation and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other speci?cally de?ned terms are to be construed in a manner consistent with the de?nitions provided herein.

The term "about" when used in reference to numerical ranges, cutoffs, or speci?c values is used to indicate that the d values may vary by up to as much as 10% from the listed value. Thus, the term "about" is used to encompass variations of i 10% or less, ions of: 5% or less, ions ofi 1% or less, variations ofi 0.5% or less, or variations ofi 0.1% or less from the speci?ed value.

As used herein, the term "biological sample" refers to a sample obtained from a t, including sample of biological tissue or ?uid origin obtained in vivo or in vitro. Such samples can be, but are not d to, body ?uid (e.g., blood, blood plasma, serum, milk, spinal ?uid, ascites, or urine), organs, tissues, fractions, and cells isolated from mammals including, humans. Biological samples also may include sections of the sample obtained from a subject ing tissues (e. g., sectional portions of an organ or tissue). Biological samples may also include ts from a sample obtained from a subject, for example, an antigen from a ical ?uid (e.g., blood or urine).

The term ng ne" refers to a free ne from solution that forms a disul?de bond with Cys80 of the light chain variable region.

The term "chimerized," "chimeric,)9 eric antibody" and like terms refer to an immunoglobulin comprising a heavy chain variable region and light chain variable region, i.e., antigen-binding region, from one source or species and at least a portion of a heavy chain constant region and light chain constant region derived from a different source or species. These portions may be joined together chemically by conventional techniques (eg., synthetic) or prepared as a contiguous polypeptide using genetic engineering techniques (e. g., DNA encoding the protein portions of the chimeric antibody may be expressed to e a contiguous polypeptide chain). Exemplary ic immunoglobulins include those sing a rabbit variable region and a human constant . Such rabbit/human chimeric immunoglobulins are the product of expressed immunoglobulin genes comprising DNA segments encoding rabbit immunoglobulin variable regions and DNA ts ng human immunoglobulin constant regions. Other forms of "chimeric immunoglobulins" encompassed by the present disclosure are those in which the class or ss has been modi?ed or d from that of the original immunoglobulin (also referred to as "class-switched immunoglobulins"). Throughout the disclosure, chimeric immunoglobulins are designated "xi." Herein, "chimeric immunoglobulin" and like terms refer to the sequence of the immunglobulin rather than the process used to generate the antibody.

As used herein, "Cys80" refers to a cysteine e at amino acid position 80 of the light chain variable region ve to a light chain variable region absent a leader ce.

For example, the light chain variable regions disclosed in Table 25 comprise a 19 amino acid (encoded by a 57 nucleotide) leader sequence. "Cys80" occurs at amino acid position 99 when the leader sequence is present and amino acid position 80 when the leader sequence is absent.

The Cys80 numbering is based upon Chothia numbering system.

The term "decapping" refers to removal of the capping cysteine using the methods provided herein under conditions that ze tion of the native intra— and inter- chain disul?des of the immunoglobulin.

The term "immunoglobulin derived from" refers to immunoglobulins, or portions thereof, having at least the CDR regions of a rabbit immunoglobulin. "Immunoglobulin derived from" includes rabbit/human chimeras or humanized rabbit immunoglobulins. The level of variability tolerated when deriving an immunoglobulin from a rabbit can be determined, for example, by the United States Adopted Names Counsel (USAN) of the American Medical Association (AMA).

As used herein, ional agent" refers to an agent having therapeutic, diagnostic, or other functional property(ies)i Various functional agents that fall within the scope of the disclosure are described elsewhere herein.

The term ized," "humanized immunoglobulin" and like terms refer to immunoglobulins of rabbit origin in which the sequence of amino acids throughout the variable s are changed to sequences having homology to a human variable region. Exemplary humanized immunoglobulins can comprise a rabbit variable domain whereby es throughout the framework region (FWR) and/or the CDRs are replaced by sequences homologous to a human immunoglobulin. In some instances, FWR residues of the rabbit globulin are not replaced by corresponding human residues. Alternatively, "humanized," "humanized immunoglobulin" and like terms can refer to immunoglobulins of human origin in which residues throughout the FWR and/or CDRs were replaced by sequences gous to a rabbit immunoglobulin. For example, humanized immunoglobulins can be human immunoglobulins in which residues from a hypervariable region of the human immunoglobulin are replaced by residues from a hypervariable region of a rabbit immunoglobulin having the desired specificity, ty, and capacity. Furthermore, humanized immunoglobulins may comprise residues that are not found in the recipient immunoglobulin or in the donor immunoglobulin. These modi?cations are made to r re?ne immunoglobulin performance. In general, the humanized immunoglobulin will comprise substantially all of at least one, and typically two, le domains, in which all or substantially all of the hypervariable loops correspond to those of a man immunoglobulin and all or substantially all of the FWRs are those of a human immunoglobulin sequence. The humanized immunoglobulin can optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See, e.g., Riechmann, L., et al., Nature 332 (1988) 323-327, and Neuberger, M. S., et al., Nature 314 (1985) 0. Throughout the disclosure, "humanized immunoglobulins" are designated "zu." Herein, "humanized immunoglobulin" and like terms refer to the sequence of the immunoglobulin rather than the process used to generate the immunoglobulin.

The term "donor immunoglobulin" refers to a non-human globulin that contributes the amino acid sequences of its variable regions, CDRs, or other functional nts or analogs thereof to the humanized immunoglobulin, and thereby provides the zed immunoglobulin with the antigenic specificity and lizing activity characteristic of the donor immunoglobulin.

The term "recipient immunoglobulin" refers to an immunoglobulin heterologous to the donor immunoglobulin, which provides the amino acid sequences of its heavy and/or light chain framework regions and/or its heavy and/or light chain constant regions to the humanized immunoglobulin. The recipient immunoglobulin may be derived from any . In preferred embodiments, the recipient immunoglobulin is non-immunogenic in humans.

Preferably the recipient immunoglobulin is a human globulin.

"Humanizing" refers to a process of generating a humanized immunoglobulin and includes any process for generating humanized immunoglobulins having the above characteristics, including, but not limited to, in silico humanization, engineering s/host CDRs into human immunoglobulins, substituting ork region residues of a chimeric immunoglobulin to match a corresponding human ork region, etc.

"Hydrophobic Amino Acid" refers to an amino acid exhibiting a hydrophobicity of greater than zero according to the normalized consensus hydrophobicity scale of Eisenberg, 1984, J. Mol. Biol. 179: 125-142. Genetically encoded hydrophobic amino acids include Pro (P), Ile (I), Phe (F), Val (V), Leu (L), Trp (W), Met (M), Ala (A), Gly (G) and Tyr (Y).

"Immunoglobulin," as used herein, refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes including the kappa and lambda light chains and the alpha, gamma, delta, epsilon and mu heavy chains. Full—length immunoglobulin "light chains" (about 25 Kd or 214 amino acids) are encoded by a variable region gene at the NH2-terminus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH - terminus. Full-length immunoglobulin "heavy chains" (about 50 Kd or 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes, e.g., gamma (encoding about 330 amino . "Immunoglobulins" include: (a) immunoglobulin polypeptides, 116., polypeptides of the globulin family that contain an antigen binding site that speci?cally binds to a speci?c antigen (e.g., MSLN, CA9, TEM1, etc), including all globulin isotypes (IgG, IgA, IgE, IgM, IgD, and IgY), classes (e.g. IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), subclasses, and various monomeric and polymeric forms of each isotype, unless otherwise speci?ed; and (b) conservatively substituted variants of such globulin ptides that immunospecifically bind to the antigen (e.g., MSLN, CA9, TEM1, etc.) globulins are generally described in, for example, Harlow & Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1988).

One form of immunoglobulin disclosed herein constitutes the basic structural unit of an antibody, For example, an dy can include a tetramer and t of two identical pairs of immunoglobulin chains, each pair having one light chain and one heavy chain.

Generally, in each pair, the light chain and heavy chain variable regions are together responsible for binding to an antigen, and the constant regions are responsible for the antibody effector In addition to antibodies, immunoglobulins may exist in a variety of other forms including, for e: antigen-binding fragments or portions of an immunoglobulin, such as Fv, Fab, (Fab')2 and Fv fragments, and alternative antibody formats such as single chain immunoglobulins (scFV and scFab), diabodies, triabodies, tetrabodies, linear antibodies, and multispecifrc dies, to name a few. See, for e, James D. Marks, Antibody Engineering, Chapter 2, Oxford University Press (1995) (Carl K. aeck, Ed.) As used herein, the term "immunospecifically" refers to the ability of an immunoglobulin to speci?cally bind to an antigen against which the immunoglobulin was generated and not specifically bind to other peptides or proteins. An immunoglobulin that immunospeci?cally binds to an n against which the immunoglobulin was generated may not bind to other polypeptides or proteins, or may bind to other polypeptides or proteins with a lower binding affinity than the antigen against which the immunoglobulin was generated as ined by, for example, immunoassays, BIAcore, or other assays known in the art. An globulin binds immunospeci?cally to an antigen against which the immunoglobulin was generated when it binds to the antigen with a higher binding affinity than to any cross-reactive n as determined using experimental techniques, such as, but not limited to, radioimmunoassays (RIA) and enzyme-linked imrnunosorbent assays s) (See, for example, Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989) for a discussion regarding antibody speci?city).

"Linker," as used , refers to a spacer, which may be a straight or branched chain, for connecting an immunoglobulin (through a thiol-reactive group on the unpaired Cys80) to a functional agent. Such linkers may be cleavable (e.g., acid labile or protease cleavable) or non-cleavable.

The term "monoclonal antibody" refers to an antibody that is derived from a single cell clone, including any eukaryotic or prokaryotic cell clone, or a phage clone, and not the method by which it is produced. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope. The term "monoclonal antibody" is not limited to antibodies produced through hybridoma technology.

"Native" refers to the wild type immunoglobulin sequence from the species in which the globulin is derived. For example, in embodiments wherein a Cys80 is present in the light chain variable region from the species from which it is derived, the CysSO is said be present in the native light chain variable region.

As used herein, "percent identity" and like terms is used to describe the sequence relationships between two or more nucleic acids, polynucleotides, proteins, or polypeptides, and is understood in the context of and in conjunction with the terms including: (a) reference sequence, (b) ison window, (c) sequence identity and (d) tage of ce identity. (a) A "reference ce" is a de?ned sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, exemplary lengths of the nce polypeptide ce include at least about 16 amino acids, at least about amino acids, at least about 25 amino acids, at least about 35 amino acids, at least about 50 amino acids, or at least about 100 amino acids. For nucleic acids, exemplary length of the reference nucleic acid sequence include at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, or at least about 300 tides, or any integer thereabout or therebetween. (b) A "comparison window" includes reference to a contiguous and ied segment of a polynucleotide or polypeptide sequence, wherein the cleotide or polypeptide sequence may be compared to a reference sequence and wherein the n of the polynucleotide or polypeptide sequence in the comparison window may comprise additions, substitutions, or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions, substitutions, or deletions) for optimal alignment of the two sequences. Exemplary comparison windows can be at least 20 contiguous nucleotides or amino acids in length, and optionally may be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a misleadingly high similarity to a reference sequence due to inclusion of gaps in the polynucleotide or polypeptide sequence a gap penalty is typically introduced and is subtracted from the number of matches.

(C) s of ent of sequences for comparison are well known in the art.

Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math, 2: 482, 1981, by the homology alignment algorithm ofNeedleman and Wunsch, J. Mol. Biol., 48: 443, 1970, by the search for similarity method of Pearson and Lipman, Proc. Natl.

Acad. Sci. USA, 8: 2444, 1988; by computerized entations of these algorithms, including, but not limited to: CLUSTAL in the PC/Gene program by igenetics, Mountain View, Calif, GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics re Package, Genetics Computer Group (GCG), 7 Science Dr., Madison, Wis, USA, the CLUSTAL program is well described by Higgins and Sharp, Gene, 7‘3: 237-244, 1988; Corpet, et al., Nucleic Acids Research, 16:881-90, 1988, Huang, et al., Computer Applications in the Biosciences, 8: 1-6, 1992; and Pearson, et al., s in Molecular Biology, 24:?- 331, 1994. The BLAST family of programs which may be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against n database sequences, BLASTP for protein query sequences against protein database sequences, TBLASTN for protein query sequences against nucleotide database sequences, and TBLASTX for nucleotide query sequences against nucleotide database sequences. See, Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., Eds, Greene Publishing and Wiley-Interscience, New York, 1995.

New versions of the above programs or new programs altogether will undoubtedly become ble in the future, and may be used with the present disclosure. (d) "Percent identity" means the value determined by comparing two lly d ces over a comparison window, wherein the n of the polynucleotide or polypeptide sequence in the comparison window may comprise additions, substitutions, or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions, substitutions, or deletions) for optimal alignment of the two sequences. The tage is ated by determining the number of positions at which the identical nucleic acid base or amino acid e occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

"Pharmaceutically ive amount" refers to an amount of an immunoglobulin that treats the subject.

"Polar Amino Acid" refers to a hydrophilic amino acid having a side chain that is uncharged at physiological pH, but which has at least one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Genetically encoded polar amino acids include Asn (N), Gln (Q) Ser (S) and Thr (T).

The term "subject" as used herein refers to a human or man organism.

Thus, the methods, immunoglobulins, and conjugated immunoglobulins described herein are applicable to both human and veterinary diseases and conditions. Subjects can be "patients," l'.e., living humans or non-human organisms that are ing medical care for a disease or condition, or humans or non-human organisms with no de?ned s who are being investigated for signs of pathology or presence/absence of a particular condition.

"Substituting" refers to the replacement of one amino acid residue for r.

"Substituting" includes, for e, missense mutations in one or more DNA base pairs encoding the amino acid residue or engineering the protein to ge one amino acid with another.

As used , "treating" and like terms refer to reducing the severity and/or frequency of e symptoms, eliminating disease symptoms and/or the underlying cause of said symptoms, reducing the frequency or likelihood of disease symptoms and/or their underlying cause, and improving or remediating damage caused, directly or ctly, by disease. Exemplary diseases include, but are not limited to, cancer.

"Thiol-reactive group" refers to a reagent or group that can form a covalent bond with the thiol group in a cysteine.

"Unpaired Cys80" refers to a Cys80 present in an immunoglobulin that has a thiol functional group that is not involved in an intramolecular or intermolecular disul?de bond.

For example, a thiol functional group of an "unpaired Cys80" is not involved in a disulfide bond with Cysl71.

As used herein "90% cal to" encompasses at least 90% identical, 91% identical, 92% identical, 93% identical, 94% identical, 95% identical, 96% identical, 97% identical, 98% identical, 99% identical, or 100% identical to the reference item (e.g., a biological sequence).

The following abbreviations are used throughout the disclosure: antibody drug ates (ADCs); drug-to-antibody (DAR); frame work region (FWR), complementary determining region (CDR); carbonic anhydrase IX (CA9), mesothelin (MSLN), auristatin F (AuF), variable heavy region (VH); variable light region (VL), variable kappa (VK); rabbit (Rb, rabb), gamma nt region (Cy); kappa constant region (CK), onal antibody (mAb); cysteine at amino acid position 80 (Cys80).

Generation ofconjugated immunoglobulins Disclosed herein are methods for generating a conjugated immunoglobulin, the methods sing: decapping a ne at amino acid position 80 ("Cys80") in a light chain variable region of an globulin derived from rabbit, wherein the immunoglobulin comprises a heavy chain variable region and the light chain variable region; and conjugating a thiol—reactive compound to the CysSO, wherein the thiol-reactive compound comprises a thiol-reactive group.

Suitable light chain le regions include, for example, a kappa light chain variable region. The light chain variable region of the sed immunglobulins are derived from rabbit. In some embodiments, the Cys80 can be present in the native light chain variable region of the rabbit immunoglobulin. Exemplary rabbits from which a light chain variable region having a Cys80 can be derived include, but are not limited to, Oryctolagus cum‘culus. In some aspects, for example, the light chain variable region can be derived from a New Zealand White (NZW) rabbit. In other aspects, the light chain variable region can be derived from a b9 rabbit.

Exemplary methods of decapping a Cys80 in a light chain variable region of an globulin include incubating the immunoglobulin with a reducing buffer followed by incubating the immunoglobulin with an oxidizing buffer. Reducing buffers comprise one or more reducing agents. Suitable reducing agents e, for example, cysteine (including L- cysteine and D-cysteine), 2-mercaptoethylamine, Tris (2-carboxyethyl) phosphine, 2- mercaptoethanesulfonic acid, 2-mercaptopropionic acid, or ations thereof. In preferred embodiments, the reducing buffer can comprise a mild reductant such as ne. The concentration of reducing agent can range from about 0.2 mM to about 100 mM, from about 1 mM to about 100 mM, from about 2 mM to about 100 mM, from about 5 mM to about 100 mM, from about 10 mM to about 100 mM, from about 20 mM to about 100 mM, from about 40 mM to about 100 mM, from about 50 mM to about 100 mM, from about 0.2 mM to about 90 mM, from about 0.2 mM to about 80 mM, from about 0.2 mM to about 70 mM, from about 0.2 mM to about 50 mM, from about 0.2 mM to about 30 mM, from about 0.2 mM to about 25 mM, from about 0.2 mM to about 10 mM, or from about 0.2 mM to about 5 mM. The concentration of reducing agent can be about 0.2 mM. The concentration of reducing agent can be about 1 mM.

The concentration of reducing agent can be about 2 mM. The concentration of reducing agent can be about 5 mM. The concentration of reducing agent can be about 10 mM. The concentration of reducing agent can be about 15 mM. The concentration of reducing agent can be about 20 mM. The concentration of reducing agent can be about 25 mM, The concentration of reducing agent can be about 30 mM. The concentration of reducing agent can be about 40 mM. The concentration of reducing agent can be about 50 mM. The concentration of reducing agent can be about 60 mM. The concentration of reducing agent can be about 70 mM. The concentration of reducing agent can be about 80 mM. The concentration of reducing agent can be about 90 mM. The concentration of reducing agent can be about 100 mM.

In some embodiments, for example, the reducing agent can comprise from about 2 mM to about 10 mM cysteine. In some ments, the reducing agent can comprise from about 2 mM to about 10 mM eine. In some embodiments, the reducing agent can comprise from about 2 mM to about 10 mM L-cysteine. In some ments, the reducing agent can comprise from about 10 mM to about 100 mM 2-mercaptoethylamine. In some embodiments, the reducing agent can comprise from about 0.2 mM to about 5 mM Tris (2- carboxyethyl) phosphine, In some embodiments, the reducing agent can comprise from about 2 mM to about 20 mM 2-mercaptoethanesulfonic acid. In some embodiments, the reducing agent can comprise from about 2 mM to about 20 mM 2-mercaptopropionic acid.

The reducing buffer can r se buffering agents such as sodium phosphate, potassium phosphate, MOPS, HEPES, sodium borate, potassium borate, or any combination thereof. Suitable buffering agent concentrations include, but are not limited to, from about 10 mM to about 100 mM, from about 15 mM to about 100 mM, from about 20 mM to about 100 mM, from about 30 mM to about 100 mM, from about 35 mM to about 100 mM, from about 40 mM to about 100 mM, from about 60 111M to about 100 mM, from about 80 mM to about 100 mM, from about 10 mM to about 90 mM, from about 10 mM to about 80 mM, from about 10 mM to about 60 mM, from about 10 mM to about 40 mM, from about 10 mM to about mM, or from about 10 mM to about 20 mM.

In some embodiments, for example, the reducing buffer can contain from about mM to about 100 mM sodium phosphate. In some ments, the reducing buffer can contain from about 10 mM to about 100 mM potassium ate. In some embodiments, the reducing buffer can contain from about 10 mM to about 100 mM MOPS. In some ments, the reducing buffer can contain from about 10 mM to about 100 mM HEPES. In some embodiments, the reducing buffer can contain from about 10 mM to about 100 mM sodium borate. In some embodiments, the reducing buffer can contain from about 10 mM to about 100 mM potassium borate.

The reducing buffer can also n a chelating agent including, but not limited to, EDTA (ethylenediaminetetraacetic acid), DTPA (diethylene triamine pentaacetic acid), or a combination thereof. Suitable concentrations of chelating agents include from about 10 mM to about 100 mM, from about 10 mM to about 80 mM, from about 10 mM to about 60 mM, from about 10 mM to about 40 mM, from about 10 mM to about 30 mM, from about 10 mM to about mM, from about 20 mM to about 100 mM, from about 30 mM to about 100 mM, from about 40 mM to about 100 mM, from about 50 mM to about 100 mM, from about 60 mM to about 100 mM, or from about 80 mM to about 100 mM.

Suitable pH ranges of the reducing buffer include from about 6.8 to about 8.0.

In some ments, the pH of the reducing buffer can be about 6.8. In some embodiments, the pH of the reducing buffer can be about 6.9. In some embodiments, the pH of the reducing buffer can be about 7.0. In some embodiments, the pH of the reducing buffer can be about 7.1.

In some embodiments, the pH of the reducing buffer can be about 7.2. In some embodiments, the pH of the reducing buffer can be about 7.3. In some ments, the pH of the ng buffer can be about 7.4. In some embodiments, the pH of the reducing buffer can be about 7.5.

In some embodiments, the pH of the ng buffer can be about 7.6. In some embodiments, the pH of the reducing buffer can be about 7.7. In some embodiments, the pH of the reducing buffer can be about 7.8. In some embodiments, the pH of the reducing buffer can be about 7.9.

In some embodiments, the pH of the reducing buffer can be about 8.0.

The immunoglobulin can be incubated with the reducing buffer for about 12 hours to about 96 hours, from about 18 hours to about 96 hours, from about 24 hours to about 96 hours, from about 30 hours to about 96 hours, from about 36 hours to about 96 hours, from about 42 hours to about 96 hours, from about 48 hours to about 96 hours, from about 54 hours to about 96 hours, from about 60 hours to about 96 hours, from about 12 hours to about 90 hours, from about 12 hours to about 84 hours, from about 12 hours to about 78 hours, from about 12 hours to about 72 hours, from about 12 hours to about 66 hours, from about 12 hours to about 60 hours, from about 12 hours to about 54 hours, from about 12 hours to about 48 hours, from about 12 hours to about 42 hours, from about 12 hours to about 36 hours, or from about 12 hours to about hours. In some embodiments, the immunoglobulin can be incubated with the reducing buffer for about 12 hours. In some embodiments, the immunoglobulin can be incubated with the ng buffer for about 18 hours. In some embodiments, the immunoglobulin can be incubated with the reducing buffer for about 24 hours. In some embodiments, the immunoglobulin can be incubated with the reducing buffer for about 30 hours. In some ments, the globulin can be incubated with the ng buffer for about 36 hours. In some embodiments, the immunoglobulin can be incubated with the reducing buffer for about 42 hours.

In some embodiments, the immunoglobulin can be incubated with the reducing buffer for about 48 hours. In some ments, the immunoglobulin can be incubated with the reducing buffer for about 54 hours. In some embodiments, the immunoglobulin can be incubated with the reducing buffer for about 60 hours. In some ments, the immunoglobulin can be incubated with the reducing buffer for about 66 hours. In some embodiments, the immunoglobulin can be incubated with the reducing buffer for about 72 hours. In some embodiments, the immunoglobulin can be incubated with the reducing buffer for about 78 hours. In some embodiments, the immunoglobulin can be incubated with the reducing buffer for about 84 hours.

In some embodiments, the immunoglobulin can be incubated with the reducing buffer for about 90 hours, In some embodiments, the immunoglobulin can be incubated with the ng buffer for about 96 hours. In some embodiments, the immunoglobulin can be incubated with the reducing buffer for greater than 96 hours.

Suitable oxidizing buffers include, but are not limited to, Tris-based, glutamine- based, arginine-based or other amino acid-based, or primary amine-based s. The concentration of oxidizing buffer can range from about 20 mM to about 100 mM, from about 40 mM to about 100 mM, from about 60 mM to about 100 mM, from about 80 mM to about 100 mM, from about 20 mM to about 80 mM, from about 20 mM to about 60 mM, or from about 20 mM to about 40 mM. The concentration of oxidizing buffer can be about 20 mM. The concentration of oxidizing buffer can be about 25 mM. The concentration of oxidizing buffer can be about 30 mM. The concentration of oxidizing buffer can be about 40 mM. The concentration of oxidizing buffer can be about 50 mM. The concentration of oxidizing buffer can be about 60 mM. The concentration of oxidizing buffer can be about 70 mM. The concentration of oxidizing buffer can be about 80 mM. The concentration of oxidizing buffer can be about 90 mM. The concentration of reducing agent can be about 100 mM, The immunoglobulin can be incubated with the oxidizing buffer for about 24 hours to about 96 hours, from about 30 hours to about 96 hours, from about 36 hours to about 96 hours, from about 42 hours to about 96 hours, from about 48 hours to about 96 hours, from about 54 hours to about 96 hours, from about 60 hours to about 96 hours, from about 24 hours to about 90 hours, from about 24 hours to about 84 hours, from about 24 hours to about 78 hours, from about 24 hours to about 72 hours, from about 24 hours to about 66 hours, from about 24 hours to about 60 hours, from about 24 hours to about 54 hours, from about 24 hours to about 48 hours, from about 24 hours to about 42 hours, or from about 24 hours to about 36 hours. In some embodiments, the immunoglobulin can be incubated with the oxidizing buffer for about 24 hours. In some embodiments, the immunoglobulin can be incubated with the oxidizing buffer for about 30 hours. In some embodiments, the immunoglobulin can be incubated with the ing buffer for about 36 hours. In some embodiments, the immunoglobulin can be incubated with the oxidizing buffer for about 42 hours. In some embodiments, the immunoglobulin can be incubated with the oxidizing buffer for about 48 hours. In some embodiments, the immunoglobulin can be incubated with the oxidizing buffer for about 54 hours. In some embodiments, the immunoglobulin can be incubated with the oxidizing buffer for about 60 hours. In some embodiments, the immunoglobulin can be incubated with the oxidizing buffer for about 66 hours. In some embodiments, the immunoglobulin can be incubated with the ing buffer for about 72 hours. In some embodiments, the immunoglobulin can be incubated with the oxidizing buffer for about 78 hours. In some embodiments, the immunoglobulin can be incubated with the oxidizing buffer for about 84 hours. In some ments, the immunoglobulin can be ted with the oxidizing buffer for about 90 hours. In some embodiments, the immunoglobulin can be incubated with the oxidizing buffer for about 96 hours. In some embodiments, the immunoglobulin can be incubated with the oxidizing buffer for greater than 96 hours. le pH ranges of the oxidizing buffer include from about 7.5 to about 9.0.

In some embodiments, the pH of the ing buffer can be about 7.5. In some embodiments, the pH of the oxidizing buffer can be about 7.6. In some embodiments, the pH of the oxidizing buffer can be about 7.7. In some embodiments, the pH of the oxidizing buffer can be about 7.8.

In some embodiments, the pH of the oxidizing buffer can be about 7.9. In some embodiments, the pH of the oxidizing buffer can be about 8.0. In some ments, the pH of the oxidizing buffer can be about 8.1. In some embodiments, the pH of the oxidizing buffer can be about 8.2.

In some ments, the pH of the oxidizing buffer can be about 8.3. In some embodiments, the pH of the oxidizing buffer can be about 8.4. In some embodiments, the pH of the oxidizing buffer can be about 8.5. In some embodiments, the pH of the oxidizing buffer can be about 8.6.

In some embodiments, the pH of the oxidizing buffer can be about 8.7. In some embodiments, the pH of the oxidizing buffer can be about 8.8. In some embodiments, the pH of the oxidizing buffer can be about 8.9. In some embodiments, the pH of the oxidizing buffer can be about 9.0.

The method can further comprise immobilizing the immunoglobulin on a matrix prior to the incubating with the reducing agent and eluting the immunoglobulin from the matrix following the incubating with the oxidizing buffer. Suitable matrices include any surface to which an immunoglobulin can be bound and eluted from including, but not limited to, protein A, protein G, protein L, ab dy, anti-Fc antibody, anti-Mab-based af?nity supports, and strong cation exchange resins. In some embodiments, the matrix can be n A. In some embodiments, the matrix can be protein G. In some embodiments, the matrix can be protein L.

In some embodiments, the matrix can comprise an anti-Fab dy. In some embodiments, the matrix can comprise an c antibody. In some embodiments, the matrix can comprise an anti-MAb. In some embodiments, the matrix can comprise a strong cation exchange resin.

The disclosed methods for decapping an immunoglobulin can comprise: brating a matrix, immobilizing the immunoglobulin onto the matrix; incubating the immobilized immunoglobulin on the matrix with a reducing buffer to remove capping group; ting the immobilized immunoglobulin on the matrix with an oxidizing buffer; g the immunoglobulin from the matrix, and neutralizing the immunoglobulin.

Those skilled in the art would recognize that the buffer, concentration, pH, and time for eluting the immunoglobulin from the matrix will depend, at least in part, on the matrix.

For example, in embodiments wherein the matrix is n A, the immunoglobulin can be eluted from the n A using glycine (for example, 0.1 M at pH 2.9). In some embodiments, the eluting can be performed in a low pH buffer.

Neutralizing the immunoglobulin can comprise incubating the immunoglobulin in a Tris-based, sodium phosphate-based, or potassium ate-based buffer n referred to as "neutralization buffer"). The neutralization buffer can have a concentration from about 0.5 M to about 2 M, and a pH from about 8.0 to about 9.5.

Conjugation can be performed by dissolving a thiol-reactive compound in a dissolution solution and ting the ved thiol-reactive compound with the immunoglobulin in a conjugation buffer.

For aqueous-insoluble thiol-reactive compounds, which may include, but are not be limited to, ide-based compounds, suitable dissolution solutions include organic, water- miscible solvents such as dimethylsulfoxide (DMSO). For aqueous—soluble thiol-reactive compounds, suitable dissolution solutions include, but are not limited to, water or buffered aqueous solutions, such as phosphate-buffered saline, pH 7.2 (1 X PBS).

Suitable concentrations of thiol-reactive compounds include from about 5 mM to about 100 mM, from about 10 mM to about 100 mM, from about 25 mM to about 100 mM, from about 40 mM to about 100 mM, from about 55 mM to about 100 mM, from about 70 mM to about 100 mM, from about 10 mM to about 90 mM, from about 10 mM to about 75 mM, from about 10 mM to about 60 mM, from about 10 mM to about 50 mM, from about 10 mM to about 40 mM, or from about 10 mM to about 30 mM. In some embodiments, the concentration of the thiol-reactive compound can be about 10 mVI. In some embodiments, the concentration of the thiol-reactive nd can be about 20 mVI. In some ments, the concentration of the thiol-reactive compound can be about 30 mVI. In some embodiments, the concentration of the thiol-reactive compound can be about 40 m\/I. In some ments, the concentration of the thiol-reactive compound can be about 50 mVI. In some embodiments, the concentration of the thiol-reactive compound can be about 60 m\/I. In some ments, the concentration of the thiol-reactive compound can be about 70 mVI. In some embodiments, the concentration of the thiol-reactive compound can be about 80 m\/I. In some embodiments, the concentration of the thiol-reactive compound can be about 90 mVI. In some embodiments, the concentration of the thiol-reactive compound can be about 100 mM.

Suitable concentrations of immunoglobulin include from about 01 mg/ml to about 20 mg/ml, from about 0.5 mg/ml to about 20 mg/ml, from about 1 mg/ml to about 20 mg/ml, from about 5 mg/ml to about 20 mg/ml, from about 10 mg/ml to about 20 mg/ml, from about 0.1 mg/ml to about 15 mg/ml, from about 0.1 mg/ml to about 12 mg/ml, from about 0.1 mg/ml to about 10 mg/ml, from about 0.1 mg/ml to about 5 mg/ml, or from about 0.1 mg/ml to about 2 mg/ml. In some embodiments, the concentration of immunoglobulin can be about 0.1 mg/ml. In some embodiments, the concentration of immunoglobulin can be about 0.5 mg/ml. In some embodiments, the concentration of globulin can be about 1 mg/ml. In some embodiments, the concentration of immunoglobulin can be about 2 mg/ml. In some embodiments, the concentration of immunoglobulin can be about 5 mg/ml. In some embodiments, the concentration of immunoglobulin can be about 10 mg/ml. In some embodiments, the concentration of immunoglobulin can be about 15 mg/ml. In some embodiments, the concentration of immunoglobulin can be about 20 mg/ml.

Suitable ratios of thiol-reactive compound:immunoglobulin include from about 3:1 to 20: 1. In some ments, the ratio of thiol-reactive compoundzimmunoglobulin can be 3: 1. In some embodiments, the ratio of thiol-reactive compound:immunoglobulin can be 4:1. In some embodiments, the ratio of thiol-reactive compound:immunoglobulin can be 5:1. In some embodiments, the ratio of thiol-reactive nd:immunoglobulin can be 6: 1. In some embodiments, the ratio of thiol-reactive compound:immunoglobulin can be 7:1. In some embodiments, the ratio of thiol-reactive compoundzimmunoglobulin can be 8:1. In some embodiments, the ratio of thiol-reactive compound:immunoglobulin can be 9:1. In some embodiments, the ratio of thiol-reactive compoundzimmunoglobulin can be 10: 1. In some embodiments, the ratio of thiol-reactive compound:immunoglobulin can be 11:1. In some embodiments, the ratio of thiol-reactive compoundzimmunoglobulin can be 12: 1. In some embodiments, the ratio of thiol-reactive compound:immunoglobulin can be 13: 1. In some embodiments, the ratio of reactive compoundzimmunoglobulin can be 14: 1. In some ments, the ratio of thiol-reactive compound:immunoglobulin can be 15: 1. In some embodiments, the ratio of thiol-reactive nd2immunoglobulin can be 16: 1. In some embodiments, the ratio of reactive compound:immunoglobulin can be 17: 1. In some embodiments, the ratio of thiol-reactive compound:immunoglobulin can be 18: 1. In some embodiments, the ratio of thiol-reactive compound:immunoglobulin can be 19: 1. In some embodiments, the ratio of thiol-reactive compound:immunoglobulin can be 20: 1.

The incubating can be performed in a number of suitable ation buffers including, for example, 1xPBS, pH 7.2, sodium phosphate, potassium phosphate, sodium borate, and HEPES, to name a few. The tration of conjugation buffer include from about 5 mM to about 100 mM, from about 10 mM to about 100 mM, from about 20 mM to about 100 mM, from about 30 mM to about 100 mM, from about 45 mM to about 100 mM, from about 60 mM to about 100 mM, from about 75 mM to about 100 mM, from about 10 mM to about 90 mM, from about 10 mM to about 75 mM, from about 10 mM to about 60 mM, from about 10 mM to about 45 mM, or from about 10 mM to about 30 mM. In some embodiments, the concentration of the conjugation buffer can be about 10 mM. In some embodiments, the concentration of the conjugation buffer can be about 20 mVI. In some embodiments, the concentration of the conjugation buffer can be about 30 mVI. In some ments, the concentration of the conjugation buffer can be about 40 mVI. In some embodiments, the concentration of the conjugation buffer can be about 50 mVI. In some embodiments, the concentration of the conjugation buffer can be about 60 mVI. In some embodiments, the concentration of the ation buffer can be about 70 mVI. In some embodiments, the concentration of the conjugation buffer can be about 80 mVI. In some embodiments, the concentration of the conjugation buffer can be about 90 mM. In some embodiments, the concentration of the conjugation buffer can be about 100 mM, The conjugation buffer can further include sodium chloride. Suitable concentrations of sodium chloride e from about 0 mM to about 500 mM, from about 25 mM to about 500 mM, from about 50 mM to about 500 mM, from about 75 mM to about 500 mM, from about 100 mM to about 500 mM, from about 150 mM to about 500 mM, from about 200 mM to about 500 mM, from about 250 mM to about 500 mM, from about 300 mM to about 500 mM, from about 350 mM to about 500 mM, from about 400 mM to about 500 mM, from about 0 mM to about 400 mM, from about 0 mM to about 350 mM, from about 0 mM to about 300 mM, from about 0 mM to about 250 mM, from about 0 mM to about 200 mM, from about 0 mM to about 150 mM, from about 0 mM to about 100 mM, from about 0 mM to about 50 mM, or from about 0 mM to about 25 mM. In some embodiments, the concentration of sodium chloride can be about 25 mM. In some embodiments, the concentration of sodium chloride can be about 50 mM. In some ments, the concentration of sodium chloride can be about 75 mM. In some embodiments, the concentration of sodium chloride can be about 100 mM. In some embodiments, the concentration of sodium de can be about 150 mM. In some embodiments, the concentration of sodium chloride can be about 200 mM, In some embodiments, the concentration of sodium chloride can be about 250 mM. In some embodiments, the concentration of sodium chloride can be about 300 mM. In some embodiments, the tration of sodium chloride can be about 350 mM. In some embodiments, the concentration of sodium chloride can be about 400 mM. In some embodiments, the concentration of sodium de can be about 500 mM.

The pH of the conjugation buffer can be from about 6.5 to about 8.5. In some embodiments, the pH of the conjugation buffer can be about 6.5. In some embodiments, the pH of the conjugation buffer can be about 6.6. In some embodiments, the pH of the conjugation buffer can be about 6.7. In some embodiments, the pH of the conjugation buffer can be about 6.8. In some embodiments, the pH of the ation buffer can be about 6.9 In some embodiments, the pH of the conjugation buffer can be about 7.0. In some embodiments, the pH of the conjugation buffer can be about 7.1. In some embodiments, the pH of the conjugation buffer can be about 7.2. In some embodiments, the pH of the conjugation buffer can be about 7.3. In some embodiments, the pH of the conjugation buffer can be about 7.4. In some ments, the pH of the conjugation buffer can be about 7.5. In some embodiments, the pH of the conjugation buffer can be about 7.6. In some embodiments, the pH of the conjugation buffer can be about 7.7. In some embodiments, the pH of the conjugation buffer can be about 7.8. In some embodiments, the pH of the conjugation buffer can be about 7.9. In some embodiments, the pH of the conjugation buffer can be about 8.0. In some embodiments, the pH of the conjugation buffer can be about 8.1. In some embodiments, the pH of the conjugation buffer can be about 8.2. In some embodiments, the pH of the conjugation buffer can be about 8.3. In some ments, the pH of the conjugation buffer can be about 8.4. In some embodiments, the pH of the ation buffer can be about 8.5.

To facilitate solubility of the thiol-reactive compound in the conjugation buffer, a ?nal tration of organic, water-miscible solvent in the conjugation buffer may be from about 0% to about 20%, from about 2% to about 20%, from about 5% to about 20%, from about 8% to about 20%, from about 11% to about 20%, from about 16% to about 20%, from about 0% to about 18%, from about 0% to about 15%, from about 0% to about 12%, from about 0% to about 10%, from about 0% to about 8%, from about 0% to about 6%, or from about 0% to about 2%.

The conjugation buffer can further comprise propylene glycol to facilitate solubility of the thiol-reactive compound in the ation buffer. Suitable concentrations of propylene glycol include from about 10% to about 50%, from about 20% to about 50%, from about 30% to about 50%, from about 40% to about 50%, from about 10% to about 40%, from about 10% to about 30%, or from about 10% to about 20%. In some embodiments, the concentration of propylene glycol can be about 10%. In some embodiments, the concentration of propylene glycol can be about 20%. In some embodiments, the tration of propylene glycol can be about 30%. In some embodiments, the concentration of propylene glycol can be about 40%. In some embodiments, the concentration of propylene glycol can be about 50%.

The conjugation buffer can r comprise a non-ionic detergent to facilitate solubility of the conjugated immunoglobulin in the conjugation buffer. Exemplary non-ionic detergents include, but are not limited to, polysorbate-20 or polysorbate-80. Suitable concentrations of non-ionic detergent include from about 0% to about 1%, from about 0.1% to about 1%, from about 0.3% to about 1%, from about 0.5% to about 1%, from about 0.7% to about 1%, from about 0% to about 0.8%, from about 0% to about 0.6%, from about 0% to about 0.4%, or from about 0% to about 0.2%. In some ments, the concentration of non-ionic detergent can be about 0.1%. In some embodiments, the tration of non-ionic detergent can be about 0.2%. In some embodiments, the concentration of non—ionic detergent can be about 0.3%. In some embodiments, the concentration of non-ionic detergent can be about 0.4%. In some ments, the concentration of non-ionic detergent can be about 0.5%. In some embodiments, the concentration of non-ionic detergent can be about 0.6%, In some embodiments, the concentration of non-ionic detergent can be about 0.7%. In some embodiments, the concentration of non-ionic detergent can be about 0.8%. In some embodiments, the concentration of non-ionic detergent can be about 0.9%. In some embodiments, the concentration of nic detergent can be about 1.0%.

The ting can be performed for about 2 hours to about 48 hours, for about 6 hours to about 48 hours, for about 12 hours to about 48 hours, for about 24 hours to about 48 hours, for about 30 hours to about 48 hours, for about 36 hours to about 48 hours, for about 42 hours to about 48 hours, for about 2 hours to about 42 hours, for about 2 hours to about 36 hours, for about 2 hours to about 30 hours, for about 2 hours to about 24 hours, for about 2 hours to about 18 hours, for about 2 hours to about 12 hours, or for about 2 hours to about 6 hours. In some embodiments, the incubating can be performed for 2 hours. In some embodiments, the incubating can be performed for 6 hours. In some embodiments, the incubating can be performed for 12 hours. In some ments, the incubating can be performed for 18 hours.

In some embodiments, the incubating can be performed for 24 hours. In some embodiments, the incubating can be performed for 30 hours. In some embodiments, the incubating can be med for 36 hours. In some embodiments, the incubating can be performed for 42 hours.

In some embodiments, the ting can be performed for 48 hours.

The temperature of the incubating can be from about 18°C to about 37°C, from about 20°C to about 37°C, from about 22°C to about 37°C, from about 24°C to about 37°C, from about 26°C to about 37°C, from about 28°C to about 37°C, from about 30°C to about 37°C, from about 32°C to about 37°C, from about 34°C to about 37°C, from about 18°C to about 34°C, from about 18°C to about 32°C, from about 18°C to about 30°C, from about 18°C to about 28°C, from about 18°C to about 26°C, or from about 18°C to about 24°C. In some ments, the incubating can be performed at 18°C. In some embodiments, the incubating can be performed at °C. In some embodiments, the incubating can be performed at 22°C. In some embodiments, the incubating can be performed at 24°C. In some embodiments, the incubating can be performed at 26°C. In some embodiments, the incubating can be performed at 28°C. In some embodiments, the incubating can be performed at 30°C. In some embodiments, the ting can be performed at 32°C. In some embodiments, the incubating can be performed at 34°C. In some embodiments, the incubating can be performed at 37°C.

Unincorporated thiol-reactive compounds can be ted from the conjugated immunoglobulin by desalting chromatography using a number of suitable resins including, but not limited to, G—25 resin, G—50 resin, Biogel P10, or other resins with exclusion limits of ranges ,000-10,000 Da. Chromatography can be performed in column format or spin-column , depending on scale. Suitable buffers for desalting include, for example, leBS, sodium phosphate, potassium phosphate, sodium borate, or HEPES—based buffers may substitute for lx In an exemplary embodiment, the conjugating can be performed by dissolving a maleimido-based thiol reactive compound in 100% dimethylsulfoxide (DMSO) at a ?nal concentration of thiol-reactive compound of 10 mM. The dissolved thiol—reactive compound can then be ted with an immunoglobulin at an immunoglobulin concentration of 5 mg/ml in leBS, pH 7.2 at a molar ratio of 5:1 thiol-reactive compoundzimmunoglobulin and mixed thoroughly. The incubating can be performed for 24 hours at 22°C. Unincorporated thiol- reactive compound can be removed from the conjugated immunoglobulin by ing chromatography using G—25 resin with 1x PBS as g buffer.

Preferably, the thiol-reactive compound is conjugated to the Cys80 Via the thiol- reactive group. Thiol-reactive groups include haloacetyls, ides, aziridines, acryloyls, arylating agents, vinylsulfones, pyridyl ides, TNB-thiols and disulfide reducing agents. In some embodiments, the thiol-reactive group can comprise a maleimide. In some embodiments, the thiol-reactive group can comprise a haloacetyl. In some embodiments, the thiol—reactive group can comprise an aziridine. In some embodiments, the thiol-reactive group can comprise an acryloyl. In some embodiments, the reactive group can se an arylating agent. In some embodiments, the reactive group can se a vinylsulfone. In some embodiments, the thiol-reactive group can comprise a pyridyl disulfide. In some embodiments, the thiol- reactive group can se a TNB-thiol. In some embodiments, the thiol-reactive group can comprise a disulfide reducing agent.

Thiol reactive groups can be derived from a number of suitable reagents including iodoacetamides, maleimides, benzylic halides and bromomethylketones, which can react by S-alkylation of thiols to generate stable thioether products.

The thiol-reactive group can be appended to a linker. Linkers can be non- ble linkers or cleavable linkers. Exemplary s include, for e, disulfide containing linkers, acetal-based linkers, and ketal-based linkers. In some aspects, the linker can be a non-cleavable linker. Suitable non-cleavable linkers include, but are not limited to, polyethylene glycol (PEG) or an alkyl. In some embodiments, the linker can se PEG. In some aspects, the linker can be a cleavable linker, Suitable cleavable linkers include, for example, valine-citrulline-para aminobenzyl. In some aspects, the linker can be a disul?de containing linker. In some aspects, the linker can be an acetal-based linker. In some aspects, the linker can be a ketal-based linker. Examples of linkers covalently appended to a thiol-reactive group are ed, for example, in US. Publ. No. 20140050746.

The thiol-reactive compound can further comprise a functional agent. Suitable functional agents include, for example, hores, ?uorescent dyes, polypeptides, immunoglobulins, antibiotics, nucleic acids, radionuclides, chemical linkers, small molecules, chelators, lipids, and drugs. In some aspects, the functional agent can comprise a ?uorophore.

In some aspects, the functional agent can comprise a ?uorescent dye. In some aspects, the functional agent can comprise a polypeptide. In some aspects, the functional agent can se an globulin. In some aspects, the functional agent can comprise an antibiotic. In some s, the functional agent can comprise a c acid (such as DNA or RNA). In some aspects, the functional agent can comprise a radionuclide. In some aspects, the functional agent can comprise a chemical linker (for example dibenzylcyclooctyne (DBCO) or azide). In some aspects, the functional agent can comprise a small molecule. In some aspects, the onal agent can comprise a chelator (for example, DOTA, CHX-A"-DTPA, NOTA, among others). In some aspects, the functional agent can comprise a lipid. In some aspects, the functional agent can se a drug. In some aspects, the functional agent can comprise a combination of any of the above listed functional agents.

The thiol-reactive compound (i.e. a ?rst thiol-reactive compound) can be bound to a second thiol-reactive compound, the second thiol-reactive compound being bound to a second immunoglobulin having a second heavy chain variable region and a second light chain variable region, the second light chain variable region having a cysteine at amino acid position 80 ("Cys802"), wherein the second thiol-reactive compound comprises a second thiol-reactive group bound to the Cy5802. For example, the ?rst thiol-reactive nd and the second thiol- reactive nds can have a ?rst and second al linker as the ?rst and second functional agents, respectively. The ?rst and second chemical linkers can be bound to each other by a number of suitable means including, for example, by click chemistry.

In red embodiments, the Cys80 can be unpaired. le means for unpairing CysSO include, for e, chimerizing a light chain variable region having Cy580 with a constant domain having an amino acid e other than cysteine at position 171.

The disclosed methods can be performed on a chimerized immunoglobulin.

Thus, in some embodiments, the immunoglobulin can be a chimerized immunoglobulin. In embodiments wherein the immunoglobulin is chimerized, the methods for generating a conjugated immunoglobulin can comprise: ing a Cys80 in a light chain variable region of a chimerized immunoglobulin, wherein the chimerized immunoglobulin comprises a heavy chain variable region and the light chain variable region; and conjugating a thiol-reactive compound to the Cys80, wherein the thiol-reactive compound comprises a thiol-reactive group.

Alternatively, the disclosed methods can r se izing an immunoglobulin prior to the decapping. For example, the methods for generating a conjugated immunoglobulin can comprise: chimerizing an immunoglobulin comprising a heavy chain le region and a light chain variable region, the light chain variable region having a Cys80, decapping the Cy580; and conjugating a thiol-reactive compound to the Cy580, wherein the thiol- reactive compound comprises a thiol-reactive group.

The disclosed s can be performed on a humanized immunoglobulin.

Thus, in some embodiments, the immunoglobulin can be a humanized immunoglobulin. In embodiments n the immunoglobulin is humanized, the methods for generating a conjugated immunoglobulin can comprise: decapping a Cys80 in a light chain variable region of a zed immunoglobulin, wherein the humanized immunoglobulin ses a heavy chain variable region and the light chain variable region; and conjugating a thiol-reactive compound to the Cys80, n the thiol-reactive compound comprises a reactive group.

Alternatively, the disclosed methods can further comprise humanizing an immunoglobulin prior to the decapping. For example, the methods for generating a conjugated immunoglobulin can comprise: humanizing an immunoglobulin comprising a heavy chain variable region and a light chain variable region, the light chain variable region having a Cys80, decapping the Cys80; and conjugating a thiol-reactive compound to the Cy580, wherein the thiol- reactive nd comprises a thiol-reactive group.

The methods can further comprise substituting an amino acid at position 83 with an amino acid e other than Phe, Lys, or Cys. In some aspects, the methods can se substituting the phenylalanine at position 83 of the light chain variable region with alanine ("Ala83"). In some aspects, the methods can comprise substituting the alanine at position 83 of the light chain variable region with valine 3"). In some aspects, the methods can comprise substituting the phenylalanine at position 83 of the light chain variable region with isoleucine ("Ile83"). In some s, the methods can comprise substituting the phenylalanine at position 83 of the light chain variable region with threonine ("Thr83"). In some aspects, the methods can comprise substituting the phenylalanine at position 83 of the light chain variable region with arginine ("Arg83"). In some aspects, the methods can comprise substituting the phenylalanine at position 83 of the light chain variable region with asparagine ("Asn83"). In some aspects, the methods can comprise substituting the phenylalanine at position 83 of the light chain variable region with aspartic acid ("Asp83"). In some aspects, the methods can comprise substituting the alanine at position 83 of the light chain variable region with glutamic acid ("Glu83"). In some aspects, the methods can comprise substituting the phenylalanine at on 83 of the light chain variable region with ine 3"). In some aspects, the methods can comprise substituting the phenylalanine at position 83 of the light chain variable region with glycine ("Gly 83"). In some aspects, the methods can comprise substituting the phenylalanine at position 83 of the light chain variable region with histidine ("His83")r In some aspects, the s can comprise substituting the alanine at position 83 of the light chain variable region with leucine ("Leu83"). In some aspects, the methods can comprise tuting the phenylalanine at position 83 of the light chain variable region with nine 3"). In some aspects, the methods can comprise substituting the phenylalanine at position 83 of the light chain variable region with e ("Pro83"). In some aspects, the methods can comprise substituting the phenylalanine at position 83 of the light chain variable region with serine ("Ser83"). In some s, the methods can comprise substituting the phenylalanine at position 83 of the light chain variable region with tryptophan ("Trp83"). In some aspects, the methods can se substituting the phenylalanine at position 83 of the light chain variable region with tyrosine 3"). In some embodiments, the methods can comprise substituting an amino acid at on 83 with a polar or hydrophobic amino acid including, but not limited to, alanine, valine, isoleucine, or threonine.

The amino acid residue other than Phe, Lys, or Cys at amino acid position 83 in ation with the disclosed decapping methods can decrease the aggregation, and increase the Cys80 conjugation ef?ciency, of the globulin. Suitable immunoglobulin aggregation achieved by the disclosed methods include, for example, less than about 5%, less than about 7%, less than about 10%, less than about 12%, less than about 15%, less than about 17%, less than about 20%, less than about 22%, or less than about 25%. Suitable conjugation ef?ciencies achieved by the disclosed methods include, for example, greater than about 70%, greater than about 73%, greater than about 76%, greater than about 79%, greater than about 82%, greater than about 85%, greater than about 88%, greater than about 91%, greater than about 94%, greater than about 97%, or greater than about 99%.

Methods ofgenerating antigen-binding molecules Also provided herein are methods for ting an antigen-binding molecule, the method comprising incubating a ?rst conjugated immunoglobulin with a second conjugated immunoglobulin to generate the antigen-binding molecule, wherein: the ?rst ated immunoglobulin ses a ?rst heavy chain variable region and a ?rst light chain variable region, the ?rst light chain variable region having a cysteine at position 80 ("Cys801") wherein Cys801 is conjugated to a ?rst thiol-reactive compound comprising a ?rst thiol-reactive group, and the second conjugated immunoglobulin comprises a second heavy chain le region and a second light chain variable region, the second light chain variable region having a ne at position 80 ("Cys802") n Cys802 is conjugated to a second thiol-reactive compound comprising a second thiol-reactive group.

Antigen-binding molecules include multivalent and/or multispeci?c antigenbinding molecules. For example, antigen-binding molecules include bivalent, trivalent, and tetravalent antigen-binding molecules that are monospeci?c or bispeci?c. In some s, the antigen-binding molecule can be bivalent and monospeci?c. In some aspects, the antigen- g molecule can be bivalent and i?c. In some s, the antigen-binding le can be trivalent and monospeci?c. In some aspects, the antigen-binding molecule can be trivalent and bispeci?c. In some aspects, the antigen-binding molecule can be tetravalent and monospeci?c. In some aspects, the antigen-binding molecule can be tetravalent and bispeci?c.

In some aspects, the valency can be greater than tetravalent. In some aspects, the speci?city can be greater than bispeci?c.

The Cys801, the Cys802, or both, can be unpaired. Suitable means for unpairing Cys80 include, for example, chimerizing a light chain variable region having Cys80 with a constant domain having an amino acid residue other than ne at position 171.

In some aspects, the methods can further comprise decapping the Cys801. In some aspects, the methods can further comprise decapping the Cys802. In other aspects, the methods can further comprise decapping the Cys801 and Cys802.

The ing can comprise incubating the ?rst immunoglobulin, the second immunoglobulin, or both, with a ng buffer followed by incubating the ?rst immunoglobulin, the second immunoglobulin, or both, with an oxidizing buffer. In some aspects of the methods for generating n-binding molecules, the decapping can further comprise lizing the ?rst immunoglobulin, the second immunoglobulin, or both on a matrix prior to the incubating with the reducing buffer and eluting the ?rst immunoglobulin, the second immunoglobulin, or both from the matrix following the incubating with the oxidizing buffer.

Suitable ing and conjugating conditions, including reducing buffers, oxidizing buffers, concentrations, pHs, times and matrices, are disclosed in the section entitled "generation ofconjugated immunoglobulins" and are equally applicable herein.

In some aspects, the methods can further comprise conjugating a ?rst thiol- reactive compound to the Cys801, n the ?rst thiol-reactive compound comprises a ?rst thiol-reactive group. In some aspects, the s can further comprise conjugating a second thiol-reactive compound to the Cys802, wherein the second thiol-reactive compound comprises a second thiol-reactive group. In yet other aspects, the methods can further comprise conjugating a ?rst reactive compound to the Cys80l and a second thiol-reactive compound to the , wherein the ?rst thiol-reactive compound ses a ?rst thiol-reactive group and the second thiol-reactive compound comprises a second thiol-reactive group.

The methods can further comprise both decapping and ating. For example, the methods can further comprise, prior to the incubating step, decapping the CysSOl, Cy5802, or both; and conjugating a ?rst thiol-reactive compound to the Cys801, a second thiol-reactive compound to the Cy5802, or both, wherein the ?rst thiol-reactive compound comprises a ?rst thiol-reactive group and the second thiol-reactive compound comprises a second thiol-reactive group.

The ?rst immunoglobulin, the second immunoglobulin, or both, can be chimerized. Conversely, the s can r comprise chimerizing the ?rst immunoglobulin, izing the second immunoglobulin, or chimerizing both the ?rst immunoglobulin and the second immunoglobulin. For e, and without intending to be limiting, the methods can further comprise, prior to the incubating step: chimerizing a ?rst immunoglobulin comprising a Cys801 to generate a ?rst chimeric immunoglobulin; chimerizing the second immunoglobulin comprising a Cys802 to te a second chimeric immunoglobulin, ing the Cys801, Cys802, or both, and conjugating a ?rst thiol-reactive compound to the Cys801, a second thiol-reactive compound to the Cys802, or both, wherein the ?rst reactive compound comprises a ?rst thiol-reactive group and the second thiol-reactive compound comprises a second thiol-reactive group.

The ?rst immunoglobulin, the second immunoglobulin, or both, can be humanized. Conversely, the s can r comprise humanizing the ?rst immunoglobulin, humanizing the second immunoglobulin, or humanizing both the ?rst immunoglobulin and the second immunoglobulin. For example, and without intending to be limiting, the methods can further comprise, prior to the ting step: humanizing a ?rst globulin comprising a Cys801 to generate a ?rst humanized immunoglobulin, humanizing the second globulin comprising a Cys802 to generate a second humanized immunoglobulin; decapping the CysSOl, Cy5802, or both, and conjugating a ?rst thiol-reactive compound to the CysSOl, a second thiol-reactive compound to the Cys802, or both, wherein the ?rst thiol-reactive compound comprises a ?rst thiol-reactive group and the second thiol-reactive compound comprises a second thiol-reactive group.

Preferably, the ?rst and second reactive nds are conjugated to the Cys801 and Cys802, respectively, via the ?rst thiol-reactive group and the second thiol-reactive group. Suitable, reactive groups include haloacetyls, maleimides, aziridines, acryloyls, arylating agents, vinylsulfones, pyridyl des, iols and de reducing agents. In some embodiments, the ?rst thiol-reactive group, the second-thiol reactive group, or both, can comprise a maleimide. In some embodiments, the ?rst thiol-reactive group, the second-thiol reactive group, or both, can comprise a haloacetyl. In some embodiments, the ?rst thiol-reactive group, the second-thiol reactive group, or both, can comprise an aziridine. In some embodiments, the ?rst thiol-reactive group, the second-thiol reactive group, or both, can comprise an acryloyl. In some embodiments, the ?rst thiol-reactive group, the second-thiol reactive group, or both, can comprise an arylating agent. In some embodiments, the ?rst thiol- reactive group, the second-thiol reactive group, or both, can comprise a Vinylsulfone. In some embodiments, the ?rst thiol-reactive group, the second-thiol reactive group, or both, can comprise a pyridyl disul?de. In some embodiments, the ?rst thiol-reactive group, the second— thiol reactive group, or both, can comprise a TNB—thiol. In some embodiments, the ?rst thiol- reactive group, the -thiol reactive group, or both, can comprise a disul?de reducing agent.

The ?rst thiol-reactive group, the second-thiol reactive group, or both can be appended to a linker. In some aspects, the ?rst thiol—reactive group can be appended to a linker ("?rst linker"). In some aspects, the second thiol-reactive group can be appended to a linker ("second linker"). In yet other aspects the ?rst thiol—reactive group can be appended to a ?rst linker and the second thiol-reactive group can be appended to a second linker. Suitable ?rst and second linkers can be non-cleavable linkers or cleavable linkers. Exemplary ?rst and second linkers include, for example, disul?de containing linkers, acetal-based linkers, and ketal-based linkers. In some aspects, the ?rst linker, second linker, or both, can be a non-cleavable linker.

Suitable non-cleavable linkers e, but are not limited to, polyethylene glycol (PEG) or an alkyl. In some embodiments, the ?rst linker, second linker, or both, can comprise PEG. In some aspects, the ?rst linker, second linker, or both, can be a cleavable linker. Suitable cleavable linkers e, for example, valine-citrulline-para aminobenzyl. In some aspects, the ?rst linker, second , or both, can be a disul?de containing linker. In some aspects, the ?rst linker, second , or both can be an acetal-based linker. In some aspects, the ?rst linker, second linker, or both, can be a based linker. Examples of s covalently ed to a thiol-reactive group are provided, for example, in US. Pub]. No. 20140050746.

The ?rst thiol-reactive compound, the second reactive compound, or both, can further comprise a functional agent. In some aspects, the ?rst thiol-reactive compound can further comprise a functional agent ("?rst functional agent"). In some aspects, the second thiol- reactive compound can further comprise a functional agent ("second functional agent"). In yet other aspects, the ?rst thiol-reactive compound can further se a ?rst functional agent and the second thiol-reactive compound can further comprise a second functional agent.

Suitable functional agents include, for example, al s. Preferably, the chemical linker of the ?rst thiol-reactive compound ("?rst chemical linker") and the chemical linker of the second thiol—reactive compound ("second chemical linker") can be d. For example, and without intent to be limiting, one of the ?rst or second chemical s can be dibenzylcyclooctyne (DBCO) and the other of the ?rst or second chemical s can be azide. In some ments, for example, the ?rst chemical linker can be DBCO and the second chemical linker can be azide. Conversely, the ?rst chemical linker can be azide and the second chemical linker can be DBCO. The DBCO and azide can be coupled, this resulting in the conjugation of the ?rst immunoglobulin and the second immunoglobulin. For example, the ?rst immunoglobulin and the second immunoglobulin can be ated to each other by click chemistry.

In an exemplary embodiment, thiol-reactive compounds can include maleimido- PEG4-azide and maleimido—PEG4—dibenzocyclooctyne. In some aspects, for example, the ?rst thiol-reactive compound can be maleimido-PEG4-azide and the second thiol-reactive compound can be maleimido-PEG4-dibenzocyclooctyne. In some aspects, the ?rst reactive compound can be maleimido—PEG4-dibenzocyclooctyne and the second thiol-reactive compound can be maleimido-PEG4-azide.

The ?rst immunoglobulin, second immunoglobulin, or both, can be Fabs. In some embodiments, the ?rst immunoglobulin can be a Fab ("?rst Fab"). In some embodiments, the second immunoglobulin can be a Fab ("second Fab"). In yet other embodiments, the ?rst immunoglobulin can be a ?rst Fab and the second immunoglobulin can be a second Fab.

In some embodiments, the methods comprise generating a ?rst Fab, a second Fab, or both, prior to the incubating. Suitable techniques for generating Fabs are known in the art and include, for example, digesting a full or partial immunoglobulin to produce a Fab or inantly sing the immunoglobulin as a Fab. For example, the methods of generating antigen-binding molecules can further comprise, prior to the incubating, ing a ?rst immunoglobulin, a second immunoglobulin, or both, with papain to generate a ?rst Fab, second Fab, or ?rst and second Fab, wherein the ?rst immunoglobulin comprises a ?rst heavy chain and a ?rst light chain, the ?rst light chain having a cysteine at position 80 ("Cys801"), and wherein the second immunoglobulin ses a second heavy chain and a second light chain, the second light chain having a cysteine at position 80 ("CysSO2"); or recombinantly expressing a ?rst Fab comprising a ?rst heavy chain and a ?rst light chain having a cysteine at position 80 (CysSOl), inantly expressing a second Fab comprising a second heavy chain and a second light chain having a cysteine at position 80 Z), or both; and conjugating the ?rst Fab at Cys801 to a ?rst thiol-reactive compound to generate a ?rst conjugated Fab, conjugating the second Fab at Cys802 to a second thiol- ve compound to generate a second conjugated Fab, or both.

The s of generating antigen-binding molecules can further comprise substituting an amino acid at position 83 of the ?rst light chain variable region with an amino acid residue other than Phe, Lys, or Cys. The methods of generating antigen-binding molecules can further comprise substituting an amino acid at on 83 of the second light chain variable region with an amino acid residue other than Phe, Lys, or Cys. The methods of generating antigen-binding molecules can further comprise substituting an amino acid at position 83 of the ?rst light chain le region with an amino acid e other than Phe, Lys, or Cys and substituting an amino acid at position 83 of the second light chain variable region with an amino acid residue other than Phe, Lys, or Cys.

In some aspects, the methods can se substituting an amino acid at position 83 of the ?rst light chain variable region, substituting an amino acid at position 83 of the second light chain variable region, or substituting an amino acid at position 83 of the ?rst light chain variable region and the second light chain variable region with alanine 3"), valine ("Val83"), isoleucine ("Ile83"), threonine ("Thr83"), arginine ("Arg83"), asparagine ("Asn83"), aspartic acid ("Asp83"), glutamic acid ("Glu83"), glutamine 3"), glycine ("Gly83"), histidine ("His83"), leucine ("Leu83"), methionine ("Met83"), proline ("Pro83"), serine ("Ser83"), tryptophan ("Trp83"), or tyrosine ("Tyr83").

The amino acid at position 83 in the ?rst light chain variable region can be the same as the amino acid at position 83 in the second light chain variable region. Conversely, theamino acid at position 83 in the ?rst light chain variable region region can be different from the polar or hydrophobic amino acid at position 83 in the second light chain variable region. The amino acid at position 83 other than Phe, Lys, or Cys in the ?rst light chain variable region and/or the amino acid at position 83 other than Phe, Lys, or Cys in the second light chain variable region can be a polar or hydrophobic amino acid including, but not limited to, alanine, , isoleucine, or threonine. In some aspects, the methods can comprise substituting an amino acid at position 83 of the ?rst light chain variable , substituting an amino acid at position 83 of the second light chain variable region, or substituting an amino acid at position 83 of the ?rst light chain variable region and the second light chain variable region with valine ("Val83"). In some aspects, the s can se substituting an amino acid at position 83 of the ?rst light chain variable region, substituting an amino acid at position 83 of the second light chain variable region, or substituting an amino acid at position 83 of the ?rst light chain variable region and the second light chain variable region with isoleucine ("Ile83"). In some aspects, the s can comprise substituting an amino acid at position 83 of the ?rst light chain variable region, substituting an amino acid at position 83 of the second light chain variable region, or substituting an amino acid at position 83 of the ?rst light chain variable region and the second light chain variable region with threonine ("Thr83"). The polar or hydrophobic amino acid at position 83 in the ?rst light chain variable region region can be the same as or different from the polar or hydrophobic amino acid at position 83 in the second light chain variable region.

Suitable light chain variable regions include, for example, a kappa light chain variable region. In some embodiments, the Cys801, , or both, can be present in the native light chain variable region. The ?rst light chain variable region and second light chain le region are d from rabbit. Exemplary rabbits from which a ?rst light chain variable region, second light chain variable region, or both, can be derived from include, but are not limited to, lagus cum‘culus. In some aspects, for example, the light chain variable region(s) can be derived from a New Zealand White (NZW) rabbit. In other s, the light chain variable region(s) can be derived from a b9 rabbit.

Immunoglobulm components ofconjugated immunoglobulins Disclosed herein are immunoglobulins comprising a heavy chain variable region and a light chain le region, the light chain variable region having a cysteine at position 80 ("Cys80") and an amino acid other than Phe, Lys, or Cys at position 83.

Suitable light chain variable regions include, for example, a kappa light chain variable region. The light chain variable region is derived from rabbit. In some ments, the Cys80 can be present in the native light chain le region of the rabbit immunoglobulin.

Exemplary rabbits from which a light chain variable region having a Cys80 can be derived include, but are not limited to, Oryctolagus cuniculus. In some aspects, for example, the light chain variable region can be derived from aNew Zealand White (NZW) rabbit. In other s, the light chain le region can be derived from a b9 rabbit.

The amino acid other than Phe, Lys, or Cys at position 83 es alanine ("Ala83"), valine ("Val83"), isoleucine ("Ile83"), threonine ("Thr83"), arginine ("Arg83"), asparagine ("Asn83"), aspartic acid ("Asp83"), glutamic acid ("Glu83"), ine ("Gln83"), glycine ("Gly83"), histidine ("His83"), leucine ("Leu83"), methionine ("Met83"), proline ("Pro83"), serine ("Ser83"), tryptophan ("Trp83"), or ne 3").

In some embodiments, the amino acid other than Phe, Lys, or Cys at position 83 can be a polar or hydrophobic amino acid including, but not limited to, alanine, valine, isoleucine, or threonine. In some aspects, the polar or hydrophobic amino acid other than Phe at position 83 is alanine ("Ala83"). In some aspects, the polar or hydrophobic amino acid other than Phe at position 83 is valine 3"). In some aspects, the polar or hydrophobic amino acid other than Phe at position 83 is isoleucine ("Ile83"). In some aspects, the polar or hydrophobic amino acid other than Phe at position 83 is threonine ("Thr83").

The Cys80 can be unpaired. For example, a light chain variable region having CysSO can be chimerized with a constant domain having an amino acid residue other than cysteine at on 171.

Preferably, the CysSO is decapped.

In some embodiments, the immunoglobulins can be chimerized. In other embodiments, the immunoglobulins can be humanized.

In some embodiment, the disclosed immunoglobulin immunospeci?cally binds to human CA9. In some ments, the immunoglobulin that immunospeci?cally binds to human CA9 comprises: a. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-141 of xi155D5HC (SEQ ID NO:52) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-130 of Xi155D5LC (SEQ ID NO:78), b. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-144 of zu155D5HC (SEQ ID NO:54) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-130 of zu155D5LC-3 (SEQ ID N084), 5LC-4 (SEQ ID NO:86), zu155D5LC-5 (SEQ ID N088), zu155D5LC-6 (SEQ ID N090), zu155D5LC-7 (SEQ ID N092), zu155D5LC-huVK2—40 (SEQ ID NO:96), zu155D5LC-huVK4-1 (SEQ ID NO: 100), zu155D5LC-huVK6-21 (SEQ ID NO: 102), zul 55D5LC-huVK6D-4l (SEQ ID NO:104); or zu155D5LC-huVK7Glu81 (SEQ ID NO:106), c. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-138 ofxi1E4HC (SEQ ID NO:58) and a light chain le region having an amino acid ce at least 90% identical to amino acids 20-130 of C (SEQ ID NO:110); d. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-140 of zu1E4HC (SEQ ID N060) and a light chain variable region having an amino acid sequence at least 90% cal to amino acids 20-130 of zu1E4LC-CXXA (SEQ ID NO:114), e. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-142 of xi166B3HC (SEQ ID N074) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-130 of xi166B3LC (SEQ ID NO:132); or f. a heavy chain variable region having an amino acid sequence at least 90% cal to amino acids 20-145 of zul 66B3HC (SEQ ID NO:76) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-130 of zul 66B3LC-CXXA (SEQ ID N01136).

In some embodiments, the immunoglobulin that immunospecifically binds to human CA9 comprises: a. a heavy chain variable region as set forth as amino acids 20-141 of Xi155D5HC (SEQ ID NO:52) and a light chain variable region as set forth as amino acids 20-130 of Xi155D5LC (SEQ ID NO:78), b. a heavy chain variable region as set forth as amino acids 20-144 of zu155D5HC (SEQ ID NO:54) and a light chain variable region as set forth as amino acids 20-130 of zu155D5LC-3 (SEQ ID N084), zu155D5LC-4 (SEQ ID NO:86), zu155D5LC-5 (SEQ ID NO:88), zu155D5LC-6 (SEQ ID N090), zu155D5LC-7 (SEQ ID N092), zu155D5LC-huVK2-40 (SEQ ID NO:96), zu155D5LC-huVK4-1 (SEQ ID NO:100), zulSSDSLC-huVK6-21 (SEQ ID NO: 102), zu155D5LC—huVK6D-4l (SEQ ID NO:104), or zulSSDSLC-huVK7Glu8l (SEQ ID NO:106), c. a heavy chain variable region as set forth as amino acids 20-138 ofxi1E4HC (SEQ ID NO:58) and a light chain variable as set forth as amino acids 20-130 of Xi1E4LC (SEQ ID ), d. a heavy chain le region as set forth as amino acids 20-140 of C (SEQ ID NO:60) and a light chain variable region as set forth as amino acids 20-130 of zu1E4LC-CXXA (SEQ ID NO:114), e. a heavy chain variable region as set forth as amino acids 20-142 of x1166B3HC (SEQ ID N074) and a light chain variable region as set forth as amino acids 20-130 of xi166B3LC (SEQ ID N02132); or f. a heavy chain variable region as set forth as amino acids 20-145 of 3HC (SEQ ID NO:76) and a light chain variable region as set forth as amino acids 20-130 of zul 66B3LC-CXXA (SEQ ID NO: 136).

In some ments, the immunoglobulin that immunospecifically binds to human CA9 comprises: a. a heavy chain CDRI, CDR2, and CDR3 of 5HC as set forth as SEQ ID NO:146, 148, and 150, respectively, and a light chain CDRl, CDR2, and CDR3 of Xi155D5LC as set forth as SEQ ID NO:224, 226, and 228, respectively; b. a heavy chain CDRl, CDR2, and CDR3 of zul 55D5HC as set forth as SEQ ID NO:152, 154, and 156, respectively, and a light chain CDRl, CDR2, and CDR3 of zu155D5LC-3 as set forth as SEQ ID NO:242, 244, and 246, respectively, 5LC-4 as set forth as SEQ ID NO:248, 250, and 252, respectively, zu155D5LC-5 as set forth as SEQ ID NO:254, 256, and 258, respectively, zu155D5LC-6 as set forth as SEQ ID NO:260, 262, and 264, respectively, zu155D5LC—7 as set forth as SEQ ID NO:266, 268, and 270, respectively, zu155D5LC-huVK2-4O as set forth as SEQ ID NO 278, 280, and 282, tively, zu155D5LC-huVK4-1 as set forth as SEQ ID NO 290, 292, and 294, respectively, zu155D5LC-huVK6-2l as set forth as SEQ ID NO 296, 298, and 300, respectively, zu155D5LC-huVK6D-4l as set forth as SEQ ID NO 302, 304, and 306, respectively, or zu155D5LC-huVK7Glu8l as set forth as SEQ ID NO 308, 310, and 312, respectively; c. a heavy chain CDRl, CDR2, and CDR3 of Xi1E4HC as set forth as SEQ ID NO 164, 166, and 168, tively and alight chain CDRl, CDR2, and CDR3 of Xi1E4LC as set forth as SEQ ID NO 320, 322, and 324, respectively; d. a heavy chain CDRl, CDR2, and CDR3 of zu1E4HC as set forth as SEQ ID , 172, and 174, respectively, and a light chain CDRl, CDR2, and CDR3 of C- CXXA as set forth as SEQ ID NO:332, 334, and 336, respectively, e. aheavy chain CDRl, CDR2, and CDR3 of xi166B3HC as set forth as SEQ ID NO:212, 214, and 216, respectively and a light chain CDRl, CDR2, and CDR3 of xi166B3LC as set forth as SEQ ID NO:386, 388, and 390, respectively; or f. aheavy chain CDRl, CDR2, and CDR3 of zu166B3HC as set forth as SEQ ID NO:218, 220, and 222, respectively, and a light chain CDRl, CDR2, and CDR3 of zu166B3LC-CXXA as set forth as SEQ ID NO:398, 400, and 402, respectively.

In some embodiments, the disclosed immunoglobulins immunospecifically bind to human TEMl. In some embodiments, the immunoglobulin that immunospecifically binds to human TEMI comprises a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-139 oinl2HC (SEQ ID N056) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-129 of Xi1 2LC (SEQ ID NO:108).

In some embodiments, the immunoglobulin that immunospecifically binds to human TEMI ses a heavy chain variable region as set forth as amino acids 20-139 of xil- 55-2HC (SEQ ID NO:56) and a light chain le region as set forth as amino acids 20-129 of Xil—55-2LC (SEQ ID NO: 108).

In some embodiments, the immunoglobulin that immunospecifically binds to human TEMI comprises a heavy chain CDRI, CDR2, and CDR3 of xil-SS-ZHC as set forth as SEQ ID NO: 158, 160, and 162, respectively, and a light chain CDR1, CDR2, and CDR3 of Xil- 55-2LC as set forth as SEQ ID NO:314, 316, and 318, respectively.

In some embodiments, the disclosed immunoglobulins immunospecifically bind to human mesothelin. In some embodiments, the immunoglobulin that immunospeci?cally binds to human mesothelin ses: a. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-142 of xi3301 1HC (SEQ ID NO:62) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-131 of xi33011LC (SEQ ID NO:116); b. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-145 of zu3301 1HC (SEQ ID NO:64) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-131 of zu330l lLC-CXXA (SEQ ID NO: 120) or zu33011LC-CXXI (SEQ ID NO: 122); c. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-137 of xi32405HC (SEQ ID NO:66) and a light chain variable region having an amino acid ce at least 90% identical to amino acids 20-127 of xi32405LC (SEQ ID NO: 124); d. a heavy chain variable region having an amino acid ce at least 90% identical to amino acids 20-137 of xi178F16HC (SEQ ID N068) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-127 of xi178F16LC (SEQ ID N02126); e. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-132 of 18HC (SEQ ID N070) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-127 of Xi237N18LC (SEQ ID NO:128); or f a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-137 of xi383118HC (SEQ ID NO:72) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-127 of Xi383118LC (SEQ ID ).

In some embodiments, the immunoglobulin that specifically binds to human mesothelin comprises: a. a heavy chain variable region as set forth as amino acids 20-142 of Xi3301 IHC (SEQ ID NO:62) and a light chain variable region as set forth as amino acids 20-131 of Xi33011LC (SEQ ID NO:116); b. a heavy chain variable region as set forth as amino acids 20-145 of zu3301 lHC (SEQ ID NO:64) and a light chain variable region as set forth as amino acids 20-131 of zu330l lLC-CXXA (SEQ ID NO: 120) or zu330l lLC-CXXI (SEQ ID N02122); c. a heavy chain variable region as set forth as amino acids 20-137 of Xi32405HC (SEQ ID NO:66) and a light chain variable region as set forth as amino acids 20-127 of Xi32405LC (SEQ ID NO:124); d. a heavy chain le region as set forth as amino acids 20-137 of Xi178F16HC (SEQ ID N068) and a light chain variable region as set forth as amino acids 20-127 of Xi178F16LC (SEQ ID NO:126), e. a heavy chain le region as set forth as amino acids 20-132 of Xi237N18HC (SEQ ID N070) and a light chain variable region as set forth as amino acids 20-127 of Xi237N18LC (SEQ ID NO:128); or f. a heavy chain variable region as set forth as amino acids 20-137 ofxi383118HC (SEQ ID NO:72) and a light chain variable region as set forth as amino acids 20-127 of Xi383118LC (SEQ ID NO:130).

In some embodiments, the immunoglobulin that immunospeci?cally binds to human mesothelin comprises: a. a heavy chain CDRI, CDR2, and CDR3 oin33011HC as set forth as SEQ ID NO: 176, 178, and 180, respectively, and a light chain CDRl, CDR2, and CDR3 of xi33011LC as set forth as SEQ ID , 340, and 342, respectively; b, a heavy chain CDRl, CDR2, and CDR3 of zu3301 lHC as set forth as SEQ ID NO:182, 184, and 186, respectively, and a light chain CDRl, CDR2, and CDR3 of zu3301 1LC-CXXA as set forth as SEQ ID NO:350, 352, and 354, tively or zu330l 1LC-CXXI as set forth as SEQ ID NO:356, 358, and 360, respectively, c. a heavy chain CDRl, CDR2, and CDR3 of 5HC as set forth as SEQ ID NO:188, 190, and 192, respectively, and a light chain CDRl, CDR2, and CDR3 of xi32405LC as set forth as SEQ ID NO:362, 364, and 366, respectively; d. a heavy chain CDRl, CDR2, and CDR3 of xi178F16HC as set forth as SEQ ID NO:194, 196, and 198, respectively, and a light chain CDRl, CDR2, and CDR3 of xi178F16LC as set forth as SEQ ID NO:368, 370, and 372, tively; e. a heavy chain CDRl, CDR2, and CDR3 of xi237N18HC as set forth as SEQ ID NO:200, 202, and 204, respectively, and a light chain CDRl, CDR2, and CDR3 of xi237N18LC as set forth as SEQ ID , 376, and 378, respectively; or f. a heavy chain CDRl, CDR2, and CDR3 ofxi383118HC as set forth as SEQ ID NO:206, 208, and 210, respectively, and a light chain CDRl, CDR2, and CDR3 of xi383Il8LC as set forth as SEQ ID , 382, and 384, respectively.

Conjugated immunoglobulins Also disclosed herein are conjugated immunoglobulins comprising any of the immunoglobulins disclosed herein, wherein the cysteine at position 80 ("Cys80") is conjugated to a thiol-reactive nd, the thiol-reactive compound comprising a thiol—reactive group.

In some embodiments, the conjugated immunoglobulins se an immunoglobulin comprising a heavy chain variable region and a light chain variable region, the light chain variable region having a Cys80 and an amino acid other than Phe, Lys, or Cys at position 83, wherein Cys80 is conjugated to a thiol-reactive compound, the thiol-reactive nd comprising a thiol-reactive group. In some embodiments, the light chain variable region can have a Cys80 and a polar or hydrophobic amino acid other than Phe, Lys, or Cys at position 83.

The immunoglobulin comprises a heavy chain variable region and a light chain variable region. Suitable light chain variable regions include, for example, a kappa light chain variable region. The light chain variable region is derived from rabbit. In some embodiments, the Cys80 can be present in the native light chain variable region of the rabbit immunoglobulin.

Exemplary s from which a light chain variable region having a Cys80 can be derived include, but is not limited to, Oryctolagus lus. In some aspects, for example, the light chain variable region can be derived from a New Zealand White (NZW) rabbit. In other s, the light chain variable region can be derived from a b9 rabbit.

The light chain le region can have a Cys80 and an amino acid other than Phe, Lys, or Cys at position 83. The amino acid other than Phe, Lys, or Cys at position 83 includes alanine ("Ala83"), valine ("Val83"), isoleucine ("Ile83"), threonine ("Thr83"), arginine ("Arg83"), gine ("Asn83"), aspartic acid ("Asp83"), ic acid ("Glu83"), glutamine ("Gln83"), glycine ("Gly83"), histidine ("His83"), leucine ("Leu83"), methionine ("Met83"), proline ("Pro83"), serine ("Ser83"), tryptophan ("Trp83"), or tyrosine ("Tyr83"). In some embodiments, the light chain variable region can have a Cys80 and a polar or hydrophobic amino acid other than Phe, Lys, or Cys at position 83. le polar or hydrophobic amino acids include, but are not limited to, e, , isoleucine, or threonine. In some s, the polar or hydrophobic amino acid other than Phe at position 83 is alanine ("Ala83"). In some aspects, the polar or hydrophobic amino acid other than Phe at position 83 is valine ("Val83").

In some aspects, the polar or hydrophobic amino acid other than Phe at position 83 is isoleucine ("Ile83"). In some s, the polar or hydrophobic amino acid other than Phe at position 83 is threonine ("Thr83").

The Cys80 can be unpaired. For example, the light chain variable region having Cys80 can be chimerized with a constant domain having an amino acid residue other than cysteine at position 171.

Preferably, the Cys80 is ed.

In some embodiments, the immunoglobulin can be chimerized. In other embodiments, the immunoglobulin can be humanized.

Preferably, the thiol—reactive compound is conjugated to the Cys80 via the thiol- ve group. Thiol-reactive groups include haloacetyls, maleimides, aziridines, acryloyls, arylating agents, ulfones, pyridyl disul?des, TNB-thiols and disul?de reducing agents. In some embodiments, the thiol-reactive group can comprise a maleimide. In some embodiments, the thiol-reactive group can se a haloacetyl. In some embodiments, the thiol-reactive group can comprise an aziridine. In some embodiments, the thiol-reactive group can comprise an acryloyl. In some ments, the thiol-reactive group can comprise an arylating agent. In some embodiments, the thiol-reactive group can comprise a vinylsulfone. In some embodiments, the thiol—reactive group can comprise a pyridyl ide. In some embodiments, the thiol- reactive group can comprise a TNB-thiol. In some embodiments, the thiol-reactive group can comprise a disul?de reducing agent.

The thiol-reactive group can be appended to a . Linkers can be non- cleavable linkers or cleavable linkers. Exemplary linkers include, for example, disul?de containing linkers, acetal—based linkers, and ketal-based linkers. In some aspects, the linker can be a non-cleavable linker. le eavable s include, but are not limited to, polyethylene glycol (PEG) or an alkyl. In some embodiments, the linker can comprise PEG. In some aspects, the linker can be a cleavable linker. Suitable cleavable linkers include, for e, valine-citrulline-para enzyl. In some aspects, the linker can be a disulfide containing linker. In some aspects, the linker can be an acetal-based linker. In some aspects, the linker can be a ketal-based linker. Examples of linkers covalently appended to a thiol-reactive group are ed, for example, in US. Publ. No. 20140050746.

The thiol-reactive compound can further comprise a functional agent. Suitable functional agents include, for example, ?uorophores, ?uorescent dyes, ptides, immunoglobulins, antibiotics, nucleic acids, radionuclides, chemical linkers, small molecules, chelators, lipids, and drugs. In some aspects, the functional agent can comprise a ?uorophore.

In some aspects, the functional agent can se a ?uorescent dye. In some s, the functional agent can comprise a polypeptide. In some aspects, the functional agent can comprise an immunoglobulin. In some aspects, the functional agent can comprise an antibiotic. In some aspects, the functional agent can comprise a c acid (such as DNA or RNA). In some aspects, the functional agent can comprise a radionuclide. In some aspects, the functional agent can comprise a chemical linker (for example dibenzylcyclooctyne (DBCO) or azide). In some aspects, the onal agent can comprise a small molecule. In some aspects, the onal agent can comprise a chelator (for example, DOTA, CHX-A"-DTPA, NOTA, among others). In some aspects, the functional agent can comprise a lipid. In some aspects, the functional agent can comprise a drug. In some aspects, the functional agent can comprise a combination of any of the above listed functional agents.

Accordingly, the sed conjugated immunoglobulins include: immunoglobulin-?uorophore Cys80 conjugates, immunoglobulin-?uorescent dye Cys80 conjugates, immunoglobulin-polypeptide Cys80 ates, immunoglobulin-immunoglobulin Cys80 conjugates, immunoglobulin-antibiotic Cys80 conjugates, immunoglobulin-nucleic acid Cys80 conjugates, immunoglobulin-radionuclide Cys80 conjugates, immunoglobulin-chemical linker Cys80 conjugates, immunoglobulin—small molecule Cys80 conjugates, immunoglobulin- chelator Cys80 conjugates, immunoglobulin-lipid Cys80 conjugates, and immunoglobulin-drug Cys80 conjugates.

Any of the immunoglobulins disclosed herein can be conjugated to any of the functional agents disclosed . For example, the conjugated immunoglobulin can comprise an immunoglobulin that immunospeci?cally binds to human CA9 and a ?uorophore, ?uorescent dye, ptide, immunoglobulin, antibiotic, nucleic acid, radionuclide, chemical linker, small molecule, chelator, lipid, or drug. In some embodiments, the conjugated immunoglobulin is a CA9-?uorophore CySSO ate. In some embodiments, the conjugated globulin is a CA9-?uorescent dye CysSO conjugate. In some embodiments, the conjugated immunoglobulin is a CA9-polypeptide Cys80 ate. In some embodiments, the conjugated immunoglobulin is a CA9-immunoglobulin Cys80 conjugate. In some embodiments, the conjugated immunoglobulin is a CA9-antibiotic CysSO conjugate. In some embodiments, the conjugated immunoglobulin is a CA9-nucleic acid Cys80 conjugate. In some embodiments, the conjugated globulin is a CA9-radionuclide Cys80 conjugate. In some embodiments, the conjugated immunoglobulin is a CA9-chemical linker CySSO conjugate. In some embodiments, the conjugated globulin is a CA9-small molecule Cys80 conjugate. In some embodiments, the conjugated immunoglobulin is a CA9-chelator Cys80 conjugate. In some embodiments, the conjugated immunoglobulin is a pid Cys80 conjugate. In some embodiments, the conjugated immunoglobulin is a CA9-drug CySSO conjugate.

Suitable immunoglobulins that immunospeci?cally bind to human CA9 that can be conjugated at Cys80 to any of the above onal agents e: a. a heavy chain variable region having an amino acid ce at least 90% identical to amino acids 20-141 of xilSSDSHC (SEQ ID N052) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-130 of xi155D5LC (SEQ ID N0278); b. a heavy chain le region having an amino acid sequence at least 90% identical to amino acids 20-144 of zu155D5HC (SEQ ID NO:54) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-130 of zu155D5LC-3 (SEQ ID N0284), zu155D5LC-4 (SEQ ID NO:86), zu155D5LC-5 (SEQ ID NO:88), zu155D5LC-6 (SEQ ID NO:90), zu155D5LC-7 (SEQ ID NO:92), zu155D5LC-huVK2-40 (SEQ ID NO:96), zu155D5LC-huVK4-1 (SEQ ID NO:100), zulSSDSLC-huVK6-21 (SEQ ID NO: 102), zulSSDSLC—huVK6D-4l (SEQ ID NO:104); or zu155D5LC-huVK7Glu81 (SEQ ID NO:106); c. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-138 of C (SEQ ID NO:58) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-130 of XilE4LC (SEQ ID NO: 1 10), d. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-140 of zulE4HC (SEQ ID NO:60) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-130 of zu1E4LC-CXXA (SEQ ID NO:114), e. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20—142 of xil66B3HC (SEQ ID NO:74) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-130 of xil66B3LC (SEQ ID NO:132); f. a heavy chain variable region having an amino acid sequence at least 90% cal to amino acids 20-145 of zu166B3HC (SEQ ID N076) and a light chain variable region having an amino acid sequence at least 90% cal to amino acids 20-130 of zu166B3LC-CXXA (SEQ ID N02136); g. a heavy chain variable region as set forth as amino acids 20-141 of XilSSDSHC (SEQ ID NO:52) and a light chain variable region as set forth as amino acids 20-130 of Xi155D5LC (SEQ ID NO:78); h. a heavy chain variable region as set forth as amino acids 20-144 of zu155D5HC (SEQ ID NO:54) and a light chain variable region as set forth as amino acids 20—130 of zu155D5LC-3 (SEQ ID N084), zu155D5LC-4 (SEQ ID , zu155D5LC-5 (SEQ ID N088), zu155D5LC-6 (SEQ ID NO:90), zu155D5LC-7 (SEQ ID NO:92), zu155D5LC-huVK2-40 (SEQ ID NO:96), zu155D5LC-huVK4-1 (SEQ ID NO:100), zu155D5LC-huVK6-21 (SEQ ID NO:102), 5LC-huVK6D—41 (SEQ ID NO:104), or zu155D5LC-huVK7Glu81 (SEQ ID ), i. a heavy chain variable region as set forth as amino acids 20-138 of xilE4HC (SEQ ID NO:58) and a light chain variable as set forth as amino acids 20-130 of Xi1E4LC (SEQ ID NO:110); j. a heavy chain variable region as set forth as amino acids 20-140 of zu1E4HC (SEQ ID NO:60) and a light chain variable region as set forth as amino acids 20-130 of zu1E4LC-CXXA (SEQ ID NO:114), k. a heavy chain le region as set forth as amino acids 20-142 of Xi166B3HC (SEQ ID NO:74) and a light chain variable region as set forth as amino acids 20-130 of X1166B3LC (SEQ ID N02132); l. a heavy chain le region as set forth as amino acids 20-145 of zu166B3HC (SEQ ID NO:76) and a light chain variable region as set forth as amino acids 20—130 of zu166B3LC-CXXA (SEQ ID NO: 136); m. a heavy chain CDR1, CDR2, and CDR3 of Xi155D5HC as set forth as SEQ ID NO:146, 148, and 150, respectively, and a light chain CDR1, CDR2, and CDR3 of xi155D5LC as set forth as SEQ ID NO:224, 226, and 228, respectively; n. a heavy chain CDR1, CDR2, and CDR3 of zu155D5HC as set forth as SEQ ID NO:152, 154, and 156, respectively, and a light chain CDR1, CDR2, and CDR3 of zu155D5LC-3 as set forth as SEQ ID NO:242, 244, and 246, respectively, zu155D5LC-4 as set forth as SEQ ID NO:248, 250, and 252, respectively, zu155D5LC-5 as set forth as SEQ ID NO:254, 256, and 258, respectively, zu155D5LC-6 as set forth as SEQ ID NO:260, 262, and 264, respectively, zu155D5LC-7 as set forth as SEQ ID NO:266, 268, and 270, respectively, zu155D5LC-huVK2-40 as set forth as SEQ ID NO 278, 280, and 282, respectively, 5LC-huVK4-1 as set forth as SEQ ID NO 290, 292, and 294, respectively, zu155D5LC-huVK6-21 as set forth as SEQ ID NO 296, 298, and 300, tively, zu155D5LC-huVK6D-41 as set forth as SEQ ID NO 302, 304, and 306, respectively, or 5LC-huVK7Glu81 as set forth as SEQ ID NO 308, 310, and 312, respectively, 0. a heavy chain CDR1, CDR2, and CDR3 of Xi1E4HC as set forth as SEQ ID NO: 164, 166, and 168, respectively, and alight chain CDR1, CDR2, and CDR3 ofXi1E4LC as set forth as SEQ ID NO:320, 322, and 324, tively; p. a heavy chain CDR1, CDR2, and CDR3 of zul E4HC as set forth as SEQ ID NO:170, 172, and 174, respectively, and a light chain CDR1, CDR2, and CDR3 of zu1E4LC- CXXA as set forth as SEQ ID NO:332, 334, and 336, respectively; q. a heavy chain CDR1, CDR2, and CDR3 of Xi166B3HC as set forth as SEQ ID NO:212, 214, and 216, respectively, and a light chain CDR1, CDR2, and CDR3 of Xi166B3LC as set forth as SEQ ID NO:386, 388, and 390, respectively; or r. a heavy chain CDR1, CDR2, and CDR3 of 3HC as set forth as SEQ ID NO:218, 220, and 222, respectively, and a light chain CDR1, CDR2, and CDR3 of zu166B3LC-CXXA as set forth as SEQ ID NO:398, 400, and 402, respectively.

The conjugated immunoglobulin can comprise an immunoglobulin that immunospecifrcally binds to human TEMI and a ?uorophore, ?uorescent dye, polypeptide, immunoglobulin, antibiotic, nucleic acid, radionuclide, chemical linker, small molecule, chelator, lipid, or drug. In some embodiments, the conjugated immunoglobulin is a TEMl-?uorophore Cys80 conjugate. In some embodiments, the conjugated immunoglobulin is a TEMl-?uorescent dye Cys80 conjugate. In some embodiments, the ated immunoglobulin is a TEMl- polypeptide Cys80 conjugate. In some embodiments, the conjugated immunoglobulin is a TEMl-immunoglobulin CysSO conjugate. In some embodiments, the conjugated immunoglobulin is a TEMl-antibiotic Cys80 conjugate. In some embodiments, the conjugated immunoglobulin is a TEMl-nucleic acid CysSO conjugate. In some embodiments, the ated immunoglobulin is a adionuclide Cys80 conjugate. In some embodiments, the conjugated immunoglobulin is a TEMl-chemical linker Cys80 conjugate. In some embodiments, the conjugated immunoglobulin is a TEMl-small molecule Cys80 conjugate. In some embodiments, the conjugated immunoglobulin is a TEMl-chelator Cys80 conjugate. In some embodiments, the conjugated immunoglobulin is a TEMl-lipid Cys80 conjugate. In some embodiments, the conjugated immunoglobulin is a TEMl-drug CysSO conjugate.

Suitable globulins that immunospecifically bind to human TEMI that can be conjugated at Cys80 to any of the above functional agents include: a. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-139 of xi12HC (SEQ ID NO:56) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-129 of xil- 55-2LC (SEQ ID NO: 108), b. a heavy chain variable region as set forth as amino acids 20-139 of xi12HC (SEQ ID NO:56) and a light chain variable region as set forth as amino acids 20-129 of xil- 55-2LC (SEQ ID NO:108); or c. a heavy chain CDRl, CDR2, and CDR3 of Xi12HC as set forth as SEQ ID NO:158, 160, and 162, respectively, and a light chain CDRl, CDR2, and CDR3 of Xi12LC as set forth as SEQ ID NO:314, 316, and 318, tively.

The conjugated immunoglobulin can comprise an globulin that specifically binds to human MSLN and a ?uorophore, ?uorescent dye, polypeptide, immunoglobulin, otic, c acid, radionuclide, chemical linker, small molecule, chelator, lipid, or drug. In some embodiments, the conjugated immunoglobulin is a MSLN-?uorophore Cys80 conjugate. In some embodiments, the conjugated immunoglobulin is a MSLN-?uorescent dye Cys80 conjugate. In some embodiments, the conjugated immunoglobulin is a MSLN- polypeptide Cys80 conjugate. In some embodiments, the ated immunoglobulin is a MSLN-immunoglobulin Cys80 conjugate. In some embodiments, the conjugated immunoglobulin is a MSLN-antibiotic Cys80 conjugate. In some ments, the conjugated immunoglobulin is a MSLN-nucleic acid Cys80 conjugate. In some embodiments, the conjugated immunoglobulin is a MSLN-radionuclide CysSO conjugate. In some embodiments, the conjugated immunoglobulin is a MSLN-chemical linker Cys80 conjugate. In some embodiments, the conjugated immunoglobulin is a MSLN-small le CysSO conjugate. In some embodiments, the conjugated immunoglobulin is a MSLN-chelator Cys80 conjugate. In some ments, the conjugated immunoglobulin is a MSLN-lipid Cy580 conjugate. In some embodiments, the conjugated immunoglobulin is a MSLN-drug Cys80 conjugate.

Suitable immunoglobulins that immunospeci?cally bind to human MSLN that can be conjugated at Cys80 to any of the above functional agents include: a. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-142 of xi330l lHC (SEQ ID NO:62) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-131 of Xi33011LC (SEQ ID NO:116); b. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-145 of zu330l lHC (SEQ ID NO:64) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-131 of zu330l XA (SEQ ID NO: 120) or zu330l lLC-CXXI (SEQ ID NO: 122); c. a heavy chain variable region having an amino acid ce at least 90% cal to amino acids 20-137 of xi32405HC (SEQ ID NO:66) and a light chain variable region having an amino acid ce at least 90% identical to amino acids 20-127 of 5LC (SEQ ID N01124); d. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-137 of xil78F16HC (SEQ ID N068) and a light chain variable region having an amino acid ce at least 90% identical to amino acids 20-127 of xi178F16LC (SEQ ID NO:126); e. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-132 of xi237N18HC (SEQ ID N070) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-127 of Xi237N18LC (SEQ ID NO:128); f, a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-137 of xi383118HC (SEQ ID NO:72) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-127 of xi383118LC (SEQ ID NO:130); g. a heavy chain variable region as set forth as amino acids 20-142 of Xi3301 lHC (SEQ ID NO:62) and a light chain variable region as set forth as amino acids 20—131 of Xi33OllLC (SEQ ID NO:116); h. a heavy chain le region as set forth as amino acids 20-145 of zu3301 IHC (SEQ ID NO:64) and a light chain variable region as set forth as amino acids 20-131 of zu330l 1LC-CXXA (SEQ ID N02120) or zu330l 1LC-CXXI (SEQ ID N02122); i. a heavy chain variable region as set forth as amino acids 20-137 of Xi324O5HC (SEQ ID NO:66) and a light chain variable region as set forth as amino acids 20-127 of Xi324O5LC (SEQ ID NO:124); j. a heavy chain variable region as set forth as amino acids 20-137 of 16HC (SEQ ID N068) and a light chain variable region as set forth as amino acids 20-127 of Xi178F16LC (SEQ ID NO:126), k, a heavy chain variable region as set forth as amino acids 20-132 of Xi237N18HC (SEQ ID N070) and a light chain variable region as set forth as amino acids 20-127 of Xi237N18LC (SEQ ID NO:128), l. a heavy chain variable region as set forth as amino acids 20-137 of Xi383118HC (SEQ ID NO:72) and a light chain variable region as set forth as amino acids 20—127 of l8LC (SEQ ID NO:130); m. aheavy chain CDRl, CDR2, and CDR3 0l IHC as set forth as SEQ ID NO: 176, 178, and 180, respectively, and a light chain CDRI, CDR2, and CDR3 of xi3301 1LC as set forth in SEQ ID NO:338, 340, and 342, tively; n. a heavy chain CDRI, CDR2, and CDR3 of zu3301 lHC as set forth as SEQ ID NO:182, 184, and 186, respectively, and a light chain CDRI, CDR2, and CDR3 of zu33011LC-CXXA as set forth as SEQ ID NO:350, 352, and 354, respectively or zu3301 1LC-CXXI as set forth as SEQ ID NO:356, 358, and 360, respectively; 0. aheavy chain CDRI, CDR2, and CDR3 of Xi324O5HC as set forth as SEQ ID NO:188, 190, and 192, respectively, and a light chain CDRl, CDR2, and CDR3 of Xi324O5LC as set forth as SEQ ID NO:362, 364, and 366, respectively; p. a heavy chain CDRl, CDR2, and CDR3 of Xi178Fl6HC as set forth as SEQ ID NO:194, 196, and 198, respectively, and a light chain CDRl, CDR2, and CDR3 of Xi178F16LC as set forth as SEQ ID NO:368, 370, and 372, respectively; q. a heavy chain CDRl, CDR2, and CDR3 of Xi237Nl8HC as set forth as SEQ ID NO:200, 202, and 204, respectively, and a light chain CDRl, CDR2, and CDR3 of Xi237N18LC as set forth as SEQ ID NO:374, 376, and 378, respectively; or r. a heavy chain CDRl, CDR2, and CDR3 of Xi383Il8HC as set forth as SEQ ID NO:206, 208, and 210, respectively, and a light chain CDRl, CDR2, and CDR3 of 18LC as set forth as SEQ ID , 382, and 384, respectively.

In some embodiments, the immunoglobulin that immunospeci?cally binds to human MSLN can be conjugated to a small molecule antineoplastic agent such as an auristatin.

In some s, the onal agent can be auristatin F (AuF). Thus, the disclosed conjugated immunoglobulins include any of the above disclosed immunoglobulins that immunospeci?cally bind to human MSLN, wherein the immunoglobulin is conjugated to auristatin F (MSLN-AuF Cys80 conjugate).

In embodiments wherein the immunoglobulin comprises two light chain variable regions, the conjugated immunoglobulin can have an immunoglobulinzfunctional agent ratio of 2: 1, with each light chain having a functional agent conjugated at Cys80.

Antigen-binding molecules Further provided herein are antigen-binding les comprising: a ?rst conjugated immunoglobulin comprising a ?rst heavy chain variable region and a ?rst light chain variable region, the ?rst light chain variable region having a cysteine at position 80 ("CysSOl"), wherein Cys801 is conjugated to a ?rst thiol-reactive nd comprising a ?rst thiol-reactive group, and a second conjugated globulin comprising a second heavy chain variable region and a second light chain variable , the second light chain variable region having a cysteine at position 80 ("Cys802") wherein Cys802 is conjugated to a second thiol-reactive compound comprising a second thiol-reactive group.

The ?rst conjugated immunoglobulin and second conjugated immunoglobulin can be any one of the ated immunoglobulins disclosed herein.

Suitable light chain variable regions include, for example, a kappa light chain variable region. The ?rst light chain variable region and the second light chain variable region are derived from rabbit. In some embodiments, the Cys801, Cys802, or both, can be present in the native light chain variable region of the rabbit immunoglobulin. Exemplary rabbits from which a ?rst light chain variable region, second light chain variable region, or both, can be derived from include, but are not limited to, Oryctofagus cum‘culus. In some aspects, for example, the light chain variable (s) can be derived from a New Zealand White (NZW) rabbit. In other aspects, the light chain le region(s) can be derived from a b9 rabbit.

The Cys801, the Cys802, or both, can be unpaired. Suitable means for unpairing Cys801 and/or Cys802 include, for e, chimerizing a light chain variable region (a ?rst light chain variable region, a second light chain variable region, or both) having a Cys80 with a constant domain having an amino acid residue other than cysteine at position 171.

The ?rst immunoglobulin, the second immunoglobulin, or both, can be chimerized. In some embodiments, the ?rst immunoglobulin can be chimerized. In some embodiments, the second immunoglobulin can be ized. In some embodiments, the ?rst immunoglobulin and the second globulin can be chimerized.

The ?rst immunoglobulin, the second immunoglobulin, or both, can be humanized. In some embodiments, the ?rst immunoglobulin can be humanized. In some embodiments, the second immunoglobulin can be humanized. In some ments, the ?rst immunoglobulin and the second immunoglobulin can be humanized.

In some embodiments, the ?rst immunoglobulin can be chimerized and the second immunoglobulin can be humanized. In some embodiments, the ?rst immunoglobulin can be humanized and the second immunoglobulin can be chimerized.

The amino acid at position 83 of the ?rst light chain variable region can be an amino acid other than Phe, Lys, or Cys if the amino acid at position 83 is Phe. The amino acid at position 83 of the second light chain variable region can be an amino acid other than Phe, Lys, or Cys if the amino acid at position 83 is Phe. The amino acid at on 83 of the ?rst light chain variable region can be an amino acid other than Phe, Lys, or Cys if the amino acid at position 83 is Phe and the amino acid at position 83 of the second light chain variable region can be an amino acid other than Phe, Lys, or Cys if the amino acid at position 83 is Phe. The amino acid at position 83 of the ?rst light chain variable region and/or second light chain variable region can be e ("Ala83"), valine ("Va183"), cine ("Ile83"), threonine ("Thr83"), arginine ("Arg83"), asparagine ("Asn83"), aspartic acid ("Asp83"), ic acid ("Glu83"), glutamine ("Gln83"), glycine ("Gly83"), histidine ("H1583"), leucine ("Leu83"), methionine ("Met83"), proline ("Pro83"), serine ("Ser83"), phan ("Trp83"), or tyrosine ("Tyr83"). The amino acid at position 83 of the ?rst light chain variable region can be the same as the amino acid at position 83 of the second light chain variable region. Conversely, the amino acid at position 83 of the ?rst light chain variable region can be different from the amino acid at position 83 of the second light chain variable region.

In some embodiments, the amino acid at position 83 of the ?rst light chain variable region can be a polar or hobic residue other than Phe if the amino acid at position 83 is Phe. In some embodiments, the amino acid at on 83 of the second light chain variable region can be a polar or hydrophobic residue other than Phe if the amino acid at position 83 is Phe. In some embodiments, the amino acid at position 83 of the ?rst light chain variable region can be a polar or hydrophobic residue other than Phe if the amino acid at position 83 is Phe and the amino acid at position 83 of the second light chain variable region can be a polar or hydrophobic residue other than Phe if the amino acid at position 83 is Phe. Suitable polar or hydrophobic amino acids include, but are not limited to alanine, valine, isoleucine, or threonine.

In some s, the amino acid at position 83 of the ?rst light chain le region, the amino acid at position 83 of the second light chain variable region, or the amino acid at position 83 of the ?rst light chain variable region and the amino acid at position 83 of the second light chain variable region can be alanine ("Ala83"). In some aspects, the amino acid at position 83 of the ?rst light chain variable region, the amino acid at position 83 of the second light chain variable region, or the amino acid at position 83 of the ?rst light chain variable region and the amino acid at on 83 of the second light chain variable region can be valine ("Val83"). In some aspects, the amino acid at position 83 of the ?rst light chain variable region, the amino acid at on 83 of the second light chain variable region, or the amino acid at position 83 of the ?rst light chain variable region and the amino acid at position 83 of the second light chain variable region can be isoleucine ("Ile83"). In some aspects, the amino acid at position 83 of the ?rst light chain variable region, the amino acid at position 83 of the second light chain variable region, or the amino acid at position 83 of the ?rst light chain variable region and the amino acid at position 83 of the second light chain variable region can be Threonine ("Thr83"). The polar or hydrophobic amino acid at position 83 in the ?rst light chain variable region can be the same as, or different from, the polar or hydrophobic amino acid at on 83 in the second light chain variable region.

The ?rst immunoglobulin and the second immunoglobulin can bind to the same ns. In some aspects, the ?rst globulin and the second immunoglobulin can bind to the same e of the same antigen. In other aspects, the ?rst immunoglobulin and the second immunoglobulin can bind to different epitopes of the same antigen. In some embodiments, for e, the ?rst immunoglobulin and the second immunoglobulin can be an immunoglobulin that immunospeci?cally binds to human CA9, wherein the ?rst immunoglobulin, second immunoglobulin, or both are conjugated to any one of a hore, ?uorescent dye, polypeptide, immunoglobulin, antibiotic, nucleic acid, radionuclide, chemical , small molecule, chelator, lipid, or drug. In some embodiments, the ?rst immunoglobulin and the second immunoglobulin can be an immunoglobulin that immunospeci?cally binds to human TEMI, n the ?rst immunoglobulin, second immunoglobulin, or both are ated to any one of a ?uorophore, ?uorescent dye, polypeptide, immunoglobulin, antibiotic, nucleic acid, radionuclide, chemical linker, small le, chelator, lipid, or drug. In some embodiments, the ?rst globulin and the second immunoglobulin can be an immunoglobulin that immunospeci?cally binds to human MSLN, wherein the ?rst immunoglobulin, second immunoglobulin, or both are conjugated to any one of a ?uorophore, ?uorescent dye, polypeptide, immunoglobulin, antibiotic, nucleic acid, radionuclide, al linker, small molecule, chelator, lipid, or drug.

The ?rst globulin and the second immunoglobulin can bind to different antigens. In some embodiments, for e, the ?rst conjugated immunoglobulin can be an immunoglobulin that immunospeci?cally binds to human CA9, wherein the ?rst immunoglobulin that binds to human CA9 is conjugated to any one of a ?uorophore, ?uorescent dye, polypeptide, immunoglobulin, antibiotic, nucleic acid, radionuclide, chemical linker, small molecule, chelator, lipid, or drug, whereas the second immunoglobulin can be an immunoglobulin that immunospeci?cally binds to human TEMI or human MSLN. In such embodiments, the second immunoglobulin can be conjugated to any one of a ?uorophore, ?uorescent dye, polypeptide, immunoglobulin, antibiotic, nucleic acid, radionuclide, chemical linker, small molecule, chelator, lipid, or drug. In some embodiments, the ?rst conjugated immunoglobulin can be an immunoglobulin that immunospeci?cally binds to human TEMI, wherein the immunoglobulin is conjugated to any one of a ?uorophore, ?uorescent dye, polypeptide, immunoglobulin, otic, nucleic acid, radionuclide, chemical linker, small molecule, chelator, lipid, or drug, whereas the second immunoglobulin can be an immunoglobulin that immunospeci?cally binds to human CA9 or human MSLN. In such embodiments, the second immunoglobulin can be ated to any one of a ?uorophore, ?uorescent dye, polypeptide, immunoglobulin, antibiotic, nucleic acid, radionuclide, chemical linker, small molecule, chelator, lipid, or drug. In some ments, the ?rst conjugated immunoglobulin can be an immunoglobulin that immunospeci?cally binds to human MSLN, wherein the immunoglobulin is conjugated to any one of a ?uorophore, ?uorescent dye, polypeptide, immunoglobulin, antibiotic, nucleic acid, radionuclide, chemical , small molecule, chelator, lipid, or drug, whereas the second immunoglobulin can be an immunoglobulin that immunospeci?cally binds to human CA9 or human TEMI. In such embodiments, the second immunoglobulin can be conjugated to any one of a ?uorophore, ?uorescent dye, polypeptide, immunoglobulin, antibiotic, nucleic acid, radionuclide, chemical linker, small molecule, chelator, lipid, or drug.

Suitable, thiol-reactive groups include haloacetyls, maleimides, aziridines, acryloyls, arylating agents, vinylsulfones, pyridyl disul?des, TNB-thiols and de reducing agents. In some ments, the ?rst thiol-reactive group, the second-thiol reactive group, or both, can comprise a maleimide. In some embodiments, the ?rst thiol-reactive group, the second-thiol reactive group, or both, can comprise ahaloacetyl. In some embodiments, the ?rst thiol-reactive group, the second-thiol reactive group, or both, can comprise an aziridine. In some embodiments, the ?rst thiol-reactive group, the second-thiol reactive group, or both, can comprise an acryloyl. In some embodiments, the ?rst thiol—reactive group, the second-thiol reactive group, or both, can comprise an arylating agent. In some embodiments, the ?rst thiol- reactive group, the -thiol reactive group, or both, can comprise a ulfone. In some embodiments, the ?rst thiol—reactive group, the second-thiol reactive group, or both, can comprise a l disul?de. In some embodiments, the ?rst reactive group, the - thiol reactive group, or both, can comprise a iol. In some embodiments, the ?rst thiol— reactive group, the second-thiol reactive group, or both, can comprise a disul?de reducing agent.

The ?rst thiol-reactive group, the second-thiol reactive group, or both can be appended to a linker. In some aspects, the ?rst thiol-reactive group can be appended to a linker ("?rst linker"). In some aspects, the second thiol-reactive group can be appended to a linker ("second linker"). In yet other s the ?rst thiol-reactive group can be appended to a ?rst linker and the second reactive group can be appended to a second linker. le ?rst and second linkers can be non-cleavable linkers or cleavable linkers. Exemplary ?rst and second linkers include, for e, disul?de containing linkers, acetal-based linkers, and ketal-based linkers. In some s, the ?rst linker, second linker, or both, can be a non-cleavable linker.

Suitable non-cleavable linkers include, but are not limited to, polyethylene glycol (PEG) or an alkyl. In some embodiments, the ?rst linker, second , or both, can comprise PEG. In some s, the ?rst linker, second linker, or both, can be a cleavable linker. Suitable cleavable linkers include, for example, valine—citrulline-para aminobenzyl. In some aspects, the ?rst linker, second linker, or both, can be a disul?de containing linker. In some aspects, the ?rst linker, second , or both can be an acetal-based linker. In some aspects, the ?rst linker, second linker, or both, can be a ketal-based linker. Examples of linkers covalently appended to a thiol-reactive group are provided, for example, in US. Pub]. No. 20140050746.

The ?rst thiol-reactive compound, the second thiol-reactive nd, or both, can further comprise a functional agent. In some aspects, the ?rst thiol-reactive compound can further comprise a functional agent ("?rst functional agent"). In some aspects, the second thiol- reactive compound can further comprise a functional agent ("second functional agent"). In yet other aspects, the ?rst thiol-reactive nd can further comprise a ?rst functional agent and the second thiol-reactive compound can further comprise a second functional agent.

Suitable functional agents include, for example, chemical s. Preferably, the al linker of the ?rst thiol-reactive compound ("?rst chemical ") and the chemical linker of the second thiol-reactive compound ("second chemical linker") can be coupled. For example, and without intent to be limiting, one of the ?rst or second chemical linkers can be dibenzylcyclooctyne (DBCO) and the other of the ?rst or second chemical linkers can be azide. In some embodiments, for e, the ?rst chemical linker can be DBCO and the second chemical linker can be azide. Conversely, the ?rst chemical linker can be azide and the second chemical linker can be DBCO. The DBCO and azide can be coupled, thus resulting in the conjugation of the ?rst globulin and the second immunoglobulin. For example, the ?rst immunoglobulin and the second immunoglobulin can be conjugated to each other by click chemistry.

In an exemplary embodiment, thiol—reactive compounds can include maleimido- PEG4-azide and maleimido-PEG4-dibenzocyclooctyne. In some aspects, for example, the ?rst thiol-reactive compound can be maleimido-PEG4-azide and the second thiol-reactive compound can be ido-PEG4-dibenzocyclooctyne. In some s, the ?rst reactive compound can be maleimido-PEG4-dibenzocyclooctyne and the second thiol-reactive compound can be maleimido-PEG4-azide. Thus, the ?rst thiol-reactive compound can differ from the second thiol-reactive compound.

The ?rst globulin, second immunoglobulin, or both, can be Fabs. In some embodiments, the ?rst immunoglobulin can be a Fab ("?rst Fab"). In some embodiments, the second immunoglobulin can be a Fab ("second Fa "). In yet other embodiments, the ?rst immunoglobulin can be a ?rst Fab and the second immunoglobulin can be a second Fab.

Methods oftreating cancer in a subject Also disclosed herein are methods of treating cancer in a subject comprising stering to the subject a pharmaceutically effective amount of a conjugated elin immunoglobulin, n the conjugated mesothelin immunoglobulin comprises: any of the conjugated mesothelin immunoglobulins disclosed herein, and a thiol-reactive compound comprising a thiol-reactive group, a linker, and a functional agent.

It is to be understood that any of the characteristics, features, and embodiments relating to the disclosed conjugated immunoglobulins are equally applicable to those conjugated immunoglobulins used in the disclosed methods of treating cancer. Accordingly, the disclosed methods can comprise administering to the subject a pharmaceutically effective amount of a conjugated mesothelin globulin, n the conjugated elin globulin comprises a heavy chain variable region and a light chain variable region, the light chain variable region having a cysteine at position 80 ("Cys80") and an amino acid other than Phe, Lys, or Cys at position 83, wherein the Cys80 is conjugated to a thiol-reactive compound, the thiol-reactive nd comprising a thiol-reactive group, a linker, and a functional agent. In some embodiments, the amino acid other than Phe, Lys, or Cys at position 83 is a polar or hydrophobic amino acid.

Preferably, the cancer is a mesothelin-expressing cancer. In some embodiments, the conjugated antibodies for use in the disclosed s can comprise: a. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-142 of x1330] 1HC (SEQ ID NO:62) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-131 of xi33011LC (SEQ ID N01116); b. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-145 of zu3301 1HC (SEQ ID N064) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-131 of zu3301 1LC-CXXA (SEQ ID NO:120) or zu330l 1LC-CXXI (SEQ ID NO: 122); c. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-137 of xi32405HC (SEQ ID NO:66) and a light chain variable region having an amino acid sequence at least 90% cal to amino acids 20-127 of 5LC (SEQ ID NO: 124); d. a heavy chain le region having an amino acid sequence at least 90% identical to amino acids 20-137 of xil78Fl6HC (SEQ ID NO:68) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-127 of Xil78Fl6LC (SEQ ID NO: 126); e. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-132 of xi237N18HC (SEQ ID N070) and a light chain le region having an amino acid sequence at least 90% identical to amino acids 20-127 of Xi237N18LC (SEQ ID NO: 128); or f. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-137 of xi38311 8HC (SEQ ID NO:72) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-127 of xi383Il8LC (SEQ ID NO:130).

Antibodies (a)-(f) can be conjugated to a number of suitable thiol-reactive compounds including, but not limited to, those having an antineoplastic agent, such as an auristatin, as the functional agent. Thus, in some embodiments, the methods can comprise administering to the subject a pharmaceutically effective amount of a conjugated immunoglobulin, wherein the conjugated immunoglobulin comprises one or more of gloublins (a)-(f), each being conjugated to a thiol-reactive compound comprising auristatin F, wherein the thiol-reactive compound is conjugated to the light chain variable region of the immunoglobulin at the Cys80.

In some embodiments, the conjugated dies for use in the disclosed s can se: a. a heavy chain variable region as set forth as amino acids 20-142 of xi3301 lHC (SEQ ID NO:62) and a light chain variable region as set forth as amino acids 20-131 of xi33011LC (SEQ ID ); b. a heavy chain variable region as set forth as amino acids 20-145 of zu3301 lHC (SEQ ID NO:64) and a light chain variable region as set forth as amino acids 20-131 of zu3301 1LC-CXXA (SEQ ID NO:120) or zu330l 1LC-CXXI (SEQ ID NO: 122); c. a heavy chain variable region as set forth as amino acids 20-137 of xi32405HC (SEQ ID NO:66) and a light chain le region as set forth as amino acids 20-127 of xi32405LC (SEQ ID NO: 124); d, a heavy chain variable region as set forth as amino acids 20-137 ofxi178F16HC (SEQ ID N068) and a light chain variable region as set forth as amino acids 20-127 8Fl6LC (SEQ ID NO:126), e. a heavy chain variable region as set forth as amino acids 20-132 of Xi237N18HC (SEQ ID N070) and a light chain variable region as set forth as amino acids 20-127 of 18LC (SEQ ID NO: 128), or f. a heavy chain variable region as set forth as amino acids 20-137 of Xi383118HC (SEQ ID NO:72) and a light chain variable region as set forth as amino acids 20-127 of Xi383118LC (SEQ ID ).

Antibodies (a)-(f) can be conjugated to a number of suitable thiol-reactive compounds including, but not limited to, those having an antineoplastic agent, such as an auristatin, as the functional agent. Thus, in some embodiments, the methods can comprise administering to the subject a pharmaceutically effective amount of a conjugated immunoglobulin, wherein the conjugated immunoglobulin comprises one or more of immunogloublins (a)-(f), each being conjugated to a thiol—reactive compound comprising auristatin F, wherein the thiol-reactive compound is conjugated to the light chain variable region of the globulin at the Cy580.

In some embodiments, the conjugated dies for use in the disclosed methods can se: a. a heavy chain CDRI, CDR2, and CDR3 oin33011HC as set forth as SEQ ID NO: 176, I78, and 180, respectively, and a light chain CDRI, CDR2, and CDR3 of xi33011LC as set forth in SEQ ID NO:338, 340, and 342, respectively; b. aheavy chain CDRI, CDR2, and CDR3 of zu33OI IHC as set forth as SEQ ID NO:182, 184, and 186, respectively, and a light chain CDRI, CDR2, and CDR3 of zu330l ILC-CXXA as set forth as SEQ ID NO:350, 352, and 354, respectively or zu3301 ILC-CXXI as set forth as SEQ ID NO:356, 358, and 360, respectively; c. a heavy chain CDRI, CDR2, and CDR3 of Xi324O5HC as set forth as SEQ ID NO:188, 190, and 192, respectively, and a light chain CDRI, CDR2, and CDR3 of Xi324O5LC as set forth as SEQ ID NO:362, 364, and 366, respectively; (1. a heavy chain CDRI, CDR2, and CDR3 of l6HC as set forth as SEQ ID NO:I94, I96, and 198, respectively, and a light chain CDRI, CDR2, and CDR3 of 16LC as set forth as SEQ ID NO:368, 370, and 372, respectively; e. a heavy chain CDRI, CDR2, and CDR3 of Xi237N18HC as set forth as SEQ ID NO:200, 202, and 204, respectively, and a light chain CDRI, CDR2, and CDR3 of xi237N18LC as set forth as SEQ ID NO:374, 376, and 378, respectively; or f a heavy chain CDRl, CDR2, and CDR3 of Xi383Il8HC as set forth as SEQ ID NO:206, 208, and 210, respectively, and a light chain CDRl, CDR2, and CDR3 of Xi383Il8LC as set forth as SEQ ID NO:380, 382, and 384, respectively. dies (a)-(f) can be ated to a number of suitable thiol-reactive compounds including, but not limited to, those having an antineoplastic agent, such as an auristatin, as the functional agent. Thus, in some embodiments, the methods can comprise administering to the subject a pharmaceutically effective amount of a conjugated immunoglobulin, wherein the conjugated globulin comprises one or more of immunogloublins (a)-(f), each being conjugated to athiol-reactive compound comprising auristatin F, wherein the thiol-reactive compound is conjugated to the light chain variable region of the immunoglobulin at the Cys80.

Methodsfor detecting cancer Also disclosed herein are methods of detecting cancer in a subject. In some embodiments, the methods can be performed on the subject. For example, the methods can comprise stering to the subject a ceutically effective amount of a conjugated globulin, wherein the conjugated immunoglobulin comprises a heavy chain variable region and a light chain variable region, the light chain variable region having a cysteine at position 80 0") and an amino acid other than Phe, Lys, or Cys at on 83, wherein the Cys80 is conjugated to a thiol-reactive compound, the thiol—reactive compound sing a thiol-reactive group, a linker, and a functional agent. In some embodiments, the amino acid other than Phe, Lys, or Cys at position 83 is a polar or hydrophobic.

Alternatively, the methods can be performed on a biological sample obtained from the subject. For example, the methods can comprise contacting a biological sample with a conjugated immunoglobulin, wherein the ated immunoglobulin comprises a heavy chain variable region and a light chain variable region, the light chain le region having a cysteine at position 80 ("Cys80") and an amino acid other than Phe, Lys, or Cys at on 83, wherein the Cys80 is conjugated to a thiol-reactive compound, the thiol-reactive compound comprising a thiol-reactive group, a linker, and a functional agent. The amino acid other than Phe, Lys, or Cys at position 83 is a polar or hydrophobic. In some embodiments, the methods can be performed ex vivo. In some embodiments, the methods can be med in viva.

The functional agent is a ?uorophore or ?uorescent dye.

Any of the immunoglobulins disclosed herein can be conjugated to a ?uorophore or ?uorescent dye and used in the disclosed methods of detecting cancer. In some embodiments, the cancer is a CA9-eXpressing cancer and the conjugated immunoglobulin is a CA9-?uorophore Cys80 conjugate or a CA9—?uorescent dye Cys80 ate comprising: a. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-141 of SHC (SEQ ID NO:52) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-130 of xi155D5LC (SEQ ID N0278); b. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-144 of 5HC (SEQ ID N054) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-130 of zu155D5LC-3 (SEQ ID N084), zu155D5LC-4 (SEQ ID N086), zu155D5LC-5 (SEQ ID N088), zu155D5LC-6 (SEQ ID N090), zu155D5LC-7 (SEQ ID N092), zu155D5LC-huVK2-40 (SEQ ID N096), zu155D5LC-huVK4-1 (SEQ ID ), zulSSDSLC-huVK6-21 (SEQ ID NO: 102), zulSSDSLC—huVK6D-4l (SEQ ID NO:104); or zu155D5LC-huVK7Glu81 (SEQ ID NO:106); c. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-138 of xi1E4HC (SEQ ID NO:58) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-130 of Xi1E4LC (SEQ ID NO:110); d. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-140 of C (SEQ ID N060) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-130 of zu1E4LC-CXXA (SEQ ID ); e. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-142 of xil66B3HC (SEQ ID N074) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-130 of xi166B3LC (SEQ ID NO:132); f. a heavy chain variable region having an amino acid ce at least 90% identical to amino acids 20-145 of zul 66B3HC (SEQ ID NO:76) and a light chain variable region having an amino acid ce at least 90% identical to amino acids 20-130 of zu166B3LC-CXXA (SEQ ID NO:136); g. a heavy chain variable region as set forth as amino acids 20-141 of Xi155D5HC (SEQ ID NO:52) and a light chain variable region as set forth as amino acids 20—130 of Xi155D5LC (SEQ ID N078), h. a heavy chain variable region as set forth as amino acids 20-144 of zu155D5HC (SEQ ID NO:54) and a light chain variable region as set forth as amino acids 20-130 of zu155D5LC-3 (SEQ ID NO:84), zu155D5LC-4 (SEQ ID , zu155D5LC-5 (SEQ ID N088), zu155D5LC-6 (SEQ ID NO:90), zul55D5LC-7 (SEQ ID NO:92), zu155D5LC-huVK2-40 (SEQ ID NO:96), zu155D5LC-huVK4-1 (SEQ ID N0100), zul55D5LC-huVK6-21 (SEQ ID NO:102), zu155D5LC-huVK6D-4l (SEQ ID NO:104); or zu155D5LC-huVK7Glu81 (SEQ ID NO: 106); i. a heavy chain le region as set forth as amino acids 20-138 of Xi1E4HC (SEQ ID NO:5 8) and a light chain variable as set forth as amino acids 20-130 of xi1E4LC (SEQ ID NO:110), j. a heavy chain variable region as set forth as amino acids 20-140 of C (SEQ ID NO:60) and a light chain variable region as set forth as amino acids 20-130 of C-CXXA (SEQ ID NO:114), k. a heavy chain variable region as set forth as amino acids 20-142 of Xi166B3HC (SEQ ID NO:74) and a light chain variable region as set forth as amino acids 20-130 of xi166B3LC (SEQ ID N0132), l. a heavy chain variable region as set forth as amino acids 20-145 of zu166B3HC (SEQ ID NO:76) and a light chain variable region as set forth as amino acids 20—130 of zu166B3LC-CXXA (SEQ ID ); m. aheavy chain CDRl, CDR2, and CDR3 ofxil55D5HC as set forth as SEQ ID NO:146, 148, and 150, respectively, and a light chain CDRl, CDR2, and CDR3 of xi155D5LC as set forth as SEQ ID NO:224, 226, and 228, respectively; n. aheavy chain CDRl, CDR2, and CDR3 of zul 55D5HC as set forth as SEQ ID NO:152, 154, and 156, respectively, and a light chain CDRl, CDR2, and CDR3 of 5LC-3 as set forth as SEQ ID NO:242, 244, and 246, respectively, zu155D5LC-4 as set forth as SEQ ID NO:248, 250, and 252, respectively, zu155D5LC-5 as set forth as SEQ ID NO:254, 256, and 258, respectively, zu155D5LC-6 as set forth as SEQ ID NO:260, 262, and 264, respectively, 5LC-7 as set forth as SEQ ID NO:266, 268, and 270, respectively, zul55D5LC-huVK2-40 as set forth as SEQ ID NO 278, 280, and 282, respectively, zu155D5LC-huVK4-1 as set forth as SEQ ID NO 290, 292, and 294, tively, zu155D5LC-huVK6-21 as set forth as SEQ ID NO 296, 298, and 300, respectively, zu155D5LC-huVK6D-41 as set forth as SEQ ID NO 302, 304, and 306, tively, or zu155D5LC-huVK7Glu81 as set forth as SEQ ID NO 308, 310, and 312, respectively, 0. a heavy chain CDRI, CDR2, and CDR3 of xi1E4HC as set forth as SEQ ID NO: 164, 166, and 168, respectively, and a light chain CDRl, CDR2, and CDR3 ofXi1E4LC as set forth as SEQ ID NO:320, 322, and 324, respectively; p. a heavy chain CDRI, CDR2, and CDR3 of zu1E4HC as set forth as SEQ ID , 172, and 174, respectively, and alight chain CDRI, CDR2, and CDR3 of zu1E4LC- CXXA as set forth as SEQ ID NO:332, 334, and 336, respectively, q. a heavy chain CDRI, CDR2, and CDR3 of Xi166B3HC as set forth as SEQ ID NO:212, 214, and 216, respectively, and a light chain CDRl, CDR2, and CDR3 of xi166B3LC as set forth as SEQ ID NO:386, 388, and 390, respectively; or r. a heavy chain CDRI, CDR2, and CDR3 of zu166B3HC as set forth as SEQ ID NO:218, 220, and 222, respectively, and a light chain CDRI, CDR2, and CDR3 of zu166B3LC-CXXA as set forth as SEQ ID , 400, and 402, respectively, In some embodiments, the cancer is a TEMl-expressing cancer and the conjugated immunoglobulin is a TEMl-?uorophore Cys80 conjugate or a TEMl-?uorescent dye Cys80 ate comprising: a. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-139 of xi12HC (SEQ ID NO:56) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-129 of xil- 55-2LC (SEQ ID NO: 108), b. a heavy chain variable region as set forth as amino acids 20-139 of xi12HC (SEQ ID NO:56) and a light chain variable region as set forth as amino acids 20-129 of xil- 55-2LC (SEQ ID NO:108); or c. a heavy chain CDRI, CDR2, and CDR3 of Xi12HC as set forth as SEQ ID NO:158, 160, and 162, respectively, and a light chain CDRl, CDR2, and CDR3 of Xi12LC as set forth as SEQ ID NO:314, 316, and 318, respectively.

In some embodiments, the cancer is a MSLN—expressing cancer and the conjugated immunoglobulin is a MSLN-?uorophore Cys80 conjugate or a MSLN—?uorescent dye Cys80 conjugate comprising: a. a heavy chain variable region having an amino acid sequence at least 90% cal to amino acids 20-142 of xi330l lHC (SEQ ID N062) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-131 of xi33011LC (SEQ ID NO:116); b. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-145 of zu3301 lHC (SEQ ID NO:64) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-131 of zu3301 1LC-CXXA (SEQ ID ) or zu3301 XI (SEQ ID N02122); c. a heavy chain le region having an amino acid sequence at least 90% identical to amino acids 20-137 of xi32405HC (SEQ ID NO:66) and a light chain variable region having an amino acid sequence at least 90% cal to amino acids 20-127 of xi32405LC (SEQ ID N02124); d. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-137 of xil78F16HC (SEQ ID N068) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-127 of Xi178F16LC (SEQ ID NO:126); e. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-132 of xi237Nl8HC (SEQ ID N070) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-127 of Xi237N18LC (SEQ ID ); f. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-137 of xi383118HC (SEQ ID NO:72) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-127 of Xi383118LC (SEQ ID NO:130); g. a heavy chain variable region as set forth as amino acids 20-142 of Xl330l lHC (SEQ ID N062) and a light chain variable region as set forth as amino acids 20-131 of xi33011LC (SEQ ID N02116); h. a heavy chain variable region as set forth as amino acids 20-145 of zu3301 IHC (SEQ ID NO:64) and a light chain variable region aS set forth as amino acids 20-131 of 2113301 1LC-CXXA (SEQ ID NO:120) or zu330l 1LC-CXXI (SEQ ID ); i. a heavy chain variable region as set forth as amino acids 20-137 of Xi32405HC (SEQ ID NO:66) and a light chain variable region as set forth as amino acids 20—127 of Xi32405LC (SEQ ID N02124); j. a heavy chain variable region as set forth as amino acids 20-137 of Xi178F16HC (SEQ ID N068) and a light chain variable region as set forth as amino acids 20-127 of Xi178F16LC (SEQ ID NO:126), k. a heavy chain variable region as set forth as amino acids 20-132 of Xi237N18HC (SEQ ID N070) and a light chain variable region as set forth as amino acids 20-127 of xi237N18LC (SEQ ID NO:128), l. a heavy chain variable region as set forth as amino acids 20-137 of Xi383II8HC (SEQ ID NO:72) and a light chain le region as set forth as amino acids 20-127 of Xi383118LC (SEQ ID NO:130); m. a heavy chain CDR1, CDR2, and CDR3 ofxi33011HC as set forth as SEQ ID NO: 176, 178, and 180, respectively, and a light chain CDRI, CDR2, and CDR3 of xi33011LC as set forth in SEQ ID NO:338, 340, and 342, respectively; n. a heavy chain CDRI, CDR2, and CDR3 of zu3301 IHC as set forth as SEQ ID NO:182, 184, and 186, respectively, and a light chain CDRI, CDR2, and CDR3 of zu33011LC-CXXA as set forth as SEQ ID NO:350, 352, and 354, respectively or zu3301 1LC-CXXI as set forth as SEQ ID NO:356, 358, and 360, respectively, 0. a heavy chain CDRI, CDR2, and CDR3 of Xi324O5HC as set forth as SEQ ID NO:188, 190, and 192, respectively, and a light chain CDRI, CDR2, and CDR3 of xi32405LC as set forth as SEQ ID NO:362, 364, and 366, respectively; p. a heavy chain CDRI, CDR2, and CDR3 of Xi178F16HC as set forth as SEQ ID NO:194, 196, and 198, respectively, and a light chain CDRI, CDR2, and CDR3 of Xi178F16LC as set forth as SEQ ID NO:368, 370, and 372, respectively; q. a heavy chain CDRl, CDR2, and CDR3 of xi237N18HC as set forth as SEQ ID NO:200, 202, and 204, respectively, and a light chain CDRI, CDR2, and CDR3 of xi237N18LC as set forth as SEQ ID NO:374, 376, and 378, tively; or r. a heavy chain CDRI, CDR2, and CDR3 of Xi383Il8HC as set forth as SEQ ID NO:206, 208, and 210, tively, and a light chain CDRI, CDR2, and CDR3 of Xi383Il8LC as set forth as SEQ ID NO:380, 382, and 384, respectively.

Exemplary ?uorophores for conjugation to the immunoglobulin include, for example, IRDye-800CW.

The methods can se stering the conjugated immunoglobulin to the subject or ting the biological sample with the conjugated immunoglobulin and detecting binding of the conjugated immunoglobulin to an antigen (CA9, TEMI, or MSLN) present in the subject or in the biological sample, respectively. Suitable methods of detection include, for example, ?uorescent imaging. Detection of binding of the conjugated immunoglobulin to the antigen (through the emission of a ?uorescent signal, for example) is indicative of cancer.

Pharmaceutical compositions Also provided herein are pharmaceutical compositions. In some embodiments, the pharmaceutical itions can comprise any of the immunoglobulins disclosed herein. In some embodiments, the pharmaceutical itions can comprise any of the conjugated immunoglobulins disclosed herein.

Administration of a ated immunoglobulin in accordance with the methods of treatment or diagnosis described herein may be by any means known in the art.

Light chains variable regions for use in conjugated immunoglobulins Provided herein are light chain variable regions for use in a conjugated immunoglobulin, the light chain le region having a cysteine at amino acid position 80 ("Cys80") and an amino acid residue other than Phe, Lys, or Cys at amino acid position 83, n the Cys80 is unpaired. In some embodiments, the amino acid other than Phe, Lys, or Cys at position 83 is a polar or hydrophobic.

In preferred embodiments, the light chain has a CysSO-Xaal—Xaag-Xaag motif, wherein Xaa3 is an amino acid other than Phe, Lys, or Cys.

Suitable light chain variable regions include, for example, a kappa light chain le region. The light chain variable region is derived from rabbit. In some embodiments, the CysSO can be present in the native light chain variable region of the rabbit immunoglobulin.

Exemplary rabbits from which a light chain variable region having a Cys80 can be derived include, but is not d to, Oryctolagus lus. In some aspects, for example, the light chain variable region can be derived from aNew d White (NZW) rabbit. In other s, the light chain variable region can be derived from a b9 rabbit.

The Cys80 can be uncapped, can be involved in an intramolecular or intermolecular disul?de bond, or can have a capping cysteine.

In some embodiments, the light chain variable region can be chimerized. In other embodiments, the light chain variable region can be humanized.

The light chain variable region can se, consist of, or consist essentially a. a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-130 5D5LC (SEQ ID NO:78), b. a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-130 of zu155D5LC-3 (SEQ ID NO:84), zu155D5LC-4 (SEQ ID NO:86), zu155D5LC-5 (SEQ ID NO:88), zu155D5LC-6 (SEQ ID NO:90), 5LC-7 (SEQ ID NO:92), zu155D5LC-huVK2-40 (SEQ ID NO:96), zu155D5LC-huVK4-I (SEQ ID NO:100), zu155D5LC-huVK6-2I (SEQ ID NO:102), zu155D5LC-huVK6D-4l (SEQ ID NO:104); or zu155D5LC-huVK7 Glu81 (SEQ ID NO:106), c. a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-130 of xilE4LC (SEQ ID NO:110), d. a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-130 of zulE4LC-CXXA (SEQ ID NO:114), e. a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-130 of xil66B3LC (SEQ ID NO:132), f. a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-130 of zul 66B3LC-CXXA (SEQ ID ), g. a light chain variable region having an amino acid sequence at least 90% cal to amino acids 20-129 of xil-SS-ZLC (SEQ ID NO:108), h. a light chain variable region having an amino acid sequence at least 90% cal to amino acids 20-131 of xi330l lLC (SEQ ID NO:116); i. a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-131 of zu330l lLC-CXXA (SEQ ID NO:120) or zu330l lLC-CXXI (SEQ ID NO:122), j. a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-127 of xi32405LC (SEQ ID NO:124), k. a light chain variable region having an amino acid sequence at least 90% cal to amino acids 20-127 of xil78Fl6LC (SEQ ID NO:126); l. a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-127 of xi237N18LC (SEQ ID NO: 128); or In. a light chain variable region having an amino acid ce at least 90% identical to amino acids 20-127 of xi383118LC (SEQ ID NO:130).

Nucleic acid molecules encoding immunoglobulins and host cells comprising the same Also provided herein are nucleic acid molecules encoding any of the above disclosed immunoglobulins. In some embodiments, the nucleic acid molecules encode an immunoglobulin comprising a heavy chain variable region and a light chain variable region, the light chain variable region having a cysteine at on 80 ("Cys80") and an amino acid other than Phe, Lys, or Cys at on 83. In some embodiments, the amino acid other than Phe, Lys, or Cys at position 83 is polar or hydrophobic.

The sed nucleic acid molecules can encode an immunoglobulin that can speci?cally bind to human CA9. In some embodiments, the nucleic acid molecule encodes: a. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-141 of 5HC (SEQ ID N052) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-130 of xi155D5LC (SEQ ID NO:78); b, a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-144 of zulSSDSHC (SEQ ID N054) and a light chain variable region having an amino acid ce at least 90% identical to amino acids 20-130 of zu155D5LC-3 (SEQ ID N084), zu155D5LC-4 (SEQ ID NO:86), zu155D5LC-5 (SEQ ID N088), zu155D5LC-6 (SEQ ID N090), zu155D5LC-7 (SEQ ID N092), zu155D5LC-huVK2-40 (SEQ ID NO:96), zu155D5LC-huVK4-1 (SEQ ID NO:100), zu155D5LC-huVK6-21 (SEQ ID NO: 102), zu155D5LC-huVK6D—41 (SEQ ID NO:104), or zu155D5LC-huVK7G1u81 (SEQ ID NO:106), c. a heavy chain variable region having an amino acid ce at least 90% identical to amino acids 20-138 of xilE4HC (SEQ ID NO:58) and a light chain variable region having an amino acid ce at least 90% identical to amino acids 20-130 of XilE4LC (SEQ ID NO:110), d. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-140 of zulE4HC (SEQ ID N060) and a light chain le region having an amino acid sequence at least 90% identical to amino acids 20-130 of zulE4LC-CXXA (SEQ ID NO:114), e. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-142 of xil66B3HC (SEQ ID N074) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-130 of Xil66B3LC (SEQ ID NO:132); or f. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-145 of zul 66B3HC (SEQ ID NO:76) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-130 of zu166B3LC-CXXA (SEQ ID N01136).

In some embodiments, the nucleic acid molecule encodes: a. a heavy chain variable region as set forth as amino acids 20-141 oin155D5HC (SEQ ID NO:52) and a light chain variable region as set forth as amino acids 20-130 of xi155D5LC (SEQ ID NO:78); b. a heavy chain variable region as set forth as amino acids 20-144 of zu155D5HC (SEQ ID NO:54) and a light chain le region as set forth as amino acids 20-130 of zulSSDSLC-3 (SEQ ID , zu155D5LC-4 (SEQ ID N0186), zulSSDSLC-S (SEQ ID NO:88), zu155D5LC-6 (SEQ ID NO:90), zu155D5LC-7 (SEQ ID NO:92), zu155D5LC-huVK2-40 (SEQ ID NO:96), zu155D5LC-huVK4-l (SEQ ID NO: 100), zu155D5LC-huVK6-21 (SEQ ID NO: 102), zu155D5LC-huVK6D-4l (SEQ ID NO:104), or zulSSDSLC-huVK7Glu81 (SEQ ID NO:106), c. a heavy chain variable region as set forth as amino acids 20-138 of C (SEQ ID NO:58) and a light chain variable as set forth as amino acids 20-130 of XllE4LC (SEQ ID NO:110), d. a heavy chain le region as set forth as amino acids 20-140 of zu1E4HC (SEQ ID NO:60) and a light chain variable region as set forth as amino acids 20-130 of zu1E4LC-CXXA (SEQ ID NO:114); e. a heavy chain le region as set forth as amino acids 20-142 of 3HC (SEQ ID NO:74) and a light chain variable region as set forth as amino acids 20-130 of Xil66B3LC (SEQ ID N01132); or f. a heavy chain variable region as set forth as amino acids 20-145 of zu166B3HC (SEQ ID NO:76) and a light chain le region as set forth as amino acids 20-130 of zu166B3LC-CXXA (SEQ ID N02136).

In some embodiments, the nucleic acid molecule encodes: a. a heavy chain CDRl, CDR2, and CDR3 of Xi155D5HC as set forth as SEQ ID NO:146, 148, and 150, respectively, and a light chain CDRl, CDR2, and CDR3 of Xi155D5LC as set forth as SEQ ID NO:224, 226, and 228, respectively; b. a heavy chain CDRI, CDR2, and CDR3 of zul 55D5HC as set forth as SEQ ID NO:152, 154, and 156, respectively, and a light chain CDRl, CDR2, and CDR3 of zu155D5LC-3 as set forth as SEQ ID NO:242, 244, and 246, respectively, zu155D5LC-4 as set forth as SEQ ID NO:248, 250, and 252, respectively, 5LC-5 as set forth as SEQ ID NO:254, 256, and 258, respectively, zu155D5LC-6 as set forth as SEQ ID NO:260, 262, and 264, respectively, zu155D5LC-7 as set forth as SEQ ID NO:266, 268, and 270, tively, zu155D5LC-huVK2-40 as set forth as SEQ ID NO 278, 280, and 282, respectively, zu155D5LC-huVK4-1 as set forth as SEQ ID NO 290, 292, and 294, respectively, 5LC—huVK6—21 as set forth as SEQ ID NO 296, 298, and 300, respectively, 5LC-huVK6D-41 as set forth as SEQ ID NO 302, 304, and 306, respectively, or zu155D5LC-huVK7Glu81 as set forth as SEQ ID NO 308, 310, and 312, respectively, c. a heavy chain CDRl, CDR2, and CDR3 of Xi1E4HC as set forth as SEQ ID NO:164, 166, and 168, respectively, and alight chain CDRl, CDR2, and CDR3 of C as set forth as SEQ ID NO:320, 322, and 324, respectively; d. a heavy chain CDRl, CDR2, and CDR3 of zu1E4HC as set forth as SEQ ID NO: 170, 172, and 174, respectively, and a light chain CDRl, CDR2, and CDR3 of zu1E4LC- CXXA as set forth as SEQ ID NO:332, 334, and 336, respectively; e. a heavy chain CDRl, CDR2, and CDR3 of Xi166B3HC as set forth as SEQ ID , 214, and 216, respectively, and a light chain CDRl, CDR2, and CDR3 of Xi166B3LC as set forth as SEQ ID NO:386, 388, and 390, respectively, or f. a heavy chain CDRl, CDR2, and CDR3 of zul 66B3HC as set forth as SEQ ID NO:218, 220, and 222, respectively, and a light chain CDRl, CDR2, and CDR3 of zu166B3LC-CXXA as set forth as SEQ ID NO:398, 400, and 402, respectively.

The disclosed c acid molecules can encode an immunoglobulin that can immunospeci?cally bind to human TEM1. In some embodiments, the nucleic acid molecule encodes a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-139 of Xi12HC (SEQ ID NO:56) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-129 of Xi1-55—2LC (SEQ ID NO: 108). In some embodiments, the nucleic acid molecule encodes a heavy chain variable region as set forth as amino acids 20-139 of xi1-55—2HC (SEQ ID NO:56) and a light chain variable region as set forth as amino acids 20-129 of Xi12LC (SEQ ID NO:108). In some embodiments, the nucleic acid molecule encodes a heavy chain CDRl, CDR2, and CDR3 of Xil- 55—2HC as set forth as SEQ ID NO:158, 160, and 162, respectively, and a light chain CDRl, CDR2, and CDR3 55-2LC as set forth as SEQ ID NO:314, 316, and 318, tively.

The disclosed nucleic acid molecules can encode an immunoglobulin that can immunospeci?cally bind to human MSLN. In some embodiments, the nucleic acid molecule encodes: a. a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-142 of xi330l 1HC (SEQ ID N062) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-131 of xi33011LC (SEQ ID N02116); a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-145 of zu3301 1HC (SEQ ID N064) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-131 of zu330l 1LC-CXXA (SEQ ID NO: 120) or zu330l 1LC-CXXI (SEQ ID NO:122); a heavy chain variable region having an amino acid ce at least 90% identical to amino acids 20-137 of xi32405HC (SEQ ID NO:66) and a light chain variable region having an amino acid sequence at least 90% identical to amino acids 20-127 of Xi32405LC (SEQ ID NO:124); a heavy chain variable region having an amino acid sequence at least 90% cal to amino acids 20-137 of xi178F16HC (SEQ ID N068) and a light chain le region having an amino acid sequence at least 90% identical to amino acids 20-127 of Xi178F16LC (SEQ ID NO:126); a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-132 of xi237N18HC (SEQ ID N070) and a light chain variable region having an amino acid ce at least 90% identical to amino acids 20-127 of Xi237N18LC (SEQ ID N01128); or a heavy chain variable region having an amino acid sequence at least 90% identical to amino acids 20-137 of xi383Il8HC (SEQ ID NO:72) and a light chain variable region having an amino acid sequence at least 90% cal to amino acids 20-127 of Xi383118LC (SEQ ID NO:130), In some embodiments, the nucleic acid molecule encodes: a. a heavy chain variable region as set forth as amino acids 20-142 of Xi33Ol 1HC (SEQ ID NO:62) and a light chain variable region as set forth as amino acids 20—131 of Xi33OllLC (SEQ ID NO:116); b. a heavy chain variable region as set forth as amino acids 20-145 of zu3301 1HC (SEQ ID NO:64) and a light chain variable region as set forth as amino acids 20-131 of zu3301 1LC-CXXA (SEQ ID NO: 120) or zu330l 1LC-CXXI (SEQ ID ); c. a heavy chain variable region as set forth as amino acids 20-137 of Xi324O5HC (SEQ ID NO:66) and a light chain variable region as set forth as amino acids 20-127 of Xi32405LC (SEQ ID NO:124); d. a heavy chain variable region as set forth as amino acids 20-137 of xi178Fl6HC (SEQ ID N068) and a light chain variable region as set forth as amino acids 20-127 of Xi178F16LC (SEQ ID NO:126); e. a heavy chain variable region as set forth as amino acids 20-132 of Xi237N18HC (SEQ ID NO:70) and a light chain variable region as set forth as amino acids 20-127 of l8LC (SEQ ID NO:128), or f. a heavy chain le region as set forth as amino acids 20-137 of Xi383118HC (SEQ ID NO:72) and a light chain variable region as set forth as amino acids 20—127 of Xi383Il8LC (SEQ ID NO:130).

In some embodiments, the nucleic acid molecule encodes: a. a heavy chain CDRI, CDR2, and CDR3 ofxi33011HC as set forth as SEQ ID NO: 176, 178, and 180, respectively, and a light chain CDRl, CDR2, and CDR3 of Xi33Ol 1LC as set forth in SEQ ID NO:338, 340, and 342, respectively, b. aheavy chain CDRl, CDR2, and CDR3 of zu33011HC as set forth as SEQ ID NO:182, 184, and 186, respectively, and a light chain CDRl, CDR2, and CDR3 of zu33011LC-CXXA as set forth as SEQ ID NO:350, 352, and 354, respectively or 1LC-CXXI as set forth as SEQ ID NO:356, 358, and 360, tively, c. a heavy chain CDRI, CDR2, and CDR3 of 5HC as set forth as SEQ ID NO:188, 190, and 192, respectively, and a light chain CDRl, CDR2, and CDR3 of xi32405LC as set forth as SEQ ID NO:362, 364, and 366, respectively; (1, aheavy chain CDRl, CDR2, and CDR3 of Xil78Fl6HC as set forth as SEQ ID NO:194, 196, and 198, respectively, and a light chain CDRl, CDR2, and CDR3 of xi178F16LC as set forth as SEQ ID NO:368, 370, and 372, respectively; e. a heavy chain CDRI, CDR2, and CDR3 of xi237N18HC as set forth as SEQ ID , 202, and 204, respectively, and a light chain CDRl, CDR2, and CDR3 of xi237N18LC as set forth as SEQ ID NO:374, 376, and 378, respectively; or f. a heavy chain CDRl, CDR2, and CDR3 of l8HC as set forth as SEQ ID NO:206, 208, and 210, respectively, and a light chain CDRl, CDR2, and CDR3 of 18LC as set forth as SEQ ID , 382, and 384, respectively.

Also disclosed are host cells comprising any of the sed nucleic acid molecules. Suitable host cells include, but are not limited to, mammalian cells, bacterial cells, yeast cells, insect cells, to name a few.

The following examples are provided to further describe some of the embodiments disclosed herein. The examples are intended to illustrate, not to limit, the disclosed embodiments.

EXAMPLES Example 1 — Exemplary Methods Generation ofrabbit mAbs speci?c to human TEM] ialin/CD248) Rabbit immunization: To generate rabbit mAbs speci?c to human TEMI (hTEMl), a soluble human endosialin extracellular domain-mouse Fc fusion protein was ed n endosialin/TE M1 extracellular domain fused to mouse IgG2b Fe"). The extracellular domain of hTEMl was cloned in-frame EcoRI/HpaI to pEF6-EK-IgG2b, which contained an enterokinase cleavage site followed by the murine IgG2b Fc gamma fragment.

CHO-K1 cells were ected with this construct and selected with 5 ug/mL blasticidin.

Secreted TEMl-Fc was electrophoresed on a 4-12% PAGE gel and Coomassie stained, followed by excision of the bands. The gel slices were emulsified in complete/incomplete nt, and injected into New Zealand White rabbits every 3 to 4 weeks, four injections. The spleen from a rabbit showing the best titers against hTEMl as assessed by ELISA was harvested for the generation of hybridomas.

Generation of hybridomas: Fusions were performed as follows: spleen cells (1.5-3 x 108) of immunized rabbits and the fusion partner 240E 12 were fused at a ratio of 2:1 with 50% PEG 4000 (EM Science, Cherry Hill, NJ) at 37°C in serum-free medium. The cells were plated in 48-well microtiter plates, at approximately 2x105 spleen cells per well, in medium with 15% FCS. After 72 hr, hypoxanthine-aminopterin-thymidine (HAT) was added. Medium was changed every 5-6 days. Supernatants were screened by ELISA for the presence of antibody speci?c for TEM-l using TEMl-Fc coated plates and counter-screened against mouse Fc.

Supernatants from hybridomas were screened for hTEMl vity by ELISA and clone 12 was chosen for recombinant cloning.

Ampli?cation of anti-hTEMl 12 light and hea? chain variable regions: RNA was isolated from rabbit hybridoma 1—55-2 using the RNeasy mini kit n, Valencia, CA). Two ug RNA was used for RT-PCR using SuperScript III One-Step RT-PCR System with Platinum Taq High ty (Invitrogen). The rabbit variable heavy chain and full length light chain gene nts were ampli?ed using primer pairs N02937/N02898 and N02937/N02347 respectively (Table l). The cycling parameters for the RT—PCR ampli?cation were as follows: 55°C 30 min; 94°C 2 min; 30 cycles of (94°C 15 sec, 55°C 30 sec, 68°C 1 min); 68°C 2 min.

These PCR products were subsequently used in a second round PCR to amplify fragments amenable to generating chimeric rabbit/human IgGs using primer pairs N02416/N02761 and /N02764 (Table 1). The cycling parameters for the second round PCR were as follows: 94°C for 2 min; 30 cycles of (94°C 30 sec, 55°C 30 sec, 68°C 1 min); 68°C 2 min, Table 1. Primers used for RT-PCR and cloning of anti-hTEMl l2 AGCTTGCCGC ATGGGCTGGTCC CATCCTGTT TCTGGTGGCGGCCGCCACCGGCGTGCACTCC N02937 Rabblt VH.

(SEQ ID N011) N02898 GTGCCTTTGGCTGGCCTGARGAGAYGGTGACCAGGGTGCC Rabbit VH (SEQ ID N022) GATCAAGCTTGCCGCCACCATGGGCTGGTCCTGCATCATCCTGTT N02937 Rabbit LC TCTGGTGGCGGCCGCCACCGGCGTGCACTCC (SEQ ID N023) N02347 GATCGGCGCGCCTCACTTGCCGGGGCTCCGG Rabbith (SEQ ID N024) N02416 GCCACCGGCGTGCACTCCCAGTCGGTGRAGGAGTCCRGGGG xi Mm HC (SEQ ID NO:5) N02761 GGGCCCTTGGTGGATGCTGARGAGAYGGTGACCAGGGTGCC xi Mm HC SEQ ID N016 N024" GCCACCGGCGTGCACTCCGAGCTCGTGATGACCCAGACTCCA xi Mm LC (SEQ ID N027) N02764 AGCCACAGTTCGTTTGACSACCACCTCGGTCCC xi rb-hu LC (SEQ ID N028) WO 05618 2016/038041 PCR products were then separated by electrophoresis in an agarose gel. PCR products having the t molecular sizes for the VL and VH products were puri?ed by QIAquick® Gel Extraction Kit (Qiagen, Valencia, CA) and cloned as described below.

Generation ofrabbit mAbs speci?c to human CA9 Rabbit Immunization: To generate rabbit antibodies speci?c to human CA9, human CA9 extracellular domain ("human CA9 extracellular domain" or "CA9-ECD") was recombinantly generated. Two b9 rabbits were immunized using CA9-ECD. Brie?y, the rabbits were subcutaneously injected with the antigens every 21 days. Each rabbit received 400 ug of CA9-ECD and Freund's te adjuvant (FCA) in the ?rst injection and 200 ug of CA9-ECD and Freund's Incomplete Adjuvant (FIA) in the subsequent boosts. The pre- and test—bleed were collected for the antibody titer testing.

The pre- and post-immunization blood was tested for CA9 binding using an -Linked Immunosorbent Assay (ELISA) as described herein. The bleeds were serial diluted and added to CA9-ECD protein-coated microplates. When the titer reached 00 after four injections, the rabbits were ?nally boosted by intravenous injection of 400 ug of CA9- ECD without adjuvant. Rabbit spleens were collected one week after the ?nal boosting. Up to 100 mL exsanguination bleeds were collected in the presence of anti-coagulant and the lymphocytes from spleens and lymph nodes were isolated from each rabbit.

Generation of hybridomas: Rabbit splenocytes were y thawed, spun down at 1200 rpm at room temperature for 5 min, and re-suspended in cIMDM plus 10% FBS ning 100 ug/mL DNase. Cells were stimulated with 2.5 ug/mL pokeweed mitogen at 37°C for at least 1 hour. After stimulation, cells were spun down at 1200 rpm at room temperature for min and re-suspended in fresh media. Cell counts and viability were determined.

Fusion partner cells CBF7 were thawed out and cultured at 37°C with 5% C02 for one week before fusion. An appropriate amount of rabbit splenocytes and fusion partner cells CBF7 were mixed at the desired ratio (1:1.55 ~ 1:4) in 50 mL tubes. The mixture of cells was spun down at 1000 rpm at room temperature for 5 min and washed twice with ice-cold 20 mL CytoPulse Fusion Medium (CPFM a C: CytoPulse Sciences #LCM-C) at 4°C. The cells were re—suspended in CPFM to 106 mL.

CytoPulse cell fusion tus CEEF-50 (CytoPulse Sciences) was used for the fusion. An appropriate volume of cells was moved to the fusion chamber and fusion was performed by ting high voltage connection. After , the cells were incubated in the chamber at RT for 5 min, gently re—suspended in Post-Fusion Medium (RPM11640 with 10% FBS, containing glutamate, te, non-essential amino acids, B-mercaptoethanol, penicillin, streptomycin, and no Phenol Red) and then transferred to a ?ask. The r was washed with the same volume of post-fusion media to obtain additional cells. The cells were incubated at room temperature for 25 min and then overnight at 37°C, 5% C02.

One day after fusion, the cells were diluted in pre-warmed seeding media (cIMDM plus 10% FBS containing 1X nthine-aminopterin-thymidine) to the desired density (35,000 cells/mL) and plated at 200 uL/well in 96-well microplates. The plates were incubated at 37°C, 5% C02 and fed with fresh medium weekly for 3-4 weeks.

Screening of anti-CA9 mAbs: B-cells from rabbit splenocytes were fused to fusion r cells CBF7 to generate hybridomas as described herein. Four weeks after plating the cells, the supematants from individual hybridoma cultures were collected and screened using a eci?c ELISA. The assay plates er Bio-One High Binding 384-well clear plate, cat #655081) were coated with 1 ug/ml CA9 ECD ght at 4°C and blocked with 1X Assay Buffer (PBS plus 1% BSA, containing 0.05% Tween-20). Then, 25 uL/well of supematants and controls were added to the blocked plates and incubated overnight at 4°C. The assay plates were washed three times and 25 uL/well of secondary antibodies (HRP-conjugated goat anti-mouse IgG, Jackson # 115146) diluted 1: 10,000 in Assay Buffer was added to the . After incubation at room temperature for one hour, the assay plates were washed three times and 25 uL/well ofTMB Substrate (KPL #5204) was added to the plates. After incubation at room temperature for 5 minutes, 25 uL/well of 1X Stop Solution (1:10 H2SO4, VWR #EM-SX1244- 75) was added. Sample absorbance at 450 nm was measured by using Paradigm (Beckman) plate reader. The positive hits from the primary screen were confirmed by a second CA9- specific ELISA.

Cloning and mutagenesz's Amplification of VH and VK regions of CA9 and hTEMl mAbs: Hybridoma cells secreting rabbit mAbs of interest were lysed to extract RNA. RNA was then used for DNA amplification of variable kappa (VK) and heavy chain le (VH) regions by using the reverse transcriptase-poly merase chain reaction (RT-PCR) method. One hundred to 10,000 cultured hybridoma cells were washed with ice cold PBS and lysed by adding 100 uL of Lysis/Binding on (Ambion, 8540G5) and pipetting. The lysed cells were quickly frozen on dry ice. RNA was isolated with Ambion RNAqueous Kit according to cture procedure. About 5 ng RNAS were subject to ?rst round of RT-PCR using the primers listed on Table 2 in each reaction.

Table 2. Primers used for ?rst round of RT-PCR Rabb.VHA1.F 5'-CAGTCGCTGCTCGAGTCCGGGGGT-3' (SEQ ID N029) Rabb.VHB1.F 5'-CTCTGGCACAGGAGCTC-3' (SEQ ID NO:10) Rabb.IgM_CH1.R 5'—GGAGACGAGCGGGTACAGAGT-3' (SEQ ID NO:1l) gG_Hinge.R 5'-CGTGGGCTTGCTGCATGTCG—3' (SEQ ID NO: 12) Rabb.V1<.F 5'-GTGATGACCCAGACTCCA-3' (SEQ ID NO: 13) Rabb.VK1B4.R 5'-ACAGTCACCCCTATTGAAGCTCTGG—3' (SEQ ID NO:14) Rabb.V1<2B4.R 5'-GCAGTCACCCCTGTTGAAGCTCTG—3' (SEQ ID NO: 15) The cycling parameters for the RT-PCR ampli?cation were as follows: 55°C 30 min; 95°C 2 min; 30 cycles of (94°C 1 min, 54°C 50 sec, 68°C 1.5 min); 68°C 10 min.

The products from the ?rst round RT—PCR were subjected to a second round of PCR cation in separate reaction for heavy chain and light chain, using the primers listed in Table 3.

Table 3. Primers used for second round of PCR ampli?cation ldr—Rabb.VHA1.F gccaccggcgtgcactccCAGTCGGTGRAGGAGTCCRGGGG (SEQ 1D NO: 16) R-Rb-VHl-hu- gggcccttggtggatgcTGARGAGAYGGTGACCAGGGTGCC gamma (SEQ ID NO‘17) gccac cactccGAGCTC A (SEQ ID N0218) R—Rb-VKI m—hu— agccacagttchTTGATCTCCAGCTCGGTCCC kappa (SEQ 1D NO: 19) agccacagttchTTGATTTCCACATTGGTGCC (SEQ ID NOIZO) R—Rb-VK3-hu-kappa agccacagttchTTGACSACCACCTCGGTCCC (SEQ ID NO:21) The cycling ters for the second round of PCR ampli?cation were as follows: 95°C 5 min; 40 cycles of (94°C 1 min, 54°C 50 sec, 68°C 1.5 min); 68°C 10 min; 4°C Soak.

PCR products were then separated by ophoresis on e gel. PCR products having the correct molecular sizes for the VL and VH products were d by QIAquick® Gel Extraction Kit (Qiagen, Valencia, CA), and the nts were subcloned into an sion plasmid ning a human gamma (Cy) or kappa (CK) constant region using an InFusion HD cloning kit ech). All clones were sequenced to con?rm the presence and ?delity of the inserts.

Gene synthesis: Humanized VH domains and zu155D5LC-l, -huVKl -39, - huVK2-40, -huVK3-11, -huVK4-l, -huVK5-2, -huVK6-21, -huVK6D-4l, -huVK7-3, zulE4LC- l, and zul66B3LC-l VK domains were codon-optimized for expression in human cells and were synthesized by DNA 2.0. The variable domains were synthesized with a Kozak sequence and an Ig leader sequence, and included 15 base-pairs at the 5’ and 3’ ends gous to the cloning site within the subcloning vector. Following excision from the DNA 2.0 vector, the fragments were subcloned into an expression plasmid ning a human Cy or CK region using an InFusion HD cloning kit. All clones were sequenced to con?rm the presence and ?delity of the inserts.

QuikChange: Mutagenesis of the codon—optimized VK domains was performed using Stratagene's QuikChange XL according to the manufacturer’s protocol. All clones were sequenced to con?rm the presence of the mutation.

Cell Culture Transfection and stable cell line generation: One day prior to transfection, 293F cells were seeded at 6.0x105 cells/mL in 293FreeStyle medium o Fisher Scienti?c) in a shake ?ask and incubated at 37°C, 8% C02, with shaking at 125 rpm. On the day of transfection, cells were seeded at 1x106 cells/mL as above. Cells were transfected using PEI (25 kDa, linear; Polysciences) or ExpiFectamine (Thermo Fisher Scienti?c). For the PEI transfections, 166.7 ng HC plasmid, 166.7 ng LC plasmid, 2.2 ug PEI, and 50 [,LL OptiPro (Thermo Fisher Scienti?c) per mL of transfected cells were incubated for 15 min at 22°C. The DNAzPEI mixture was added to the cells while ng and incubated at 37°C, 8% C02, shaking at 125 rpm. After 48-72 h, cells were fed at a ?nal concentration of 10 g/L late (BD Biosciences), 5 mM valeric acid (Sigma h), and 1:100 CD Lipid Concentrate (Thermo Fisher Scienti?c).

For each mL of cells to be transfected with ExpiFectamine, 333.3 ng HC plasmid and 333.3 ng LC plasmid were incubated for 10 min in 50 [L EM (Thermo Fisher Scienti?c). Likewise, 2.67 uL ExpiFectamine was incubated in 50 [LL Opti-MEM. The ExpiFectamine solution was added to the DNA mixture, and incubated for 30 min at 22°C. The DNAzExpiFectamine mixture was added to the cells while swirling and ted at 37°C, 8% C02, shaking at 125 rpm. The following day, 3 uL of er l and 30 uL of enhancer 2 per mL of cells were added to the transfection with continued to incubate for another 7 or 10 days, depending on cell density.

Antibody-expressing stable pools were selected by adding 3 mL of transfectants to 12 mL DMEM in a T75 ?ask with 5 ug/mL blasticidin and 400 ug/mL zeocin (Thermo Fisher Scienti?c) one to three days after transfection. After drug—resistant cells grew to ncy, the medium was replaced with FreeStyle 293 expression medium. After 24 or 48 h, cells were physically dislodged by tapping the ?ask (trypsinization resulted in low Viability, data not shown) and were then seeded at 6x105 cells/mL in 30 mL FreeStyle 293 expression medium in a 125-mL shake ?ask. Cultures were incubated at 37°C in 8% C02 with shaking at 125 rpm. mAb production: Antibody production from stable pools was performed by one of two methods: 1. Stable-transfected cell line pools were seeded at 0.6 to 1x106 mL in 293FreeStyle medium. Two days after the culture reached a density of 1x106 cells/mL, cultures were fed as described , or 2. Stable-transfected cell line pools were centrifuged at 1000 rpm in a Beckman Allegra 6 centrifuge for 5 min. The supernatant was removed, and the cells were resuspended in 1 L 3 medium ) at 0.5-0.8x106 cells/mL in a 2.8-L shake ?ask. Cells were incubated at 37 °C, 8% C02, shaking at 125 rpm.

For both s, the cultures were incubated at 37°C in 8% C02 with shaking at 125 rpm for 7-10 days, depending on when cell viability dropped to about 50%, at which time the cultures were centrifuged for 1 h at 8000 rpm in a Beckman JLA8.1000 rotor. The supernatant was then ?ltered through a 0.2 um PES ?lter and stored at 4°C or -20°C until puri?cation. mAb purification Antibody puri?cation by n A af?ni? chromatography: Using an AKTA Explorer (GE Healthcare), a protein A column (GE Healthcare) was equilibrated with 10 column volumes (CV) of 20 mM sodium phosphate, 10 mM EDTA, pH 7.2. The sample was then loaded, followed by washing unbound material with 10 CV of equilibration buffer. The sample was eluted using 5 CV of 0.1 M e pH 2.9. The fractions containing the mAb were pooled and dialyzed in Dulbecco’s phosphate buffer (DPBS) using a MWCO 20K Slide-A-Lyzer o Fisher Scienti?c).

Cysteine decapping: Using an AKTA Explorer (GE Healthcare), a protein A column (GE Healthcare) was equilibrated with 10 CV of 20 mM sodium phosphate, 10 mM EDTA, pH 7.2 (equilibration buffer). The sample was then loaded, followed by washing unbound material with 10 CV of equilibration buffer, The column was washed with 16 CV of 20 mM sodium phosphate, 10 mM EDTA, 5 mM cysteine, pH 7.2 at 0.5 mL/min for 16 h at 4°C to remove capping group. The column was then washed with 60 CV of 20 mM Tris, pH 7.5 at 0.5 mL/min for 60 h at 4°C. The sample was eluted using 5 CV of 0.1 M glycine pH 2.9 and immediately neutralized using 5% volume of 2M Tris, pH 9.0. The fractions containing mAb were pooled and dialyzed in DPBS using a MWCO 20K Slide-A-Lyzer (Thermo Fisher Scienti?c).

LC—MS/MS cysteinylaz‘l'on and disufflde bond mapping analyses The mAb was buffer-exchanged to 50 mM ammonium bicarbonate buffer, pH 7.8 using aZeba spin desalting column (Thermo-Fisher). The concentration was adjusted to 1 mg/mL and RapiGest s) was added to 0.1%. The mAb was then digested with Glu-C (New England BioLabs) (25:1 w/w) at 37 0C for 4 h, followed by digestion with Asp-N (New d BioLabs) (25:1 w/w) at 37°C for 18 h. Following digestion, 5% tri?uoroacetic acid (TFA) was added to 0.5% and incubated at 37°C for 90 min. The sample was centrifuged at 13,000 rpm for 30 min to remove pellets and analyzed by LC-MS/MS using MSE methodology in the second ionization phase. MSE methodology uses a ramped voltage rather than a ?xed e in the second ionization phase to generate a more complete ion profile. Samples were analyzed using a Waters y UPLC and Q-Tof Premier mass spectrometer. s were injected onto a Waters BEH 300 C18, 1.7 pm pore size, 2.1 x 100 mm, eluted from the column with a 3 min equilibration in 97% of mobile phase A (0.1% formic acid in H20), a 55 min linear gradient (3-45% mobile phase B (0.1% formic acid in acetonitrile)), a 5 min linear gradient 0% mobile phase B), a 5 min isocratic phase (90% mobile phase B), a 5 min linear gradient (90%-3% mobile phase B), and a 5 min ilibration in 97% of mobile phase A, at 0.05 mL/min. The Q-Tof mass spectrometer was run in positive ion, V-mode with detection in the range of 200-2000 m/z. The source parameters were as follows: capillary voltage, 3.0 kV, sampling cone voltage, 40 V, source temperature, 120°C, desolvation temperature, 25 0°C, desolvation gas ?ow, 600 L/hr. Lockspray mass reference standard was glu-flb. MSE method was as follows: acquisition time, 3-70 mins, data range, 200-2000m/z, scan time, 1.5 sec, expression, low energy 6V, ramp high energy from .

The antibody aggregation was ed by size-exclusion, high-performance liquid tography method (SEC-HPLC) using an Agilent 1100. The mAb was diluted to 1 mg/mL in DPBS. The antibody (20 uL) was injected onto a TSngl SuperSW guard column (4.6 mm x 3.5 cm, 4 mm pore size, Tosoh Bioscience), followed by a TSngl SuperSW3000 column (4.6 mm X 30 cm, 4 um pore size), eluted from the column with 0.1 M PBS containing 0.15 M NaCl and 0.05% NaN3, at pH 7.4, at a ?ow rate of 0.3 mL/min for 20 min. All data were analyzed using Agilent ChemStation software. Percent ation was calculated as [PAaggregate/PAmelOO, where PA = integrated peak area.

UPLC/ESI-MS analysis ofmalemz'de-biotin:mAb conjugation Purified antibodies were diluted to 1 mg/mL in DPBS (samples were left at original concentration if below 1.0 mg/mL). Maleimide-PEG2-Biotin ((ma1)-PEG2-Biotin) (Thermo Fisher Scienti?c) was ved in DPBS to yield a 20 mM stock solution, followed by dilution to 1 mM in DPBS. Mal-PEG2-Biotin was added to 1 mL of decapped mAb at a 5:1 conjugation ratio and ted at 22°C with gentle rotating for 2 hr. The reaction was ed using a Zeba spin desalting column. The mAbs were then deglycosylated using PNGase F (New England BioLabs). G7 buffer (10 uL) and PNGase F (2 uL) were added to the mAb (90 uL).

The reaction was incubated in a er microwave (CEM) for 2 cycles: 1) microwave power W, 37°C, 10 min, followed by a 5—min pause; and 2) ave power 2 W, 37°C, 10 min.

A portion of the sample was reduced by adding dithiothreitol (DTT) to a final concentration of mM, followed by incubation at 60°C for 3 min.

Samples were then analyzed using a Waters Acquity UPLC and Q-Tof Premier mass spectrometer. Samples (0.5—2 ug each) were injected onto a MassPrep micro desalting column at 65°C, eluted from the column with a 5 min equilibration in 95% of mobile phase A, a min gradient (5-90% B), and a 10 min re-equilibration in 95% of mobile phase A, at 0.05 mL/min. Mobile phase A was 0.1% formic acid in water. Mobile phase B was 0.1% formic acid in acetonitrile. The Q-Tof mass spectrometer was run in positive ion, V-mode with detection in the range of 500-4000 m/z. The source parameters were as follows: capillary voltage, 2.25 kV (intact antibody)-2.50 kV (reduced antibody); sampling cone voltage, 65.0 V (intact antibody) or 50.0 V (reduced antibody); source temperature, 100°C, desolvation temperature, 250°C, desolvation gas ?ow, 550 L/hr. The protein peak was deconvoluted using the MassLynx MaXEnt 1 function. Conjugation ency was calculated as [Ibiotmylated/(Ibio?nylated+1unmod~l?ed)]*100 of the deconvoluted mass spectrum, where I = mass peak intensity.

BIAcore analysis ofmAbzaniigen af?nity Antibody concentrations were ed to te 30-40 RU signal when bound to the antigen. Humanized mAbs purified by standard protein A affinity chromatography or by the decapping method were injected over an anti-human IgG sensor on a BIAcore T100 (GE Healthcare) for 1 min at a ?ow rate of 10 uL/min. The sensor surface was washed by injecting HBS-P buffer for 1 min at a ?ow rate of 50 uL/min. To record the n association to the captured mAb, a series of increasing concentrations of antigen was injected for 60 sec at a ?ow rate of 50 uL/min. The dissociation of antigen was monitored for 30 min at the same flow rate. The sensor surface was regenerated by injecting 3 M MgClz for 1 min and then 30 sec at a ?ow rate of 30 uL/min. Sensograms were ed with Biacore T100 Evaluation Software using a 1:1 ir g model.

Bivaleni/Bispeci?c Fab preparation mAb-derived Fab fragments were prepared separately using immobilized , followed by isolation of the pure Fab nts from Fc/undigested mAb using Protein A chromatography. Maleimido-PEG4-azide was synthesized by combining NHS-maleimide and azido-PEG4-amine in DMSO for 1 hr in a 1:1 molar ratio. Unreacted NHS was quenched by the addition of Tris-HCl buffer to prevent homodimerization. Fabs were conjugated to either maleimido-PEG4-azide or maleimido-PEG4-dibenzocyclooctyne (DBCO) at a 5:1 molar ratio of maleimidezFab and reacted for 4 hr at 22°C. The modified Fab fragments were desalted twice each in DPBS to remove all unreacted products, and the Fab fragments were ed at a molar ratio of 1:1 at 2 mg/mL final concentration and allowed to form dimers overnight at 22°C.

The reaction was ed by SDS-PAGE and dimerization efficiency was estimated at 20%.

The dimer preparation was puri?ed from unreacted monomer by S-200 gel ?ltration chromatography, nt/Bispeci?c Octet assay Biotinylated human CA9 was captured on streptavidin Biosensor tips (Pall) for 4 min. Following tion in PBS for 2 min, the tips were incubated with the bivalent/bispeci?c Fabs, mAb alone, or Fab alone for 5 min. Following incubation in PBS for 2 min, the tips were ted with human endosialin/TEM-l for 5 min. Finally, the tips were ted in PBS for another 2 min. Association and disassociation protein to the tips was measured throughout.

Example 2 — Cy580 conjugation Objective Site-speci?c conjugation logies are desirable to produce a homogeneous t with a de?ned drug-to-antibody ratio (DAR). The VK domain of a rabbit mAb, such as that derived from Oryctolagus cuniculus, may contain a cysteine in position 80 (referred to as "Cys80") () and the CK region may contain a cysteine in position 171 ("Cys171") (). ico modelling predicted that Cys80 and Cys171 might be forming a disul?de bond, as the two S atoms are predicted to be approximately 1.6 A apart (). Human mAbs have proline, , or alanine residues in position 80 (), and serine in position 171 (), thus there is no disul?de bridge between the variable and constant region ().

The crystal structure closest to rabbit or human VK and CK sequences was identi?ed using BLAST pdb database and used as a template for modeling 155D5 mAb structure. Models were generated using Discovery Studio's "Build Homology Models" tool (Accelrys). The model with the lowest total energy was selected, typed with the CHARMm force?eld, and the energy was further minimized through two rounds of energy minimization using the "Minimize" tool. The CDR loops were then re?ned using the "Model Antibody Loops" tool. The model with the lowest total energy was selected, typed with the CHARMm force?eld, and the energy was further minimized as above. The proximity of CysSO and Cys171 () ts that these cysteines may be forming a disul?de bond. The "Build Mutants" tool was used to represent this disul?de bond.

Since disul?de bonds are critical for maintaining secondary and tertiary structural integrity, which in turn is necessary for an antibody’s biological activity, it was important to prove whether the predicted Cys80-Cys171 bond actually existed. Therefore, ad hoc experiments were conducted that unequivocally demonstrated that the rabbit mAbs contained such a bond (Table 4).

Table 4. Demonstration of the existence of Cys80-Cys171 disulfide bond 'DCTYNLSSTLSLTK (170-183) (SEQ ID N022) 1545.7465 Not observed ———(SEQIDN023) 82 Not observed (SEQ ID N022) (SEQ ID N023) (disulfide-linked Hetides as above) 2741.3285 2740.2659 LC-MS/MS analysis was performed on a Glu—C/Asp-N digest of rabbit 155D5 MAb (from NZW rabbit). Only masses corresponding to ide-linked cys80-cysl71 were found, indicating that cysSO forms a disulfide bond with cysl71 in rabbit IgG. A similar analysis was performed using 12 mAb (from b9 rabbit).

A Species-human chimerized mAb is made through the fusion between: i) the le region from the Species where the mAb was generated; and ii) the human nt region. This s is called chimerization. A humanized mAb is mostly made of human variable and constant s, except for those residues necessary for antigen binding, which are from the same species of the host from which the mAb was generated. This process is called humanization. To engineer human chimerized or humanized mAbs, whereby the mAbs were generated in hosts belonging to the species Oryctolagus cuniculus the entire constant domains as well as most of the variable regions (if zed) were genetically replaced with the human variable and human constant ces. After either chimerization or humanization, the Cys80 in the VK no longer formed a disulfide bond with position 171 in the CK (), and is therefore ed.

Germline NZW rabbit VK families have a ne at position 80 as shown in (the CDR s were deleted, and frameworks (FWR) 1, 2, and 3 were aligned).

Discovery and characterization ofan unpaired cysteine at position 80 Rabbit constant regions of 155D5 and 1E4 (anti-CA9), 12 (anti-hTEMl), as well as 33011 (anti-MSLN), all of which contain Cys80 and generated as described in Example 1, were replaced with the human constant regions of an IgGlK to generate rabbit/human chimerized mAb, as described elsewhere herein. Speci?cally, the rabbit VH region of 155D5 was fused with the human Cy region to generate Xi155D5HC, and the rabbit VK region of 155D5 was fused with the human CK region to generate Xi155D5LC. The rabbit/human chimerized 155D5 mAb with the unpaired CysSO is referred to herein as Xi155D5.

VH region of 12 was fused with the human Cy region to generate Xi1 2H, and the rabbit VK region of 1-55—2 was fused with the human CK region to generate - 2LC. The rabbit/human chimerized 12 mAb with the unpaired Cys80 is referred to herein as Xil2.

The rabbit VH region of 1E4 was fused with the human Cy region to generate xi1E4HC, and the rabbit VK region of 1E4 was fused with the human CK region to generate Xi1E4LC. The /human chimerized 1E4 mAb with the unpaired Cys80 is ed to herein as xi1E4.

The rabbit VH region of 33011 was fused with the human Cy region to generate xi33011HC, and the rabbit VK region of 33011 was fused with the human CK region to generate Xi33011LC. The rabbit/human chimerized 33011 mAb (Xi33011) with the unpaired Cys80 is ed to herein as 1.

Because the Cys171 was substituted with Ser171 during chimerization, the ized antibodies (Xi155D5, -2, Xi1E4, and Xi33011) ned an unpaired cysteine at position 80 in the VK (referred to as "Cy580"), When reduced using harsh conditions (20 mM DTT at 60 0C for 5 min), the molecular weight (mass) of the protein A-puri?ed mAb Xi155D5 light chain was 23,382 Da (). However, when subjected to mild reducing conditions (100 M DTT, RT, 30 min) the mass increased by 120 Da (). This mass increase suggested that CysSO might be forming a disul?de bond with a free cysteine, referred to as "capping" cysteine. This molecular structure, which results from a reaction called "cysteinylation", is referred to as "capped" CysSO. To con?rm this hypothesis, xi155D5 mAb was digested with Asp-N and Glu-C, and the masses of the peptides were analyzed. Mass spectrometry analysis of peptide fragments corresponding to residues 71 through Cys80 (FTLTITGVQC) (SEQ ID N0:24) ted an increased molecular weight by 119 Da (Table 5), thereby ming that Cys80 was capped.

Table 5. Mass spectrometry of X1155D5 peptide 71—Cys80 fragments FTLTITGVQC (SEQ 10 N024) 1082.555 12015544 118.9994 (SEQ ID N0225) 935.4866 1054.4946 119.008 (SEQ ID N0226) 834.4389 953.4397 119.0008 TITGVQC (SEQ 10 NO:27) 721.3549 840.3552 119.0003 ITGVQC (SEQ ID N028) 6203072 739.3067 118.9995 (SEQ ID NO:29) 31 626.2235 119.0004 (SEQ ID N0230) 405.47 525.1809 119.7109 (SEQ ID NO:31) 249.28 369.0883 119.8083 Because the lack of the Cysl71 disul?de bond could have led to antibody instability, disruption of antigen g, or both, antibody stability and n binding tests were conducted. The stability of Xi155D5 was tested using a SE-HPLC assay. This assay tests whether the lack of Cys80-Cysl71 disul?de bond could lead to aggregation (due to possible olecular Cys80-Cys80 bonds), or degradation (due to sed sensitivity to proteases).

Puri?ed antibody at 1 mg/mL in 1X PBS was stored at -80°C or 37°C for 1 week. Ten uL of Xi155D5 was injected onto a SuperSW3000 column (TOSOH Biosciences, 4.6 mm X 30 cm, 4 pm particle size) equipped with an in-line TSngl 4.6 mm X 3.5 cm guard column at a ?ow rate at 0.3 mL/min with 0.1 M sodium phosphate, 0.15 M NaCl, 0.05% NaN3 as mobile phase. No cant change in aggregation was observed between the two storage conditions ( and 5B). The level of aggregation was in the 3-4% range and hence within the normal range of a typical human IgG1 (data not shown). Little or no degradation ts were observed in any storage conditions (. These results suggest that Xi155D5 lacking the Cys80-Cys171 disul?de bond is a stable protein under the storage conditions tested.

To determine if chimerization, and ore the loss of CysSO-Cysl71 disul?de bond, results in structural perturbations leading to loss of antigen binding, the binding af?nity of mAbs 155D5, xi155D5, 12, and xi12 by surface plasmon resonance was evaluated.

Biotinylated ligand (biotin-hTEMI for 12, -CA9 for 155D5) was captured on a coated biotin CAP e chip (GE Healthcare, Piscataway, NJ) using HBS-EP as running buffer.

Final antigen capture levels were 130 RU and 280 RU, respectively, for biotin-TEMl and biotin- CA9. Serial dilutions of antibody (120 uL of 0-50 nM) were passed over the ligand-coated chip.

Dissociation was observed for 25 min. The chip e was regenerated with 6 M GuHCl, 250 mM NaOH. Sensograms were double referenced and kinetic ters were determined using BIAEvaluations software (ver. 4.1). Little or no loss of binding ty was observed due to chimerization of two different mAb (Table 6), ting that the lack of the CysSO-Cysl7l disulfide bond does not lead to disruption of the binding .

Table 6. c constants of chimerized and rabbit mAbs 12 3.7 x106 5.8 x 10'5 1.5 x 10'11 xi12 1.5 x106 '5 4.6 x10'11 155D5 5.1x 105 2.1 x 10'5 4.1x10'11 xilSSDS 4.6 x105 2.4 x10'5 5.1 x10'11 Assessment ofthe utility ofCys80for conjugations offunctional agents After having established that the lack of Cys80-Cys17l disulfide bond does not lead to structural perturbations, the possibility of replacing the capping cysteine with a thiol- reactive compound was explored. A thiol-reactive group can be attached to a linker, which in turn can be attached to a molecule of stic or eutic utility, referred to herein as "functional agent." Functional agents may include ?uorophores, ?uorescent dyes, polypeptides, immunoglobulins, antibiotics, nucleic acids, radionuclides, chemical linkers, small molecules (such as chemotherapeutic agents), ors, lipids, and drugs.

To substitute the capping cysteine with a onal agent, the capping cysteine was first removed. Exposing purified mAbs to reducing conditions could break the disulfide bond between Cys80 and the capping cysteine, referred to herein as "decapping." However, suboptimal ng conditions, for example harsh reducing conditions, could also break the inter— and intra—chain disul?de bonds, thereby compromising the mAb ure and activity.

Therefore, a decapping method involving removal of the capping cysteine using mild reduction, followed by reoxidation with Tris—containing buffer that does not alter the mAb structure and activity, while still allowing removal of the capping cysteine, was developed. A number of reducing agents were lly evaluated, including reduced glutathione, cysteine, TCEP, and DTT. Glutathione did not efficiently remove the capping cysteine (data not shown). Both DTT and TCEP efficiently removed the capping cysteine, but higher concentrations also resulted in the near-complete breakage of inter—chain disulfldes and likely some intra—chain disulfides as well (data not shown). The mild reductant cysteine efficiently d the capping cysteine and only limited inter-chain breakage was observed. Reoxidation was examined using phosphate , Tris buffer, and the strong oxidant CuSO4, No reoxidation of the disrupted chain disul?des was observed with phosphate buffer, while CuSO4 ef?ciently and rapidly reformed the disul?des, but was not evaluated further, due to its inherent toxicity compared with Tris.

Optimized conditions were adapted to a column format to allow for sequential puri?cation and decapping from feedstock. With this method, the antibody was bound to protein A resin and incubated with limited ?ow (0.5 mL/min) with a buffer containing 5 mM cysteine for 16 h to reduce (break) the CysSO-cysteine disul?de bond, followed by washing with a ne-free Tris- containing buffer for 60 h to remove the cysteines released by this treatment and re-oxidize any reduced hain disul?de bonds. The mAb was then eluted in a low pH glycine buffer. In an exemplary experiment whereby the decapping method was d to xi155D5, the mass of the non-reduced, puri?ed mAb was determined and ~99% of the mAb was found decapped, as demonstrated by the drop in mass equivalent to two free nes ( and 6B). Free thiol assay con?rmed the presence of two thiol groups per mAb, also demonstrating ef?cient re- oxidation (data not shown).

Decapped Cys80 can be conjugated to maleimide Cysteine is an (x-amino acid with anonpolar side chain (thiol; -SH). The reduced thiol side chain in an unpaired cysteine could serve as a nucleophile that can react with an electrophile molecule such as maleimide, a chemical compound with the a H2C2(CO)2NH. The electrophile double bond in maleimide readily reacts with the nucleophile thiol group found on cysteine to form a stable carbon-sulfur thioether bond. The arity of the thiol side chain, depending on the surrounding residues, might confer a hydrophobic property to a cysteine that may prevent solvent exposure necessary for chemical ations. In addition, the location of the cysteine in the context of the secondary structure of the peptide in which it is located may r prevent access of thiol-reactive molecule. Experimental testing to determine whether Cys80 could react with a thiol-reactive molecule after decapping was performed. The ed xi155D5 was incubated with maleimide-PEG2-biotin as described elsewhere herein. Mass spectrometry is showed that 94% of the mAb was conjugated with maleimide—PEGZ-biotin as indicated by an increase in molecular mass by 526 Da (, ponding to the onal agent mass. As each light chain was found conjugated (single mass peak, , the maleimide—PEGZ-biotin to xi155D5 ratio was homogeneously equal to 2:1. One malein1ide—PEG2-biotin was conjugated to a Cys80 in each of the two light chains in the chimerized mAb (CysSO1 and Cys802).

These results trate that Cys80 and Cys171 form a disulfide bond that links the VK and CK regions of a rabbit mAb. When rabbit mAbs were chimerized, Cys171 was substituted by Ser17l present in the human CK region. This tution abolished the Cys80- Cysl71 disul?de bond. When the effects of losing this disul?de bridge on the structural stability and activity of the resulting chimerized mAb compared with the parental rabbit mAb were evaluated, it was observed that the chimerized mAb was stable and active. It was discovered that both Cys801 and Cys802, which remained unpaired in the chimerized mAb, were capped by a free cysteine (capping cysteine). Subsequently, a method to remove the capping cysteine (decapping), while maintaining structural stability and activity of the resulting chimera mAb, was developed. Additionally, it was demonstrated that high yields of mAb conjugated to malein1ide—PEG2-biotin could be ed with a functional agent to mAb ratio equal to 2: 1.

Humanization ofrabbit mAbs Chimerized mAbs could be immunogenic when administered to humans and therefore it is ble to ze rabbit mAbs by substituting rabbit sequences with human sequences in the VK and VH regions. The amino acid sequence of mAb 155D5 was analyzed using a BLAST search t a human variable domain se at /wwwncbinlmnih.gov/igblast/ to identify the human sequence with highest homology to the rabbit sequence. IGHV3-64*04 and IGKVl-5*03 were identi?ed as the best sequences for humanization, as their use would result in the least number of rabbit residue substitutions (FIG.

The 155D5 sequences corresponding to the antigen binding domains as identi?ed by Kabat and Chothia CDRHl, Chothia CDRHZ, CDRH3, CDRLl, CDRLZ, and CDRL3 were ed into the framework (FWR) regions ofhuman IGHV3-64*04 or IGKVl- *03 to generate the humanized 155D5 mAb, named zu155D5-1 (Table 7 and Table 8).

During the humanization of 155D5LC (zu155D5LC), Cys80 was maintained, which was unpaired since the human kappa sequence has Ser17l as opposed to . zu155D5-1 was produced and purified using rd protein A purification, and found to be , as evidenced by the change of mass after decapping by 233 Da, approximately ponding to two capping cysteines (. As observed with xi155D5, zu155D5-1 could also be decapped with efficiency close to 100% (Table 9). However, the decapping led to massive levels of aggregation (70%) versus only 14% in Xi155D5. When zu155D5-1 was reacted with maleirr1ide—PEG2-biotin, 0% conjugation was observed while xi155D5 was 93% conjugated (Table 9). These results were surprising, as they suggest that: 1) having a cysteine in position 80, although necessary, is not sufficient to allow for site-specific conjugation of a functional agent; and 2) in some conditions, attempting conjugation on Cys80 could lead to high aggregation not compatible with drug manufacturability, However, since the disclosed studies, which characterized 5, demonstrated that at least in some conditions conjugation of a functional agent on Cys80 could be very efficient, it was next investigated how residues surrounding Cys80 may in?uence conjugation efficiency. uently, structural models of chimerized 5 were generated (), which indicated that residues Vall 1, Ala14, Glyl7, Thr18, Lys63, Thr76, Gly77, Val78, Ala83, Glu103, and Leu104 were in close proximity (within 18 A) to Cys80 and therefore could ially be affecting the efficiency of its conjugation.

Two versions of FWR1 (FWR1 a-b), one version of FWR2, three versions of FWR3 —c), and two version of FWR4 (FWR4a—b) were designed based on the aforementioned residues (Table 7).

Table 7. Versions of frameworks derived from human VK family IGKVl-S DIQMTQSPSTLSA W Y QQKPGKAP SGSGSGTEFTL TITC FGGGTKVEIK A KLLIY TISSLQCDDFATYYC (SEQ ID NO‘32' ) (SEQ ID NO‘35) (SEQ ID N033) (SEQ ID N034) ' DIQMTQSPSTVSA GVPSRFSGSGSGTEFTL AVGGTVTITC TITGVQCDDFATYYC Egggl?lggg) (SEQ ID N036) (SEQ ID N037) ' GVPSRFKGSGSGTEFT IGKV1-5 C n/a LTITGVQCDDAATYYC (SEQ ID NO:39) Residues in bold font te differences between the framework variants. n/a, not applicable.

A series of humanized 155D5 variants were generated that contained combinations of these frameworks and either Cys80—Xaa1-Xaa2-Phe83 (also ed to as C—X- X-F or CXXF) or Cys80-Xaa1-Xaa2-Ala83 (also referred to as C-X—X-A or CXXA), whereby "Xaa" or "X" indicates amino acids in position 81 and 82 (Table 8).

Table 8. Humanized 155D5 ts derived from human VK family IGKVl-S It was observed that, irrespective of the FWR version used, humanized mAbs having the C-X-X-F motif showed high ation (after decapping) and poor conjugation (Table 9). Conversely, 4 of the 5 mAb variants containing C-X-X-A motif, and irrespective of the FWR version used, showed high percent of conjugation ef?ciency (280%) and low aggregation (after ing) of <1 8% (Table 9). zu155D5-4 is an outlier that exhibited a low percentage of conjugation ef?ciency and a high aggregation after decapping. It is noted that the zu155D5-4 antibody has a propensity to aggregate independently of ing, which may account for the observed results. These data suggested that Phe83 is involved in causing high aggregation after decapping and is not conducive to ation on Cys80.

Table 9. Aggregation levels and conjugation efficiency of Xi155D5 versus different variants of 5 _%Aggl‘egates ___ mAb name ProA Decapped 0A) Decapped aa 80 83— Con'uated zu155D5-4 10.80% 28.30% 100. 00% 25.90% CXXA zulSSDS-S 7.40% 13.30% 100.00% 86.90% CXXA zu155D5-6 1.60% 6.70% 100.00% 88.20% CXXA zu155D5-7 4.90% 12.30% 100.00% 89.30% CXXA In bold are indicated the C—X-X-F motif and values not meeting the conjugation specifications.

It is desirable to achieve aggregation of 25% or less as a ng point of downstream process optimization, whereby further optimization of fermentation parameters, puri?cation conditions and drug formulations can achieve a more desirable ation level of % or less. It is also desirable to achieve 70% or higher conjugation ency to minimize product waste, cost of goods, and maximize product homogeneity. Henceforth, the investigation focused on meeting and ing these speci?cations by extrapolating rules to apply to the humanization methods of rabbit mAbs. 155D5-l was ted by following a standard practice, which involves utilizing the human germline sequences most homologous to the parent sequence. Because of this practice, the human VK subfamily IGKV1-5 was used for humanizing 155D5, having an percent identity of 70.5% (data not shown) and containing Phe83. The alternative VK ilies, which have similar percent of identity (data not shown), also contained Phe83 (. As this residue appeared to vely in?uence Cys80 conjugation efficiency and cause high aggregation, the presence or absence of Phe83 in other human VK families was evaluated, despite the fact that the highest identity found in these VK families was lower (68.4%) and therefore they would not typically be used for humanizing 155D5. Since all of the human VK families except IGKV4, IGKV5, and IGKV7 have multiple subfamilies, all subfamilies were aligned within their family (data not . After removal of redundant sequences, only one sequence remained for each of the IGKV—4, -5, -6, —6D, and -7 families, which could be used for humanization. For families IGKV-l, -2, and —3, the subfamily with closest homology to the sus was chosen as the framework to humanize 155D5. A preliminary analysis indicated that while some of these VK families contained Phe83 others did not (and Table 10).

To study the effect of the presence or absence of Phe83 in the context of these VK es, the CDR regions of 155D5 were genetically grafted onto the human frameworks IGKV1-39*01, IGKV2-40*01, IGKV3-11*01, IGKV4-1*01, IGKV5-2*01, IGKV6-21*01, IGKV6D-4l*01, and IGKV7-3*Ol. IGKV5-2*Ol Asn20, which contains an N—linked glycosylation site at residues 20, and its Thr20 t were not included in this analysis because the former could not be analyzed by mass spectrometry due to heterogeneity, and the latter did not express well. IGKV7—3*Ol Asn8l was not included in the analysis because it could not be analyzed by mass spectrometry. However, the t 3*01—Glu81 was included in the analysis. The ing results were obtained from the analysis (Table 10): l. The zed mAb with the huIGKVl-39 sequence and containing Phe83 showed an aggregation increase from 0 to 26% after decapping; the ation efficiency was borderline acceptable (68%) but aggregation being >25% was not; 2. The zed mAb with the huIGKV2-40 sequence contained a human germline Val83, this mAb showed aggregation <25% after decapping (11%) and conjugation ef?ciency >70% (92%). These parameters were acceptable, suggesting that the human germline Va183 is conducive to pairing with Cys80 to allow Cys80 conjugation; 3. The humanized mAb with the huIGKV3-11 sequence contained a human germline Phe83, while ation was below 25%, ation ncy was 55%, therefore not meeting the criterion of >70%, 4. The humanized mAb with the huIGKV4-1 sequence contained a human germline Va183, this mAb showed aggregation <25% after decapping (6%) and conjugation ef?ciency >70% (82%). These parameters were acceptable, suggesting that the human Va183 is conducive to pairing with Cys80 to allow Cys80 conjugation, as seen with the huIGKV2- 40 sequence, . The humanized mAbs with the huIGKV6-21 or huIGKV6D-41 sequences both contained a human germline Ala83; these mAbs showed aggregation <25% after decapping and ation ef?ciency >70%. These parameters were acceptable, suggesting that the human germline Ala83 is conducive to pairing with CysSO to allow CysSO conjugation, as seen with the Xi155D5 ce; and 6. The humanized mAb with the huIGKV7Glu81 sequence contain a human germline Thr83, this mAb showed aggregation <25% after decapping (6%) and conjugation ef?ciency close to 100%. These parameters were acceptable, suggesting that the human germline Thr83 is conducive to g with Cys80 to allow Cys80 conjugation.

Table 10. Aggregation levels and ation ef?ciency of different variants of zu155D5 ted by using various human VK subfamilies _ % Aggregates % % humanized mAb aa 80-83 Decapped Deca ed Con'uated zu155D5-1 (huVKl-S) CXXF 1.10% 70.10% 100.00% 0.00% zu155D5-huVKl-39 CXXF 0.00% 26.00% 100. 00% 68.70% zu155D5-huVK2-40 CXXV 9. 60% 11.70% 100. 00% 92. 70% 100.00% 55-00% zu155D5-huVK4-1 100.00% 82.70% .00% 61.70% 31.50% 11.90% 62.00% "80% "155133;:K7'3' 0.00% 6.30% 100.00% 100.00% In bold are indicated the F motif and values not meeting the conjugation speci?cations.

These results t the discovery that position 83 in?uences Cys80 conjugation efficiency negatively when occupied by phenylalanine, and indicate that, in addition to alanine, valine and threonine can substitute Phe83 to allow Cys80 conjugation.

To con?rm that, in the context of other mAbs, Phe83 is involved with causing high aggregation after ing and is not conducive to ation on Cy580, humanized mAb variants of 1E4 (anti-CA9), 166B3 (anti-CA9), and 33011 (anti-MSLN) were generated containing either C-X-X-F or C-X-X-A.

Monoclonal antibody variants having C-X—X-F motif met the conjugation specifications but not the aggregation specifications, whereas all humanized mAb variants having C-X-X-A showed aggregation less than 25% and conjugation efficiency greater than 70% (Tables 11 and 12). These studies demonstrate that the C-X-X-(non) F or K is a motif that allows meeting conjugation speci?cations.

Table 11. Aggregation levels and ation efficiency of different variants of zed mAbs comparing C-X-X-F versus C-X-X-A and C-X-X-I motifs h m ° % % u anized mAb ProA Decapped Deca ed C0n°u_ated zulSSDS-CXXF (zulSSDS-l) 1.10% 70.10% 100.00% 0.00% -CXXA (zu155D5-3) 0. 00% 17.30% 100.00% 80.10% In bold are indicated the F motif and values not meeting the conjugation specifications. m?cosvé?v?o WXQGO .msoENoEoomm @5088 38 3.3.85 :cwswohwwn of 83 91 m0; mva soumms?oo 34%: Ho vaSN $ "Swami—EU .52 $8.: $8.8 $8.8 $2.; $05.3 sosmmoammw $525; 2w .52 $8.2: $8.2: $8.2: $0002 $00.03 $ wig—30n— WEBB: 3200ch hoEcac—z 8: mo *oz $8.8 $8.8 $8.8 $0905 $05.3 338, 556000 coma—50D was $ mowswohww< .52 $8.8 $8.2 $8.8 $0m.0N $0m.£ $508 coumws?oo MQAQQU was $ 3:55.: $8.8 $8.8 $8.8 can < $8.8 $0v$w $0w.ww 232 .5895 woeswohww< U :o?mBmmxw $ $0: $8.2 $03 $8.2 géééééé EEEEEEE Héééééa gééééaé a é EEEEEEE EEEEEEE HEEEEEE Hééagég Héééééé $00.9 $0N.: 2: @3865 .NH $508 became—ZN En Bash wxxoé? 308.82 808.0% >xxoém? 808-83 Exxoé? 506+": 50848 808-83 €08-me 808-0% 285-82 808-82 80848 889% MUOmoém: $0-3: Eon In addition to Ala83, Va183, and Thr83, which can be found in VIC sequences belonging to huIGKV1-7 germline subfamilies, Ile83 can also be found, albeit rarely, in the huIGKVl germline family. Because Ala83, Val83 and Thr83 were already found conducive for Cys80 conjugation (Table 9, and Table 11), it remained to be determined whether Ile83 would be a favorable or unfavorable e with respect to Cys80 conjugation. Hence, the humanized mAb variant of 33011 was generated containing the Xaa1-Xaa2-Ile83 (also referred to as C-X-X-I or CXXI) motif, which showed aggregation less than 25% and conjugation ef?ciency greater than 70% (Table 11), consistent with previous C-X-X-(non)F motifs tested. This result supports the discovery that, in addition to Ala83, Va183 and Thr83, Ile83 can also substitute Phe83 to allow Cys80 conjugation while meeting ation speci?cations.

The disclosed studies te that, while chimerized rabbit mAbs are le for site-speci?c conjugation on Cys80 only after ng the disclosed decapping method as discussed above, humanized rabbit mAbs having the C-X-X-F motif or C-X-X-K motif are not well suited for such modi?cations due to high aggregation after decapping and/or low conjugation ef?ciency. It was hypothesized that the residues surrounding Cys80 may play a role in this phenomenon. Because the VK region encompasses more than 100 residues, understanding the interplay between key surrounding residues and Cys80 required the use of structural modelling paired with experimental testing. It was discovered that among the residues in close proximity to Cys80, Phe83 exerted a negative effect on Cys80 ation ef?ciency. It was also observed that all of the rabbit mAbs contained Phe83 after humanization using a classical humanization approach (), despite the fact that human VK sequences can also contain other amino acids in position 83, including e, ine, valine, and isoleucine. When these VK families were used for humanization, it was con?rmed that Phe83 and Ly583 are suf?cient to endow the humanized mAb with undesirable properties, such as high aggregation and/or low conjugation ef?ciency, while the remaining amino acid es (with the exception of Cys, which was not tested due to the desire to obtain a single Cys for conjugation) were conducive to Cys80 conjugation.

These results suggest that the C-X—X-F motif and C-X-X-K motif are to be avoided when conjugating at CysSO. Using a C-X-X-(not)F or K motif, for e the motif C- X-X-A, C-X-X-T, C-X-X-V, and C-X-X-I through the substitution of Phe83, chimerized as well as humanized mAbs were generated that met the desired conjugation speci?cations.

Affinity ofxi]55D5 and the humanized variants xi155D5 and the humanized variants were d by standard protein A chromatography or the decapping method, and their af?nity was analyzed using BIAcore (Table 13). There was less than a 2-fold difference in the KD between chimeric and humanized 155D5, and little difference between the samples puri?ed by the two different methods.

Table 13. BIAcore analysis of antigen g of humanized and chimeric 155D5 mAbs Xi155D5 4.58E--05 1.49E-04 3.24E-10—- zu155D5-l l.OlE--06 4.95E-04 4.98E-10 7.70E+05 3.55E-04 5.00E-10 zu155D5-2 05 3.76E-04 5.69E-10 4.58E+05 2.70E-04 5.89E-10 Example 3 - Generation of mesothelin-auristatin conjugated monoclonal antibodies Mesothelin (MSLN) is a cell-surface protein expressed in cancer, including certain ovarian, lung, pancreatic, and mesothelioma tumors. To improve the potency of agents targeting MSLN, de novo anti-MSLN rabbit monoclonal antibodies (mAbs) were developed and subsequently ered and conjugated with auristatin F (AuF) at Cys80 to generate a panel of MSLN-AuF conjugated mAbs.

Generation and characterization ofrabbit anti-MSLN mAbs New Zealand rabbits (Oryctolagus cuniculus) were immunized at on (Germany) using plasmid DNA encoding the human MSLN protein ("MSLN"). On day 52, animal sera were collected and later tested for MSLN binding by ?ow cytometry using mammalian cells ently sing human MSLN. -D shows that sera from both animals contained MSLN—binding dies. An ELISA assay later con?rmed this result (E). Then, animals were sacri?ced and PBMCs from blood as well as the lymphocytes from spleens and lymph nodes were collected and cryopreserved.

The lymphocytes from lymph nodes previously frozen were recovered and treated with DNase I and Pokeweed mitogen for one hour at 37°C/5% C02. Cells were counted and seeded at 5 cells/40 uL/well in complete IMDM medium ning 10% fetal bovine serum (FBS) and cytokines (IL-2 and IL-21 at 10.5 ng/mL) in 384-well plates pre-seeded with CHO—K1 cells expressing CD154 as feeder cells. Cells were fed again by adding 20 uL/well of the same medium as above after one week. Two weeks after seeding, B cell culture supematants were collected for ing to fy clones with specific reactivity to human MSLN. The plates with cells were frozen and stored at -80°C for future RNA ion and gene ampli?cation. B cell culture supematants were first screened for IgG production by rabbit IgG FRET assay. IgG- producing clones (5,775) were selected and screened by using ELISA to determine binding to human MSLN (1St ing). Some of the anti-MSLN—reactive clones were re-tested (211d screening) for reactivity to MSLN but not to a control antigen (human CD73). Five mAbs were selected and are shown in Table 14.

Table 14. Clones with specific reactivity to human MSLN 1St ing 2nd screening rabbit mAb Reading (Optical Density) g (Optical y) production reactivity reactivity reactivity The plates containing the selected mAbs were thawed, and the B cells were lysed to isolate RNAs using RNAqueous Kit (Ambion). The RNAs were used to set up RT-PCR reactions to amplify the antibody variable regions. The resulting DNAs were sequenced, and primers were designed for compatibility with the InFusion HD® cloning system as described by the manufacturer (Clontech, Mountain View, CA). The variable region PCR fragments were cloned into an expression plasmid ning either a human gamma or kappa constant region.

These plasmids were transfected using the FreeStyle 293 sion system (Thermo Fisher Scienti?c) to produce mAbs as described elsewhere herein.

Generation and terization ofMSLN-AuF Cys80 conjugated mAbs ized mAbs were generated as disclosed in Example 2, n xi33011 is one of the ?ve SLN mAbs and the other four mAbs were chimerized following the same method. Hence, these SLN mAbs contain unpaired Cys80, speci?cally, the motif C- X-X-A. They are herein referred to as xi32405, xi178Fl6, xi237N18, Xi33011, and xi383118.

After their production, the selected ?ve chimerized mAbs were ated to auristatin F (AuF) according to the following methods to generate MSLN—AuF Cy580 conjugated mAbs.

Antibody puri?cation with "decapping": Decapping rabbit/human mAbs is a step required for conjugation to CysSO. Using an AKTA Explorer (GE Healthcare), a protein A column (GE Healthcare) was equilibrated with 10 CV of 20 mM sodium phosphate, 10 mM EDTA, pH 7.2. The sample was then loaded, followed by washing unbound material with 10 CV of bration buffer. The column was washed with 16 CV of 20 mM sodium phosphate, mM EDTA, 5 mM cysteine, pH 7.2 at 0.5 mL/min for 16 h. The column was then washed with 60 CV of 20 mM Tris, pH 7.5 at 0.5 mL/min for 60 h. The sample was eluted using 5 CV of 0.1 M Glycine pH 2.9. The fractions containing the mAb were pooled and ed in DPBS using a MWCO 20K Slide-A-Lyzer (Thermo Fisher Scienti?c). Protein recovery was determined by BCA assay.

Auristatin F conjugation: Puri?ed and decapped chimerized MSLN-mAbs containing the C-X-X-A motif were incubated with maleimido-PEGZ-auristatin F (mal-PEG2- AuF) (structure shown below), diluted from a 10 mM stock in DMSO (Concortis Biosystems, San Diego, CA) at a 5:1 molar ratio (AuF:MAb) at a ?nal antibody concentration of 5 mg/mL in 1x PBS. Conjugation was performed for 2 hr at 22°C. Unreacted mal-PEG2-AuF was removed by desalting puri?cation on an AKTA FPLC ?tted with a 26/10 desalting column (GE Healthcare) using 1x PBS as running buffer. Antibody-containing fractions were pooled and n concentration ined by BCA assay.

Maleimido-PEG2—auristatin F: \T o'-T/\\0000 0 K/Ov\o/\/N \ SI-MS analysis: Samples were reduced by adding DTT to a ?nal concentration of 20 mM, followed by incubation at 60°C for 3 min. Samples were then analyzed using a Waters Acquity UPLC and Q-Tof Premier mass ometer. Samples (0.5-2 ug each) were injected onto a MassPrep micro desalting column at 65°C, eluted from the column with a 5 min equilibration in 95% of mobile phase A, a 10 min gradient (5-90% B), and a 10 min re- equilibration in 95% of mobile phase A, at 0.05 mL/min. Mobile phase A was 0.1% formic acid in water. Mobile phase B was 0.1% formic acid in acetonitrile. The Q-Tof mass spectrometer was run in positive ion, V-mode with detection in the range of 500-4000 m/z. The source parameters were as follows: capillary e, 2.25 kV t antibody)—2.50 kV (reduced antibody); sampling cone voltage, 65.0 V (intact antibody) or 50.0 V (reduced antibody), source temperature, 100°C, desolvation temperature, 250 °C, desolvation gas ?ow, 550 L/hr. The protein peak was deconvoluted using the MassLynx MaxEnt 1 function. A representative analysis is shown in . Typically, 390% of conjugated mAbs had an antibody-to- functional agent ratio of 2, signifying that each chimerized anti-MSLN mAbs carried an AuF molecule conjugated to each of Cy5801 and Cys802.

In vitro cytotoxicity A431-MSLN are cells derived from A431 cells (ATCC® 55TM) that stably express human MSLN. A431-MSLN cells were sub-cultured and seeded in 96-well plates at 10,000 cells/well/ 100 uL in RPMI medium containing 10% FBS and incubated at 37°C, 5% C02 for 16 hour. MSLN—AuF Cy580 conjugated mAbs were serially diluted 1:4 in 2 mL deep- well dilution plates. Diluted compounds (100 uL) were added to the cell , with ?nal concentrations ranging 0.28-75 ug/mL. Mal-PEG2—AuF was used at an equimolar concentration of the conjugated mAbs. For e, 10 ug/mL of MSLN-AuF Cys80 conjugated mAb (DAR=2) equates to 0.14 ug/mL of mal-PEG2-AuF. Plates were incubated at 37°C, 5% C02 for an additional 48 hours. Medium was discarded, plates were washed once with 200 uL DPBS, d with 50 uL of 0.2% crystal violet on at 22°C for 15 minutes, and then washed extensively with tap water. Plates were air-dried, and crystal violet was dissolved with 200 uL of 1% SDS solution. Colorimetric optical density was determined at 570 nm. Excel was used to extrapolate cell number from optical densities and GraphPad Prism 6 was used to plot the percent cytotoxicity.

When MSLN-negative A431 cells were treated with MSLN—AuF CysSO conjugated mAbs, no signi?cant xicity was observed, while mal-PEG2-AuF was cytotoxic only at the t concentration tested (A). When MSLN-positive A431-MSLN cells were treated with MSLN—AuF Cys80 conjugated mAbs, cant cytotoxicity was observed.

Based on the dose-response curves (B), MSLN-AuF CysSO conjugated mAbs could be categorized into 2 groups: Medium cytotoxicity — xi32405-AuF, and xi178F16-AuF; and High cytotoxicity — xi237N18—AuF, xi330l 1—AuF, and xi383118-AuF.

In vivo evaluation - Initial selection ofMSLN-AuF Cys80 conjugated mAbs The in vivo ef?cacy of the MSLN-AuF Cys80 conjugated mAbs was tested against tumor expressing MSLN, with the objective of selecting a few compounds with high ef?cacy and low toxicity.

The A431-MSLN cells were propagated in cell culture, ded in serum-free growth medium, mixed 1:1 with MatrigelTM, and 5 million 0,2 mL/mouse were implanted subcutaneously (so) into athymic NCr nu/nu mice. Tumor volume was determined by caliper measurements (mm) and using the a for an ellipsoid sphere: Length x Width2/2 = Volume (mm3). When tumor volume ranged 100-250 mm3, mice were randomized into treatment groups.

The animal body weights and tumor size were recorded twice . The overall design of this study is summarized in Table 15. The MSLN—AuF Cys80 conjugated mAbs were administered intravenously (i.v.) Q7D starting on randomization day (day 1), two doses total Table 15. Study design , _Dose # mice ent Re imen. (mg/kg) $33011qu Q7D" mam Q7D" xi178F16-AuF Q7D x 2 xi237N18—AuF Q7D x 2 6 8 xi383118-AuF 10 Q7D x 2 7 8 mal-PEGZ-AuF 10 Q7D x 2 shows the average tumor volumes among different treatment groups, up to day 18 post-implantation, when 3 of 8 animals had to be euthanized in the saline-treated group due to high tumor burden and/or tumor ulceration. Day 4 post-implantation corresponds to the day when mice were randomized into different treatment groups, with average tumor volume ranging 157-160 mm3 across all . On this day, the ?rst dose of MSLN-AuF Cys80 conjugated mAbs was administered followed by a second dose on day 11.

All MSLN-AuF Cys80 conjugated mAbs showed anti-tumor response, as evidenced by the fact that the tumor volume on day 18 was 20% or less compared to the saline- treated group (Table 16). In contrast, mal-PEGZ-AuF showed no anti-tumor response.

Table 16. Percent of tumor volume vs. saline group Number of on- 0A) tumor volume vs.

Treatment study animals on. saline group day 18 Saline Xi330l l-AuF xi32405-AuF Xll AuF xi237N18-AuF Xi38311 8-AuF mal-PEGZ-AuF Toxicity was also evaluated by observing any body weight loss on day 18 (based on average weight of on-study animals in each group) compared to day 4, as well as by observing any dead or moribund animals (Table 17). In the Xi32405-AuF-treated group, a body weight loss of 11% was ed in surviving animals as well as two oribund animals. In the Xi178F16-AuF, Xi237N18-AuF, and Xi383118-AuF-treated groups, no signi?cant body loss was ed, but one or two dead/moribund animals were ed. All the other treatment groups showed neither body weight loss nor dead/moribund animals.

Table 17. Assessment of gross toxicity % body weight Number of mice dead Treatment change on day 18 or moribund on day vs. day 4 18 Saline 106% Xi330ll-AuF 102% Xi32405-AuF Xil78F16-AuF Xi237N18-AuF Xi383ll8-AuF 107% 1 mal-PEGZ-AuF 1 11% Based on the umor responses as well as the minimal toxicity, mAbs xi33011-AuF and xi237N18-AuF were chosen for further evaluation.

Assessment oftarget speci?city ofanti-tumor activity ed by MSLN-AuF Cys80 conjugated The method used for this study was the same as described above (In vivo evaluation - Initial selection ofMSLN-AuF Cys80 conjugated mAbs). In addition to A431- MSLN cells, which were implanted on the left ?ank of each mouse on Day 4, MSLN—negative A431 cells were implanted in the same mouse on the te (right) ?ank on Day 1. The former cells grow tumors faster than the latter, and hence were implanted 3 days later so that the ?rst dose of test drug were given when the tumor in both ?anks were similar in volume. The overall design of this study is summarized in Table 18.

Table 18. Study Design -_. Dose . rou # mice treatment Re imen (mg/kg) ..m—07D x2 xi12-AuF Q7D x 2 ositive tumors: A shows the average tumor volumes among different treatment groups, up to day 18 post-implantation, when 4 of 8 animals had to be euthanized in the saline-treated group due to high tumor burden and/or tumor ulceration. Day 4 post-implantation corresponds to the day when mice were randomized into different treatment , with average A431-MSLN tumor volume ranging 8 mm3 across all groups. On this day, the ?rst dose of MSLN—AuF Cys80 conjugated mAbs was administered followed by a second dose on day 11. xi33011-AuF mediated anti-tumor responses that reduced tumor volume on day 18 to 12% compared to the saline-treated group (Table 19). Xi237N18-AuF mediated umor responses that reduced tumor volume to 24% compared to the saline—treated group (Table 19).

An unpaired, two-tailed t test indicated a p value of 0.00039 and 0.00197, respectively, suggesting that these anti-tumor responses versus saline-treated group were statistically significant. In st, mal-PEG2-AuF or Xi12-AuF, which targets endosialin/TEMl, showed no signi?cant anti-tumor responses.

Table 19. Percent of A431-MSLN tumor volume vs. saline group § MWFm:3: 2'4 .- mamam m asWmw, MSLN—negative tumors: B shows the average tumor volumes among different treatment , up to day 21 post-implantation; this day corresponds to Day 18 of A431-MSLN post—implantation, as described above, and Day 7 post-implantation ponds to the day when mice were ized into different treatment groups, with average A431 tumor volume ranging 174-184 mm3 across all groups. Xi33011-AuF mediated anti-tumor responses that reduced tumor volume on day 21 to 78% compared to the saline—treated group (Table 20).

Xi237N18-AuF Cys80 conjugated mAb mediated anti-tumor ses that reduced tumor volume on day 21 to 64% ed to the saline-treated group (Table 20). An unpaired, two- tailed t test indicated a p value of 0.317 and 0.091, respectively, ting that these anti-tumor responses versus saline-treated group were not statistically signi?cant. These responses were similar to those observed with mal-PEGZ-AuF or xi12-AuF.

Table 20. Percent of A431 (MSLN—negative) tumor volume vs. saline group % Tumor Volume Number of Mice on Treatment on Day 21 vs.

Study on Day 21 Saline Group 100% — xi33011-AuF x1237N18-AuF —— Xil-SS—Z-AUF 76% 5 mal-PEGZ-AuF 78% 8 Toxicity was also evaluated by observing any body weight loss on Day 21 post implantation of A431 cells compared to Day 7, as well as by observing any dead or moribund animals (Table 21). No body weight loss 310% was observed in any of the treatment groups.

Two deaths were observed in both xi33011-AuF and 18-AuF-treated group.

Table 21. Assessment of gross toxicity 0A’ Body Weight.

Number of Mice Dead or Treatment Change on Day 21 Moribund on Day 21 vs. Day 7 X133011 AuF x1237N18-AuF X11 2-AUF mal-PEG2-AuF Conclusion MSLN-AuF Cys80 conjugated mAb were ted and screened based on in vitro cytotoxicity and in viva anti-tumor activity. The in vitro cytotoxicity analysis indicated that these nds were targeting and killing MSLN—positive but not MSLN-negative tumor cells.

All MSLN—AuF Cys80 conjugated mAbs tested had anti-tumor activity, some of which appeared to be potentially more toxic than others. The nature of this toxicity was not r characterized. It was observed that both the MSLN-AuF Cys80 conjugated mAbs tested in viva could target MSLN—positive tumors and inhibit their growth, whereas no signi?cant effect was observed against egative tumors in the opposite ?ank. While the toxicity pro?le of xi237N18 was similar in both studies, xi33011-AuF treatment showed no toxicity in the ?rst study but was associated with two deaths in the second study. The nature of this toxicity was not further characterized; however, as x1330] l-AuF-treated mice still d a large MSLN- ve tumor on the other ?ank and were therefore sicker than the animals in the ?rst study, these mice may have been more sensitive to the effect of the massive tumor cell lysis t the MSLN—positive tumor.

Example 4 - Generation of dy-?uorescent dye conjugates The xi155D5 mAb containing the C-X-X-A motif was conjugated to the 800CW dye (LI-COR Biotechnology, Lincoln, NE) to generate a xi155D5-800CW CysSO conjugated mAb having two dye molecules ated to Cys801 and Cys802.

Conjugation of 800CW onto CysSO was carried out using maleimide-(CH2)2- 800CW (LiCor), whereby (CH2)2 is an alkyl linker. Brie?y, ide-(CH2)2- 800CW was dissolved into 100% DMSO at a ?nal tration of 10 mM. Maleimide-(CH2)2- 800CW was added to xi155D5 (5 mg/ml in 1x PBS) at a 5:1 molar ratio of b and incubated for 4 hr at room temp. Unincorporated dye was removed by desalting on PD-10 columns (Millipore). xi155D5-800CW was r polished via size-exclusion chromatography on Superdex 75. Void volume material was pooled, aliquotted, and frozen at -80 until use. SDS-PAGE and imaging analyses of reduced xi155D5-800CW indicated that the dye was conjugated only on the light chain but not the heavy chain ().

NCR-nude female mice were injected with either 0010205 or HT-29 human tumor cells subcutaneously to the right hind limbs. Tumor growth was monitored by caliper measurement. When the tumor volume was 200-300 mm3, xi155D5-800CW was injected through the tail veils at 0.1 mg/200uL/mouse. For monitoring xi155D5-800CW distribution via ?uorescent living imaging, animals were placed into an anesthesia chamber for approximately 3- 4 minutes using iso?uorane/Oz until the animals were unconscious. Animals were imaged using the ?uorescence setting of 745 excitation and 840 emission in a IVIS® Lumina II-Kinetic instrument (PerkinElmer, Waltham, MA). Images of the dorsal, right, ventral, and left side were taken at different time points as indicated in . After each successive image the animal were allowed to regain ousness in a recovery chamber receiving 100% oxygen ?ush followed by normal air.

Using the S or HT-29 models, it was observed that xi155D5—800CW ef?ciently targeted human tumors, as trated by the tumor-speci?c localization of its ?uorescent signal ().

These results demonstrated that a mAb containing the C-X-X-(not)F, K, or C motif can be conjugated to a dye and that the conjugated mAb can be used to identify and monitor tumor status.

Example 5 - Generation of bivalent/bispecific antigen-binding molecules When a mAb containing the (not)F, K, or C motif is digested with papain, or is inantly expressed as a Fab fragment, it will contain a single unpaired Cys80 since the Fab ns only one VK region. Using orthogonal conjugation chemistry, Cys80- containing Fabs can be used to generate chemically-conjugated bivalent/bispecific antigenbinding molecules, such as bivalent/bispecific Fab-Fab, that can be utilized for ing two independent disease-relevant targets, including two ligands (cytokines, chemokines), two membrane receptors, or ligand/receptor combinations, to name a few.

As an example, Fabs were generated from xi155D5 and xi12 using limited papain digestion, followed by protein A chromatography to remove the Fc fragments and undigested mAb. Fabs were shown to be fully decapped using mass spectrometry (data not shown). Subsequently, Xi155D5 and -2 Fabs were conjugated separately using maleimido-PEG4-dibenzylcyclooctyne (DBCO) and maleimido-PEG4-azide, respectively.

Unconjugated compound was removed by desalting chromatography and complete occupancy of the Cys80 sites was confirmed by mass spectrometry (data not shown). Then, xi155D5- maleimido-PEG4-DBCO and xi12-maleimido-PEG4-azide fragments were conjugated to each other via strain-promoted copper-free click chemistry by incubation in PBS at 22°C for 16 hours. Conjugated products were onated by using gel-?ltration chromatography (A) and the different species were identified by SDS-PAGE based on their ed molecular size (B). Fractions containing the xi155D5/xi12 bivalent/bispecific antigen-binding molecule were pooled and the xi155D5/xi1—55-2 bivalent/bispecific n-binding molecule identity was con?rmed by mass spectrometry based on its ed mass (95,939 Da, C).

The bispeci?city of XilSSDS/Xil2 bivalent/bispeci?c antigen-binding molecule was con?rmed via biolayer inferometry (BLI) analysis using an inverse sandwich assay. This analysis trated binding to immobilized human CA9 (bound by the Xi155D5 Fab ) followed by binding of soluble TEM-l (bound by -2 Fab moiety) ().

As expected, Xi155D5 mAb, Xi155D5 Fab, and the XilSSDS/Xil2 bivalent/bispeci?c antigen- binding molecule bound to immobilized CA9. Only the CA9-immobilized xilSSDS/xil-SS-Z bivalent/bispeci?c antigen-binding molecule was able to bind also human alin/TEM-l, as demonstrated by the additional response shift observed (, double arrow). e plasmon resonance analysis trated that the af?nity of XilSSDS/Xi12 for CA9 or TEM-l was the same or slightly reduced, respectively (Table 22).

These results demonstrate that: l) a mAb containing the C-X-X-(not)Fcan be conjugated to polypeptides, such as an antibody fragment or a Fab; and 2) when two mAbs or Fabs, of ent speci?city, containing the C-X-X—(not)F are orthogonally conjugated, a bivalent/bispeci?c compound can be generated.

Table 22. Af?nity of xi155D5/xi12 bivalent/bispeci?c antigen-binding molecule to CA9 and TEM-l . . Rmax Chi2 Antibody Flowcell ka (1/Ms) kd (1/s) (RU) (RU2) xi12 (Fab) TEMI' 3'76E' Fc4 8.65E+05 3.26E—04 38 5.89 Fe 10 nt/ c bispecific TEFl‘fl_ Fc4 1.02E+06 1.89E-04 96.8 1.1131:_ 31.6 05 2.19E—05 65.1 31:3? 8.86 xi155D5 (Fab) 4.99E- Fc4 CA9 5.19E+05 2.59E-05 63.9 8.24 bivalent/ bispeci?c 1 50E- Fc4 CA9 2.94E+05 4.40E—05 131.8 ' 24.3 Example 6 - Generation of antibody-peptide conjugates xi33011 and X11 2 mAbs containing the C-X-X—A motif were conjugated to azide-modi?ed peptide A[3(1-16) (SEQ ID NO:40) (Table 23).

Table 23. A[3(1-16) Peptide Sequence human amyloid-beta NH2-DAEFRHDSGYEVHHQK(PEG8—N3)—COOH peptide AMI-16) (SEQ ID NO:40) ACCESSION lBA6 A Conjugation of peptide A[3(1-16) onto Cys80 was carried out using a two-step conjugation procedure, whereby Cys80 was ?rst conjugated with maleimidodibenzylcyclooctyne (mal—DBCO). Azido-modi?ed peptide AB(1-16) was then conjugated to the DBCO-modi?ed mAbs using strain-promoted copper-free click chemistry. Brie?y, mAb (20 mgs) was incubated with mal—DBCO (Click Chemistry Tools, cat A108) at a mal-DBCO:MAb molar ratio of 5:1 for 16 hrs at 22°C in 1X DPBS. Unincorporated CO was removed from conjugated mAb by desalting tography using a HiPrep 26/10 column with 1x DPBS as running buffer. ation ef?ciency of 100% (no evidence of unconj ugated light chain) was con?rmed for both mAbs by LC-MS ( and Table 24). Each mAb (50 uL/each, 95 pg and 70 ug of -2 and 1, respectively) was ted with peptide 16) at peptidezMAb molar ratio of 20:1 in 1X DPBS for 16 hrs at 22°C. Conjugations were analyzed by SDS-PAGE. Samples were run under ng ions, with 20 mM DTT as reductant and heating to 75°C for 10 min prior to tion.

Analysis of the SDS-PAGE indicated retardation of the peptide-conjugated light chain migration accompanied by no detectable unconj ugated light chain, indicating ef?cient conjugation (). No change in heavy chain mobility was ed. Conjugations were then desalted using 0.5 ml Zeba 40k MWCO spin desalting columns (Thermo-Fisher) to remove unconjugated peptide and were analyzed by LC-MS. Mass spectrometry analysis (A-F) indicated that the peptide was conjugated to the light chains of Xi12 and Xi33011 with ef?ciencies of 85%-100% (Table 24).

These results demonstrated that a mAb containing the C-X-X-(not)F, K, or C motif can be ef?ciently conjugated to peptides.

Table 24. Conjugation summary predicted Amass -- 429 Da - 2833 Da Xi12 measured Amass -- 425 Da - 2829 Da conjugation ef?ciency 100% 100% 1 measured Amass + 432 Da + 2834 Da conjugation ef?ciency 100% 85% Table 25. Monoclonal antibodies and corresponding LC and HC Xi12LC Xi12HC xi12 (SEQ ID NO: 108) (SEQ ID N0256) xi155D5LC xi155D5HC (SEQ ID \10278) (SEQ ID \10252) zu155D5LC-1 zu155D5HC zu155D5-1 ( ' ) (SEO ID N0154) zu155D5HC (SEQ ID NO:54) zu155D5HC -3 (SEQ ID N084) (SEQ ID \10154) zu155D5HC zu155D5 4 (SEQ ID N086) (SEQ ID \10254) zul55D5LC-5 zu155D5HC zul55D5-5 (SEQ ID N088) (SE 0 ID N054) zu155D5LC-6 zu155D5HC zu155D5-6 (SEQ ID \IO:90) (SEQ ID \IO:54) zu155D5LC—7 zu155D5HC zu155D5-7 (SEQ ID N092) (SEQ ID N054) zu155D5LC-huVK1-39 zu155D5HC zu155D5-huVK1-39 (SEQ ID N094) (SEQ ID N054) zu155D5LC-huVK2-40 zu155D5HC zu155D5—huVK2-40 (SEQ ID N096) (SEQ ID N054) zu155D5LC-huVK3-11 zu155D5HC -huVK3-ll (SEQ ID \IO:98) (SEQ ID N054) -zu155D5LC—huVK4-1 zu155D5HC zulSSDS-huVK4—1 (SEQ ID NO: 100) (SEQ ID N054) —zu155D5LC-huVK6-21 zu155D5HC zu155D5-huVK6-21 (SEQ ID NO: 102) (SEQ ID \IO:54) —zu155D5LC-huVK6D-41 zu155D5HC zu155D5-huVK6D-41 (SEQ ID N0:104) (SEQ ID \10254) —zu155D5LC-huVK7G1u81 5HC zulSSDS-huVK7Glu81 (SEQ ID NO; 106) (SEQ ID N054) xilE4LC xilE4HC inE4 (SEQ ID N0:110) (SEQ ID N058) zu1E4LC-CXXF zu1E4HC zu1E4-CXXF (SEQ ID \102112) (SEQ ID \10260) _zu1E4LC-CXXA zu1E4HC zu1E4-CXXA (SEQ ID N0:114) (SEQ ID N0160) —xil66B3LC xil66B3HC 3 (SEQ ID N0:132) (SEQ ID N0;74) -zu166B3LC-CXXF zu166B3HC B3-CXXF (SEQ ID N0:134) (SEQ ID \10276) —zu166B3LC-CXXA zu166B3HC zu166B3-CXXA (SEQ ID NO: 136) (SEQ ID ) _X133011LC X133011HC xi33011 (8130 ID ) (SEQ ID N0162) —zu33011LC-CXXF zu33011HC zu3301 l-CXXF (SEQ ID N0:118) (SEQ ID NO:64) —zu33011LC-CXXA 2113301 1HC zu33011-CXXA (SEQ ID \102120) (SEQ ID \10164) —zu33011LC-CXXI zu3301 1HC zu33011-CXXI (SEQ ID NO: 122) (SEQ ID \10264) —xi32405LC xi32405HC xi32405 (SEQ ID NO: 124) (SEQ ID NO:66) —xil78F16LC X1178F16HC xil78F16 (SEQ ID NO: 126) (SEQ ID \IO:68) —x1237N18LC X1237N18HC x1237N18 (SEQ ID NO: 128) (SEQ ID \IO:70) 118LC 18HC x1383118 (SEQ ID NO: 130) (SEQ ID N0:72) 4ue1suO3 <2 "0141506 atqersz °d <2 xepeeq "0141305 HmIH 00G05U0m OGHEG DOGQEmw<<>mwum13? mw0wmmdx>o?qm >Ommmowwzommowew1OQ <<émuw;90mom>mwm>?? m000m0m4040m?mom?wa émomomww0mwwm OO>®UmImQQ mmwmmowommww>wz>020 m<0>>mm9>md>mmmwm> mmOmmHOQOmQ OZdeOmOW< 00:05U0m GZQ oowww 80:25me wEwZ IWOHommmv Amm%#OMH nwv Izm—H.

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Claims (37)

What is Claimed:

1. A method for generating a ated immunoglobulin, the method comprising: decapping a cysteine at amino acid position 80 0”) in a light chain variable region of an immunoglobulin d from rabbit, wherein the Cys80 is based upon the Kabat or Chothia numbering system, and wherein the immunoglobulin comprises a heavy chain variable region and the light chain variable region; and conjugating a thiol-reactive compound to the Cys80, wherein the thiol-reactive compound comprises a thiol-reactive group.

2. The method of claim 1, wherein the decapping comprises ting the globulin with a ng buffer followed by incubating the immunoglobulin with an oxidizing buffer.

3. The method of claim 2, further comprising immobilizing the immunoglobulin on a matrix prior to the incubating with the reducing buffer and eluting the immunoglobulin from the matrix following the incubating with the oxidizing buffer.

4. The method of any one of claims 1-3, wherein the thiol-reactive compound is attached to a functional agent.

5. The method of claim 4, wherein the functional agent comprises a phore, fluorescent dye, polypeptide, immunoglobulin, antibiotic, nucleic acid, radionuclide, chemical linker, small molecule, chelator, lipid, or drug.

6. The method of any one of claims 1-5, wherein the thiol-reactive nd is bound to a second thiol-reactive compound, the second thiol-reactive compound being bound to a second immunoglobulin having a second heavy chain le region and a second light chain variable region, the second light chain variable region having a cysteine at amino acid position 80 (“Cys802”), wherein the Cys802 is based upon the Kabat or Chothia numbering system, and wherein the second thiol-reactive compound comprises a second thiol-reactive group bound to the Cys802.

7. The method of any one of claims 1-6, n the Cys80 is unpaired.

8. The method of any one of claims 1-7, further sing substituting an amino acid at position 83 with an amino acid residue other than Phe, Lys, or Cys, wherein position 83 is based upon the Kabat or Chothia numbering system.

9. A method for generating an antigen-binding molecule, the method sing incubating a first conjugated immunoglobulin with a second conjugated globulin to te the antigen-binding molecule, wherein: the first conjugated immunoglobulin comprises a first heavy chain variable region and a first light chain variable region, the first light chain variable region having a cysteine at position 80 (“Cys801”) wherein the Cys801 is conjugated to a first thiol-reactive compound comprising a first thiol-reactive group, wherein the first immunoglobulin is derived from rabbit and the Cys801 is based upon the Kabat or Chothia numbering system; and the second conjugated immunoglobulin comprises a second heavy chain variable region and a second light chain variable region, the second light chain variable region having a cysteine at position 80 (“Cys802”) wherein the Cys802 is conjugated to a second thiolreactive nd comprising a second thiol-reactive group, wherein the second immunoglobulin is derived from rabbit and the Cys802 is based upon the Kabat or Chothia numbering system.

10. The method of claim 9, wherein the Cys801, the Cys802, or both, is unpaired.

11. The method of claim 9 or 10, further comprising, prior to the incubating step, decapping the Cys801, Cys802, or both; and conjugating a first reactive compound to the , a second thiol-reactive compound to the , or both, wherein the first thiol-reactive compound comprises a first thiol-reactive group and the second thiol-reactive compound comprises a second thiolreactive group.

12. The method of any one of claims 9-11, wherein the first thiol-reactive compound further comprises a first functional agent, the second thiol-reactive compound r ses a second functional agent, or both.

13. The method of any one of claims 9-12, wherein the first immunoglobulin is a first Fab, the second immunoglobulin is a second Fab, or both.

14. The method of any one of claims 9-13, further comprising substituting an amino acid at position 83 of the first light chain variable region with an amino acid residue other than Phe, Lys, or Cys, substituting an amino acid at position 83 of the second light chain variable region with an amino acid residue other than Phe, Lys, or Cys, or both, wherein position 83 is based upon the Kabat or Chothia numbering .

15. An antigen-binding molecule produced ing to the method of any one of claims 9-14.

16. An immunoglobulin derived from rabbit comprising a heavy chain variable region and a light chain variable region, the light chain variable region having a cysteine at position 80 (“Cys80”), wherein the Cys80 is unpaired, and an amino acid other than Phe, Lys, or Cys at on 83, wherein the Cys80 and position 83 are based upon the Kabat or a numbering system.

17. The immunoglobulin of claim 16, wherein the Cys80 is decapped.

18. The immunoglobulin of claim 16 or 17, comprising: a. a heavy chain variable region comprising amino acids 20-141 of xi155D5HC (SEQ ID NO:52) and a light chain variable region comprising amino acids 20-130 of xi155D5LC (SEQ ID NO:78); b. a heavy chain variable region comprising amino acids 20-144 of zu155D5HC (SEQ ID NO:54) and a light chain variable region comprising amino acids 20-130 of zu155D5LC- 3 (SEQ ID NO:84), zu155D5LC-4 (SEQ ID NO:86), zu155D5LC-5 (SEQ ID NO:88), zu155D5LC-6 (SEQ ID NO:90), zu155D5LC-7 (SEQ ID NO:92), zu155D5LC-huVK2- 40 (SEQ ID , 5LC-huVK4-1 (SEQ ID NO:100), zu155D5LC-huVK6-21 (SEQ ID NO:102), zu155D5LC-huVK6D-41 (SEQ ID NO:104); or zu155D5LC-huVK7- 3-Glu81 (SEQ ID NO:106); c. a heavy chain variable region comprising amino acids 20-138 of xi1E4HC (SEQ ID NO:58) and a light chain variable region comprising amino acids 20-130 of xi1E4LC (SEQ ID NO:110); d. a heavy chain variable region comprising amino acids 20-140 of zu1E4HC (SEQ ID NO:60) and a light chain variable region comprising amino acids 20-130 of zu1E4LCCXXA (SEQ ID NO:114); e. a heavy chain variable region sing amino acids 20-142 of xi166B3HC (SEQ ID NO:74) and a light chain variable region sing amino acids 20-130 of xi166B3LC (SEQ ID NO:132); or f. a heavy chain variable region comprising amino acids 20-145 of zu166B3HC (SEQ ID NO:76) and a light chain variable region comprising amino acids 20-130 of zu166B3LCCXXA (SEQ ID NO:136).

19. The immunoglobulin of claim 18, comprising: a. a heavy chain CDR1, CDR2, and CDR3 of xi155D5HC as set forth as SEQ ID NO:146, 148, and 150, respectively, and a light chain CDR1, CDR2, and CDR3 of xi155D5LC as set forth as SEQ ID NO:224, 226, and 228, respectively; b. a heavy chain CDR1, CDR2, and CDR3 of zu155D5HC as set forth as SEQ ID NO:152, 154, and 156, respectively, and a light chain CDR1, CDR2, and CDR3 of zu155D5LC-3 as set forth as SEQ ID NO:242, 244, and 246, respectively, zu155D5LC-4 as set forth as SEQ ID NO:248, 250, and 252, respectively, zu155D5LC-5 as set forth as SEQ ID , 256, and 258, respectively, zu155D5LC-6 as set forth as SEQ ID NO:260, 262, and 264, respectively, zu155D5LC-7 as set forth as SEQ ID NO:266, 268, and 270, tively, 5LC-huVK2-40 as set forth as SEQ ID NO 278, 280, and 282, respectively, zu155D5LC-huVK4-1 as set forth as SEQ ID NO 290, 292, and 294, respectively, zu155D5LC-huVK6-21 as set forth as SEQ ID NO 296, 298, and 300, respectively, 5LC-huVK6D-41 as set forth as SEQ ID NO 302, 304, and 306, respectively; or zu155D5LC-huVK7Glu81 as set forth as SEQ ID NO 308, 310, and 312, respectively; c. a heavy chain CDR1, CDR2, and CDR3 of xi1E4HC as set forth as SEQ ID NO:164, 166, and 168, respectively, and a light chain CDR1, CDR2, and CDR3 of xi1E4LC as set forth as SEQ ID NO:320, 322, and 324, respectively; d. a heavy chain CDR1, CDR2, and CDR3 of zu1E4HC as set forth as SEQ ID NO:170, 172, and 174, respectively, and a light chain CDR1, CDR2, and CDR3 of zu1E4LCCXXA as set forth as SEQ ID , 334, and 336, respectively; e. a heavy chain CDR1, CDR2, and CDR3 of xi166B3HC as set forth as SEQ ID NO:212, 214, and 216, respectively, and a light chain CDR1, CDR2, and CDR3 of xi166B3LC as set forth as SEQ ID NO:386, 388, and 390, respectively; or f. a heavy chain CDR1, CDR2, and CDR3 of zu166B3HC as set forth as SEQ ID NO:218, 220, and 222, respectively, and a light chain CDR1, CDR2, and CDR3 of zu166B3LCCXXA as set forth as SEQ ID , 400, and 402, respectively.

20. The globulin of claim 16 or 17, comprising: a heavy chain variable region comprising amino acids 20-139 of xi12HC (SEQ ID NO:56) and a light chain variable region comprising amino acids 20-129 of xi12LC (SEQ ID ).

21. The immunoglobulin of claim 20, comprising: a heavy chain CDR1, CDR2, and CDR3 of xi12HC as set forth as SEQ ID NO:158, 160, and 162, respectively, and a light chain CDR1, CDR2, and CDR3 of - 2LC as set forth as SEQ ID NO:314, 316, and 318, respectively.

22. The immunoglobulin of claim 16 or 17, comprising: a. a heavy chain variable region comprising amino acids 20-142 of xi33O11HC (SEQ ID NO:62) and a light chain variable region comprising amino acids 20-131 of xi33O11LC (SEQ ID NO:116); b. a heavy chain variable region comprising amino acids 20-145 of zu33O11HC (SEQ ID NO:64) and a light chain variable region comprising amino acids 20-131 of zu33O11LCCXXA (SEQ ID NO:120) or zu33O11LC-CXXI (SEQ ID NO:122); c. a heavy chain variable region comprising amino acids 20-137 of xi324O5HC (SEQ ID NO:66) and a light chain variable region comprising amino acids 20-127 of xi324O5LC (SEQ ID NO:124); d. a heavy chain variable region comprising amino acids 20-137 of xi178F16HC (SEQ ID NO:68) and a light chain le region comprising amino acids 20-127 of xi178F16LC (SEQ ID NO:126); e. a heavy chain variable region comprising amino acids 20-132 of xi237N18HC (SEQ ID NO:70) and a light chain variable region comprising amino acids 20-127 of 18LC (SEQ ID NO:128); or f. a heavy chain variable region comprising amino acids 20-137 of xi383I18HC (SEQ ID NO:72) and a light chain variable region sing amino acids 20-127 of xi383I18LC (SEQ ID NO:130).

23. The immunoglobulin of claim 22, comprising: a. a heavy chain CDR1, CDR2, and CDR3 of xi33O11HC as set forth as SEQ ID NO: 176, 178, and 180, respectively, and a light chain CDR1, CDR2, and CDR3 of xi33O11LC as set forth in SEQ ID NO:338, 340, and 342, respectively; b. a heavy chain CDR1, CDR2, and CDR3 of zu33O11HC as set forth as SEQ ID NO:182, 184, and 186, respectively, and a light chain CDR1, CDR2, and CDR3 of zu33O11LCCXXA as set forth as SEQ ID , 352, and 354, respectively, or zu33O11LC-CXXI as set forth as SEQ ID NO:356, 358, and 360, respectively; c. a heavy chain CDR1, CDR2, and CDR3 of 5HC as set forth as SEQ ID NO:188, 190, and 192, respectively, and a light chain CDR1, CDR2, and CDR3 of xi324O5LC as set forth as SEQ ID NO:362, 364, and 366, respectively; d. a heavy chain CDR1, CDR2, and CDR3 of xi178F16HC as set forth as SEQ ID NO:194, 196, and 198, respectively, and a light chain CDR1, CDR2, and CDR3 of xi178F16LC as set forth as SEQ ID , 370, and 372, respectively; e. a heavy chain CDR1, CDR2, and CDR3 of xi237N18HC as set forth as SEQ ID NO:200, 202, and 204, respectively, and a light chain CDR1, CDR2, and CDR3 of xi237N18LC as set forth as SEQ ID NO:374, 376, and 378, respectively; or f. a heavy chain CDR1, CDR2, and CDR3 of xi383I18HC as set forth as SEQ ID NO:206, 208, and 210, respectively, and a light chain CDR1, CDR2, and CDR3 of xi383I18LC as set forth as SEQ ID NO:380, 382, and 384, respectively.

24. A conjugated immunoglobulin comprising: the immunoglobulin of any one of claims 16-23, wherein the cysteine at position 80 is conjugated to a thiol-reactive compound, the thiol-reactive compound sing a eactive group.

25. The conjugated immunoglobulin of claim 24, wherein the thiol-reactive compound further comprises a functional agent.

26. The ated immunoglobulin of claim 25, wherein the functional agent comprises a fluorophore, fluorescent dye, polypeptide, immunoglobulin, antibiotic, nucleic acid, uclide, chemical linker, small molecule, chelator, lipid, or drug.

27. Use of a pharmaceutically effective amount of a conjugated mesothelin immunoglobulin in the manufacture of a medicament for the ent of a mesothelin-expressing cancer, wherein the conjugated mesothelin immunoglobulin comprises: the immunoglobulin of claim 22 or 23, and a thiol-reactive compound comprising a reactive group, a linker, and a functional agent.

28. The use of claim 27, wherein the functional agent is auristatin F.

29. An antigen-binding le comprising: a first conjugated immunoglobulin comprising a first heavy chain le region and a first light chain variable region, the first light chain le region having a cysteine at position 80 (“Cys801”), n the Cys801 is conjugated to a first thiol-reactive compound comprising a first thiol-reactive group, and wherein the immunoglobulin is derived from rabbit and the Cys801 is based upon the Kabat or Chothia numbering system, and a second conjugated immunoglobulin comprising a second heavy chain variable region and a second light chain variable region, the second light chain variable region having a cysteine at position 80 (“Cys802”) wherein the Cys802 is conjugated to a second thiolreactive compound comprising a second thiol-reactive group, and wherein the immunoglobulin is derived from rabbit and the Cys802 is based upon the Kabat or Chothia numbering system.

30. The antigen-binding molecule of claim 29, wherein Cys801, Cys802, or both, is unpaired.

31. The antigen-binding molecule of claim 29 or 30, wherein the amino acid at position 83 of the first immunoglobulin, the amino acid at position 83 of the second immunoglobulin, or both, is an amino acid residue other than Phe, Lys, or Cys.

32. The antigen-binding molecule of any one of claims 29-31, n the first thiol-reactive nd further comprises a first functional agent, the second thiol-reactive compound further comprises a second functional agent, or both.

33. The antigen-binding molecule of any one of claims 29-32, wherein the first immunoglobulin, second immunoglobulin, or both is a Fab.

34. A light chain variable region derived from rabbit for use in a ated immunoglobulin, the light chain variable region having a ne at amino acid position 80 (“Cys80”) and an amino acid residue other than Phe, Lys, or Cys at amino acid position 83, wherein the Cys80 is ed.

35. The light chain variable region of claim 34, wherein the light chain variable region has a Cys80-Xaa1-Xaa2-Xaa3 motif, wherein Xaa3 is any amino acid other than Phe, Lys, or Cys.

36. A nucleic acid molecule encoding the immunoglobulin of any one of claims 16-23.

37. An isolated recombinant host cell comprising the nucleic acid molecule of claim 36. “movuoz Awowuoz Amowuoz Awowuoz OH OMmV OHmV OMWV OHmV Umeth 00%WH< U?wmzmm0>>mHHIGOE>MUHWM¥IMZMOHmHHHAHmmHZWHUQMEB>H>QImmwxz<§U>HH>HGH<>OQmmmeOmE>mo?w>xmxmwomm0mzwmOH?ZQ>EBO>¥>mmm<€>Em MgwmmwmeOUVWH¢wmHHHHmOmeUmwwhmmm>wmWOmwa>mmmmHOHH>O¢ IllmeZNOOOWWHwmm?mmme>m mHEQEm mQHAHmmH mmmm>wm WE m .05.— m meOO>3 Hmdm? QU>Hm HE> me mOHH >mm< HO«NWH>MOH\QH >KwHI3£ mSmQOWQOU Ho*Homelnu Ho*owa\d£ mSmQOmCOU N m SUBSTITUTE SHEET (RULE 26) SUBSTITUTE SHEET (RULE 26) <—————————FWRl———————— > <——————FWRZ---> < —————————————— FWR3----( ------> IGKVlSl*0 ALVMTQ'PSPVSAAVGG’VTISC GQPP LLIY GVPSRF GSGSG"QF‘L'ISGVECDDAA'YYC (SEQ ID NO:407) IGKVlSZ*0 AQVLTQrESPVSA?VGG'VTINC WYQQKPGQPP LLIY GVPSRFSGSGSG“QF‘LrISGVQCDDAArYYC (SEQ ID NO 408) IGKV152*02 AQVLTQrESPVSA?VGGrVTINC GQPP LLIY GVPSRFSGSGSG“QF‘LrISGVQCDDAArYYC (SEQ ID NO 409) IGKVlS3*0 AQVLTQ'PASVSAAVGG'VTINC WYQQKLGQPE 3*02 AQVMTQ'EASVSAAVGG'VTIIC NYQQKLGQPE IGKVlS4*0 'EASVEAAVGG'ITINC WYQQKPGQPP LLIY GVPSRFSGSGSG"QF‘L'ISGVQCDDAA'YYC (SEQ ID NO:412) IGKVlSS*0 DPVMTQrPSSTSAAVGG'VTINC WFQQKPGQPP LLIY GVPSRFSGSGSG QF‘LrISGVQCDDAArYYC (SEQ ID No:413) 6*0 DGVMTQrPAPVSAAVGGrVTINC WYQQKPGQPP LLIY GVPSRF GSGSG“QF‘LrISGVQCDDAArYYC (SEQ ID No:4I4) IGKVlS7*Ol DVVMTQTPSPVSAAVGGTVTINC WYQQKPGQPEKLLIY GVPSRFSGSGSGTQFTLTISGVQCDDAATYYC (SEQ ID No:415) B*0 DPMLTQTASPVSAAVGSTVTISC WFQQKPGQPPKLLIY GVPSRFKGSGSGTQFTLTINGVQCDDAATYYC (SEQ ID NO 416) IGKV159*0 AAVLTQTPSPVSVAVGGTVTINC GQPP LLIY GVSSRF GSGSGTQF‘LTISGVQCDDAATYYC (SEQ ID No:4I7) IGKV 510*01 'PSSKSAAVGD'VTIKC GQPP LLIY GVPSRF GSGSG"QF‘L'ISDLECADAA'YYC (SEQ ID N0:418) IGKV 511*0 DPML“Q’ASPVSAAVGS'VTISC WFQQ PGQPP LLIY GVPSRF GSGSG“QF‘L’IKGVQCDDAA’YYC (SEQ I) NO 419) IGKV 512*0 DPVLnQ’ASPVSAAVGG’VT SC WYQQ II Y GVPSR" GSGSGnQF‘I’ SGVQCDDAA’YYC (SEQ D NO:420) IGKV 513*0 ALVMTQ'PSPVSAAVGS'VTISC WFQ* LLIY GVPSRF GSGFG"QF‘L'ISGAQCDDAA'YYC (SEQ ID NO:42l;SEQ ID No:422) IGKV 514*0 DPMLTQ'ASPVSAAVGS'VT SC WYQQ >GQPP IIIY GVPSR‘ GSGSG"QF‘I’ISGVQCDDAA’YYC (SEQ I) N02423) IGKv 515*0 AQGP“Q’PSSVSAAVGG’VT NC WYQQ >GQPP II Y GVPSRE QF‘Ir SDVQCDDAA’YYC (SEQ ) NO:424) IGKV 816*0 DPML“QrASPVSAAVGSrVT SC WFQQ GQPP II Y GVSSR) GSGSGmQF‘I’ SGVQCDDAArYYC (SEQ ) NQ:425) IGKV 517*0 'PASVEAAVGG'VT KC WYQQ DGQPPKII Y GVSSRF GSGSG?EY‘Ir SGVQCADAA'YYC (SEQ D No:426) IGKV 518*0 AAVMTQ'PSPVSVAVGG'VTINC WFQQ PGQPPKLLIY GVSSRF GSGSG"QF‘L'ISGVQCDDAA'YYC (SEQ ID N02427) IGKV 319*0 AAVLTQ'PSPVSAAVGG’VT KC WYQQ >GQPP IIIY GVPDR‘SGSGSG"QF‘I'ISGVQCDDAA'YYC (SEQ I) N02428) IGKv 520*0 DPML“QrASPVSAAVGS'VT SC WYQQ JGQPP II Y GVPSRE GSGSG“QF‘I’ SGVQCDDAArYYC (SEQ ) No:429) IG IG IG 164v 524*0 DPMLTQ'ASPVSAAVGS'VT SC WFQQ >GQPP IIIY GVPSR‘ GSGSG"QF‘I'INGVQCDDAA'YYC (SEQ I) ) IGGQPP II Y GVPSRE GSGSG"QF‘I’ SGVQCDDAArYYC (SEQ ) No:434) IG IG IG IGKV 529*0 DVVMTQTPSPVSAAVGGTVTINC WYQQKPGQPP LLIY GVPSRF GSGSGTQF‘LTINGVQCDDAATYYC (SEQ ID N02438) IGKV 530*01 DPMLTQ'ASPVSAAVGSTVTISC WFQQKPGQPP LLIY GVPSRF GSGSG"QF‘L'INGVQCDDAA'YYC (SEQ ID No:439) IG IGGQRP IG 164v 534*0 va TQ'PSPVSAAVGG’VT C WYQQ >GQPP ILIY GVPSR‘SGSGSG"EF‘I'ISDLECADAA'YYC (SEQ I) ) IGGQPP II Y EGSGSG"QFIIF SGMKAEDVA'YYC (SEQ ) NQ:444) IGGQPP II Y GVPSEE GSRSG“EF‘I' SDLECADAA'YYC (SEQ ) NQ:445) 1G IGKV 539*0 DVVMTQ'PSPVSAAVGG'VTI C NYQQ PGQPPKLLIY GVPSRFSGSGSG"QF‘L'ISGVQCDDAA'YYC (SEQ ID N0:447) IGKv 540*0 DPVITQ'ASPVSAAVGG'VT SC WYQQ DGQPP ILIY GVPSR" GSGSG"QF‘I'ISGVQCDDAA'YYC (SEQ I) N02448) IGKV 541*0 'PSPVSVAVGG'VT C WFQQ JGQPP II Y GVSSRE GSGSG"QF‘I’ SGVQCDDAA'YYC (SEQ ) NO 449) IGGQPP II Y GVPPEE GSGSG“EF‘I’ SDLECADAArYYC (SEQ ) NO 450) IG IG 164v 546*0 DPVITQ'ASPVSAAVGG'VT SC WFQQ >GQPP ILIY GVSSR" GSGSG"QF‘I'ISGVQCDDAA'YYC (SEQ I) N02453) IsGQP> II Y GVPSEE GSGSG"QF‘I’ NGVQCDDAA'YYC (SEQ ) No:454) IGGQP> II Y GVSSEE GSGSGmgF‘I’ SGVQCDDAA’YYC (SEQ ) No:455) IG IGKV_550*01 DPMLTQTASPVSAAVGSTVTISC WFQQKPGQPPKLLIY GVSSRFKGSGSGTQFTLTISGVQCDDAATYYC (SEQ ID NO:457) IGKV 551*0 YVMMTQTPSSVSEAVGGTVTIYC WYQQKPGQPP LLIY GVPSRFKGSGSGTQF‘LTINGVQCDDAATYYC (SEQ ID N02458) IG IGKV 353*0 YVMM“Q’PSSVSEAVGG’VT SC NEQQ JGQPJ II Y GVPSEE GSGSGHQEIIF KGVQCDDAA'YYC (SEQ ) NO:460) IG IG 164v 856*0 DPVLTQ'ASPVSAAVGG’VT SC W“QQ DGQPP ILIY GVPSR“ GSGSG"Q“‘L'IKGVQCDDAA'YYC (SEQ ID NO 463) IGGQP> II Y GVPSNVSSSGSG"EEIIFISGVQPG)AAFYYC (SEQ ) NQ:464) IGKV 558*0 DVVM“Q’PSPVSAAVGG’VT NC WYQQ >GQP> II Y GVPSRESGSGSGHQEIIFISGVQCD)AAFYYC (SEQ ) ) IG IGKV 560*0 DPVLTQ'PSPVSVAVGG'VTINC WFQQ PGQPPKLLIY GVSSRF GSGSG"QF‘L'ISGVQCDDAA'YYC (SEQ ID NO:467) IGKV 361*0 ALVMTQ'PSSTSEPVGG'VT NC W“QQ >GQPD LLIY GVSSR‘ Q“‘L'ISGVQCDDAA'YYC (SEQ ID N02468) IGKV 362*0 DVVM“Q'ASPVSAAVGS'VT SC NEQQ JGQP> II Y GVPSRE GSGSG"QE‘IIINGVQCDDAAFYYC (SEQ D No:469) IGKV 363*0 AAVL“QrPSPVSVAVGGrVT NC GQPP II Y GVPSRE GSGSGHQE‘IIINGVQCDDAAIYYC (SEQ D NO 470) IGKV 564*0 rASPVSAAVGGrVT SC NEQQ =GQEE II Y GVESRE GSGSGHQE‘IIINGVQCDDAAIYYC (SEQ D NO 471) IGKV 565*0 YVMMTQ'ESSVSEAVGG'VTIYC WYQQ PGQPEKLLIY GVPSRFRGSGSG"QF‘L'INGVQCDDAA'YYC (SEQ ID NO:472) IGKV 566*0 DPMLTQ'ASPVSAAVGS'VT SC W“QQ >GQPE LLIY GVPSR‘ GSGSG"Q“‘L'INGVQCDDAA'YYC (SEQ ID N02473) IGKV 567*0 DVVM“Q'PSSKSAAVGD'VT KC WYQQ >GQPP II Y GVPSRE GSGSG"QE‘IFINGVQCDDAAFYYC (SEQ D NO 474) IGKV 568*01 AAVL“QrPSPVSVAVGGTVTINC NEQQ >GQEE IIIY GVSSRE GSGSGTQE‘IIINGVQCDDAATYYC (SEQ D N0:475) SUBSTITUTE SHEET (RULE 26) FIG;4 .A B §\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\