NZ623756B2 - Antibodies, variable domains & chains tailored for human use - Google Patents

Abstract

Disclosed is an isolated antibody-producing cell that is a progeny of a non-human vertebrate cell whose genome comprises an immunoglobulin heavy chain locus comprising human gene segment JH6*02, one or more human VH gene segments and one or more human D gene segments upstream of a constant region; wherein the JH6*02 gene segment comprises the following nucleotide sequence: ATTACTA CTACTACTAC GGTATGGACG TCTGGGGCCA AGGGACCACG GTCACCGTCT CCTCAG, wherein the antibody-producing cell produces an antibody heavy chain specific for a target antigen which is an antigen of an infectious disease pathogen, and wherein the variable domain of the heavy chain is the product of recombination between a human VH, human D and human JH6*02 and wherein the HCDR3 length is at least 20 amino acids. Further disclosed are antibodies which bind an infectious disease pathogen which comprise said JH6*02 segment and have a HCDR3 of at least 20 amino acids and methods of making said antibodies. herein the JH6*02 gene segment comprises the following nucleotide sequence: ATTACTA CTACTACTAC GGTATGGACG TCTGGGGCCA AGGGACCACG GTCACCGTCT CCTCAG, wherein the antibody-producing cell produces an antibody heavy chain specific for a target antigen which is an antigen of an infectious disease pathogen, and wherein the variable domain of the heavy chain is the product of recombination between a human VH, human D and human JH6*02 and wherein the HCDR3 length is at least 20 amino acids. Further disclosed are antibodies which bind an infectious disease pathogen which comprise said JH6*02 segment and have a HCDR3 of at least 20 amino acids and methods of making said antibodies.

Description

PCT/GBZOIZ/052296 FID T IVNTI The present ion relates to the provision of antibody therapeutics and prophylactics that are tailored specifically for human use.

The present invention provides libraries, rates and cells, such as transgenic mice or rats or transgenic mouse or rat cells. rmore, the invention relates to methods of using the vertebrates to isolate antibodies or nucleotide sequences encoding antibodies. dies, heavy chains, polypeptides, tide sequences, pharmaceutical compositions and uses are also ed by the invention.

The state of the art provides non-human vertebrates (eg, mice and rats) and cells comprising enic immunoglobulin loci, such loci comprising human variable (V), diversity (D) and/orjoining (J) segments, and ally human nt regions. Alternatively, endogenous constant regions of the host vertebrate (eg, mouse or rat constant s) are provided in the transgenic loci. Methods of constructing such transgenic vertebrates and use of these to generate antibodies and nucleic acids thereof following antigen immunisation are known in the art, eg, see 552 (Medarex), US$939598 (Abgenix), U56130364 (Abgenix), W002/066630 (Regeneron), w02011004192 (Genome Research Limited), W02009076464, W02009143472 and W02010039900 (Ablexis), the disclosures of which are explicitly incorporated herein. Such transgenic loci in the art include varying amounts of the human V(D) J repertoire. Existing transgenic immunoglobulin loci are based on a single human DNA source. The potential diversity of human antibody variable regions in non-human vertebrates bearing such transgenic loci is thus confined.

The inventors considered that it would be desirable to tailor the genomes of these transgenic non- human vertebrates (and thus antibody and antibody chain products of these) to address the variability — and commonality — in the natural dy gene usage of humans. The inventors wanted to do this in order to better address human use of antibody-based therapeutic and prophylactic drugs. 2012l052296 It would be desirable also to provide for novel and potentially expanded repertoire and diversity of human le regions in transgenic immunoglobulin loci and non-human vertebrates harbouring these, as well as in dies produced ing immunisation of such animals.

M Y F I VENTI The present invention has been developed from extensive bioinformatics analysis of l antibody gene segment distributions across a myriad of different human tions and across more than two thousand samples from human individuals. The inventors have undertaken this huge task to more thoroughly understand and design non-human vertebrate systems and ant antibodies to better address human medical therapeutics as a whole, as well as to enable rational design to address specific ethnic populations of humans. Using such rational design, the inventors have ucted transgenic non-human vertebrates and isolated antibodies, antibody chains and cells expressing these in a way that yields products that utilise gene segments that have been purposely included on the basis of the human bioinformatics is. The examples illustrate worked experiments where the inventors isolated many cells and antibodies to this effect.

The invention also relates to synthetically-extended & ethnically-diverse uman immunoglobulin gene repertoires. The present invention thus provides for novel and potentially expanded synthetic immunoglobulin diversities, thus providing a pool of diversity from which human antibody therapeutic leads can be selected. This expanded pool is useful when seeking to find antibodies with desirable teristics, such as relatively high affinity to target antigen without the need for r affinity maturation (eg, using laborious in vitro ques such as phage or ribosome display), or improved biophysical characteristics, or to address targets and new epitopes that have previously been difficult to address with antibodies are not reached by prior antibody binding sites.

The invention also provides for diversity that is potentially biased towards variable gene usage common to members of a specific human population, which is useful for generating antibodies for treating and/or preventing diseases or conditions within such population, This ability to bias the PCT/G82012/052296 antibody oire allows one to tailor antibody therapeutics with the aim of more effectively ng and/or preventing disease or medical conditions in specific human populations.

The present inventors realised the possibility of providing immunoglobulin gene segments from disparate sources in transgenic loci, in order to provide for novel and potentially-expanded dy ities from which antibody therapeutics (and antibody tool reagents) could be generated. This- opens up the potential of transgenic human-mouse/rat technologies to the possibility of interrogating different and possibly larger antibody ce-spaces than has hitherto been possible.

In rationally designing transgenic dy loci, as well as antibodies and antibody chains, the ors also realised that a relatively long HCDR3 length (at least 20 amino acids) is often desirable to address epitopes. For example, naturally-occurring antibodies have been isolated from humans infected with infectious disease pathogens, such antibodies having a long HCDR3 length.

Neutralising antibodies have been found in this respect. A long HCDR3 length would be desirable to address other antigens (eg, receptor clefts or enzyme active sites), not just limited to infectious disease pathogens, and thus the inventors ed the general desirability of the possibility of engineering transgenic loci to be able to produce long HCDR3 antibodies and heavy chains. The inventors, through laborious execution of bioinformatics on in excess of 2000 human DNA samples via the 1000 Genomes project together with rational sequence choices, identified that the ion of the specific human gene segment variant JH6*02 is desirable for ing long HCDR3 antibodies and chains.

Additional rational design and bioinformatics has led the inventors to realise that specific human constant region ts are conserved across many diverse human populations. The inventors realised that this opens up the possibility of making a choice to se dies, chains and variable domains by using such specific constant regions in products, rather than arbitrarily choosing the human constant region (or a synthetic version of a human constant region). This aSpect of the invention also s one to tailor antibody-based drugs to specific human ethnic populations, thereby more closely matching drug to patient (and thus disease setting) than has hitherto been performed. It can be a problem in the state of the art that antibodies are humanised with an ary choice of human constant region (presumably derived from one (often unknown) ethnic population or non-naturally occurring) that does not function as well in patients of a different human ethnic tion. This is important, since the constant region has the major role in providing antibody effector functions, eg, for antibody recycling, cellular and complement recruitment and for cell killing.

A first aspect of the invention provides a non-human vertebrate or non-human vertebrate cell whose genome comprises an immunoglobulin heavy chain locus comprising unrearranged human gene segment JH6*02, one or more VH gene segments and one or more D gene segments upstream of a constant region; wherein the gene segments in the heavy chain locus are operably linked to the constant region thereof so that the vertebrate is capable of producing an antibody heavy chain ed by recombination of the human JH6*02 with a human D segment and a human VH segment, wherein the JH6*02 gene segment comprises the following nucleotide sequence: A CTACTACTAC GGTATGGACG TCTGGGGCCA AGGGACCACG GTCACCGTCT , wherein the vertebrate has been sed with a target antigen and produces an antibody heavy chain specific for the target antigen, wherein the target antigen is an antigen of an ious e pathogen, and wherein the variable domain of the heavy chain is the product of recombination between a human VH, D and JH6*02 and wherein the HCDR3 length is at least 20 amino acids.

A second aspect of the invention provides an isolated antibody-producing cell that is a progeny of a non-human vertebrate cell whose genome comprises an immunoglobulin heavy chain locus comprising human gene segment JH6*02, one or more human VH gene segments and one or more human D gene segments upstream of a constant region; n the gene segments in the heavy chain locus are operably linked to the constant region f for ing an antibody heavy chain produced by ination of the human JH6*02 with a D segment and a VH segment, wherein the JH6*02 gene segment comprises the following nucleotide sequence: ATTACTA CTACTACTAC GGTATGGACG TCTGGGGCCA AGGGACCACG GTCACCGTCT CCTCAG wherein the antibody-producing cell comprises a heavy chain locus comprising a rearranged variable region produced by ination of human JH6*02 with a human D segment and a human VH segment, wherein the antibody-producing cell produces an antibody heavy chain specific for a target antigen, wherein the target antigen is an n of an infectious disease pathogen, and wherein the variable domain of the heavy chain is the product of recombination between a human VH, human D and human JH6*02 and n the HCDR3 length is at least 20 amino acids. 7782292_1 ters) P96492.NZ 24-May-16 A third aspect of the ion provides a method for producing a heavy chain, a VH domain, or an antibody specific to a target antigen, the method comprising isolating from the vertebrate of any one of the first aspect or second aspect, the heavy chain, VH domain or antibody specific to the target antigen or a cell producing the heavy chain, VH domain or antibody, wherein the heavy chain, VH domain or an dy comprises a HCDR3 that is derived from the recombination of human JH6*02 with a human VH gene segment and a human D gene segment.

A fourth aspect of the invention provides a heavy chain, a VH domain, or an dy produced by the method of the third aspect.

A fifth aspect of the invention es a B-cell or hybridoma expressing a VH domain that is identical to a VH domain of the heavy chain of the fourth aspect or the VH domain of the fourth aspect.

A sixth aspect of the ion provides a nucleic acid encoding a VH domain of the heavy chain of the fourth aspect, the VH domain of the fourth aspect, or the heavy chain of the fourth aspect.

A seventh aspect of the invention provides a vector comprising the nucleic acid of the sixth aspect.

An eighth aspect of the invention provides a non-human vertebrate or isolated host cell, comprising the vector of the seventh aspect.

A ninth aspect of the invention provides a heavy chain or an antibody ed by the method of the third aspect, wherein the HCDR3 length is at least 20 amino acids and the constant region is mouse or rat.

A tenth aspect of the invention provides use of human JH6*02 for generating a HCDR3 of at least 20 amino acids in a non-human vertebrate whose genome comprises an immunoglobulin heavy chain locus comprising human gene segment JH6*02, one or more human VH gene segments and one or more human D gene segments upstream of a constant region; n the gene segments in the heavy chain locus are operably linked to the constant region thereof so that the vertebrate is capable of producing an antibody heavy chain produced by recombination of the human JH6*02 with a human D segment and a human VH t and wherein the JH6*02 gene segment ses the following nucleotide ce: ATTACTA CTACTACTAC GGTATGGACG TCTGGGGCCA AGGGACCACG GTCACCGTCT CCTCAG, wherein the antibody heavy chain specifically binds a target antigen of an infectious disease pathogen.

An eleventh aspect of the invention provides a heavy chain, a VH domain, or an antibody sing the HCDR3 generated by the use of the tenth aspect. 7782292_1 (GHMatters) P96492.NZ 24-May-16 A twelfth aspect of the invention provides an antibody sing the heavy chain of the eleventh aspect.

A thirteenth aspect of the invention provides a pharmaceutical composition comprising the, heavy chain, VH domain, or antibody of the fourth aspect or the eleventh aspect or the twelfth aspect, together with pharmaceutically-acceptable excipient, diluent or a medicament.

To this end, further configurations of the invention are disclosed.

First Configuration A non-human vertebrate or vertebrate cell (optionally an ES cell or antibody-producing cell) comprising a genome having a superhuman immunoglobulin heavy chain human VH and/or D and/or J gene repertoire.

A non-human vertebrate or vertebrate cell (optionally an ES cell or antibody-producing cell) sing a genome having a superhuman immunoglobulin light chain human VL gene repertoire; optionally n the vertebrate or cell is according to the first configuration.

A non—human vertebrate or vertebrate cell (optionally an ES cell or antibody-producing cell) whose genome comprises a transgenic immunoglobulin locus (eg, a heavy chain locus or a light chain locus), said locus comprising immunoglobulin gene segments according to the first and second human globulin gene ts (optionally V segments) as mentioned below operably connected upstream of an immunoglobulin constant region; optionally wherein the genome is homozygous for said transgenic immunoglobulin locus; optionally wherein the immunoglobulin locus comprises more than the l human complement of functional V gene segments; and/or optionally n the immunoglobulin locus comprises more than the natural human complement of functional D gene segments; and/or optionally wherein the immunoglobulin locus comprises more than the natural human complement of functional J gene segments. 8809877_1 [GHMallels) PN‘DZ NZ LEOWNR 19-Auo15 A transgenic non—human vertebrate (eg, a mouse or rat) or vertebrate cell (optionally an ES cell or dy-producing cell) whose genome comprises a transgenic immunoglobulin locus comprising a plurality of human immunoglobulin gene segments operably connected upstream of a non-human vertebrate constant region for the production of a repertoire of chimaeric dies, or chimaeric light or heavy chains, having a man vertebrate constant region and a human variable ; wherein the transgenic locus comprises one or more human immunoglobulin V gene segments, one or more human J gene segments and optionally one or more human D gene segments, a first (optionally a V segment) of said gene segments and a second (optionally a V segment) of said gene segments being different and derived from the genomes of first and second human individuals reSpectively, wherein the individuals are different; and optionally not related; optionally wherein the globulin locus comprises more than the natural human ment of functional V gene segments; and/or optionally n the immunoglobulin locus comprises more than the natural human complement of functional D gene segments,- and/or optionally wherein the immunoglobulin locus comprises more than the l human complement of functional J gene segments.

A enic non-human vertebrate (eg, a mouse or rat) or vertebrate cell (optionally an ES cell or antibody-producing cell) whose genome comprises first and second transgenic immunoglobulin loci, each locus comprising a plurality of human immunoglobulin gene segments operably connected upstream of a non-human vertebrate constant region for the production of a repertoire of chimaeric antibodies, or chimaeric light or heavy chains, having a man vertebrate constant region and a human variable region; wherein (i) the first transgenic locus comprises one or more human immunoglobulin V gene segments, one or more human J gene segments and optionally one or more human D gene segments, (ii) the second transgenic locus comprises one or more human immunoglobulin V gene segments, one or more human J gene segments and Optionally one or more human D gene segments; and (iii) wherein a first (optionally a V) gene segment of said first locus and a second (optionally a V) gene segment of said second gene locus are ent and derived from the genomes of first and second human individuals respectively, wherein the individuals are different; and optionally not related; ally wherein the first and second loci are on different chromosomes (optionally chromosomes with the same chromosome ) in said genome; optionally wherein each immunoglobulin locus comprises more than the natural human complement of functional V gene segments; and/or optionally wherein each immunoglobulin locus comprises more than the natural human complement of functional D gene segments; and/or ally wherein each immunoglobulin locus comprises more than the natural human complement of functional J gene segments.

A method of constructing a cell (eg, an ES cell) according to the invention, the method comprising (a) identifying functional V and J (and optionally D) gene segments ofthe genome sequence of a (or said) first human individual; (b) identifying one or more functional V and/or D and/orJ gene segments of the genome sequence of a (or said) second human individual, wherein these additional gene ts are not found in the genome sequence of the first individual; (c) and constructing a transgenic immunoglobulin locus in the cell, wherein the gene segments of (a) and (b) are provided in the locus operably ted am of a constant region.

In one embodiment, the gene segment(s) in step (b) are identified from an immunoglobulin gene database selected from the 1000 s, Ensembl, Genbank and IMGT databases.

Throughout this text, Genbank is a reference to k release number 185.0 or 191.0; the 1000 Genomes database is Phase 1, release v3, 16‘h March 2012; the Ensembl database is assembly GRCh37.p8 (10/04/2012); the IMGT database is available at www.imgt.org .

In one ment, the first and second human individuals are members of first and second ethnic populations respectively, wherein the populations are different, optionally wherein the human PCT/G32012/052296 immunoglobulin gene segment derived from the genome sequence of the second individual is low— frequency (optionally rare) within the second ethnic population.

This configuration of the invention also provides a method of making a transgenic non-human vertebrate (eg, a mouse or rat), the method comprising (a) ucting an ES cell (eg, a mouse C57BL/6N, 6J, 12955 or 129$v strain ES cell) by carrying out the method above; (b) injecting the ES cell into a donor man vertebrate blastocyst (eg, a mouse C57BL/6N, CS7BL/61, 12955 or 1295v strain blastocyst); (c) ting the blastocyst into a foster non—human vertebrate mother (eg, a C57BL/6N, C57BL/6], 12985 or 129Sv strain mouse); and (d) obtaining a child from said mother, wherein the child genome comprises a transgenic immunoglobulin locus.

In one embodiment, the invention provides a method of ing an antibody that binds a predetermined antigen (eg, a ial or viral pathogen antigen), the method comprising immunising a non-human vertebrate according to the invention.

Second Configuration A library of antibody-producing transgenic cells whose genomes collectively encode a repertoire of antibodies, wherein (a) a first transgenic cell expresses a first antibody having a chain encoded by a first immunoglobulin gene, the gene comprising a first variable domain nucleotide sequence ed following recombination of a first human unrearranged immunoglobulin gene segment; (b) a second enic cell expresses a second antibody having a chain encoded by a second immunoglobulin gene, the second gene comprising a second variable domain nucleotide sequence produced following recombination of a second human unrearranged immunoglobulin gene segment, the first and second antibodies being non-identical; (c) the first and second gene segments are different and derived from the genome sequences of first and second human duals respectively, wherein the individuals are different,- and ally not related; PCT/GBZOIZ/052296 (d) wherein the cells are non—human vertebrate (eg, mouse or rat) cells.

In one embodiment, the first and second human individuals are members of first and second ethnic populations tively, wherein the populations are different; optionally wherein the ethnic populations are selected from those identified in the 1000 Genomes database.

In another embodiment, the second human immunoglobulin gene segment is a polymorphic variant of the first human immunoglobulin gene segment,- optionally n the second gene segment is selected from the group consisting of a gene segment in any of Tables 1 to 7 and 9 to 14 below (eg, ed from Table 13 or Table 14), eg, the second gene segment is a polymorphic variant of VH1— Il'li E' .

An isolated antibody having (a) a heavy chain d by a nucleotide ce produced following recombination in a transgenic non-human vertebrate cell of an unrearranged human immunoglobulin V gene t with a human D and humanJ segment, optionally with affinity maturation in said cell, wherein one of the gene segments is derived from the genome of an individual from a first human ethnic population; and the other two gene segments are derived from the genome of an individual from a , different, human ethnic population, and wherein the antibody comprises heavy chain constant regions of said non-human vertebrate (eg, rodent, mouse or rat heavy chain constant regions); and/or (b) a light chain encoded by a tide sequence produced ing recombination in a transgenic non-human vertebrate cell of an unrearranged human immunoglobulin V gene segment with a humanJ segment, optionally with affinity maturation in said cell, wherein one of the gene segments is derived from the genome of an individual from a first human ethnic population (optionally the same as the first population in (3)); and the other gene segment is derived from the genome of an individual from a second, different, human ethnic tion (optionally the same as the second population in (a)), and wherein the antibody comprises light chain nt regions of said non-human vertebrate (eg, rodent, mouse or rat heavy light constant regions); PCT/G82012l052296 (c) Optionally wherein each variable domain of the antibody is a human variable domain. (d) Optionally wherein the heavy chain constant regions are type constant regions.

The invention also provides an isolated nucleotide sequence encoding the antibody, optionally wherein the sequence is provided in an antibody expression vector, optionally in a host cell.

The invention also provides a method of producing a human antibody, the method comprising replacing the non-human vertebrate constant regions of the antibody of the third configuration with human antibody constant regions.

The invention also provides a pharmaceutical composition comprising an antibody according to the third configuration, or an antibody produced according to the method above and a diluent, excipient or carrier; ally wherein the composition is provided in a container connected to an IV needle or syringe or in an IV bag.

The invention also es an antibody-producing cell that expresses the second antibody recited in any one of the configurations.

In an alternative configuration, the invention contemplates the combination of nucleotide sequences of first and second immunoglobulin gene segments (eg, two or more polymorphic variants of a particular human germline VH or VL gene segment) to provide a synthetic gene segment. Such tic gene segment is used, in one embodiment, to build a enic globulin locus, wherein the synthetic gene segment is provided in combination with one or more human variable and J regions (and optionally one or more human D regions) operably connected upstream of a nt region. When provided in the genome of a non-human vertebrate or cell (eg, mouse or rat celi, eg, ES cell), the ion provides for superhuman gene segment diversity. The sequences to be combined can be selected from gene ts that have been ed to be commonly used in human antibodies raised against a particular n (eg, a flu antigen, such as haemaglutinin). By ing the sequences, the synthetic gene segment may recombine in vivo to produce an antibody that is well suited to the treatment and/or prevention of a disease or ion (eg, nza) mediated by said antigen.

Fourth Configuration A non-human vertebrate (optionally a mouse or a rat) or vertebrate cell whose genome comprises an immunoglobulin heavy chain locus comprising human gene segment JH6*02, one or more VH gene segments and one or more D gene segments upstream of a constant region; wherein the gene segments in the heavy chain locus are operably linked to the constant region thereof so that the mouse is capable of producing an dy heavy chain produced by recombination of the human JH6*02 with a D segment and a VH segment.

A non-human vertebrate cell nally a mouse cell or a rat cell) whose genome comprises an immunoglobulin heavy chain locus comprising human gene t JH6*02, one or more VH gene segments and one or more D gene segments am of a constant region; wherein the gene segments in the heavy chain locus are operably linked to the nt region thereof for producing (eg, in a subsequent progeny cell) an dy heavy chain produced by recombination of the human JH6*02 with a D t and a VH segment.

A heavy chain (eg, comprised by an antibody) isolated from a vertebrate of the invention wherein the heavy chain comprises a HCDR3 of at least 20 amino acids.

A method for producing a heavy chain, VH domain or an antibody specific to a target antigen, the method comprising immunizing a non-human vertebrate according to the invention with the antigen and isolating the heavy chain, VH domain or an antibody specific to a target antigen or a cell producing the heavy chain, VH domain or an antibody, wherein the heavy chain, VH domain or an antibody comprises a HCDR3 that is derived from the recombination of human JH6*02 with a VH gene segment and a D gene segment.

PCT/G32012/052296 A heavy chain, VH domain or an antibody produced by the method.

A B-cell or hybridoma expressing a heavy chain VH domain that is identical to the VH domain ofthe heavy chain.

A nucleic acid encoding the VH domain of the heavy chain of claim 22, 23 or 28, or encoding the heavy chain.

A vector (eg, a CHO cell or HEK293 cell vector) comprising the c acid; ally wherein the vector is in a host cell (eg, a CHO cell or HEK293 cell).

A pharmaceutical composition comprising the antibody, heavy chain or VH domain (eg, comprised by an antibody), together with a pharmaceutically-acceptable excipient, diluent or a medicament (eg, a further antigen-specific variable , heavy chain or antibody).

The antibody, heavy chain or VH domain (eg, comprised by an antibody) as above for use in medicine.

The use of an antibody, heavy chain or VH domain (eg, comprised by an antibody) as above in the manufacture of a medicament for treating and/or preventing a medical ion in a human.

'EII E' .

A method of producing an dy heavy chain, the method comprising (a) providing an antigen-specific heavy chain variable domain; and (b) combining the variable domain with a human heavy chain constant region to produce an dy heavy chain comprising (in N- to C-terminal direction) the variable domain and the constant region; wherein PCT/G32012/052296 the human heavy chain constant region is an IGHGlref, lGHGZref, IGHGZa, IGHGBref, |GHG3a, IGHGBb, lGHG4ref or lGHG4a constant region.

An antibody comprising a human heavy chain, the heavy chain comprising a le domain that is specific for an antigen and a constant region that is an IGHGlref, IGHGZref, IGHGZa, lGHGSref, lGHG3a, lGHG3b, IGHG4ref or IGHG4a constant region . Optionally, the variable domain ses mouse—pattern AID somatic mutations.

A ptide comprising (in N- to C- terminal direction) a leader sequence, a human variable domain that is specific for an antigen and a human constant region that is an IGHGlref, IGHGZref, IGHGZa, IGHG3ref, IGHGBa, lGHG3b, IGHG4ref or IGHG4a constant region wherein (i) the leader sequence is not the native human variable domain leader sequence; and/or (ii) the variable domain comprises mouse AID—pattern somatic mutations and/or mouse Terminal deoxynucleotidyl transferase (TdT)— pattern junctional mutations.

A nucleotide sequence encoding (in S' to 3’ direction) a leader ce and a human dy heavy chain, the heavy chain comprising a variable domain that is specific for an antigen and a constant region that is an IGHGlref, ef, lGHGZa, IGHGSref, IGHGBa, IGHG3b, IGHG4refor lGHG4a constant ; and the leader sequence being operable for expression of the heavy chain and wherein the leader sequence is not the native human variable domain leader ce.

A nucleotide sequence encoding (in 5’ to 3’ direction) a promoter and a human antibody heavy chain, the heavy chain comprising a variable domain that is specific for an antigen and a constant region that is an IGHGlref, IGHGZref, lGHGZa, IGHGBref, |GHG3a, IGHGBb, lGHG4ref or IGHG4a nt region; and the promoter being operable for expression of the heavy chain and wherein the promoter is not the native human promoter.

A vector (eg, a CHO cell or HEK293 cell vector) comprising a IGHGlref, IGHGZref, IGHGZa, IGHGSref, IGHGBa, , IGHG4ref or lGHG4a constant region tide sequence that is 3’ of a g site for the insertion of a human antibody heavy chain variable domain nucleotide sequence, such that upon insertion of such a le domain sequence the vector comprises (in 5’ to 3’ direction) a promoter, a leader sequence, the le domain sequence and the constant region sequence so that the vector is capable of expressing a human antibody heavy chain when t in a host cell.

Sixth Configuration A non-human vertebrate (eg, a mouse or rat) or a non-human vertebrate cell (eg, an ES cell or a B- cell) having a genome comprising at least 3 human variable region gene segments of the same type (eg, at least 3 human VHS-1 gene segments, at least 3 human JH6 gene segments, at least 3 human VK1-39 gene segments, at least 3 human DZ-Z gene segments or at least 3 human JKl gene segments), wherein at least two of the human gene segments are variants that are not identical to each other.

A non-human vertebrate (eg, a mouse or rat) or a non-human vertebrate cell (eg, an ES cell or a B- cell) having a genome comprising at least 2 different non-endogenous le region gene segments of the same type (eg, at least 2 human VHS-1 gene segments, at least 3 human JH6 gene segments, at least 2 human VKl-39 gene ts, at least 2 human 02-2 gene segments or at least 2 human 1x1 gene segments) cis at the same lg locus.

A man vertebrate (eg, a mouse or rat) or a non-human vertebrate cell (eg, an ES cell or a B— cell) having a genome comprising at least 2 different human variable region gene segments of the same type (eg, at least 2 human VH6-1 gene segments, at least 2 human JH6 gene segments, at least 2 human VK1—39 gene segments, at least 2 human D2-2 gene segments or at least 2 human JKl gene segments) trans at the same lg locus; and optionally a third human gene segment of the same type, wherein the third gene segment is cis with one of said 2 different gene segments.

A population of nonvhuman vertebrates (eg, mice or rats) comprising a repertoire of human variable region gene segments, wherein the ity comprises at least 2 human variable region gene segments of the same type (eg, at least 2 human VH6-1 gene ts, at least 2 human 1H6 gene segments, at least 2 human VK1—39 gene segments, at least 2 human D2-2 gene segments or at least 2 human JKl gene segments), a first of said different gene segments is provided in the genome of a W0 41844 PCT/G82012/052296 first vertebrate of the population, and a second of said different gene segments being ed in the genome of a second vertebrate of the population, wherein the genome of the first vertebrate does not comprise the second gene segment.

A non-human vertebrate (eg, a mouse or rat) or a non-human rate cell (eg, an ES cell or a B- cell) having a genome comprising at least 2 different dogenous variable region gene segments ofthe same type (eg, at least 2 human VHS-1 gene segments, at least 2 human JH6 gene segments, at least 2 human VK1—39 gene segments, at least 2 human 02—2 gene segments or at least 2 human JKl gene segments), wherein the gene segments are derived from the genome sequence of different human individuals that are not genetically related over at least 3 generations.

A method of enhancing the human globulin gene diversity of a non-human vertebrate (eg, a mouse or rat), the method comprising ing the vertebrate with a genome comprising at least 3 human variable region gene segments of the same type (eg, at least 3 human VH6-1 gene segments, at least 3 human JH6 gene ts, at least 3 human VKl-39 gene segments, at least 3 human 02-2 gene segments or at least 3 human JKl gene segments), wherein at least two of the human gene segments are variants that are not identical to each other.

A method of enhancing the immunoglobulin gene diversity of a non—human vertebrate (eg, a mouse or rat), the method comprising providing the vertebrate with a genome sing at least 2 different non-endogenous variable region gene segments of the same type (eg, at least 2 human VHS-1 gene segments, at least 2 human JH6 gene segments, at least 2 human VK1-39 gene segments, at least 2 human 02-2 gene segments or at least 2 human JKl gene segments) cis at the same lg locus.

A method of enhancing the immunoglobulin gene diversity of a non-human vertebrate (eg, a mouse or rat), the method comprising providing the vertebrate with a genome comprising at least 2 different human variable region gene segments of the same type (eg, at least 2 human VH6-1 gene segments, at least 2 human JH6 gene segments, at least 2 human VK1-39 gene segments, at least 2 human 02-2 gene segments or at least 2 human JKl gene segments) trans at the same lg locus; and PCT/G32012/052296 optionally a third human gene segment of the same type, wherein the third gene segment is cis with one of said 2 different gene segments.

A method of providing an enhanced human immunoglobulin variable region gene segment repertoire, the method comprising providing a population of non—human vertebrates (eg, a mouse or rat) comprising a oire of human variable region gene segments, wherein the method comprises providing at least 2 different human variable region gene segments of the same type (eg, at least 2 human VH6—1 gene segments, at least 2 human JH6 gene segments, at least 2 human VKl- 39 gene segments, at least 2 human D2—2 gene segments or at least 2 human JKl gene segments), n a first of said different gene segments is provided in the genome of a first vertebrate of the population, and a second of said different gene segments is provided in the genome of a second vertebrate of the population, wherein the genome of the first rate does not comprise the second gene t.

A method of ing the human immunoglobulin gene diversity of a man vertebrate (eg, a mouse or rat), the method comprising providing the vertebrate with a genome comprising at least 2 different non-endogenous variable region gene segments of the same type (eg, at least 2 human VH6-1 gene segments, at least 2 human JH6 gene segments, at least 2 human VKl-39 gene segments, at least 2 human 02—2 gene ts or at least 2 human Ji<1 gene segments), wherein the gene segments are derived from the genome sequence of different human individuals that are not cally related over at least 3 generations.

A method of enhancing the human immunoglobulin gene diversity of a non-human vertebrate (eg, a mouse or rat), the method comprising ing the vertebrate with a genome comprising at least 2 human variable region gene segments of the same type (eg, at least 2 human VH6-1 gene segments, at least 2 human JH6 gene segments, at least 2 human VK1~39 gene segments, at least 2 human D2-2 gene segments or at least 2 human JKl gene segments), wherein the gene segments are derived from the genome sequence of different human individuals that are not genetically related over at least 3 generations; optionally n at least 2 or 3 of said different gene segments are provided at the same lg locus in said genome.

PCT/G82012/052296 A non-human vertebrate (eg, a mouse or rat) or a non-human rate cell (eg, an ES cell or a B- cell) having a genome sing first and second human lg locus gene segments of the same type (eg, first and second human JH6 gene segments; or first and second lgGZ gene segments; or first and second human J17 gene segments), wherein the first gene segment is a gene segment selected from any one of Tables 1 and 9 to 14 (eg, selected from Table 13 or Table 14) (eg, lGHJ6-a) and the second gene segment is the corresponding reference sequence, A tion of non-human vertebrates (eg, mice or rats) comprising first and second human lg locus gene ts of the same type (eg, first and second human JH6 gene segments; or first and second lgGZ gene segments; or first and second human 117 gene segments), wherein the first gene segment is a gene segment selected from any one of Tables 1 and 9 to 14 (eg, selected from Table 13 or Table 14) (eg, lGHJ6-a) and the second gene segment is the corresponding reference sequence, wherein the first gene t is provided in the genome of a first vertebrate of the population, and the second gene segment is provided in the genome of a second vertebrate of the population.

A method of enhancing the human immunoglobulin gene diversity of a non-human vertebrate (eg, a mouse or rat), the method comprising providing the vertebrate with a genome comprising first and second human lg locus gene segments of the same type (eg, first and second human JH6 gene ts; or first and second lgGZ gene segments; or first and second human 1A7 gene segments), wherein the first gene segment is a gene segment selected from any one of Tables 1 and 9 to 14 (eg, selected from Table 13 or Table 14) (eg, lGHJ6-a) and the second gene segment is the corresponding nce sequence.

In one aspect of this configuration, the invention relates to human D gene t variants as described further below. in one aspect of this configuration, the invention relates to human V gene segment variants as described further below.

PCT/G82012/052296 In one aspect of this configuration, the ion relates to humanJ gene segment variants as described further below.

BBIEF DE§CR|P|lQfl QE |fl§ Elfiuflgfi Figures 1 to 3: Schematic rating a protocol for producing recombineered BAC vectors to add V gene segments into a mouse genome; Figure 4: Schematic illustrating a protocol for adding V gene ts to a mouse genome using sequential recombinase mediated cassette exchange (sRMCE); and Figure 5 (in 4 parts): Alignment of 13 IGHV1—69 variants showing the variable (V) coding region only.

Nucleotides that differ from VH1—69 variant *01 are indicated at the appropriate position whereas cal nucleotides are marked with a dash. Where nucleotide changes result in amino acid differences, the encoded amino acid is shown above the corresponding triplet. Boxed regions correspond to COM, CDR2 and CDRB as indicated.

Figure 6 is a schematic illustrating gene segment diversity and the effect of including variant variants in cis ing to the ion:- (a) Situation in a normal person: ination on the same chromosome limits combinations of variants, for instance the antibody gene V4-4 can only be recombined within t 1 to form for instance for instance V4-4—D-16 or V4-4—D-JZ". Similarly the variant V4-4“ can’t be recombined with either 16 or 12A from variant 1 and can only be joined with s from variant 2 to form V4-4“-D-16A and V4-4A-D—J2. V412/16 complexity = 4. (b) Situation in a transgenic mouse: Only one variant is provided so the genome is limited. V4- 4—16/12 complexity = 2. (c) Supra mouse of the invention: The variants are added in cis and thus can be recombined in every combination, expanding the repertoire. For instance V4-4 can be combined with 16A, J6, JZA or 12 and similarly V4-4A can be ined with these same J-genes. The V4J6/J2 complexity = 8, which in this simple example is double that ofa person and 4X that of a mouse with a single variant.

PCT/GBZOIZ/052296 Figure 7: Alignment of human JH6*02 variants. Nucleotides that differ from JH6 *01 are indicated at the appropriate position whereas identical nucleotides are marked with a dash. Where nucleotide changes result in amino acid differences, the encoded amino acid is shown above. Accession numbers (eg, 100256) are shown to the left of the IMGT variant name.

Figure 8: Alignment ofJH sequences from various species.

Figure 9: Codon Table Figure 10: BAC database extract BRIEF DE§CRIPTIQN 9E ”1E TABLEfi Table 1: Human lgH V rphic Variants m Human lgH D Polymorphic Variants m Human lgHJ Polymorphic Variants M Human lg Vk Polymorphic Variants Tab—e; Human lg VA Polymorphic Variants E.—ble 6: Human IgH Jk Polymorphic Variants EM Human lgH JA Polymorphic Variants MTale : 1000 Genomes Project Human Populations I§b|§ 9 lmmunoglobulin Gene Usage in Human Antibody Responses to ious Disease Pathogens MHuman IgH JHS Variant Occurrences Lid—LE.a e 0 : Non-Synonymous Human lgHJHS ts Lemma Human lgH JH6 Variant Occurrences Lam Non-Synonymous Human lgH JH6 Variants Ta e : Human lgH JH2 Variant ences Table 128: Non-Synonymous Human lgH JH2 Variants Table 13: Variant ncy Analyses & Human Population Distributions Table 14: Frequent Human Variant Distributions Table 15: Human Gene Segment Usage: Heavy Chain Repertoires From Naive Non-Human Vertebrates Table 16: Human Gene t Usage: Heavy Chain Repertoires From lmmunised Non- Human Vertebrates Table 17: Human Gene Segment Usage: Heavy Chain Repertoires From Antigen-Specific Hybridomas Table 18: Sequence Correlation Table Table 19: Summary Of Function Correlated With Human Gamma Constant Region Sub-Type Table 20: Gene Segments Prevalent In Few Human Populations Table 21: Genomic and sequence ation A suitable source ofJH6*02 and other human DNA sequences for use in the ion will be readily apparent to the skilled . For example, it is possible to t a DNA sample from a consenting human donor (eg, a cheek swab sample as per the Example herein) from which can be obtained suitable DNA sequences for use in constructing a locus of the invention. Other sources of human DNA are commercially available, as will be known to the skilled . Alternatively, the skilled person is able to construct gene segment sequence by referring to one or more databases of human lg gene segment sequences disclosed herein.

An example source for human V, D and] gene segments according to the ion are Bacterial Artificial Chromosomes (RPCI-ll BACs) obtained from Roswell Park Cancer institute (RPCI)/Invitrogen. See http',“bagpgcchoriorgfihmaig;1.h;m, which describes the BACs as follows:- ”RPC/ - 11 Human Male BAC Libragy 2012/052296 The RPCI-ll Human Male BAC Library (Osoegawa et ai., 2001) was constructed using improved cloning ques (Osoegawa et al., 1998) developed by Kazutoyo Osoegawa. The library was generated by Kazutoyo Osoegawa. Construction was funded by a grantfrom the National Human Genome Research Institute (NHGRI, NIH) (#1R01R601165-03). This library was generated according to the new NHGRl/DOE "Guidance on Human Subjects in Large-Scale DNA Sequencing...

”Ma/e blood was obtained via a double-blind selection protocol. Male blood DNA was isolated from one randomly chosen donor (out of 10 male donors)”. 0 Osoegawa K, Mammoser AG, Wu C, Frengen E, Zeng C, se JJ, de Jong PJ; Genome Res. 2001 Mar;11(3):483-96; “A bacterial artificial chromosome library for sequencing the complete human genome"; - Osoegawa, K., Woon, P.Y., Zhao, 8., Frengen, E., Tateno, M., Catanese, JJ, and de Jong, P.J.

; ”An Improved Approach for Construction of Bacterial cial some Libraries”; Genomics 52, 1-8. il lll'iE .

The invention relates to synthetically-extended & ethnically-diverse superhuman immunoglobulin gene repertoires. The human immunoglobulin repertoires are beyond those found in nature (ie, ”Superhuman"), for example, they are more diverse than a natural human repertoire or they se combinations of human immunoglobulin gene segments from disparate sources in a way that is non-natural. Thus, the repertoires of the invention are ”superhuman" immunoglobulin oires, and the invention relates to the application of these in transgenic cells and non-human vertebrates for utility in producing chimaeric antibodies (with the possibility of converting these into fully-human, isolated dies using recombinant DNA technology). The present invention thus provides for novel and potentially expanded synthetic immunoglobulin ities, which provides for a pool of diversity from which antibody eutic leads (antibody therapeutics and antibody tool reagents) can be ed. This opens up the potential of transgenic human—mouse/rat technologies to the possibility of ogating different and possibly larger antibody sequence- spaces than has hitherto been possible. To this end, in one embodiment, the invention provides a SUPERHUMAN mouseM (aka SUPRA-MOUSE“) and a SUPERHUMAN RATTM (aka SUPRA-RAT”) 2012/052296 In developing this thinking, the present inventors have realised the possibility of mining the huge genetics resources now available to the skilled person thanks to s such as the HapMap Project, 1000 Genomes Project and sundry other immunoglobulin gene ses (see below for more details). Thus, in some embodiments, the inventors realised the application of these genome sequencing developments in the present invention to te synthetically-produced and ethnically-diverse artificial immunoglobulin gene repertoires. In one aspect, the inventors realised that such repertoires are useful for the production of antibodies having improved affinity and/or biophysical characteristics, and/or wherein the range of epitope specificities produced by means of such repertoire is novel, es for antibodies to epitopes that have hitherto been intractable by prior transgenic immunoglobulin loci or difficult to address.

The present invention provides libraries, vertebrates and cells, such as transgenic mice or rats or transgenic mouse or rat cells. Furthermore, the ion relates to methods of using the rates to isolate antibodies or nucleotide sequences ng dies. Antibodies, nucleotide sequences, pharmaceutical compositions and uses are also provided by the invention, ll"l|' The present inventors have realized methods and antibody loci designs that harness the power of genetic variation analysis. The reference human genome provides a foundation for experimental work and genetic analysis of human samples. The reference human is a compilation of the genomes from a small number of individuals and for any one segment of the genome a high quality single reference genome for one of the two chromosomes is available. Because the reference genome was assembled from a series of very large insert clones, the identity of these clones is known.

Accordingly, experimental work with human genomic DNA is usually conducted on the clones from which the reference sequence was derived. individual humans differ in their sequence and recently several individuals have had their s sequenced, for ce James Watson and Craig Venter. ison of the genome sequence of these individuals has revealed differences between their sequences and the reference genome in both coding and non-coding parts of the genome, approximately 1 in 1000 bases are different.

Some variants will be significant and contribute to differences between individuals. In extreme cases PCT/G82012/052296 these will reSult in genetic disease. Variation can be implicated in differing responses to drugs stered to human patients, eg, yielding an undesirable lowering of patient response to treatment.

The 1000—Genomes Project has the objective of identifying the most frequent variations in the human genome. This public domain project involved sequencing the genomes of more than 1000 individuals from diverse ethnic groups, comparing these sequences to the nce and assembling a catalogue of variants. This has enabled the annotation of variants in coding regions, but e this sequence wasn't derived from large clones of DNA, the is of the sequence from diploid individuals can’t discriminate the distribution of the variation between the maternal and paternally inherited chromosomes. Where more than one variant is identified in a protein coding gene, it is not possible to illuminate the distribution of the pattern of variants in each version of the protein. For example, if two variants are detected in different positions of the same protein in an dual, this could have resulted from one copy with two variants and none in the other or each copy could have just one variant. To illuminate the sequence of real ns, the 1000-Genome t has sequenced mother-father-child trios. This allows one to “phase” the sequence variants, in other words identify blocks of sequence that are inherited from one or other parent and deconvolute the variants.

To further understand the variation within the enome set a tool has been developed that can identify the significant variants (defined as non-synonymous amino acid changes) from a region of DNA from the phased data in the 1000-genome data set. This tool has been made available online WThis tool allows an investigator to download non-synonymous variation delimited n specific coordinates. The aded files are configured as individual genotypes, but the data is phased so the haplotype ation and the frequencies of specific halotypes in different populations can be extracted.

The inventors' is of the 1000-genome data for the individual human coding segments of the C, V D and] genes from the heavy and light chains s that there is significant variation in these segments. Individuals will usually have two different heavy chain alleles and also different light chain alleles at both kappa and lambda loci. The repertoire of antibodies that can be generated W0 2013/041844 from each allele will be different. This variation will bute to a better or differing immune response to certain antigens.

Humanized mice that have hitherto been generated with immunoglobulin heavy and light chain loci contain just one type of immunoglobulin locus. Even if these mice contain a full human heavy chain locus, the variation will be less than contained in a typical human because only one set of C, V, D and J genes are available, while atypical human would have two sets.

The inventors have devised ways to improve on this tion when constructing enic non- human rates and cells for human antibody and variable region production in vivo.

Mice can be generated with two different loci, each engineered to have a different repertoire of V, D and J ts. This could be in a single mouse or two or more separate mouse strains and would be analogous to or beyond the repertoire found in a normal human. The engineering of such a mouse would go beyond the repertoire described in humanized mice to date which only have one set of alleles.

However, the inventors also realized that this also has limitations, because the different loci would not normally interact to shuffle V, D and J variants between loci. This same limitation is also inherent in a human, thus this system does not utilize the advantage of recombining variants in all ations.

To go beyond the normal repertoire in humans and take advantage of combinations of C, V, D and J variants the inventors decided, in one embodiment, to provide these on the same chromosome in cis. See figure 6. These loci would be characterized by having more than the normal number ofJ, D or V genes, For example n:6 for theJ genes, but including one 16 variant and one 12 variant would increase this to n=8. This could be combined with additional variants for the D and V genes, for example. By detailed is of the 1000- Genomes database, the inventors have devised a collection of candidate rphic human variant gene ts, eg, JH gene segments (eg, see the examples), that can be built into the design of transgenic heavy and light chain loci in mice for expressing increasingly diverse and new, synthetic repertoires of human variable regions. Moreover, by utilizing naturally-occurring human t gene segments, as per embodiments of the ion, this addresses compatibility with human ts since the inventor’s analysis has drawn out candidate variants that are naturally conserved and sometimes very prevalent amongst human ethnic populations. Additionally this enables one to tailor the urations of the invention to provide for antibody-based drugs that better address specific human ethnic populations.

In an example according to any configuration of the invention, loci (and cells and vertebrates comprising these) are provided in which gene segments from ent human populations are used.

This is desirable to increase antibody gene diversity to better address more diverse human patients.

In an example, the gene segments are from first and second different human populations respectively, and thus the second gene segment is found in the second human population, but not so (or ) in the first human population. Rarely means, for example, that the gene segment is found in 5, 4, 3, 2, or 1 or zero individuals in the first population in the 1000 Genomes se. For example, the first gene segment may be shown as present in a first population by reference to Table 13 or 14 herein, the second gene segment may be shown as present in the second population by nce to Table 13 and not in the first population. Optionally, the first gene segment may also be shown as being present in the second population by reference to Table 13 or 14.

In any uration or aspect of the invention, where a V gene segment is used, this may be used optionally with the native leader sequence. For example, use of genomic DNA (eg, from BACs as in the examples) will mean that the native leader will be used for each V gene t incorporated into the locus and genomes of the invention. In an alternative, the skilled person may wish to inert a non-native leader sequence together with one or more of the V gene segments. Similarly, in any configuration or aspect of the invention, where a V gene segment is used, this may be used optionally with the native 5’ UTR sequence. For example, use of genomic DNA (eg, from BACs as in the es) will mean that the native 5’ UTR sequence will be used for each V gene segment incorporated into the locus and genomes of the ion. In an alternative, the skilled person may wish to exclude the native 5’ UTR sequence. i ‘ ' e inafirst confi ura n PCT/G82012/052296 (a) Superhuman heavy chain gene repertoires A non-human vertebrate or vertebrate cell (optionally an ES cell or antibody-producing cell) comprising a genome having a superhuman immunoglobulin heavy chain human VH and/or D and/or J gene repertoire.

In one aspect the cell of the invention is an embryonic stem cell. For example, the ES cell is derived from the mouse 6N, C57BL/6J, 12955 or 129Sv strain. In one aspect the non—human vertebrate is a rodent, suitably a mouse, and cells of the invention, are rodent cells or ES cells, ly mouse ES cells. The ES cells ofthe present invention can be used to generate s using techniques well known in the art, which comprise injection of the ES cell into a blastocyst followed by implantation of chimaeric blastocystys into females to produce offspring which can be bred and selected for homozygous recombinants having the required insertion. In one aspect the invention relates to a transgenic animal comprised of ES cell-derived tissue and host embryo derived . In one aspect the invention relates to genetically-altered subsequent tion s, which include animals having a homozygous recombinants for the VDJ and/or VJ regions.

The l human immunoglobulin gene segment repertoire consists of (see eg,W): VH: total~125 ; functional-41 DH: total-27; functional-23 JH: total-8; functional-6 Vk: total-77; functional-38 Jk: total-5; functional-5 V lambda: total-75; functional-31 Z012/052296 J lambda: total-7; functional-5 In one embodiment, the vertebrate or cell genome comprises a transgenic immunoglobulin heavy chain locus comprising a plurality of human immunoglobulin VH gene segments, one or more human D gene segments and one or more human J gene segments, wherein the ity of VH gene segments consists of more than the natural human repertoire of functional VH gene segments; optionally wherein the genome is homozygous for said transgenic heavy chain locus.

In one embodiment of the vertebrate or cell, the VH gene repertoire consists of a ity of VH gene segments derived from the genome ce of a first human individual, supplemented with one or more different VH gene segments derived from the genome sequence of a second, different human individual. Optionally the D and J segments are d from the genome ce of the first human dual. Optionally the VH gene segments from the genome sequence of the second individual are ed from the VH gene segments listed in Table 1, 13 or 14. In this way, the locus provides a superhuman repertoire of D gene segments.

Optionally the individuals are not related. duals are ”not related” in the context of any configuration or aspect of the ion, for example, if one of the individuals does not appear in a family tree of the other individual in the same generation or going back one, two, three or four generations. Alternatively, are not related, for example, if they do not share a common ancestor in the present generation or going back one, two, three or four generations.

In one embodiment of the vertebrate or cell, the transgenic locus comprises more than 41 functional human VH gene segment species, and thus more than the natural human functional repertoire.

Optionally the locus comprises at least 42, 43, 44, 45, 46, 47, 48, 49 or 50 functional human VH gene segment species (eg, wherein the locus comprises the full functional VH repertoire of said first individual supplemented with one or more VH gene segments derived from the genome sequence of the second human individual and optionally with one or more VH gene segments derived from the genome sequence of a third human individual). In this way, the locus provides a uman repertoire ofVH gene segments that is useful for generating a novel gene and antibody diversity for 2012/052296 use in therapeutic and tool antibody selection.

In one embodiment of the vertebrate or cell, the enic locus comprises a first VH gene t derived from the genome sequence of the first individual and a second VH gene segment derived from the genome sequence of the second individual, wherein the second VH gene segment is a polymorphic t of the first VH gene segment. For example, the VH gene segments are polymorphic ts of VH1-69 as illustrated in the es below. Optionally the locus comprises a further polymorphic variant of the first VH gene segment (eg, a variant derived from the genome sequence ofa third human individual). In this way, the locus provides a superhuman repertoire of VH gene segments.

In one embodiment of the vertebrate or cell, the genome (alternatively or additionally to the superhuman VH diversity) comprises a transgenic immunoglobulin heavy chain locus comprising a plurality of human immunoglobulin VH gene segments, a plurality of human D gene segments and one or more human J gene segments, wherein the plurality of D gene segments consists of more than the natural human repertoire of functional D gene segments. Optionally the genome is homozygous for said transgenic heavy chain locus. in one embodiment of the rate or cell, the D gene repertoire consists of a plurality of D gene segments derived from the genome sequence of a (or said) first human individual, supplemented with one or more different D gene segments derived from the genome sequence of a (or said) second, different human individual. ally the individuals are not related. Optionally the] segments are derived from the genome sequence of the first human individual. Optionally the D gene segments from the genome sequence of the second individual are ed from the D gene segments listed in Table 2, 13 or 14. In this way, the locus provides a superhuman repertoire of D gene segments.

In one embodiment of the vertebrate or cell, the transgenic locus comprises more than 23 functional human D gene segment species; optionally wherein the locus comprises at least 24, 25, 26, 27, 28, 29, 30 or 31 functional human D gene segment species (eg, wherein the locus comprises the full functional D oire of said first individual supplemented with one or more D gene segments derived from the genome ce of the second human individual and optionally with one or more WO 41844 PCT/G82012/052296 D gene segments derived from the genome sequence of a third human individual). In this way, the locus provides a superhuman repertoire of D gene segments.

In one embodiment of the vertebrate or cell, the transgenic locus comprises a first D gene segment derived from the genome sequence of the first individual and a second D gene segment d from the genome sequence of the second individual, wherein the second D gene segment is a polymorphic variant of the first D gene segment. Optionally the locus comprises a further polymorphic t of the first D gene segment (eg, a variant d from the genome sequence of a third human individual). In this way, the locus provides a superhuman repertoire of D gene In one ment of the vertebrate or cell (alternatively or additionally to the uman VH and/or JH diversity), the genome comprises a (or said) transgenic immunoglobulin heavy chain locus comprising a plurality of human immunoglobulin VH gene segments, one or more human D gene segments and a piurality of human JH gene segments, wherein the plurality ofJ gene segments ts of more than the natural human repertoire of functional J gene segments; optionally wherein the genome is gous for said transgenic heavy chain locus.

In one embodiment of the vertebrate or cell, the JH gene repertoire consists of a plurality ofJ gene segments derived from the genome sequence of a (or said) first human individual, supplemented with one or more differentJ gene segments derived from the genome sequence of a (or said) second, different human individual. Optionally the individuals are not related. Optionally D segments are derived from the genome sequence of the first human individual. Optionally the} gene segments from the genome sequence of the second individual are selected from the J gene segments listed in Table 3 13 or 14. In this way, the locus provides a superhuman repertoire ofJH gene segments.

In one embodiment of the vertebrate or cell, the transgenic locus comprises more than 6 functional human JH gene t segments. Optionally the locus comprises at least 7, 8, 9, 10, 11, 12, 13, 14, , or 16 onal human JH gene segments (eg, wherein the locus comprises the full functional JH repertoire of said first individual supplemented with one or more JH gene segments derived from WO 41844 PCT/G82012/052296 the genome sequence of the second human individual and optionally with one or more JH gene segments derived from the genome sequence of a third human individual). In this way, the locus provides a superhuman repertoire of JH gene segments.

In one embodiment of the vertebrate or cell, the transgenic locus comprises a first JH gene segment derived from the genome ce of the first individual and a second JH gene segment derived from the genome sequence of the second individual, wherein the second JH gene segment is a polymorphic variant of the first JH gene segment. ally the locus ses a further polymorphic t of the first JH gene t (eg, a variant derived from the genome sequence of a third human individual). In this way, the locus provides a superhuman repertoire of JH gene segments. (b) Superhuman light chain gene repertoires The first configuration of the invention also provides:- A non-human vertebrate or vertebrate cell nally an ES cell or antibody-producing cell) comprising a genome having a superhuman immunoglobulin light chain human VL gene repertoire.

Optionally the vertebrate or cell comprises a heavy chain transgene according to aspect (a) of the first uration. Thus, superhuman diversity is provided in both the heavy and light chain globulin gene segments in the cell and vertebrate. For example, the genome of the cell or vertebrate is homozygous for the heavy and light chain transgenes and endogenous antibody expression is inactivated. Such a vertebrate is useful for immunisation with a predetermined n to produce one or more selected antibodies that bind the antigen and have human variable regions resulting from recombination within the superhuman gene segment repertoire. This provides potentially for a novel antibody and gene sequence space from which to select therapeutic, prophylactic and tool antibodies.

In one embodiment of aspect (b) of the first configuration, the vertebrate or cell genome comprises (i) a transgenic immunoglobulin kappa light chain locus comprising a plurality of human PCT/G82012/052296 immunoglobulin VK gene segments and one or more human J gene segments, wherein the plurality of VK gene segments consists of more than the natural human repertoire of functional VK gene segments; optionally wherein the genome is homozygous for said transgenic kappa light chain locus; and/or (ii) a transgenic globulin lambda light chain locus sing a plurality of human immunoglobulin VA gene segments and one or more human J gene segments, wherein the plurality of VA gene segments consists of more than the natural human repertoire of functional VA gene segments; optionally wherein the genome is homozygous for said transgenic lambda light chain locus.

In this way, the locus provides a uman oire of VL gene segments.

In one ment of the vertebrate or cell, (i) the VK gene repertoire consists of a plurality of VK gene segments derived from the genome sequence of a first human individual, supplemented with one or more VK gene segments derived from the genome sequence of a second, different human individual; optionally wherein the individuals are not related; optionally wherein the] segments are derived from the genome sequence of the first human individual; and optionally wherein the VK gene segments from the genome sequence of the second individual are selected from the VK gene segments listed in Table 4, 13 or 14; and (i) the VA gene repertoire consists of a plurality of VA gene segments derived from the genome sequence of a first human individual, supplemented with one or more VA gene segments derived from the genome sequence of a second, different human dual; optionally wherein the individuals are not related; optionally wherein the] segments are derived from the genome sequence of the first human individual; and optionally n the VA gene segments from the genome ce of the second individual are selected from the VA gene segments listed in Table 5, 13 or 14.

In this way, the locus provides a superhuman repertoire of VL gene segments.

WO 41844 In one embodiment of the vertebrate or cell, -the kappa light transgenic locus comprises more than 38 functional human VK gene segment species; ally wherein the locus comprises at least 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 functional human Vx gene segment species (eg, n the locus comprises the full functional VK repertoire of said first individual supplemented with one or more VK gene segments derived from the genome sequence of the second human individual and optionally with one or more VK gene segments derived from the genome sequence ofa third human dual); and -the lambda light transgenic locus comprises more than 31 functional human VA gene t species; optionally wherein the locus comprises at least 32, 33, 34, 35, 36, 37, 38, 39, 40 or 41 onal human VA gene segment species (eg, wherein the locus comprises the full functional VA repertoire of said first dual supplemented with one or more VA gene segments derived from the genome sequence of the second human dual and optionally with one or more VA gene segments derived from the genome sequence of a third human individual).

In this way, the locus provides a superhuman repertoire ofVL gene segments.

In one embodiment of the vertebrate or cell, -the kappa light transgenic locus comprises a first VK gene segment derived from the genome sequence of the first individual and a second VK gene segment derived from the genome sequence of the second individual, wherein the second VK gene segment is a polymorphic variant of the first VK gene segment; optionally wherein the locus comprises a further polymorphic variant of the first VK gene segment (eg, a variant derived from the genome ce of a third human individual); and ’the lambda light transgenic locus comprises a first VA gene segment derived from the genome sequence of the first individual and a second VA gene segment derived from the genome sequence of the second individual, wherein the second VA gene segment is a polymorphic t of the first VA gene segment; optionally wherein the locus comprises a r polymorphic variant of the first VA gene segment (eg, a variant derived from the genome sequence ofa third human individual).

PCT/G820 l 2/052296 In this way, the locus provides a superhuman repertoire ofVL gene segments.

In one embodiment of the vertebrate or cell, the genome comprises a (or said) transgenic immunoglobulin light chain locus comprising a plurality of human immunoglobulin VL gene segments and a plurality of human JL gene segments, wherein the plurality ofJ gene segments consists of more than the natural human repertoire of functional 1 gene segments; optionally wherein the genome is homozygous for said transgenic heavy chain locus.

In one ment ofthe vertebrate or cell, (i) the JK gene repertoire ts of a plurality of JK gene ts derived from the genome sequence of a (or said) first human individual, supplemented with one or more JK gene segments derived from the genome sequence of a (or said) , different human dual; optionally wherein the individuals are not related; optionally wherein the Vx segments are derived from the genome sequence of the first human individual; optionally wherein the Jl< gene segments from the genome sequence of the second individual are selected from the JK gene segments listed in Table 6, 13 or 14; and (ii) the JK gene repertoire consists of a plurality ofJA gene segments derived from the genome ce of a (or said) first human individual, supplemented with one or more JA gene segments derived from the genome sequence of a (or said) second, different human individual; optionally wherein the individuals are not related; optionally wherein the VA segments are derived from the genome sequence of the first human individual; optionally wherein the JA gene segments from the genome sequence of the second individual are ed from the JA gene ts listed in Table 7, 13 or 14.

In this way, the locus provides a superhuman repertoire ofJL gene segments. in one ment of the vertebrate or cell, (i) the transgenic light chain locus comprises more than 5 functional human .lK gene segment species; optionally wherein the locus comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 functional human JK gene segment species (eg, n the locus comprises the full functional JK repertoire of said first individual supplemented with one or more JK gene segments d from the genome sequence of the second human individual and optionally with one or more JK gene segments derived from the genome sequence of a third human individual); and/or (i) the transgenic light chain locus ses more than 5 functional human lit gene segment species; optionally wherein the locus ses at least 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 functional human M gene segment species (eg, wherein the locus comprises the full functional JA repertoire of said first individual supplemented with one or more JA gene segments derived from the genome sequence of the second human individual and optionally with one or more JA gene segments derived from the genome sequence of a third human individual).

In this way, the locus provides a superhuman repertoire of JL gene segments.

In one embodiment of the vertebrate or cell, (i) the kappa light transgenic locus comprises a first JK gene segment derived from the genome sequence of the first individual and a second JK gene segment derived from the genome sequence of the second individual, wherein the second JK gene segment is a polymorphic variant of the first JK gene segment; ally wherein the locus comprises a further polymorphic variant of the first JK gene segment (eg, a t derived from the genome sequence of a third human individual); and (ii) the lambda light transgenic locus comprises a first M gene segment derived from the genome sequence of the first individual and a second M gene segment derived from the genome sequence of the second individual, wherein the second JK gene t is a rphic variant of the first JA gene segment; optionally wherein the locus comprises a further polymorphic t of the first JA gene segment (eg, a variant derived from the genome sequence of a third human individual).

In this way, the locus provides a superhuman repertoire ofJLgene segments.

Further aspects of the first configuration are described below.

A library of antibody—producing transgenic cells whose genomes tively encode a repertoire of antibodies, wherein WO 41844 2012/052296 (a) a first transgenic cell expresses a first antibody having a chain (eg, heavy chain) encoded by a first immunoglobulin gene, the gene comprising a first variable domain nucleotide sequence produced following recombination of a first human ranged immunoglobulin gene segment (eg, a VH); (b) a second transgenic cell ses a second antibody having a chain (eg, a heavy chain) encoded by a second immunoglobulin gene, the second gene comprising a second variable domain nucleotide sequence produced following recombination of a second human unrearranged immunoglobulin gene t (eg, a VH), the first and second antibodies being non—identical; (c) the first and second gene segments are different and derived from the genome sequences of first and second human individuals respectively, wherein the individuals are different; and optionally not d; (d) wherein the cells are non-human rate (eg, mouse or rat) cells (eg, B-cells or hybridomas).

In one embodiment, the library is provided in vitro. In another embodiment, the library is provided in vivo by one or a ity of transgenic non-human vertebrates. For example, the or each vertebrate is according to any aspect of the first configuration of the ion.

In one embodiment, the library encodes an antibody repertoire of from 10 to 109 antibodies, for example, 10, 20, 30, 40, 50, 100 or 1000 to 108; or 10, 20, 30, 40, 50, 100 or 1000 to 107; or 10, 20, , 40, 50, 100 or 1000 to 10“; or 10, 20, 30, 40, 50, 100 or 1000 to 105; or 10, 20, 30,40, 50, 100 or 1000 to 10‘ antibodies. In an example, library encodes an antibody repertoire of at least 103, 104, 105, 106, 107, 103, 109'or 101° antibodies.

The first variable domain nucleotide sequence is produced following ination of the first human unrearranged immunoglobulin gene segment with one or more other immunoglobulin gene ts (for example, human immunoglobulin gene segments). For example, where the first gene segment is a VH, the first variable domain nucleotide sequence (a VH domain) is produced following recombination ofthe VH with a human D and JH segments in vivo, optionally with somatic hypermutation, in the first transgenic cell or an ancestor thereof. For e, where the first gene segment is a VL, the first variable domain nucleotide sequence (a VL domain) is produced following recombination of the VL with a human JL segment in vivo, optionally with somatic hypermutation, in the first transgenic cell or an ancestor thereof.

The second variable domain nucleotide sequence is produced following recombination of the second human unrearranged immunoglobulin gene segment with one or more other immunoglobulin gene segments (for example, human immunoglobulin gene segments). For e, where the second gene segment is a VH, the second variable domain nucleotide sequence (a VI-l domain) is produced following recombination of the VH with a human D and JH segments in vivo, optionally with somatic hypermutation, in the second transgenic cell or an ancestor thereof. For e, where the second gene segment is a VL, the second variable domain nucleotide ce (a VL ) is produced following recombination of the VL with a human JL segment in vivo, optionally with somatic hypermutation, in the second transgenic cell or an ancestor thereof.

The first and second gene segments are respectively d from genome sequences of first and second human individuals. In one example, such a gene segment is isolated or cloned from a sample cell taken from said individual using standard molecular biology techniques as know to the skilled person. The sequence of the gene segment may be d (eg, by the introduction of up to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 nucleotide changes) prior to use in the present invention. In another example, a gene segment is derived by identifying a ate human immunoglobulin gene segment in a database (see guidance below) and a nucleotide sequence encoding a gene segment for use in the present invention is made by reference (eg, to be identical or a mutant with up to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 nucleotide changes to the reference sequence) to the database sequence. The skilled person will be aware of s of obtaining nucleotide ces by reference to ses or by obtaining from cellular samples. in one embodiment of the vertebrate, cell or library of any configuration of the invention, the first and second human individuals are members of first and second ethnic populations respectively, wherein the populations are ent. This, therefore, provides for superhuman gene diversity in transgenic loci, cells and vertebrates as per the invention.

PCT/GBZOl2/052296 Optionally the ethnic populations are selected from those fied in the 1000 Genomes Project of database. In this respect, see Table 8 which provides details of the ethnic populations on which the 1000 Genomes database is based.

N A Rosenberg et a/ (Science 20 December 2002: vol. 298 no. 5602 2342-2343) studied the genetic structure of human populations of differing geographical ancestry. In total, 52 populations were sampled, these being populations with: African ancestry (Mbuti Pygmies, Biaka Pygmies, San peoples, and speakers of Niger-Kordofanian ges (Bantu, Yoruba or Mandenka populations), Eurasian ancestry (European ancestry (Orcadian, Adygel, Basque, French, ns, Italians, Sardinian, ), Middle Eastern ancestry (Mozabite, Bedouin, Druze, Palestinians), Central/South Asian ancestry (Balochl, Brahul, Makrani, Sindhi, Pathan, Burusho, Hazara, Uygur, Kalash)), East Asian ancestry (Han, Dal, Daur, Hezhen, Lahu, Miao, Oroqen, She, Tujia, Tu, Xibo, Yi, Mongola, Naxi, Cambodian, Japanese, Yakut), Oceanic ry (Melanesian, Papuan); or Americas ancestry (Karitiana, Surui, Colombian, Maya, Pima).

The International HapMap Project, Nature, 2003 Dec 18;426(6968):789-96, ses that goal of the HapMap Project: to determine the common patterns of DNA sequence variation in the human genome by ining the genotypes of one n or more sequence variants, their frequencies and the degree of association between them in DNA s from populations with ry from parts of Africa, Asia and Europe. The relevant human tions of differing geographical ancestry include Yoruba, Japanese, Chinese, Northern European and n European populations. More specifically:- PCT/GBZOIZ/052296 Utah population with Northern or Western European ancestry (samples collected in 1980 by the Centre d’Etude du Polymorphisme Humain (CEPH)); population with ry of Yoruba people from lbadan, Nigeria; tion with Japanese ancestry; and population with ancestry of Han Chinese from China.

The authors, citing earlier publications, suggest that ancestral geography is a reasonable basis for sampling human populations.

A suitable sample of human populations from which the populations used in the present invention are selected is as follows:- (a) European LZ (b) rn European ancestry; Western European ancestry; Toscani ancestry; British ancestry, Finnish ancestry or Iberian ancestry. (c) More specifically, population of Utah residents with Northern and/or n European ancestry; Toscani population in Italia; British population in England and/or Scotland; h population in Finland; or Iberian population in Spain. (a) 505; Asian ancestry (b) Japanese ancestry; Chinese ry or Vietnamese ancestry. (c) More specifically, Japanese population in Toyko, Japan; Han Chinese population in Beijing, China; Chinese Dai population in Xishuangbanna; Kinh population in Ho Chi Minh City, Vietnam; or Chinese population in Denver, Colorado, USA. (a) WestAfin'can ancestry (b) Yoruba ry; Luhya ancestry; n ancestry; or Malawian ancestry. (c) More specifically, Yoruba population in lbadan, Nigeria; Luhya population in Webuye, Kenya; n tion in Western Division, The Gambia; or Malawian population in Blantyre, Malawi. (0) tion at The Americas (b) Native American ancestry; Afro-Caribbean ancestry; Mexican ancestry; Puerto Rican ancestry; Columbian ancestry; or Peruvian ancestry. (c) More specifically, population of African Ancestry in Southwest US; population of African American in Jackson, MS; population of African Caribbean in Barbados; population of Mexican Ancestry in Los Angeles, CA; population of Puerto Rican in Puerto Rico; population of Colombian in Medellin, Colombia; or population of Peruvian in Lima, Peru. {0) éguth Asign ancestry (b) Ahom ancestry; Kayadtha ancestry; Reddy ry; Maratha; or Punjabi ancestry. (c) More ically, Ahom population in the State of Assam, India; Kayadtha population in Calcutta, India; Reddy population in Hyderabad, India; Maratha tion in Bombay, lndia; or i population in Lahore, Pakistan.

In any configuration of the invention, in one embodiment, each human population is selected from a population marked ”(a)" above.

In any configuration of the invention, in another embodiment, each human population is selected from a population marked ”(b)" above.

In any configuration of the invention, in another ment, each human population is selected from a population marked “(c)” above.

In one ment of the library of the vertebrate, cell or y of the invention, the first and second ethnic populations are selected from the group consisting of an ethnic population with PCT/G82012/052296 European ancestry, an ethnic population with East Asian, an ethnic population with West African ancestry, an ethnic population with Americas ancestry and an ethnic population with South Asian ancestry.

In one embodiment of the library of the vertebrate, cell or y of the invention, the first and second ethnic populations are selected from the group consisting of an ethnic tion with Northern European ancestry; or an ethnic population with n European ancestry; or an ethnic population with Toscani ry; or an ethnic tion with h ancestry; or an ethnic population with Icelandic ancestry; or an ethnic population with Finnish ancestry; or an ethnic population with Iberian ancestry; or an ethnic population with Japanese ancestry; or an ethnic population with Chinese ry; or an ethnic population Vietnamese ancestry; or an ethnic population with Yoruba ancestry; or an ethnic population with Luhya ancestry; or an ethnic population with Gambian ancestry; or an ethnic population with Malawian ancestry; or an ethnic population with Native American ancestry; or an ethnic population with Afro-Caribbean ancestry; or an ethnic tion with Mexican ancestry; or an ethnic population with Puerto Rican ancestry; or an ethnic population with Columbian ancestry; or an ethnic population with Peruvian ancestry; or an ethnic population with Ahom ancestry; or an ethnic population with Kayadtha ancestry; or an ethnic population with Reddy ancestry; or an ethnic population with Maratha; or an ethnic population with Punjabi ancestry.

In one embodiment of any uration of the vertebrate, cell or library of the ion, the human globulin gene segment derived from the genome sequence of the second individual is low-frequency (optionally rare) within the second ethnic population. Optionally human globulin gene segment has a Minor Allele Frequency (MAF) (cumulative frequency) of between 0.5% - 5%, optionally less than 0.5%, in the second human population, eg, as in the 1000 Genomes database.

In one embodiment of any configuration of the vertebrate, cell or library of the invention, the first variable region nucleotide sequence is produced by recombination of the first human immunoglobulin gene t with a first] gene segment and optionally a first D gene segment, wherein the first human immunoglobulin gene segment is a V gene segment and the V, D and J segments are derived from the first human population, optionally from the genome of one individual W0 20131041844 PCT/032012/052296 of the first human tion. in one embodiment of the library of the vertebrate, cell or y of the invention, the second variable region nucleotide sequence is produced by recombination of the second human immunoglobulin gene segment with a second J gene segment and optionally a second D gene segment, wherein the second human immunoglobulin gene segment is a V gene t derived from the second tion and the D and/or] segments are derived from the first human population, optionally the D and J gene ts being from the genome of one dual ofthe first human population.

In one embodiment of the y of the vertebrate, cell or library of the invention, all of the D and J segments that have been recombined with the first and second V gene segments are D and J segments derived from the first human population, optionally the D and J gene segments being from the genome of one individual of the first human population.

In one embodiment of the library, the second human immunoglobulin gene segment is a polymorphic variant of the first human immunoglobulin gene segment; Optionally n the second gene segment is selected from the group consisting of a gene t in any of Tables 1 to 7 and 9 to 14 (eg, selected from Table 13 or 14).

In one embodiment of the library, the first and second human immunoglobulin gene segments are both (i) VH gene segments; (ii) D segments; (iii) J segments (optionally both in segments, both 1‘ segments or both 1; segments); (iv) constant regions (optionally both a gamma constant region, optionally both a C gamma-1 constant region); (v) CH1 regions; (vi) CH2 regions; or (vii) CH3 regions.

The library is, for example, a naive and optionally has a library size of from 10 or 102 to 109 cells.

For example, from 10, 20, 30, 40, 50, 100 or 1000 to 108; or 10, 20, 30, 40, 50, 100 or 1000 to 107; or , 20, 30, 40, 50, 100 or 1000 to 106; or 10, 20, 30, 40, 50, 100 or 1000 to 105; or 10, 20, 30, 40, 50, 100 or 1000 to 10“ cells.

PCT/G82012/052296 The library has, for example, been selected t a predetermined antigen and optionally has a library size of from 10 or 102 to :109 cells. For example, from 10, 20, 30, 40, so, 100 or 1000 to 108; or 10, 20, 30, 40, 50, 100 or 1000 to 107; or 10, 20, 30, 40, 50, 100 or 1000 to 106; or 10, 20, 30, 40, 50, 100 or 1000 to 105; or 10, 20, 30, 40, 50, 100 or 1000 to 104 cells.

In one embodiment of the library of the invention, said first and second cells are progeny of first and second ancestor non-human vertebrate cells respectively, wherein the first ancestor cell comprises a genome comprising said first human immunoglobulin gene segment; and the second ancestor cell comprises a genome comprising said second human immunoglobulin gene segment.

The invention further provides a library of antibody-producing enic cells whose genomes tively encode a repertoire of antibodies, wherein the library comprises the first and second or cells described above.

The invention further provides a library of oma cells produced by fusion of the library of the invention (eg, a B-cell library) with fusion partner cells and optionally has a library size of from 10 or 102 to 109 cells. For example, from 10,20, 30, 40, 50, 100 or 1000 to 108; or 10, 20, 30, 4o, 50, 100 or 1000 to 107; or 10, 20, 30, 40, 50, 100 or 1000 to 106; or 10, 20, 30, 40, 50, 100 or 1000 to 105; or , 20, 30, 40, 50, 100 or 1000 to 10“ cells. Production of hybridomas is well known to the d person Examples of fusion rs are SP2/0-g14 (obtainable from ECACC), P3X63-Ag8.653 (obtainable from LGC rds; CRL-1580), N51 and N50 cells. PEG fusion or electrofusion can be carried out, as is conventional.

Il'l 'l'l'lll An ed antibody having (a) a heavy chain encoded by a nucleotide sequence produced following recombination in a transgenic non-human vertebrate cell of an unrearranged human immunoglobulin V gene segment with a human D and human] segment, optionally with affinity maturation in said cell, wherein one of the gene segments (eg, VH) is derived from the genome of an individual from a first human ethnic population; and the other two gene segments (eg, D and JH) are derived from the genome of an individual from a second (eg, a second and third respectively), different, human ethnic population, PCT/G820 12/052296 and wherein the antibody comprises heavy chain constant regions (eg, C gamma) of said man vertebrate (eg, rodent, mouse or rat heavy chain constant regions); and/or (b) alight chain encoded by a nucleotide sequence produced following recombination in a transgenic man vertebrate cell of an unrearranged human immunoglobulin V gene segment with a human J segment, optionally with affinity maturation in said cell, wherein one of the gene segments (eg, VL) is derived from the genome of an dual from a first human ethnic population (optionally the same as the first population in (a)); and the other gene segment (eg, JL) is derived from the genome of an dual from a second, different, human ethnic tion (optionally the same as the second population in (a)), and wherein the antibody comprises light chain constant s of said non-human rate (eg, rodent, mouse or rat heavy light constant regions); (c) Optionally wherein each variable domain of the antibody is a human variable domain. (d) Optionally wherein the heavy chain constant regions are mu- or gamma—type constant regions.

The invention also provides an ed nucleotide sequence encoding the antibody of the third configuration, optionally n the sequence is provided in an antibody expression vector, optionally in a host cell. Suitable vectors are mammalian expression vectors (eg, CHO cell vectors or HEK293 cell vectors), yeast s (eg, a vector for expression in Picchia pastoris, or a bacterial expression vector, eg, a vector for E. coli expression.

The invention also provides a method of producing a human antibody, the method comprising replacing the non-human vertebrate nt s of the antibody of the third configuration with human dy constant regions (eg, a C variant disclosed in table 13 or 18). The skilled person will be aware of standard molecular biology techniques to do this. For example, see Harlow, E. & Lane, D. 1998, 5"1 edition, Antibodies: A Laboratory Manual, Cold Spring Harbor Lab. Press, Plainview, NY; and Pasqualini and Arap, Proceedings of the National Academy of Sciences (2004) 101:257-259 for standard immunisation. Joining of the variable regions of an antibody to a human constant region can be effected by techniques readily available in the art, such as using conventional inant DNA and RNA technology as will be apparent to the skilled person. See e.g. Sambrook,J and l, D. (2001, 3'd edition) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab.

Press, Plainview, NY).

In one embodiment, the method ses further making a mutant or derivative of the antibody.

The invention also provides a pharmaceutical composition comprising an antibody ing to the third configuration, or a human antibody of the invention and a t, excipient or carrier; optionally wherein the composition is provided in a container connected to an IV needle or syringe or in an IV bag.

The invention also provides an antibody-producing cell (eg, a mammalian cell, eg, CHO or HEK293; a yeast cell, eg, P pastoris; a bacterial cell, eg, E coli; 3 B-cell; or a hybridoma) that expresses the second antibody of the third configuration or the isolated dy of the invention. i ' ‘ r 'onoft invninl r ~- A man vertebrate or vertebrate cell (optionally an ES cell or antibody-producing cell) whose genome ses a transgenic immunoglobulin locus (eg, a heavy chain locus or a light chain locus), said locus comprising immunoglobulin gene segments ing to the first and second human immunoglobulin gene segments (optionally V segments) described above in connection with the third configuration. The gene segments are operably connected upstream of an immunoglobulin constant region; optionally wherein the genome is homozygous for said transgenic immunoglobulin locus.

Optionally the immunoglobulin locus comprises more than the l human complement of functional V gene segments; and/or Optionally wherein the immunoglobulin locus comprises more than the natural human complement of onal D gene segments; and/or ally wherein the immunoglobulin locus comprises more than the natural human complement of functional J gene segments.

In this way, a superhuman immunoglobulin gene repertoire is provided in a transgenic non-human vertebrate or vertebrate cell according to the invention.

W0 2013/041844 PCT/G82012/052296 The first configuration also provides:- A transgenic non-human vertebrate (eg, a mouse or rat) or vertebrate cell (optionally an ES cell or antibody~producing cell) whose genome comprises a transgenic immunoglobulin locus comprising a plurality of human immunoglobulin gene segments operably connected upstream of a non-human vertebrate constant region for the production of a repertoire of chimaeric antibodies, or chimaeric light or heavy chains, having a non-human vertebrate constant region and a human variable region; wherein the transgenic locus ses one or more human immunoglobulin V gene segments, one or more human J gene segments and optionally one or more human D gene segments, a first (optionally a V segment) of said gene segments and a second nally a V segment) of said gene segments being different and derived from the genomes of first and second human individuals tively, wherein the individuals are different; and ally not related; ally wherein the globulin locus comprises more than the natural human complement of functional V gene segments; and/or optionally wherein the immunoglobulin locus comprises more than the natural human complement of functional D gene segments; and/or optionally n the immunoglobulin locus comprises more than the natural human complement of functional J gene ts.

In this way, a superhuman immunoglobulin gene repertoire is provided in a transgenic non-human vertebrate or vertebrate cell according to the invention. l f ll . | 'l __ A transgenic non-human rate (eg, a mouse or rat) or vertebrate cell (optionally an ES cell or antibody—producing cell) whose genome comprises first and second transgenic immunoglobulin loci, each locus comprising a plurality of human globulin gene segments operably connected upstream of a non—human vertebrate constant region for the production of a oire of chimaeric antibodies, or chimaeric light or heavy chains, having a man vertebrate constant region and a human variable region; 2012l052296 n (i) the first transgenic locus comprises one or more human immunoglobulin V gene ts, one or more human J gene segments and optionally one or more human 0 gene segments, (ii) the second transgenic locus comprises one or more human immunoglobulin V gene segments, one or more human J gene segments and ally one or more human D gene segments; and (iii) n a first (optionally a V) gene segment of said first locus and a second (optionally a V) gene segment of said second gene locus are different and derived from the genomes of first and second human individuals respectively, wherein the individuals are different; and optionally not related; optionally wherein the first and second loci are on ent chromosomes (optionally chromosomes with the same chromosome ) in said genome; optionally wherein each immunoglobulin locus comprises more than the natural human complement of functional V gene segments; and/or optionally wherein each immunoglobulin locus comprises more than the natural human complement of functional D gene segments; and/or optionally wherein each immunoglobulin locus comprises more than the natural human ment of functional 1 gene ts.

In this way, a superhuman immunoglobulin gene oire is provided in a transgenic non -human vertebrate or vertebrate cell according to the invention.

In these embodiments of the first configuration, the immunoglobulin gene segments are optionally as described for the third configuration.

In these embodiments of the first configuration, the genome optionally comprises a third immunoglobulin gene segment (optionally a V segment), the third gene segment being derived from a human individual that is different from the individual from which the first (and optionally also the second) gene segment is derived; optionally wherein the first, second and third gene segments are polymorphic variants of a human immunoglobulin gene segment (eg, VH1-69 — see the examples for WO 41844 further description).

In these embodiments of the first configuration, the genome of the vertebrate or cell is optionally homozygous for the first, second and optional third gene segment, wherein a copy of the first, second and optional third gene segments are provided together on the same chromosome operably connected upstream of a common non-human vertebrate constant region.

For example, each first, second and optional third gene segment is a V gene segment.

In one example, the library of the invention is provided by a collection of non-human vertebrates (optionally a collection of rodents, mice or rats); optionally, wherein a first member of said collection produces said first antibody but not said second antibody, and a second member of the tion produces said second antibody (but optionally not said first antibody). It is therefore contemplated to make non-human vertebrates where different human genomes have been used as a source for building the enic loci in the vertebrates. For example, a first rate comprises a transgenic heavy chain locus having gene ts only from a first (and optionally a ) human population or individual; a second vertebrate comprises a transgenic heavy chain locus having gene segments only from a third (and optionally a ) human population or individual; and optionally third and more vertebrates can be built rly based on unique or overlapping human population genomes. However, when provided as a mixed population of transgenic vertebrates, the mixed tion provides a collective pool of human immunoglobulin genes that is greater than found in a natural human repertoire. This is useful to extend the dy and gene sequence space beyond those possible with prior transgenic mice and rats bearing human immunoglobulin loci. As explained above, these have been based on a single human genome.

In one embodiment, the collection of non-human vertebrates bear human immunoglobulin genes confined to human populations that are together grouped under the same population genus ”(a)” mentioned above. This provides for a gene oire that is biased to producing human antibody variable regions prevalent in the tion genus (a) and thus useful for generating antibody therapeutics/prophylactics for members of said population. Alternatively, where gene segments from different human populations are provided in a single transgene according to the invention (not necessarily in a collection of rates), the different human populations are for example together grouped under the same population genus ”(a)" mentioned above.

The invention also provides a repertoire of antibodies expressed from a y of cells according to the ion.

In the non—human vertebrate or cell of any configuration of the invention, the constant region of the transgenic locus is, in one example, an endogenous constant region of said vertebrate (eg, endogenous mouse or rat constant region, eg, from the same strain of mouse or rat as the non- human vertebrate itself).

The invention also provides a method of constructing a cell (eg, an ES cell) according to the invention, the method comprising (3) fying onal V and J (and optionally D) gene segments of the genome sequence of a (or said) first human individual; (b) identifying one or more functional V and/or D and/orJ gene segments of the genome sequence of a (or said) second human individual, wherein these additional gene segments are not found in the genome ce of the first individual; (c) and constructing a transgenic immunoglobulin locus in the cell, wherein the gene ts of (a) and (b) are provided in the locus operably connected upstream of a constant region.

Optionally the cell ses a heavy chain locus constructed according to steps (a) to (c) and/or a light chain locus (kappa and/or lambda loci) constructed according to steps (a) to (c).

Optionally the cell is homozygous for the or each transgenic locus; optionally wherein antibody expression from loci endogenous to said cell has been inactivated. This is useful for confining the functional antibody gene oire, and thus antibody production, to antibodies bearing human le regions.

Optionally the gene segment(s) in step (b) are identified from an immunoglobulin gene database selected from the 1000 Genomes, Ensembl, Genbank and lMGT databases.

Optionally the first and second human individuals are members of first and second ethnic populations respectively, wherein the populations are different, optionally wherein the human immunoglobulin gene segment derived from the genome sequence of the second individual is low- frequency (optionally rare) within the second ethnic p0pulation.

The invention also provides a method of making a transgenic non-human vertebrate (eg, a mouse or rat), the method comprising (a) constructing an ES cell (eg, a mouse C57BL/6N,CS7BL/61, 12955 or 129Sv strain ES cell) by ng out the method above; (b) injecting the ES cell into a donor non-human vertebrate blastocyst (eg, a mouse C57BL/6N, C57BL/6J, 12955 or 1295v strain blastocyst); (c) implanting the blastocyst into a foster non-human vertebrate mother (eg, a C57BL/6N, C57BL/6], 12955 or 1295v strain mouse); and (d) obtaining a child from said mother, wherein the child genome comprises a transgenic globulin locus.

The invention provides a transgenic non-human vertebrate (eg, a mouse or rat} made by the method or a progeny thereof. The invention also provides a population of such man vertebrates.

Microinjection of ES cells into blastocysts and generation of transgenic mice ter are conventional practices in the state of the art, and the skilled person is aware of techniques useful to effect this. C57BL/6N, C57BL/61, 12955 or 129Sv mouse strains and ES cells are readily and ly available.

The ion also provides a method of isolating an antibody that binds a predetermined antigen (eg, a bacterial or viral pathogen antigen), the method comprising (a) providing a vertebrate (optionally a mouse or rat) ing to the invention; (b) immunising (eg, using a standard prime-boost method) said vertebrate with said antigen nally n the antigen is an antigen of an infectious disease pathogen); (c) removing B lymphocytes from the vertebrate and selecting one or more B lymphocytes expressing antibodies that bind to the antigen; (d) optionally immortalising said selected B cytes or progeny thereof, optionally by PCT/GBZOIZ/052296 producing hybridomas therefrom; and (e) isolating an antibody (eg, and IgG-type antibody) sed by the B lymphocytes; and (f) optionally producing a derivative or variant of the antibody.

This method optionally further comprises after step (e) the step of isolating from said B lymphocytes nucleic acid encoding said antibody that binds said antigen; optionally exchanging the heavy chain constant region nucleotide sequence of the antibody with a nucleotide sequence encoding a human or humanised heavy chain constant region and optionally affinity maturing the variable region of said antibody; and optionally inserting said nucleic acid into an expression vector and optionally a host.

See also the discussion on variation analysis above.

The skilled person will know of sources of human antibody gene sequences, such as IMGT (wwwjmgtorgl, GenBank (www.ncbi.n|m.nih.gov/genbank) Bioinformatics tools for database manipulation are also readily available and known to the d , eg, as publicly available from the 1000 Genomes Project/EBI (www.3000ggnomesorgi As a source of antibody gene segment ces, the skilled person will also be aware of the following available ses and resources (including s thereof):- 1.1. The Kabat Database (6. Johnson and T. T.Wu, .

Created by E. A. Kabat and T. T. Wu in 1966, the Kabat database publishes d sequences of antibodies, T-cell receptors, major histocompatibility x (MHC) class I and II molecules, and other ns of immunological interest. A able interface is provided by the Seqhuntll tool, and a range of utilities is available for sequence alignment, sequence subgroup classification, and the generation of variability plots. See also Kabat, E. A.,Wu, T. T., Perry, H., Gottesman, K., and Foeller, C. (1991) Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication No. 91— 3242,8ethesda, MD, which is incorporated herein by reference, in particular with reference to human gene segments for use in the present invention. 1.2. KabatMan (A. C. R. , 2002; [mg-luau!bioinforg.uk‘abszsimggbhlmli. This is a web interface to make simple queries to the Kabat sequence database. 1.3. IMGT, the International GeneTics Information System”; M. -P. Lefranc, 2002; httg.{Zimgt.cines.[rl. IMGT is an integrated information system that specializes in antibodies, T cell receptors, and MHC molecules of all vertebrate species. It provides a common portal to standardized data that include nucleotide and protein sequences, oligonucleotide primers, gene maps, genetic polymorphisms, specificities, and mensional (20) and three-dimensional (3D) structures. IMGT includes three sequence databases (iMGT/LiGM-DB, lMGT/MHC-DB, IMGT/PRIMERDB), one genome database (lMGT/GENE—DB), one 30 structure database (IMGT/3Dstructure-DB), and a range of web resources ("lMGT Marie~Pau|e page”) and interactive tools. 1.4. V-BASE (I. M. Tomlinson, 2002,-http://www.mrc-cpe.cam.ac.uk/vbase). V-BASE is a hensive directory of all human antibody germline variable region sequences ed from more than one thousand published sequences. It es a version of the alignment software T (developed by Hans-Helmar Althaus and Werner Muller) that allows the assignment of rearranged antibody V genes to their closest germline gene segments. 1.5. Antibodies—Structure and $equence(A. C. R. Martin, 2002; haQZwawbioint.orgiukfigggl.

This page summarizes useful information on antibody structure and sequence. It provides a query interface to the Kabat antibody sequence data, general information on antibodies, crystal structures, and links to other dy-related information. It also butes an automated summary of all antibody structures deposited in the Protein Databank (PDB). Of particular interest is a thorough description and comparison of the s numbering s for antibody variable regions. 1.6. AAAAA—AHo’s Amazing Atlas ofAntibody Anatomy (A. Honegger, 2001; htt : www.unizh.ch ~antibod . This ce es tools for structural analysis, modeling, and engineering. It adopts a unifying scheme for hensive structural alignment of antibody and T- PCT/G32012/052296 cell-receptor sequences, and includes Excel macros for antibody analysis and graphical representation. 1.7. WAM-Web Antibody Modeling (N. Whitelegg and A. R. Rees, 2001; hag-(Zantibodybath.acuk). Hosted by the Centre for n Analysis and Design at the University of Bath, United Kingdom. Based on the AbM package (formerly marketed by Oxford Molecular) to construct 3D models of dy Fv sequences using a combination of established theoretical methods, this site also includes the latest dy structural information. 1.8. Mike’s g/obu/in Structure/Function Page (M. R. Clark, 2001,- mmiflwwmggthxamoc.uk‘Nmrc7‘migfl’mgggglmm"These pages provide educational materials on globulin structure and function, and are illustrated by many colour images, models, and animations. Additional ation is available on antibody humanization and Mike Clark’s Therapeutic Antibody Human gy Project, which aims to correlate clinical efficacy and anti- immunoglobulin ses with variable region sequences of therapeutic antibodies. 1.9. The Antibody Resource Page (The Antibody Resource Page, 2000; Mtg-(memgntibodyresourcgromi. This site describes itself as the "complete guide to antibody research and suppliers.” Links to amino acid sequencing tools, nucleotide antibody sequencing tools, and hybridoma/cell-culture data bases are provided. 1.9. Humanization bY Design (1.50/danha, 2000; bttg:(Zg§ggigiggy§;igbk.acukfubcggysi. This ce provides an overview on antibody humanization technology. The most useful feature is a searchable database (by sequence and text) of more than 40 published humanized antibodies including information on design , framework choice, framework back-mutations, and binding affinity of the humanized constructs. 2012/052296 See also Antibody Engineering Methods and Protocols, Ed. Benny K C Lo, Methods in Molecular Biologym, Human Press. Also at - lo sua.com df antibod - I in -m h — nd- - - - col.f As a source of genomic sequence variation data, the skilled person will also be aware of the following available databases and ces (including updates f):- 1. HapMap (The International HapMap Consortium. 2003; http://hapmap.ncbi.n|m.nih.gov/index.html.en). The HapMap Project is an international project that aims to e the genetic sequences of different individuals to identify chromosomal regions containing shared genetic variants. The HapMap www site es tools to identify chromosomal regions and the variant therein, with options to drill down to population level frequency data. 1000 Genomes (The ;090 angmgs Prgiect ansortium 2010; http://www.1000genomes.org/).

This resource es complete genomic sequence for 2500 unidentified individuals from one of 25 distinct population groups, with the aim of identifying genomic variants of >1%. The site provides the ability to interrogate data utilizing online tools (e.g. tion Pattern Finder’) and to download variant data for individual population groups. se SNP Database (H.Haga et al. 2002; hm“(sngjms.u-to|§¥o.ag.igzingggimmll. Based on a study identifying 190,562 human genetic variants this site catalogues genomic variants with useful features for searching and izing data.

It is possible to identify variants in globulin genes classed as low-frequency or rare variants that segregate with specific human ethnic populations. For the purpose of this analysis, a low- frequency immunoglobulin gene segment is classed as one with ’Minor Allele Frequency’ (MAF) (cumulative frequency) of between 0.5% - 5%, rare variants are those classed as having a MAF of less than 0.5% in a ular human population.

PCT/G32012/052296 The ing bioinformatics protocol is envisaged to indentify human immunoglobulin gene segments for use in the present invention: (a) Identify one or more genomic regions containing gene segments of interest (’target genomic regions’) and ate the c coordinates, using coordinates that match the sequence assembly build used by either the 1000 Genomes t or International HapMap t (or another selected human gene database of ). (b) Identify genomic variants mapped to the genomic regions previously identified in (a). ve variant frequencies for variants for each super tion and preferably sub- population where such data is available. Tools readily available on the HapMap WWW site and the VWC tools for the 10006enomes Project are useful for this step. (c) Filter list of genomic variants from target genomic regions to contain only variants classed as either ’Non-synonymous’ single nucleotide polymorphisms (SNPs) or genomic ’insertions or delections’ (indels). Filter further to include those that are present in exonic sequences only. (d) ate population frequency data for each of the identified variants for each of the super populations (for e 'European Ancestry’, 'East Asian ancestry', ’West African ancestrV, 'Americas’, and ’South Asian ancestry’) to identify those variants that segregate with less than two super-populations. Further correlate all identified variants with each of the sub- populations (for example, ’European ancestry’ super—population might be subdivided into groups such as ’CEU — Utah residents with Northern or Western European ancestry’, 'TSI Toscani in Italia’ and ’British from England and Scotland’) and produce a second score for rarity of variants in within a super—population. (e) Collect one or more gene segments that show segregation to specific pulations for construction of tic loci according to the invention.

In one embodiment throughout the present text, "germline" refers to the canonical germline gene segment sequence.

By detailed analysis of the 1000 Genomes database, the inventors have d a collection of candidate polymorphic antibody gene segment variants, eg, human variant JH gene segments (eg, see Example 4), that can be built into the design of transgenic heavy chain loci in mice for expressing increasingly diverse and new, synthetic oires of human variable regions. To this end, the invention provides the ing embodiments. seninvn‘ ' ‘ r i -mVr _'i a As explained above, in designing transgenic Ig heavy chain loci the present ors have considered the huge amount of data available from the 1000 Genomes project (see Wthatanalyses gene distributions amongst many human populations, and in particular data on lg gene segments. The inventors were also aware of human gene ts disclosed in the IMGT database (seeW and in Ensembl (see www.ensembl.org). The inventors needed to make choices about which human gene segments to include t the large number of human gene segments presented in these databases and the other sources of human lg gene segment information known in the art, ing those other databases sed . When choosing human JH gene segments, the inventors were aware that human JH6 encodes a relatively long amino acid sequence, and thus the inventors thought it desirable to include this for increasing the chances of producing lgH chains with relatively long HCDR3 regions. dies with long HCDR3 (at least 20 amino acids according to IMGT nomenclature) have been shown to neutralise a variety of pathogens effectively including HIV, Influenza virus, malaria and Africa trypanosomes. Reference is also made to naturally-occurring Camelid (eg, llama or camel) heavy chain-only antibodies which bear long HCDR3S for reaching relatively inaccessible epitopes (see, eg, EP0937140). Long HCDR3$ can form unique stable subdomains with extended loop structure that towers above the antibody WO 41844 PCT/G82012/052296 surface to confer fine specificity. In some cases, the long HCDR3 itself is sufficient for epitope binding and neutralization (Liu, L et al; Journal of Virology. 2011. 85: 8467-8476, incorporated herein by reference). The unique structure of the long HCDR3 allows it to bind to cognate epitopes within inaccessible structure or extensive glycosylation on a pathogen e. In human peripheral blood, there is around 3.5% of naive B antibodies or 1.9% of memory 8 lgG antibodies containing the HCDR3s with lengths of more than 24 amino acids (PLoS One. 2012;7(5):e36750. Epub 2012 May 9; “Human peripheral blood antibodies with long HCDR3s are established primarily at original recombination using a limited subset of germline genes”,- Briney BS 9 of, incorporated herein by reference) (Fig. 1). The usage analysis indicates that these antibodies have the preference to use human JH6 with human 02-2, D3-3 or D2-15 (Brinley, BS et 0/, Figs. 2-5). See also PLoS One. 2011 Mar 30;6(3):e16857; Comparison of antibody repertoires produced by HIV-1 infection, other chronic and acute infections, and ic autoimmune disease”; Breden F et al, incorporated herein by reference. Around 20% of all HCDR3 of antibodies use JH6. r, in those antibodies with HCDR3 of more than 24 amino acids, 70% use JH6 (Brinley, BS et 0/, Fig.2).

There is a need in the art for cally ed non-human vertebrates and cells that can make antibodies and heavy chains that have long human HCDR3S, as well as antibodies, chains and VH domains that can be selected from such vertebrates and cells wherein these can address target epitopes better ed by long .

The inventors, therefore, chose in this configuration of the invention to include a human JH6 gene segment as a mandatory human gene t in their IgH locus design. Several different natural|y~ occurring human JH6 variants are known (eg, JH6*01 to *04 as well as others; IMGT nomenclature).

The inventors considered this when deciding upon which human JH6 variant should be included in the transgenic IgH locus design. An ent of some human JH6 variants is shown in Figure 7 (fromW dashes indicate identical tides; nucleotide changes versus the *01 variant are shown by underlined nucleotides and corresponding amino acid changes are shown by underlined amino acids; Genbank accession numbers se 185.0) are shown prefixed byJ, X, M or A). The ors used sequencing of human genomic DNA samples, inspection of public igH DNA databases as well as informed choices on the basis of variant sequences as means to arrive at a rational choice of which JH6 variant to use.

PCT/G32012/052296 The 1000 Genomes se uses human JH6*O3 as the reference sequence, which would be a possible choice for the skilled person wishing to construct a transgenic lgH locus. The inventors noticed (eg, Figure 7 herein) that position 6 in JH6*03 is a tyrosine (Y) encoded by a TAC codon, whereas some other naturally-occurring human variants have a glycine (G) encoded by a GGT codon (the glycine being present as a YYG motif, forming part of a larger YYGXDX motif). To understand the ial significance of this, the inventors carried out analysis of JH sequences from other vertebrate species. The inventors surprisingly noticed that YYG and YYGXDX motifs are conserved across many vertebrate species (see Figures 7 & 8). This suggested to the ors, therefore, that preservation of this motif might be desirable, which could guide the choice ofJH6 variant for use in the present invention.

Another pointer arose when the ors considered the TAC codon versus the GGT codon encoding Y or 6 respectively. The inventors considered the impact of these tide sequences on the action of activation-induced cytidine deaminase (AID). The inventors knew that activationv induced cytidine deaminase (AID) is believed to initiate lg somatic hypermutation (SH M) in a multi- step ism and they addressed this activity when rationally designing the locus. AID catalyses the deamination of C to U in DNA, ting mutations at C bases. Cytidines d within hotspot motifs are preferentially ated. Certain motifs are hotspots for AID acitivity (DGYW,m w,w, RGYW, AGY,m WGCW, wherein W=A or T, Y=C or T, D=A, G or T, H:A or C or T, and R=A or G). The presence of a TAC codon encoding Y at position 6 in JH6*03 s AID mutation hotspots (the cytidine being the substrate of AID), these hotspots being the underlined motifs in the previous sentence. The inventors considered the impact of this and in doing so they considered possible mutants created by AID activity at the cytidine. Reference is made to Figure 9. The inventors noticed that a mutation at the third base of the TAC codon would yield 3 possible outcomes: Y, stop or stop. Thus, out of the three stop codons possible in the genetic code (the other being encoded by TGA u see Figure 9), two of them would be provided by mutation of the cytidine in the TAC codon ng position 6 in JH6*03. The inventors, therefore, considered that this might increase the chances of non-productive lgH variable region production in transgenic loci based on JH6*03. Moreover, the inventors noticed that ion of a GGT codon d (as per the other human JH6 variants) seemed preferable since mutation of the third base would never yield a stop codon (see Figure 9), and furthermore would retain coding, and thus conservation, of glycine at position 6, which the ors also d was is in the YYG and YYGXDX motifs conserved across species.

PCT/G82012/052296 Having decided against using JH6*03, the inventors needed to make a choice from other possible human variants. The MDV motif is at the C-terminus of HCDR3 based on human JH6, the adjacent framework 4 (FW4) starting with the WGQ motif (with reference to the sequence shown encoded by JH6*01; Figure 7). In making their choices for locus design, the inventors wished to maximise conservation of this HCDR3/FW4 junction in product lgH chains and antibodies including these. The inventors believed this to be ble for heavy chain variable domain functionality and conformation. The inventors thought that this might in some cases be desirable to minimise immunogenicity (suitable for human ceutical use). tent with these considerations, the inventors wanted to make a choice that would minimise mutation around the HCDR3/FW4 junction as a result of SHM in vivo to conserve junction configuration. See Rogozin & Diaz; "Cutting Edge: DGYW/WRCH Is a Better Predictor of Mutability at G:C Bases in lg Hypermutation Than the Widely Accepted RGYW/WRCY Motif and Probably Reflects a Two-Step Activation-Induced Cytidine Deaminase-Triggered Process”; Journal of logy; March 15, 2004 vol. 172 no. 6 3382-3384.

An example of a DGYW motif is GGCA. The inventors had this in mind when analysing the variant sequences.

With these considerations in mind, the ors decided specifically to use human JH6*02 as the mandatory human JH6 for their lgH locus design. JH6*01 was ed as the mandatory JH6 gene segment since the tide sequence GGG CAA ing G and Q) contains a GGCA motif which is an AID recognition hotspot. The ors realised that JH6*04 also contains such a motif due to the presence of the ce GGC AAA encoding G and K (positions 11 and 12 respectively). The inventors also realised that the *02 variant has a C instead of a G that is in the *01 variant, the C desirably being a synonymous change (ie, not changing the encoded amino acid sequence around the CDR3/FW4junction) and also this does not provide a GGCA AID hotspot motif. The inventors, ore, decided that the mandatoryJH6 should have this C base and this too pointed them to using the human JH6*02 variant. in one example of any configuration of the invention , the only JH6 species included in the locus or genome is human JH6*02.

The inventors ed 9 anonymised DNA samples from cheek swabs of 9 ting human adults.

Sequencing was performed on lgH locus DNA to confirm natural JH6 variant usage. It was found that the genome of all 9 humans contained a JH6*02 variant gene segment. In 7 out of the 9 humans, the genome was homozygous for JH6*02 (ie, each chromosome 14 had JH6*02 as its JH6 gene t in the IgH . The inventors also inspected the publicly-available sequence information from the genomes of well-known scientists Craig Venter and Jim . Both of these genomes contain JH6*02 too. This ted to the inventors that this variant is common in humans.

So, the inventors made a choice of human JH6*02 on the basis of (i) Containing the W6 and YYGXDX motifs that is conserved across several vertebrate species; (ii) Provision of one less TAC codon (an AID hotspot that risks stop codons) and a choice instead of a codon that preserves the W6 and YYGXDX ; (iii) Avoidance of a GGCA AID hotspot in the region of the HCDR3/FW4 junction; and (iv) Common occurrence (and thus conservation and acceptability) in humans of the JH6*02 variant.

This rationale was tested by the inventors in laboratory examples, in order to see if human JH6*02 could desirably ipate in antibody gene segment ination and heavy chain production in a foreign (non—human vertebrate) setting, and moreover to assess if long HCDR3S based on human JH6*02 could be produced in vivo (in naive and immunised settings) in such non-human systems. It was noted that in some non-human settings, such as a mouse, the W6 and YYGXDX motifs are not conserved, and thus the inventors decided that it was important to test r or not JH6*02 (having the W6 and YYGXDX motifs) could function properly in such a foreign setting to participate in VDJ recombination and selection against antigen.

Thus, as explained further in the examples, the inventors constructed transgenic JH6‘OZ-containing IgH loci in ES cells, generated transgenic man vertebrates from the ES cells (both naive and immunised with a range of different target n types), isolated antibodies and heavy chain sequences based on JH6*02 as well as B-cells expressing these and made hybridomas expressing antigen-specific antibodies that are based on the chosen JH6*02 variant. The inventors found that the JH6*02 variant was extensively used and could contribute to the production of HCDR3 of at least PCT/GBZOIZ/052296 amino acids in many ent heavy chains (including antigen-specific heavy chains). The chosen variant was preferably used over other JH gene segments in all settings (naive, immunised and antigen-specific) for the production of HCDR3 of at least 20 amino acids.

Thus, the present invention provides an lgH locus including human JH6*02 (IMGT nomenclature) as a mandatory JH gene segment. In one embodiment, the locus comprises non-human vertebrate (eg, mouse or rat) constant region gene segments downstream (ie, 3‘ of) the human JH6*02; and one or more VH gene segments (eg, a plurality of human VH gene segments) and one or more D gene segments (eg, a plurality of human D gene segments) upstream of (ie, 5’ of) the human JH6*02. For example, the locus is comprised by a vector (eg, a DNA vector, eg, a yeast artificial chromosome (YAC), BAC or PAC). Such a vector (eg, YAC) can be introduced into a non-human vertebrate (eg, mouse or rat) cell using standard ques (eg, pronuclear ion) so that the locus is integrated into the cell genome for expression of lgH chains comprising at least one chain whose le domain is a product of the ination of human JH6*02 with a VH and a D gene segment.

In another example, the locus (eg, with a tely human, rat or mouse nt region, or a human/mouse chimaeric constant region) can be provided in the genome of a non—human vertebrate (eg, mouse or rat) cell. For example, the cell is an ES cell or an antibody-producing cell (eg, an isolated B—cell, an iPS cell or a oma).

In another example, the invention provides a non-human vertebrate (eg, a mouse or a rat) comprising an lgH locus of the invention which comprises a human JH6*02 gene segment, wherein the locus can s an lgH chain whose variable domain is a product of the recombination of human JH6*02 with a VH and a D gene segment. As shown in the examples, the inventors have successfully produced such mice which produce such lgH chains with VH domains based on human JH6*02. The inventors isolated and sequenced lgH chains from the mice before (naive) and after (immunised) exposure to a range of target antigens and confirmed by comparison to IMGT lgH gene segment sequences that the ed chains (and antibodies containing these) were produced based on JH6*02. Such chains were found in naive mice, as well as in antigen-specific dies from immunised mice. B-cells were isolated from immunised mice, wherein the B-cells express antibodies based on JH6*02 and omas were generated from the B-cells, the hybridomas expressing antigen-Specific dies based on JH6*02. The inventors, therefore, provided the locus, vertebrate, cell and hybridoma of the invention based on the use of human JH6*02 and showed that antibodies based on JH6*02 and B-cells expressing these can be successfully produced and isolated following immunisation of the rates, corresponding hybridomas being a good source of antibodies whose VH domains are based on , eg for administration to a patient, eg, for human medicine. Furthermore, it was found possible to produce and isolated antigen—specific antibodies whose VH domains are based on JH6*02 and which had a relatively long HCDR3 (eg, 20 amino acids).

Thus, the present invention provides ments as in the following clauses:— 1. A non-human vertebrate (optionally a mouse or a rat) or vertebrate cell whose genome comprises an immunoglobulin heavy chain locus comprising human gene segment JH6*02, one or more VH gene segments and one or more D gene segments upstream of a constant region; wherein the gene segments in the heavy chain locus are operably linked to the constant region f so that the mouse is capable of producing an antibody heavy chain produced by recombination of the human JH6*02 with a 0 segment and a VH segment.

In another example, the invention provides A non-human vertebrate (optionally a mouse or a rat) or rate cell whose genome comprises an globulin heavy chain locus comprising one, more or all of human IGHV gene segments selected from V3-21, V3—13, V3-7, V6-1, V1-8, V1—2, V7—4—1, V1-3, V1-18, V4-4, V3‘9, V3-23, V3-11 and V3-20 (eg, one, more or all of V3-21‘03, V3-13*01, V3-7*01, V6-1*01, V1-8*01, Vl—2*02, V71‘01, V1-3*01, 01, V4-4*01, V3-9*Ol and 04). These segments were found in naive repertoires to be productive to produce HCDR3s of at least 20 amino acids in length. In an embodiment, the locus comprises a human 1H6, eg, JH6*02.

The invention also provides a HCDR3, VH domain, antibody heavy chain or antibody having a HCDR3 size of at least 20 amino acids. Optionally, the HCDR3 or VH domain (or VH domain of the heavy chain or antibody) ses mouse AID-pattern somatic hypermutations and/or mouse de-pattern mutations. This can be provided, for e, wherein VH domain is produced in a mouse comprising mouse AID and/or mouse TdT (eg, endogenous AID or TdT).

See also Annu. Rev. Biochem. 2007. 76:1~22; Javier M. Di Noia and Michael S. Neuberger, ”Molecular Mechanisms of Antibody Somatic Hypermutation” (in particular figure 1 and PCT/GBZOIZ/052296 ated discussion on AID hotspots in mouse); and Curr Opin Immunol. 1995 Apr;7(2):248—54, "Somatic hypermutation”, Neuberger MS and Milstein C (in particular, discussion on hotspots in mouse), the disclosures of which are orated herein by reference.

These segments were found in naive repertoires to be productive in recombination with human JH6*02 to produce HCDR3S of at least 20 amino acids in length.

In an example, the vertebrate is naive. In another embodiment, the vertebrate instead is immunised with a target antigen.

In an example, the vertebrate or cell mentioned below is capable of so producing an antibody heavy chain upon immunisation with a target antigen In an example, the rate is an immunised rate that produces antibody heavy chains specific for a target antigen and wherein the variable s of the heavy chains are the product of recombination between a VH, D and JH6*02. For example, the D is selected from human 03-3, 02—15, 03-9; 04-17; D3-10; 02—2; 05-24; 06-19; D3-22; D6-13; D5-12; D1-26; 01-20; 05-18; D3-16; 02-21; 01-14; 07-27; 01- 1; D6—25; D2-14 and D4-23 (eg, selected from 03-9*01; D4-17*01; D3—10*01; D2-2*02; DS- 24*01; D6-19*01; D3-22*01; 06-13*01; 01; 01; D1-20*01; DS~18*01; D3-16*02; DZ—Zl*02; Dl-l4*01; D7-27*02;Dl-1*01; D6-25*01; D2-15*01; and D4-23‘01). For example, the D is human 03-9 or 03-10. In an example, the HCDR3 length is at least 20 amino acids (eg, , 21, 23 or 24).

In an example of the vertebrate or cell, the genome comprises additional human JH gene segments (eg, 1H2, 3, 4 and 5 gene segments).

In an e of the vertebrate or cell, the genome comprises an immunoglobulin light chain locus comprising one or more human V gene segments and one or more human I gene segments upstream of a constant region (eg, a human or a mouse lambda or kappa nt region).

For rearrangement and sion of heavy chains, the locus comprises control elements, such as an Ep and Sp between the J gene segment(s) and the constant region as is known by the skilled person. In one e, a mouse Ep and Sp is included in the heavy chain locus between the JH6*02 and the constant region (ie, in 5' to 3’ order the locus comprises the JH6*02, Ep and Sp and constant region). In an example, the Ep and Sp are Ep and Sp of a mouse 129-derived PCT/G82012/052296 genome (eg, a 129Sv-derived genome, eg, 129Sv/EV (such as 12957Sv/Ev (such as from ABZ.1 or A322 cells obtainable from Baylor College of Medicine, Texas, USA) or 129$6$v/Ev))); in another example, the Eu and Su are Eu and Sp of a mouse C57BL/6 ~derived genome. In this respect, the locus can be constructed in the lgH locus of the genome of a cell selected from A821, A822, VGF1, CJ7 and FH14. VGFl cells were established and described in Auerbach W, Dunmore 1H, FairchiId-Huntress V, et 0/; Establishment and chimera analysis of 129/Sva- and 6-derived mouse embryonic stem cell lines. Biotechniques 2000; 29:1024—8, 30, 32, incorporated herein by reference.

Additionally or alternatively, the nt region (or at least a Cu; or Cu and gamma constant regions thereof) is a constant region (or Cu; or Cu and gamma nt regions thereof) is of a genome described in the paragraph immediately above.

A suitable source ofJH6*02 and other human DNA sequences will be readily apparent to the skilled person. For example, it is possible to collect a DNA sample from a ting human donor (eg, a cheek swab sample as per the Example herein) from which can be obtained suitable DNA sequences for use in constructing a locus of the invention. Other sources of human DNA are commercially available, as will be known to the skilled person. Alternatively, the skilled person is able to construct gene segment sequence by referring to one or more databases of human lg gene segment sequences sed herein.

The vertebrate of clause 1, wherein the vertebrate has been immunised with a target antigen and wherein the variable domain of the heavy chain is the product of ination between a VH, D and JH6*02 and n the HCDR3 length is at least 20 amino acids (eg, 20, 21, 23 or 24).

Optionally, the immunised vertebrate es an antibody heavy chain specific for a target antigen and wherein the variable domain of the heavy chain is the product of recombination between a VH, D and JH6‘02 and wherein the HCDR3 length is at least 20 amino acids (eg, 20, 21, 23 or 24).

A non—human vertebrate cell (optionally a mouse cell or a rat cell) whose genome ses an immunoglobulin heavy chain locus comprising human gene segment , one or more VH gene segments and one or more D gene segments upstream of a nt region; n the gene segments in the heavy chain locus are operably linked to the constant region thereof for PCT/G82012/052296 producing (eg, in a uent progeny cell) an dy heavy chain produced by recombination of the human JH6*02 with a D segment and a VH segment. 4. The cell of clause 3, which is an ES cell e of differentiation into a progeny antibody- producing cell that expresses said heavy chain.

. The vertebrate or cell of any preceding clause, wherein the heavy chain locus comprises a human JH6*02 recombination signal sequence (RSS) operably connected 5’ to the JH6*02 gene segment.

For example, the native RSS-JH6*02 sequence can be used to advantageously maintain the natural pairing n RSS and theis JH gene segment. In this respect, the following sequence is used:- nggtgaggatggacattctgcmgattactactactactacggtatggacgtctggggccaagggaccacggtcaccg tctcctcag (SEQ lD NO: 238) R555 have a common architecture: 9mer (eg, first underlined ce above) followed by a 22bp spacer and then a 7mer (eg, second underlined sequence above). Spacers are 23bp +/- 1 normally, while the 9 and 7mer are more conserved. 6. The rate or cell of clause 5, n the RSS is SEQ ID NO: 238 or a sequence having an identical 9mer and 7mer sequence flanking a sequence that is at least 70% identical to the 22mer sequence of SEQ ID NO: 238. 7. The vertebrate or cell of clause 6, wherein the RSS and JH6*02 are provided as SEQ ID NO: 237. 8. The vertebrate or cell of any preceding clause, wherein the JH6*02 is the only JH6-type gene segment in the genome. 9. The vertebrate or cell of any preceding clause, wherein the JH6*02 is the closest JH gene segment to the constant region in the locus.

PCT/GBZOl2/052296 . The vertebrate or cell of any preceding clause, wherein the locus comprises one, more or all human D gene segments D3-9; D4-17; D3-10; 02-2; 05-24; 06-19; D3-22; D6-13; D5-12; 01-26; D1-20; 05-18; 03-16; 02-21; D1-14; D7-27; Dl-l; D6-25; D2-14; and 04-23.

For example, the locus comprises one, more or all of human D gene segments D3-9*01; D4- 17*01; DB-lO‘Ol; D2-2‘02; 01; D6-19*01; 03-22‘01; D6-13*01; 01; D1-26*01; Dl-ZO*01; DS-l8*01; 03-16*02; D2-21*02; Dl-l4*01; D7-27*02; Dl-l*01; 06-25*01,' DZ—lS*01; and 01. 11. The vertebrate or cell of clause 10, wherein the locus comprises one, more or all human D gene ts D3-9, D3—10, D6-19, D4—17, D6—13, 03-22, 02-2, 02-25 and D3—3.

These D segments were found to be productive in recombination with human JH6*02 to produce HCDR3s of at least 20 amino acids in length.

In an example, the locus comprises one, more or all human D gene segments 03-9, 03-10, DS- 19, 04-17, 06-13 and D3‘22 (for example one, more or all of D3-9*01, 03—10*01, 06—19*01, D4- 17*01, D6-13*01 and DS-ZZ‘Ol). These D segments were found in naive repertoires to be productive in recombination with human JH6*02 to e HCDR35 of at least 20 amino acids in length.

In an example, the locus comprises one, more or all human D gene ts 03-10, 06-19 and 01-26 (for example, one, more or all of D3-10*01, 01 and D1—26*01). These D segments were found in immunised repertoires to be productive in recombination with human JH6*02 to produce HCDR3s of at least 20 amino acids in length.

In an example, the locus comprises one, more or all human D gene segments 038 and 03—10 (for example, one, more or all of D3-9*01 and D3-10*01). These D segments were found in antigen-specific repertoires to be productive in recombination with human JH6*02 to produce HCDRSs of at least 20 amino acids in length. 12. The vertebrate or cell of any preceding clause, wherein the locus comprises a plurality of human D gene segments and the JH6*02 is in human germline configuration with respect to the 3’-most human D gene segment (or all of the human D segments comprised by the locus).

W0 2013/041844 PCT/G32012/052296 In an example, the 3'-most D gene t is 07-27. In an example, the locus comprises all of human D gene ts from Dl-l to D7-27 as present in a germline human lgH locus (eg, as shown in the IMGT database).

Alternatively or additionally, the JH6*02 is in human ne configuration with respect to one, more or all of the Eu, Sp and nt region (eg, Cu). 13. The vertebrate or cell of any preceding clause, wherein the locus comprises one, more or all of IGHV gene segments selected from V3-21, V3-13, V3-7, V6-1, V1—8, Vl-Z, V7-4—1, V1-3, V1-18, V4-4, V3-9, V3-23, V3-11 and V3-20.

In an example, the locus comprises one, more or all human IGHV gene segments V3-21, V3—13, V3-7, V6-1, V1-8, V1-2, V71, V1-3, V1-18, V4-4, V3-9, V3—23 (for example, one, more or all of V3-21*03, V3-13*01, V3-7*01, V6—1*01, V1-8*01, Vl-2*02, V71‘01, V1-3*Ol, 01, V4- 4*01, V3-9*01 and V3-23‘04). These segments were found in naive repertoires to be productive in recombination with human JH6*02 to produce HCDR35 of at least 20 amino acids in length.

In an example, the locus comprises one, more or all human IGHV gene ts V3-7, V3—11 and V4-4 (for e, one, more or all of V3-7*01, V3-11*01 and V4-4*02). These segments were found in immunised repertoires to be productive in recombination with human JH6‘02 to produce HCDR3s of at least 20 amino acids in length.

In an example, the locus ses one, more or all human IGHV gene segments V4-4, V1-8, V3- 9, V3-11 and V3—20 (for example, one, more or all of V4-4*02, V1-8*01, 1, V3-11*01 and V3-20 [eg, *d01). These segments were found in antigen-specific repertoires to be productive in recombination with human JH6‘02 to produce HCDR3S of at least 20 amino acids in length. 14. The vertebrate or cell of any preceding clause, wherein the locus comprises one, more or all of human D3-9*01, 03—10*01, D6-19‘01, DS-13‘01, D1—26*01, IGHV1-8‘01, IGHV4-61‘01, iGHV6- 1*01, lGHV4-4‘02, IGHV1~3*Ol, |GHV3-66*03, lGHV3-7*Ol and lGHV3-9*01.

These are gene segments that very frequently combine with JH6*02 to produce productive heavy chains and antibodies.

WO 41844 PCT/G82012/052296 For example, the locus comprises one, more or all of human IGHV1-8*01, D3—9*01 and D3- *01. These gene ts were productive with JH6*02 to produce HCDR3s of at least 20 amino acids in more than 10 dies.

. An antibody—producing cell (eg, a B-cell) that is a y of the cell of any one of clauses 3 to 14, wherein the antibody-producing cell comprises a heavy chain locus comprising a rearranged variable region produced by recombination of human JH6*02 with a D segment and a VH segment (eg, JH6*02 with human VH3-11(eg, VH3—11*01) and 03-9; VHS-20 (eg, VH3—20*01) and 03-10; VH4-4 (eg, VH4-4*02) and 03-10; VH3-9 (eg, VH3-9*Ol) and 03-10; or VH1—8 (eg, VH1-8*01) and 0310).

Such a variable region would be the product of in vivo somatic hypermutation in a non—hman vertebrate or cell of the invention. 16. The cell of clause 15, which is a B-cell or hybridoma that expresses a target antigen-specific antibody comprising a heavy chain that comprises a rearranged variable region produced by recombination of human JH6*02 with a D segment and a VH segment (eg, JH6*02 with human VH3—11(eg, *01) and 03-9; VH3-20 (eg, VH3-20*01) and VH4-4(eg,VH4-4*02) and 03-10; VH3-9 (eg, VH3-9‘01) and 03-10; or VH1-8 (eg, VH1-8*01) and 0310).

Such a variable region would be the product of in vivo somatic hypermutation in a non-hman vertebrate or cell of the invention 17. The vertebrate or cell of any preceding clause, wherein the antibody heavy chain specifically binds a target antigen. 18. The vertebrate or cell of any preceding clause, n the antibody heavy chain has a HCDR3 length of at least 20 amino acids.

Optionally, the HCDR3 length is at least 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids.

Additionally, in one example the length is no more than 35, 34, 33, 32 or 31 amino acids. For example, the HCOR3 length is 20, 21, 22, 23 or 24 amino acids. 19. The vertebrate or cell of any preceding clause, wherein the antibody heavy chain is a product of the recombination of JH6*02 with a human VH gene segment recited in clause 13 or 14 and/or a D gene t recited in clause 10, 11 or 14.

. The vertebrate or cell of any preceding clause, wherein all endogenous non-human vertebrate heavy chain variable region gene segments have been inactivated in the genome (Eeg, by gene t deletion or inversion). 21. The vertebrate or cell of any preceding , wherein the genome is gous for said heavy chain locus. 22. A heavy chain (eg, comprised by an dy) ed from a vertebrate of any one of clauses 1, 2, 5 to 14 and 17 to 21 wherein the heavy chain comprises a HCDR3 of at least 20 amino acids. 23. The heavy chain of clause 22, wherein the HCDR3 is the product of recombination of human JH6*02 with a human VH gene t recited in clause 13 or 14 and/or a D gene segment recited in clause 10, 11 or 14.

In an example, the heavy chain is chimaeric where the C region is non-human. In an e, the heavy chain is human where the C region is human. 24. A heavy chain (eg, comprised by an antibody) whose VH variable domain is identical to the VH variable domain of the heavy chain of clause 22 or 23, and which comprises a human constant region or a human-mouse chimaeric constant region (eg, CH1 is human and the other constant domains are mouse).

. The heavy chain of clause 22, 23 or 24, whose VH variable domain is specific for a target antigen. 26. A method for producing a heavy chain, VH domain or an antibody specific to a target antigen, the method comprising immunizing a non human vertebrate according to any one of clauses 1, 2, 5 to 14 and 17 to 21 with the antigen and isolating the heavy chain, VH domain or an antibody specific to a target antigen or a cell producing the heavy chain, VH domain or an antibody, wherein the heavy chain, VH domain or an antibody comprises a HCDR3 that is derived from the recombination of human JH6*02 with a VH gene segment and a D gene segment. 27. A method for producing a human heavy chain or antibody comprising carrying out the method of clause 26, wherein the constant region ofthe locus is a non-human vertebrate (eg, mouse or rat) constant region, and then replacing the non-human nt region of the isolated heavy chain or antibody with a human constant region (eg, by engineering of the nucleic acid encoding the antibody). 28. A heavy chain, VH domain or an dy produced by the method of clause 26 or 27.

Optionally the HCDR3 length is at least 20 amino acids as herein described. 29, A B—cell or hybridoma expressing a heavy chain VH domain that is cal to the VH domain of the heavy chain of clause 22, 23 or 28.

. A nucleic acid encoding the VH domain of the heavy chain of clause 22, 23 or 28, or encoding the heavy chain of clause 22, 23, 24, 25 or 28. 31. A vector (eg, a CHO cell or HEK293 cell vector) comprising the nucleic acid of clause 30; optionally wherein the vector is in a host cell (eg, a CHO cell or HEK293 cell). 32. A pharmaceutical composition comprising the antibody, heavy chain or VH domain (eg, comprised by an antibody) of any one of s 22 to 25 and 28, together with a ceutically-acceptable excipient, diluent or a medicament (eg, a further antigen-specific variable domain, heavy chain or dy). 33. The antibody, heavy chain or VH domain (eg, sed by an antibody) of any one of clauses 22 to 25 and 28 for use in medicine (eg, human medicine).

For example, the locus comprises the following human VH gene segments IGHV6-1 IGHV3-7 IGHV1-8 lGHV3-9 IGHV3-11 lGHV3-13 lGHV1-18 lGHV3-30 lGHV4-31 IGHV4-39 IGHV4-59 Optionally also (i) and/or (ii) IGHV1-2 IGHVZ-S and lGHV3-21 (ii) lGHVl—Z IGHV2-5 IGHV3-21 IGHV1-24 For example, the locus comprises the following human VH gene segment variants IGHV6-1*Ol |GHV3-7*01 |GHV1-8*01 9*01 lGHV3-11*01 lGHV3-13*01 |GHV1-18*01 *18 |GHV4-31*03 IGHV4—39 *01 and |GHV4-59*01; Optionally also (iii) or (iv) (ii) |GHV1-2*04 ’10 and lGHV3—21*03 (iV) |GHV1-2*02 IGHV2-5*01 21*01 and IGHV1—24*01 For example, the locus comprises the following human JH gene segment variants IGHJ2*01 |GHJ3*02 W0 20131041844 lGHJ4*02 IGHJ5*02 and lGHJ6*02 For example, the locus comprises the following human D gene segments lGHDl-l lGHD2-2 lGHD3—10 lGHDS-lZ lGHDG-13 lGHDl-l4 lGHDZ-IS lGHD4—17 lGHDfi-lQ lGHDZ-Zl lGHDS-24 lGHD1-26 and lGHD7-27 and optionally also (v) or (vi) IGHoae |GHD$3 lGHD44 lGHDES lGHD&6 IGHDL7 lGHDZ8and IGHDZ—8 1h . . ‘l . ffl E .

Constant Re i ' n T i t Huma Humanisat' PCT/GBZOIZ/052296 Additional rational design and bioinformatics has led the ors to realise that specific human constant region variants are conserved across many diverse human populations. The inventors realised that this opens up the possibility of making a choice to humanise antibodies, chains and variable domains by using such specific nt regions in products, rather than arbitrarily choosing the human constant region (or a synthetic version of a human constant region). This aspect of the invention also enables one to tailor antibody-based drugs to specific human ethnic populations, thereby more closely matching drug to patient (and thus e setting) than has to been performed. It can be a problem in the state of the art that dies are humanised with an ary choice of human nt region (presumably d from one (often unknown) ethnic population or non-naturally ing) that does not function as well in patients of a different human ethnic population. This is important, since the constant region has the major role in providing antibody effector functions, eg, for antibody recycling, ar and complement recruitment and for cell killing.

As discussed r in W02011066501, human IgG sub-types lgGl, IgGZ, gGB and lgG4 exhibit differential capacity to recruit immune functions, such as antibody-dependent cellular cytotoxicity (ADCC, e.g., lgGl and IgG3), antibody—dependent cellular phagocytosis (ADCP, e.g., IgG], IgGZ, IgGB and lgG4), and complement dependent cytotoxicity (CDC, e.g., IgGl, igGB). Sub-type-specific engagement of such immune functions is based on selectivity for Fc receptors on distinct immune cells and the ability to bind Clq and activate the assembly of a membrane attack complex (MAC).

Among the various types, relative affinity for FcY receptors (e.gi, FcYRI, FcYRIIa/b/c, FcYRllla/b) is high for lgGl and lgG3, however, there is minimal affinity for lgGZ (restricted to the FcYRIIa 131H polymorphism), and IgG4 only has able affinity for FcYRI. Using comparative sequence analysis and co-crystal structures, the key contact residues for receptor binding have been mapped to the amino acid residues spanning the lower hinge and CH2 region. Using standard protein engineering techniques, some success in enhancing or reducing the affinity of an antibody preparation for Fc ors and the Clq component ofcomplement has been achieved.

Among the isotypes, lgGZ is least capable of binding the family of Fc ors. Using IgGZ as the starting point, efforts have been made to find a mutant with diminished effector functions but which retains FcRn binding, ged stability, and low immunogenicity. Improved mutants of this nature may provide improved antibody therapeutics with retained safety. Human IgGl therapeutic PCT/GBZOIZ/052296 antibodies that bind to cell surface targets are able to engage effector cells that may mediate cell lysis of the target cell by antibody-dependent cellular cytotoxicity (ADCC) or complement ent cytotoxicity (CDC). These mechanisms occur through interaction ofthe CH2 region of the antibody Fc domain to FcyR receptors on immune effector cells or with Clq, the first component of the complement cascade. Table 19 shows the activities of ent human gamma sub—types. The skilled person may choose accordingly to promote or dampen-down activity depending upon the disease setting in humans of interest. For example, use of a human 1 constant region is desirable when one wishes to isolated totally human heavy chains and antibodies that have relatively high complement tion activity by the classical pathway and FcYRl recognition in human patients. See also Mol Immunol. 2003 (9):585—93; “Differential binding to human chamma Rlla and chamma Rllb receptors by human lgG wild type and mutant antibodies",- Armour KL et al, which is incorporated herein by nce.

IgGZ constant regions are well suited to producing antibodies and heavy chains according to the invention for binding to cytokines or soluble targets in humans, since lgGZ is essentially FcYRI,|II- silent, a—active and has little Complement activity.

IgGl constant regions have wide utility for human therapeutics, since IgGl antibodies and heavy chains are ll,l|l- active and have complement activity. This can be enhanced by using a human gamma-1 constant region that has been activated by engineering as is known in the art.

The work of the inventors has therefore identified a collection of human constant region of different isotypes from which an informed choice can be made when humanising chimaeric antibody chains (or conjugating V s, such as dAbs or Came/id VH H, to constant regions). The collection was identified on the basis of bioinformatics analysis of the 1000 Genomes database, the ors selecting constant region variants that are frequently occurring across several human ethnic populations, as well as those that appear with relatively high frequency within individual populations (as assessed by the number of individuals whose genomes comprise the variant). By sorting through the myriad possible sequences on this basis, the ors have ed a collection of human constant region ts that are naturally-occuring and which can be used when rationally designing PCT/GBZOIZ/052296 dies, heavy chains and other antibody-based formats that bear a human constant region. In particular, this is useful when humanising chimaeric heavy chains to produce totally human chains in which both the variable and constant regions are human. This is useful for compatibility with human patients receiving antibody-based drugs.

To this end, the invention provides the following aspects:- 1. A method of producing an antibody heavy chain, the method comprising (a) providing an antigen-specific heavy chain variable domain (eg, VH (such as a human VH or dAb) or VHH or a humanised heavy chain le domain); and (b) combining the variable domain with a human heavy chain constant region to produce an antibody heavy chain sing (in N- to C-terminal direction) the variable domain and the constant ; wherein the human heavy chain constant region is an f, IGHAla, IGHAZa, IGHAZb, lGHGlref, ef, IGHGZa, IGHGBref, IGHGBa, IGHGSb, ef, iGHG4a, lGHDref, lGHEref, IGHMref, IGH Ma or IGHMb constant region.

Step (b) can be carried out, eg, using recombinant DNA technology using the corresponding tide sequences.

For the constant region according to any aspect of this configuration, either genomic DNA or equivalent (ie, having introns and exons and optionally also 5' UTR sequences, eg, with native or a non-native leader sequence) can be used for the nt region. For example, any of the ”GENOMIC” sequences disclosed as SEQ ID NO: 365 onwards herein. Alternatively, an intronless sequence can be used, for example any of the ”CDS" sequences disclosed as SEQ ID NO: 365 onwards herein (eg, with native or a non-native leader sequence).

Optionally for any aspect of this uration of the invention, the human heavy chain constant region is an lGHAref constant region.

PCT/GBZO 12/052296 Optionally for any aspect of this configuration of the invention, the human heavy chain constant region is an IGHAla constant region.

Optionally for any aspect of this configuration of the invention, the human heavy chain constant region is an IGHAZB constant region.

Optionally for any aspect of this configuration of the ion, the human heavy chain nt region is an IGHAZb constant region.

Optionally for any aspect of this configuration of the invention, the human heavy chain constant region is IGHGIref constant region.

Optionally for any aspect of this configuration of the invention, the human heavy chain constant region is an lGHGZref constant region.

Optionally for any aspect ofthis configuration of the invention, the human heavy chain constant region is an IGHGZa constant region.

Optionally for any aspect of this configuration of the invention, the human heavy chain constant region is an lGHGBref constant region.

Optionally for any aspect of this configuration of the invention, the human heavy chain constant region is an IGHGBa constant region. ally for any aspect of this uration of the invention, the human heavy chain nt region is an IGHG3b constant region.

PCT/GBZOl2/052296 Optionally for any aspect of this configuration ofthe invention, the human heavy chain constant region is an ef constant region.

Optionally for any aspect of this configuration of the ion, the human heavy chain constant region is an lGHG4a constant region.

Optionally for any aspect of this configuration ofthe invention, the human heavy chain constant region is an IGHDref constant region.

Optionally for any aspect of this uration of the ion, the human heavy chain constant region is an IGHEref constant region.

Optionally for any aspect of this configuration ofthe invention, the human heavy chain constant region is an IGHMref constant region.

Optionally for any aspect of this configuration of the invention, the human heavy chain constant region is an IGHMa constant region.

Optionally for any aspect of this configuration of the ion, the human heavy chain constant region is an IGHMb constant region.

Optionally, a derivative (eg, a mutant or conjugate) of the heavy chain or an antibody ning the heavy chain is produced. For e, a toxic payload can be conjugated (eg, for oncology applications). For example, one or more mutations can be introduced, as is known in the art, to inactivate or enhance Fc effector function.

The method of aspect 1, n the variable domain is a human variable domain.

A human variable domain is, for example, the product of recombination in a transgenic non- human vertebrate of human VH, D and JH gene segments. Alternatively, the variable domain is identified using in vitro display technology from a human VH library, eg, using phage display, ribosome display or yeast display, as is known in the art.

PCT/G32012/052296 In another embodiment, the variable domain is a humanised variable domain, eg, comprising human frameworks with non—human (eg, mouse or rat) CDRs). Humanisation technology is conventional in the art, and will be readily known to the skilled person.

The method of any preceding , wherein the variable domain has previously been selected from a non-human rate that has been immunised with the antigen.

For example, the rate (such as a mouse or rat) genome comprises a chimaeric heavy chain locus comprising a human variable region (human V, D and JH gene segments) operably connected am of a non-human vertebrate constant region so that the locus is able to rearrange for the expression of heavy chains comprising human variable domains and non- human rate constant regions.

In alternative embodiments, the variable domain is selected using an in vitro technology such as phage display, ribosome y or yeast display, In this case the variable domain may be displayed with or without an constant region, provided that it is later combined with a human constant region as per the invention.

The method of any preceding aspect, sing providing an expression vector (Eg, a ian expression vector, such as a CHO or HEK293 vector) comprising a nucleotide sequence encoding the constant region; inserting a nucleotide ce encoding the variable domain into the vector 5’ of the constant region sequence; inserting the vector into a host cell and expressing the heavy chain by the host cell; the method further comprising isolating a heavy chain (eg, as part of an antibody) comprising the variable domain and the human constant region.

The vector ses regulatory elements sufficient to effect expression of the heavy chain when the vector is harboured by a host cell, eg, a CHO or HEK293 cell.

The method of any preceding aspect, further comprising obtaining a nucleotide sequence encoding the heavy chain.

PCT/G32012/052296 6. An antibody comprising a human heavy chain, the heavy chain comprising a variable domain that is specific for an antigen and a constant region that is an IGHAref, IGHAla, lGHAZa, |GHA2b, lGHGlref, |GHGZref, lGHGZa, |GHGBref, lGHGSa, lGHGSb, |GHG4ref, |GHG4a, |GHDref, |GHEref, lGHMref, lGHMa or IGH Mb constant region. 7. A polypeptide comprising (in N- to C- terminal direction) a leader sequence, a human variable domain that is specific for an n and a human constant region that is an lGHAref, |GHA1a, , |GHA2b, lGHGlref, lGHGlref, IGHGZa, |GHG3ref, |GH63a, lGHGBb, |GHG4ref, |GHG4a, lGHDref, f, lGHMref, lGHMa or lGH Mb constant region; wherein (i) the leader ce is not the native human variable domain leader sequence (eg, the leader ce is another human leader sequence or a non—human leader sequence); and/or (ii) the variable domain ses mouse ttern somatic mutations or mouse terminal deoxynucleotidyl transferase (TdT)- pattern junctional mutations. 8‘ A nucleotide sequence encoding (in 5’ to 3’ direction) a leader sequence and a human antibody heavy chain, the heavy chain comprising a variable domain that is specific for an antigen and a nt region that is an lGHAref, |GHA1a, lGHAZa, , lGHGlref, lGHGZref, |GHGZa, |GHG3ref, |GHGBa, |GHGBb, IGHG4ref, |GHG4a, IGHDref, f, lGHMref, IGHMa or IGHMb constant region; and the leader sequence being operable for expression (eg, in a mammalian CHO or HEK293 cell) of the heavy chain and wherein the leader sequence is not the native human variable domain leader sequence (eg, the leader sequence is another human leader sequence or a non-human leader sequence).

In an example, the leader sequence is TGGTCCTGCATCATCCTGTI‘I‘CTGGTGGCCACCGCCACCGGCGTGCACAGC Which translates to MGWSCIILFLVATATGVHS 9. A nucleotide ce encoding (in 5' to 3’ direction) a promoter and a human antibody heavy chain, the heavy chain comprising a variable domain that is ic for an antigen and a constant region that is an lGHAref, |GHAla, |GHA2a, lGHAZb, lGHGlref, lGHGZref, |GHGZa, |GHGSref, IGHGSa, IGHGBb, |GHG4ref, IGHG4a, IGHDref, f, IGHMref, IGHMa or IGHMb constant region; and the promoter being operable for expression (eg, in a mammalian CHO or HEK293 cell) of the heavy chain and wherein the promoter is not the native human promoter.

In one embodiment, the promoter sequence is a human IGK 3-15 promoter.

. The antibody, polypeptide or nucleotide sequence of any one of aspects 6 to 9, wherein the variable domain ses mouse AID-pattern c ons and/or mouse terminal deoxynucleotidyl transferase (TdT)- pattern junctional mutations.

For example, one way, in any aspect of this configuration of the invention, to provide mouse AID-pattern somatic mutations and/or mouse terminal deoxynucleotidyl transferase (TdT)- pattern junctional mutations is to select a variable domain from a non-human vertebrate or cell.

For example, a vertebrate or cell as disclosed . 11. A vector (eg, a CHO cell or HEK293 cell vector) comprising the nucleic acid of aspect 8, 9 or 10; optionally wherein the vector is in a host cell (eg, a CHO cell or HEK293 cell). 12. A pharmaceutical composition comprising the antibody or polypeptide of any one of aspects 6, 7 and 10, together with a pharmaceutically-acceptable excipient, t or a medicament (eg, a further antigen—specific variable domain, antibody chain or antibody). 13. The antibody or polypeptide of any one of aspects 6, 7 and 10 for use in treating and/or preventing a l condition in a human patient. 14. Use of the antibody or polypeptide of any one of aspects 6, 7 and 10 for the cture of a ment for treating and/or preventing a medical condition in a human patient.

. The antibody, polypeptide or use of aspect 13 or 14, wherein the human is a member of a human population selected from population numbers 1-14, n the populations are numbered as follows (population labels being according to 1000 Genomes Project nomenclature) I: ASW,‘ 2= CEU; 3=CHB; 4=CHS; =CLM; 6=F|N; 7=GBR; 8=|BS; 9=JPT,' =LWK; 11=MXL; 12=PUR; 13=TS|; 14=YR|.

. The antibody, ptide or use of aspect 15, wherein the nt region is a (i) lGHAla constant region and the human population is selected from any population number 1-14; (ii) IGHAZa constant region and the human population is selected from any population number 1-14; (ill) IGHAZb nt region and the human population is selected from any population number 1-14; (iv) IGHGZa constant region and the human population is selected from any population number 1-9 and 11-13; (v) IGHGBa constant region and the human population is selected from any population number 1—14; (vi) IGHGBb constant region and the human population is selected from any population number 1-8 and 11-13; (vii) IGHG4a constant region and the human population is selected from any population number 1-9 and 11-13; (viii)|GHMa constant region and the human population is selected from any population number 1-14; or (ix) IGHMb constant region and the human population is selected from any population number 1-14; PCT/G32012/052296 Wherein the populations are numbered as s (population labels being according to 1000 s Project lature) 1= ASW; 2= CEU; 3=CH B; 4=CHS; =CLM; 6=FIN; 7=GBR; 8=|BS; 9=JPT; 11=MXL; 12=PUR; 13:TSI; 14=YRL 17. A vector (eg, a CHO cell or HEK293 cell vector) comprising a lGHGlref, IGHGZref, lGHGZa, lGHGBref, |GH63a, lGHGBb, |GHG4ref or |GHG4a constant region nucleotide sequence that is 3’ of a cloning site for the insertion of a human antibody heavy chain variable domain nucleotide sequence, such that upon insertion of such a variable domain sequence the vector comprises (in ’ to 3’ direction) a er, a leader sequence, the le domain sequence and the constant region sequence so that the vector is capable of expressing a human antibody heavy chain when present in a host cell.

The inventors’ analysis has revealed groupings of naturally—occurring human antibody gene segment variants as set out in Table 13 and Table 14. This revealed the possibility of producing transgenic genomes in non—human vertebrates and cells wherein the genomes contain more than the natural PCT/GBZOl2/052296 human complement of specific human gene segments. In one example, this can be achieved by providing more than the natural human complement of a specific gene segment type on one or both of the respective lg locus (eg, one or both chromosomes harbouring IgH in a mouse genome or mouse cell genome).

To this end, this configuration of the invention provides the following (as set out in ed paragraphs):- 1. A non-human vertebrate (eg, a mouse or rat) or a non-human vertebrate cell (eg, an ES cell or a B-cell) having a genome comprising at least 3 human variable region gene ts of the same type (eg, at least 3 human VH6-1 gene segments, at least 3 human JH6 gene segments, at least 3 human VK1-39 gene ts, at least 3 human D2-2 gene segments or at least 3 human 1x1 gene segments), wherein at least two of the human gene segments are variants that are not identical to each other.

For example, the genome comprises a variable region that ses V, D and J gene segments (for the le region of a heavy chain locus) or V and J gene segments (for the variable region of a light chain locus) upstream of a constant region for expression of heavy or light chains respectively.

In an alternative, the skilled person can choose to provide more than the wild type human ment of a specific gene segment type by providing several copies of one variant type of the human gene segment. Thus, there is provided A non-human vertebrate (eg, a mouse or rat) or a non-human vertebrate cell (eg, an ES cell or a B-cell) having a genome comprising at least 3 human variable region gene segments of the same type (eg, at least 3 human VH6-1 gene segments, at least 3 human JH6 gene segments, at least 3 human VK1-39 gene segments, at least 3 human 02-2 gene segments or at least 3 human JK]. gene segments), wherein the human gene ts are cal variants.

For example, the genome comprises a variable region that comprises V, D and J gene segments (for the variable region of a heavy chain locus) or V and J gene ts (for the variable region of a light chain locus) upstream of a constant region for expression of heavy or light chains respectively. 2012/052296 A non-human vertebrate (eg, a mouse or rat) or a man vertebrate cell (eg, an ES cell or a B-cell) having a genome comprising at least 2 different non-endogenous variable region gene segments of the same type (eg, at least 2 human VH6—1 gene segments, at least 3 human JH6 gene segments, at least 2 human VKl-39 gene segments, at least 2 human 02-2 gene segments or at least 2 human JKl gene segments) cis at the same lg locus.

In an alternative, the skilled person can choose to provide more than the wild type human complement of a specific gene segment type by providing several copies of one variant type of the human gene t. Thus, there is provided A non—human vertebrate (eg, a mouse or rat) or a non-human vertebrate cell (eg, an ES cell or a Bvcell) having a genome sing at least 2 dogenous variable region gene segments of the same variant type (eg, at least 2 human JH6*02 gene segments) cis at the same lg locus.

A man vertebrate (eg, a mouse or rat) or a non-human vertebrate cell (eg, an ES cell or a B-cell) having a genome comprising at least 2 different human variable region gene segments of the same type (eg, at least 2 human VH6-1 gene segments, at least 2 human JH6 gene segments, at least 2 human VK1-39 gene segments, at least 2 human D2—2 gene segments or at least 2 human JKl gene segments) trans at the same lg locus; and optionally a third human gene segment of the same type, wherein the third gene segment is cis with one of said 2 different gene segments.

In an alternative, the skilled person can choose to provide more than the wild type human complement of a specific gene segment type by providing several copies of one variant type of the human gene segment. Thus, there is provided A non—human vertebrate (eg, a mouse or rat) or a man vertebrate cell (eg, an ES cell or a B-cell) having a genome comprising at least 2 ent human variable region gene segments of the same variant type (eg, at least 2 human JH6*02 gene segments) trans at the same lg locus; and optionally a third human gene segment ofthe same variant type, wherein the third gene segment is 0'5 with one of said 2 different gene segments. 4. A population of man rates (eg, mice or rats) comprising a repertoire of human variable region gene segments, wherein the plurality comprises at least 2 human variable region gene segments of the same type (eg, at least 2 human VH6-1 gene segments, at least 2 human JH6 gene segments, at least 2 human VK1-39 gene segments, at least 2 human 02-2 gene ts or at least 2 human Ji<1 gene segments), a first of said different gene segments is provided in the genome of a first vertebrate of the population, and a second of said different gene segments being provided in the genome of a second vertebrate of the population, wherein the genome of the first vertebrate does not comprise the second gene segment.

. A non-human vertebrate (eg, a mouse or rat) or a non-human vertebrate cell (eg, an ES cell or a B—cell) having a genome comprising at least 2 different non-endogenous variable region gene segments of the same type (eg, at least 2 human VH6-1 gene segments, at least 2 human JH6 gene segments, at least 2 human VK1-39 gene segments, at least 2 human D2-2 gene segments or at least 2 human 1x1 gene segments), wherein the gene ts are derived from the genome sequence of different human individuals that are not genetically d over at least 3 generations. 6. A method of ing the human immunoglobulin gene diversity of a non-human vertebrate (eg, a mouse or rat), the method comprising providing the vertebrate with a genome comprising at least 3 human variable region gene segments of the same type (eg, at least 3 human VH6-1 gene segments, at least 3 human JH6 gene segments, at least 3 human VKl-39 gene segments, at least 3 human 02-2 gene segments or at least 3 human Jxl gene segments), wherein at least two of the human gene segments are variants that are not identical to each other. 7. A method of ing the immunoglobulin gene diversity of a non-human vertebrate (eg, a mouse or rat), the method comprising ing the vertebrate with a genome comprising at least 2 different non-endogenous variable region gene segments of the same type (eg, at least 2 human VH6-1 gene segments, at least 2 human JH6 gene segments, at least 2 human VK1-39 gene segments, at least 2 human 02-2 gene ts or at least 2 human 3K1 gene segments) cis at the same lg locus.

ZOIZI052296 8. A method of enhancing the immunoglobulin gene diversity of a non-human vertebrate (eg, a mouse or rat), the method comprising providing the vertebrate with a genome comprising at least 2 different human variable region gene segments of the same type (eg, at least 2 human VH6—1 gene segments, at least 2 human JH6 gene segments, at least 2 human Via-39 gene segments, at least 2 human D2-2 gene ts or at least 2 human 1K1 gene segments) trans at the same lg locus; and optionally a third human gene segment of the same type, wherein the third gene segment is cis with one of said 2 different gene segments.

A method of providing an enhanced human immunoglobulin variable region gene t repertoire, the method comprising providing a population of non-human rates (eg, a mouse or rat) comprising a oire of human variable region gene segments, wherein the method comprises providing at least 2 different human variable region gene segments of the same type (eg, at least 2 human VHS-1 gene segments, at least 2 human JH6 gene segments, at least 2 human VK1-39 gene segments, at least 2 human D2-2 gene segments or at least 2 human JKl gene segments), wherein a first of said different gene segments is provided in the genome of a first vertebrate of the population, and a second of said different gene segments is ed in the genome of a second vertebrate of the population, wherein the genome of the first vertebrate does not comprise the second gene segment.

. A method of enhancing the human globulin gene ity of a non-human vertebrate (eg, a mouse or rat), the method comprising providing the rate with a genome comprising at least 2 different non—endogenous variable region gene ts of the same type (eg, at least 2 human VH6-1 gene segments, at least 2 human JH6 gene segments, at least 2 human VK1—39 gene segments, at least 2 human 02—2 gene segments or at least 2 human JKl gene segments), wherein the gene segments are derived from the genome sequence of different human individuals that are not genetically related over at least 3 generations. 11. The vertebrate, cell or method of any preceding paragraph, wherein at least 2 or 3 of said different gene segments are provided cis at the same lg locus in said genome. 12. The rate, cell or method of any preceding paragraph, wherein the gene segments are derived from the genome sequence of different human individuals that are not genetically WO 41844 PCT/G82012/052296 related over at [east 3 generations. 13. The vertebrate, cell or method of any preceding aph, wherein the gene segments are d from the genome sequence of two or more different human individuals; optionally wherein the different human individuals are from different human populations. 14. The vertebrate, cell or method of paragraph 13, wherein the individuals are not genetically related.

. A method of enhancing the human immunoglobulin gene diversity ofa non-human vertebrate (eg, a mouse or rat), the method comprising providing the vertebrate with a genome comprising at least 2 human variable region gene segments of the same type (eg, at least 2 human VH6-1 gene segments, at least 2 human JH6 gene segments, at least 2 human VK1-39 gene ts, at least 2 human 02-2 gene segments or at least 2 human JKl gene segments), wherein the gene segments are derived from the genome sequence of different human individuals that are not genetically related over at least 3 generations,- optionally wherein at least 2 or 3 of said ent gene segments are provided at the same lg locus in said genome. 16. The method of paragraph 15, wherein the different human individuals are from different human populations. 17. The method of paragraph 15, wherein the individuals are not genetically related. 18. The vertebrate, cell or method of preceding paragraph, wherein at least one of the different segments is a synthetic mutant of a human germline gene segment. 19. The vertebrate, cell or method of any ing paragraph, wherein each of said gene segments occurs in 10 or more different human populations.

. The vertebrate, cell or method of preceding paragraph, wherein each of said gene segments has a human frequency of 5% or greater (eg, 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% or greater).

In this respect, the skilled person can be guided by the information provided in Table 14. ncy can, for example, be cumulative frequency in the 1000 Genomes database. 21. The rate, cell or method of paragraph 20, wherein each of said gene segments occurs in or more different human tions. 22. The vertebrate, cell or method of any preceding paragraph, wherein each of said gene segments occurs in the 1000 s database in more than 50 individuals. 23. The vertebrate, cell or method of preceding paragraph, n each of said gene segments (i) has a human frequency of 5% or greater (eg, 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% or greater); and (ii) occurs in 10 or more different human populations.

In this respect, the skilled person can be guided by the information provided in Table 14.

Frequency can, for example, be cumulative frequency in the 1000 Genomes database. 24. A non-human vertebrate (eg, a mouse or rat) or a non—human vertebrate cell (eg, an ES cell or a B-cell) having a genome comprising first and second human lg locus gene segments of the same type (eg, first and second human JH6 gene segments; or first and second lgGZ gene segments; or first and second human M7 gene segments), wherein the first gene segment is a gene segment selected from Table 14(eg, IGHJS—a) and the second gene segment is the ponding reference ce (eg, IGHJG ref; SEQ ID NO: 244).

Table 14 lists commonly-occurring natural human ts. It can be seen that these occur across many human populations and thus usefully have wide applicability for human antibody- based drugs.

PCT/GB201 2/052296 For example, the gene segments are provided as targeted insertions into an endogenous non- human vertebrate lg locus. Alternatively, random integration (eg, using YACs) as is know in the art can be performed.

For example, the genome comprises a variable region that comprises V, D and J gene segments (for the variable region of a heavy chain locus) or V and 1 gene segments (for the variable region of a light chain locus) upstream of a constant region for expression of heavy or light chains respectively.

In another embodiment, the invention enables the skilled person to select two or more different naturally-occurring human gene segment variants for combination into the genome of a non- human vertebrate or cell. A reference sequence need not be included. It may be desirable to use one or more rare gene segments to increase diversity of the repertoire. Additionally or atively, it may be desirable to include a e of nt and rare variants of the same type to provide repertoire diversity. The variants may be chosen additionally or alternatively to tailor the gene t ion to one or more ic human populations as indicated by the information ed in Table 13 or Table 14.

Thus, the invention provides A non-human vertebrate (eg, a mouse or rat) or a non-human vertebrate cell (eg, an ES cell or a B-cell) having a genome comprising first and second human lg locus gene segments of the same type (eg, first and second human .lH6 gene segments; or first and second lgGZ gene segments; or first and second human 1A7 gene segments), wherein the gene segments are gene segments selected from Table 13 or Table 14; and optionally n one or more of the gene segments appears in Table 14 (eg, lGHJG—a) or is a reference sequence (eg, IGHJ6 ref; SEQ ID NO: 244).

. The vertebrate or cell of paragraph 24, wherein the genome comprises a third human gene segment of said type, the third gene segment being different from the first and second gene segments. 26. The vertebrate or cell of paragraph 24 or 25, wherein the first and second gene ts are cis on the same chromosome; and optionally the third gene t is also cis on said chromosome. 27. The vertebrate or cell of paragraph 26, n the gene segments are targeted insertions into an endogenous non-human lg locus.

For example, the gene segments are heavy chain gene segments and the non-human locus is an IgH locus. For example, the gene segments are light chain (kappa or lambda) gene segments and the non-human locus is an lgL locus. 28. The vertebrate or cell of aph 24 or 25, wherein the first and second gene segments are trans on different somes.

Thus, the chromosomes are the same type (eg, both mouse chromosome 6 or rat chromosome 29. The vertebrate or cell of any one of paragraphs 24 to 28, wherein the first gene segment is a gene segment selected from any one of Tables 1 to 7 and 9 to 14 (eg, selected from Table 13 or 14) and the second gene segment is the corresponding reference sequence.

. A population of non~human vertebrates (eg, mice or rats) comprising first and second human lg locus gene ts of the same type (eg, first and second human JH6 gene segments; or first and second IgGZ gene segments; or first and second human 1A7 gene segments), wherein the first gene segment is a gene segment selected from any one of Tables 1 to 7 and 9 to 14 (eg, Table 13 or 14) (eg, lGHJ6—a) and the second gene segment is the corresponding reference sequence (eg, SEQ ID NO: 7), wherein the first gene segment is provided in the genome of a first vertebrate of the tion, and the second gene segment is provided in the genome ofa second vertebrate of the population.

PCT/G32012/052296 31. The population of paragraph 30, wherein the genome of the first vertebrate does not comprise the second gene segment. 32. The population of paragraph 30 or 31, wherein the population comprises a third human gene segment of said type, the third gene segment being different from the first and second gene segments and optionally wherein the first and third gene segments are present in the genome of the first vertebrate. 33. The population of paragraph 30, 31 or 32, n the gene segments are targeted ions into an endogenous non-human lg locus in the respective genome.

For example, the gene segments are heavy chain gene segments and the man locus is an IgH locus. For example, the gene ts are light chain (kappa or lambda) gene segments and the non-human locus is an lgL locus. 34. The population of of any one of paragraphs 30 to 33, wherein the first gene segment is a gene segment selected from any one of Tables 1 to 7 and 9 to 14 (eg, Table 13 or 14) and the second gene segment is the corresponding reference sequence.

. A method of enhancing the human immunoglobulin gene ity of a non-human vertebrate (eg, a mouse or rat), the method comprising providing the vertebrate with a genome sing first and second human lg locus gene segments of the same type (eg, first and second human JH6 gene segments; or first and second IgGZ gene segments; or first and second human M7 gene segments), wherein the first gene segment is a gene segment ed from any one of Tables 1 to 7 and 9 to 14 (eg, Table 13 or 14) (eg, lGHJG-a) and the second gene segment is the corresponding reference sequence (eg, SEQ ID NO: 7). 36. A method of providing an enhanced human immunogolobulin gene segment repertoire, the method comprising ing a population according to any one of paragraphs 30 to 33.

Variants Prevalent in Few Populations PCT/GBZOIZ/052296 In another aspect, it is of note that certain human gene segment variants may appear relatively frequently in one or a small number of populations, but is not found prevalently across many different human populations. There is thinking that specific germline gene segment repertoires have evolved in dual human ethnic populations due to iterative exposure to antigens (eg, disease pathogen antigens) to which the population is often d. Repeated exposure and mutation may have lead to the evolution of gene segment variants that can provide an effective response to the antigen (pathogen) in the population, and this may explain the conservation of the gene ts in those populations (as d to other human ethnic populations that may not have frequently encountered the antigen). With this in mind, the inventors identified gene segment variants from their analysis that are relatively ent in a small number of human populations, and not across many populations. The inventors realized that inclusion of one or more of such gene segments in the configurations of the invention (eg, in transgenic lg loci, vertebrates and cells) would be useful for producing antibodies, lg chains and variable domains that can address antigens (eg, disease- g antigens or pathogens) to which the small number of human populations may become exposed. Such products would be useful for treating and/or preventing disease or medical conditions in members of such a population. This aspect could also be useful for sing infectious disease pathogens that may have been common in the small number of populations, but which in the future or relatively recently in evolution has become a more ent e-causing pathogen in other human populations (ie, those not listed in Table 13 against the gene segment variant(s) in question). To this end, from the 1000 Genomes database the inventors have identified the gene segment ts listed in Table 20.

Thus, ing to any configuration or aspect described herein, one, more or all of the gene segments used in the present ion can be a gene segment listed in Table 20A, 203, 20C or 20D.

Multiple JH Gene t yan'ants A specific application of this configuration is the provision of multiple human JH gene segments as follows.

A non-human vertebrate (eg, a mouse or rat) or a non-human vertebrate cell (eg, an ES cell or a B- cell) having a genome comprising at least 3 human JH gene segments of the same type (JH1,JH2, 1H3, JH4, JH5 or JH6), wherein at least two of the human JH gene segments are variants that are not WO 41844 identical to each other.

In an example, any cell of the invention is an isolated cell. An ”isolated" cell is one that has been identified, separated and/or recovered from a component of its production environment (eg, naturally or recombinantly). Preferably, the isolated cell is free of association with all other components from its production environment, eg, so that the cell can produce an antibody to an FDA-approvable or approved standard. Contaminant components of its tion environment, such as that resulting from inant transfected cells, are materials that would typically interfere with research, diagnostic or eutic uses for the resultant antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In red embodiments, the polypeptide will be purified: (1) to greater than 95% by weight of antibody as ined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N—terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to neity by SDS-PAGE under non- reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Ordinarily, however, an isolated cell will be prepared by at least one purification step.

A man vertebrate (eg, a mouse or rat) or a non-human vertebrate cell (eg, an ES cell or a B- cell) having a genome comprising at least 2 different non-endogenous JH gene segments (eg, human gene segments) of the same type (1H1, JH2, JH3, 1H4, JHS or JH6) cis at the same lg (eg, lgH, eg, endogenous lgH, eg, mouse or rat lgH) locus. In an example, the genome comprises a human VH, D and JH repertoire sing said different JH gene segments. Optionally the non-endogenous JH gene segments are non-mouse or non-rat, eg, human JH gene segments. In an example one or more or all of the non-endogenous gene segments are synthetic.

A non-human vertebrate (eg, a mouse or rat) or a non-human vertebrate cell (eg, an ES cell or a B- cell) having a genome comprising at least 2 different human JH gene ts of the same type (JH1,JH2,JH3,JH4,JH5 or JH6) trans at the same lg (eg, lgH, eg, endogenous lgH, eg, mouse or rat lgH) locus; and optionally a third human JH gene segments of the same type, wherein the third JH is cis with one of said 2 different JH gene segments.

A population of non-human rates (eg, mice or rats) comprising a repertoire of human JH gene segments, wherein the plurality comprises at least 2 different human JH gene ts of the same type (JH1, 1H2, JHS, JH4, JH5 or 1H6), a first of said different JH gene segments is provided in the genome of a first vertebrate of the population, and a second of said different JH gene segments being provided in the genome of a second vertebrate of the population, wherein the genome of the first vertebrate does not comprise the second JH gene segment.

A non-human vertebrate (eg, a mouse or rat) or a non—human vertebrate cell {eg, an ES cell or a B- cell) having a genome comprising at least 2 different non-endogenous (eg, human) JH gene segments of the same type (JH1, JHZ, 1H3, JH4, JH5 or 1H6), wherein the JH gene segments are derived from the genome sequence of different human individuals that are not genetically related over at least 3 generations (eg, 3, 4, 5 or 6 generations). ally the non-endogenous JH gene segments are human JH gene segments. In an example one or more or all of the non-endogenous gene segments are synthetic.

A method of enhancing the human immunoglobulin gene diversity of a non-human vertebrate (eg, a mouse or rat), the method sing providing the vertebrate with a genome comprising at least 3 human JH gene segments of the same type (JH1, 1H2, 1H3, JH4, JHS , wherein at least two of the human JH gene segments are variants that are not identical to each other.

A method of enhancing the immunoglobulin gene ity of a non-human vertebrate (eg, a mouse or rat), the method comprising providing the vertebrate with a genome comprising at least 2 different non-endogenous (eg, human) JH gene segments of the same type (JH1, 1H2, JH3, JH4, JH5 or 1H6) cis at the same lg (eg, IgH, eg, endogenous lgH, eg, mouse or rat IgH) locus). Optionally the non-endogenous JH gene segments are non-mouse or non-rat, eg, human JH gene segments. In an e one or more or all of the non-endogenous gene segments are synthetic.

A method of enhancing the immunoglobulin gene diversity of a non—human vertebrate (eg, a mouse or rat), the method sing providing the vertebrate with a genome comprising at least 2 different human JH gene segments of the same type (JH1, 1H2, 1H3, JH4, JHS or JH6) trans at the same lg (eg, lgH, eg, endogenous lgH, eg, mouse or rat IgH) locus; and optionally a third human JH WO 41844 2012/052296 gene segments of the same type, wherein the third JH is cis with one of said 2 different JH gene A method of providing an enhanced human immunoglobulin JH gene segment repertoire, the method comprising providing a population of non—human vertebrates (eg, a mouse or rat) comprising a repertoire of human JH gene segments, wherein the method comprises providing at least 2 different human JH gene segments ofthe same type (JHl, 1H2, 1H3, 1H4, JHS or JH6), wherein a first of said different JH gene segments is provided in the genome of a first vertebrate of the tion, and a second of said differentJH gene segments is provided in the genome of a second vertebrate of the population, n the genome of the first vertebrate does not comprise the second JH gene segment.

A method of enhancing the human globulin gene diversity of a non-human vertebrate (eg, a mouse or rat), the method comprising providing the vertebrate with a genome comprising at least 2 different non-endogenous (eg, human) JH gene segments of the same type (JHl, JH2, 1H3, 1H4, JHS or JH6), wherein the JH gene segments are derived from the genome sequence of different human individuals that are not genetically related over at least 3 generations (eg, 3, 4, 5, or 6 generations).

Optionally the non-endogenous JH gene segments are human JH gene segments. In an example one or more or all of the non-endogenous gene segments are tic.

In an e of the vertebrate or cell or the method of the invention at least 2 or 3 of said ent gene segments are provided cis at the same lg locus in said genome.

In an example of the vertebrate or cell or the method of the invention the JH gene segments are derived from the genome ce of different human individuals that are not genetically related over at least 3 generations (eg, 3, 4, 5, or 6 generations).

In an example of the vertebrate or cell or the method of the invention the JH gene segments are derived from the genome sequence of two or more different human individuals; optionally wherein the different human individuals are from different human populations.

PCT/GBZO]2/052296 In an example of the vertebrate or cell or the method of the invention the individuals are not genetically related (eg, going back 3, 4, 5, or 6 generations).

In an example of the vertebrate or cell or the method of the invention at least one of the ent JH segments is a synthetic mutant ofa human germline JH gene segment.

The invention also provides a method of enhancing the human immunoglobulin gene diversity of a non—human vertebrate (eg, a mouse or rat), the method comprising providing the vertebrate with a genome comprising at least 2 human JH gene segments of the same type (1H1, JHZ, JH3, JH4, JHS or JH6), wherein the JH gene segments are d from the genome sequence of different human individuals that are not genetically related over at least 3 generations (eg, 3, 4, 5, or 6 generations); optionally wherein at least 2 or 3 of said different gene segments are provided at the same lgH locus in said genome. in an e of the rate or cell or the method of this embodiment of the invention the genome comprises a substantially complete functional repertoire of human JH gene segment types supplemented with one, two or more human JH gene segments, wherein said ntially complete onal repertoire and the supplementary JH gene segments are not found together in the germline genome of a human individual.

In an example of the population of the ion, the population comprises a substantially te functional repertoire of human JH gene segment types supplemented with one, two or more human JH gene segments, wherein said substantially complete functional repertoire and the supplementary JH gene segments are not found together in the germline genome of a human individual.

A non-human vertebrate (eg, a mouse or rat) or a non-human cell (eg, an ES cell or a B-cell) having a genome comprising a ntially complete functional repertoire of human JH gene segment types supplemented with one, two or more human JH gene segments, wherein said substantially te functional repertoire and the supplementary JH gene segments are not found together in the germline genome of a human individual.

PCT/082012/052296 A population of non-human vertebrates (eg, mice or rats) comprising a substantially complete functional repertoire of human JH gene segment types supplemented with one, two or more human JH gene segments, wherein said substantially complete functional oire and the supplementary JH gene segments are not found together in the ne genome of a human individual.

In an example ofthe vertebrate or the population, at least one of said JH gene segments is SEQ ID NO: 1, 2, 3 or 4. For example, at least one of said JH gene segments is SEQ ID NO: 1 and at least one, two or more of said supplementary JH gene ts is a variant according to any example above.

For example, at least one of said JH gene segments is SEQ ID NO: 2 and at least one, two or more of said supplementary ll-l gene segments is a variant according to any one of the examples above. For example, at least one of said JH gene segments is SEQ ID NO: 2 and at least one, two or more of said supplementary JH gene segments is a variant according to any one of the examples above.

In an embodiment, the non—human vertebrate or vertebrate cell of the ion comprises a genome that comprises VH, D and JH gene repertoires sing human gene segments, the JH gene repertoire (eg, a human JH gene segment repertoire) comprising a plurality ofJHl gene segments provided by at least 2 different 1H1 gene ts in cis at the same lg locus in said genome; a plurality ofJHZ gene segments provided by at least 2 different JHZ gene segments in cis at the same lg locus in said genome; a plurality ofJH3 gene segments provided by at least 2 different JH3 gene segments in cis at the same lg locus in said genome; a plurality ofJH4 gene segments provided by at least 2 different JH4 gene segments in cis at the same lg locus in said genome; a ity ofJH5 gene segments provided by at least 2 different JH5 gene segments in cis at the same lg locus in said genome; and/or a pluraiity of JH6 gene segments provided by at least 2 different JH6 gene segments in cis at the same lg locus in said genome; optionally wherein the JH gene ts are derived from the genome ce of two or more ent human individuals.

Optionally said at least 2 different JH gene segments are human gene segments or synthetic gene segments derived from human gene segments.

Optionally, the Ig locus is a lgH locus, eg, an endogenous locus, eg, a mouse or rat lgH locus.

In an embodiment, the man vertebrate or vertebrate cell of the ion comprises a genome that comprises VH, D and JH gene oires comprising human gene segments, the JH gene repertoire (eg, a human JH gene segment repertoire) comprising a plurality ofJHl gene segments provided by at least 3 different JH1 gene segments; a plurality ofJHZ gene segments ed by at least 3 different JHZ gene segments; a plurality ofJH3 gene segments provided by at least 3 different JH3 gene segments; a plurality of JH4 gene segments provided by at least 3 different JH4 gene segments; a plurality ofJHS gene segments provided by at least 3 different JHS gene segments; and/or a ity of JH6 gene segments provided by at least 3 different JH6 gene segments; optionally wherein the JH gene segments are derived from the genome sequence of two or three different human individuals; optionally wherein at least 2 or 3 of said different gene ts are provided in cis at the same lg locus in said genome.

Optionally said at least 3 different JH gene segments are human gene ts or synthetic gene ts derived from human gene segments.

Optionally, the lg locus is a lgH locus, eg, an endogenous locus, eg, a mouse or rat lgH locus.

Optionally in the vertebrate or cell the different human individuals are from different human populations.

Optionally in the vertebrate or cell the individuals are not genetically related (eg, Going back 3, 4, 5 or 6generations).

PCT/G82012/052296 Optionally in the vertebrate or cell at least one of the different JH segments is a synthetic mutant of a human germline Jl-l gene segment.

In an embodiment of a non-human vertebrate or vertebrate cell (optionally an ES cell or B-cell) according to the ion, the vertebrate or cell genome comprises human VH, D and JH gene repertoires, the JH gene repertoire (eg, a human JH gene repertoire) comprising a plurality ofJHl gene segments provided by at least 2 different human JHl gene segments, optionally in cis at the same lg locus in said genome; a plurality ofJHZ gene segments provided by at least 2 different human JHZ gene ts, optionally in cis at the same lg locus in said genome; a plurality ofJH3 gene segments provided by at least 2 different human JH3 gene segments, optionally in cis at the same lg locus in said genome; a plurality ofJH4 gene ts ed by at least 2 different human JH4 gene segments, optionally in cis at the same lg locus in said genome; a plurality oleS gene segments provided by at least 2 different human JHS gene segments, optionally in cis at the same lg locus in said genome; and/or a plurality ofJH6 gene segments provided by at least 2 different human JH6 gene ts, optionally in cis at the same lg locus in said genome; wherein the JH gene segments are d from the genome sequence of different human individuals that are not genetically related over at least 3 generations (eg, 3, 4, 5 or 6 generations).

Optionally said at least 2 different JH gene segments are human gene segments or synthetic gene segments derived from human gene segments.

Optionally, the Ig locus is a lgH locus, eg, an endogenous locus, eg, a mouse or rat lgH locus.

Optionally in the vertebrate or cell the human individuals are from different human populations.

An embodiment es a rate, cell or population of the invention whose genome ses a ity of JHS gene segments, wherein the plurality comprises a human 1H5 gene variant of SEQ ID NO: 1, wherein the variant comprises a nucleotide mutation at one or more positions WO 41844 PCT/GBZOl2/052296 corresponding to positions 106,330,024 106,330,027 106,330,032 106,330,041 106,330,044 106,330,045 106,330,062 106,330,063 106,330,065 106,330,066 106,330,067 106,330,068 and 106,330,071 on human chromosome 14.

In the vertebrate, cell or population optionally the plurality comprises a human JHS gene variant of SEQ ID NO: 1, wherein the variant comprises a guanine at a position ponding to position 106,330,067 on human chromosome 14; and optionally no further mutation from the sequence of SEQID NO: 1.

Optionally the variant comprises additionally a mutation at a on corresponding to (i) position 106,330,071 on human some 14 (optionally the additional mutation being a guanine); (ii) position 106,330,066 on human chromosome 14 nally the additional mutation being a guanine); and/or (iii) position 106,330,068 on human some 14 (optionally the additional mutation being a thymine).

Optionally the plurality comprises a human JHS gene variant of SEQ ID NO: 1, wherein the variant comprises 3 guanine at a position corresponding to position 106,330,071 on human chromosome 14; and optionally no further on from the sequence of SEQ ID NO: 1.

PCT/GBZOIZ/052296 Optionally the variant comprises additionally a mutation at a position corresponding to (i) position 106,330,063 on human chromosome 14 (optionally the additional mutation being an adenine); and/or (ii) position 106,330,067 on human chromosome 14 (optionally the additional mutation being a guanine).

Optionally the plurality comprises a human JHS gene variant of SEQ ID NO: 1, wherein the variant comprises a cytosine at a position corresponding to position 106,330,045 on human chromosome 14; and optionally no further mutation from the sequence of SEQ ID NO: 1. ally the plurality ses a human JH5 gene variant of SEQ ID NO: 1, wherein the t comprises an adenine at a position corresponding to position 106,330,044 on human chromosome 14; and optionally no r mutation from the sequence of SEQ ID N0:1.

Optionally the variant comprises additionally a mutation at a position corresponding to (i) position 106,330,066 on human chromosome 14 nally the additional mutation being a e); and/or (ii) position 106,330,068 on human chromosome 14 (optionally the additional mutation being a thymine).

Optionaly the plurality comprises a human JHS gene variant of SEQ ID NO: 1, wherein the variant comprises 3 guanine at a position corresponding to position 106,330,066 on human chromosome 14; and optionally no further mutation from the ce ofSEQ iD NO: 1.

Optionally the t comprises additionally a mutation at a position corresponding to (i) position 0,067 on human chromosome 14 nally the additional mutation being a guanine); and/or (ii) position 106,330,068 on human chromosome 14 nally the additional mutation being a thymine).

Optionally the plurality comprises a human JH5 gene variant of SEQ ID NO: 1, wherein the variant comprises a thymine at a position corresponding to position 106,330,068 on human chromosome 14; and optionally no r mutation from the sequence of SEQ 10 NO: 1.

PCT/G32012/052296 Optionally the variant comprises additionally a mutation at a position corresponding to (i) position 106,330,067 on human chromosome 14 (optionally the additional mutation being a guanine); and/or (ii) position 106,330,066 on human chromosome 14 (optionally the additional mutation being a Optionally the ity ses a human JHS gene t of SEQ ID NO: 1, wherein the variant comprises a cytosine at a position corresponding to position 106,330,027 on human chromosome 14; and optionally no further mutation from the sequence of SEQ ID NO: 1.

Optionally the ity comprises a human JHS gene variant of SEQ ID NO: 1, wherein the variant comprises an adenine at a position corresponding to position 106,330,024 on human some 14; and optionally no further mutation from the sequence of SEQ ID NO: 1.

Optionally the plurality comprises a human JHS gene variant of SEQ ID NO: 1, wherein the variant comprises a thymine at a position corresponding to position 106,330,032 on human chromosome 14; and optionally no further mutation from the sequence of SEQ ID NO: 1.

Optionally the plurality comprises a human JHS gene variant of SEQ ID NO: 1, wherein the variant comprises a thymine at a position corresponding to position 106,330,041 on human chromosome 14; and optionally no further on from the sequence of SEQ ID NO: 1. ally the plurality comprises a human JH5 gene variant of SEQ ID NO: 1, wherein the variant comprises an e or thymine at a position ponding to position 106,330,063 on human chromosome 14; and optionally no further mutation from the sequence of SEQ ID NO: 1.

Optionally the variant comprises additionally a mutation at a position corresponding to position 106,330,071 on human chromosome 14 (optionally the onal mutation being a e).

Optionally the plurality comprises a human JH5 gene variant of SEQ ID NO: 1,wherein the variant comprises a cytosine at a position corresponding to position 106,330,062 on human chromosome 14; and optionally no further mutation from the sequence of SEQ ID NO: 1.

Optionally the genome comprises SEQ ID N011; optionally in cis at the same lg locus as one, two or more of the variants.

An embodiment provides a vertebrate, cell or population of the invention whose genome comprises a plurality of JH6 gene segments, wherein the plurality ses a human JH6 gene variant of SEQ ID NO: 2, wherein the variant comprises a nucleotide mutation at one or more positions corresponding to positions 106,329,411 106,329,413 106,329,414 106,329,417 106,329,419 106,329,426 106,329,434 9,435, and 106,329,468 on human chromosome 14.

Optionally the genome of the vertebrate, cell or population comprises a plurality ofJH6 gene segments, wherein the plurality comprises a human JH6 gene variant of SEQ ID NO: 2, wherein the t comprises 3 guanine at a on ponding to position 106,329,435 on human some 14; and optionally no further mutation from the sequence of SEQ ID NO: 2.

Optionally the variant comprises additionally a mutation at a position corresponding to (i) position 106,329,468 on human chromosome 14 (optionally the additional mutation being a guanine); (ii) position 9,419 on human chromosome 14 (optionally the additional mutation being an adenine); (iii) position 106,329,434 on human some 14 nally the additional mutation PCT/G32012/052296 being a ne) and/or position 106,329,414 on human chromosome 14 (optionally the additional mutation being a guanine); (iv) position 106,329,426 on human chromosome 14 (optionally the additional mutation being an adenine); (v) position 106,329,413 on human chromosome 14 (optionally the additional mutation being an adenine); (vi) position 9,417 on human chromosome 14 (optionally the additional mutation being a thymine); (vii) position 106,329,411 on human chromosome 14 (optionally the onal mutation being a thymine); (viii) position 106,329,451 on human chromosome 14 (optionally the additional mutation being an adenine); (ix) position 106,329,452 on human chromosome 14 (optionally the additional mutation being a cytosine); and/or (x) position 106,329,453 on human chromosome 14 (optionally the additional mutation being a cytosine).

Optionally the t comprises onally mutations at positions ponding to position 106,329,451 on human chromosome 14, the additional mutation being an adenine; position 106,329,452 on human chromosome 14, the additional mutation being a cytosine; and position 106,329,453 on human chromosome 14, the additional mutation being a cytosine, The vertebrate, cell or population ally comprises a plurality ofJH6 gene segments,, n the plurality comprises a human JH6 gene variant ofSEQ lD NO: 2, wherein the variant comprises a guanine at a position corresponding to on 106,329,468 on human chromosome 14; and optionally no further mutation from the sequence of SEQ ID NO: 2.

Optionally the variant comprises additionally a mutation at a position corresponding to position 106,329,435 on human chromosome 14 (optionally the additional mutation being 3 guanine). ally the vertebrate, cell or population comprises a ity of JH6 gene segments,, wherein the plurality comprises a human JH6 gene t of SEQ ID NO: 2, n the variant comprises a thymine at a on corresponding to position 106,329,417 on human some 14; and optionally no further mutation from the sequence ofSEQ ID NO: 2. ally the variant comprises additionally a mutation at a position corresponding to position 106,329,435 on human chromosome 14 nally the additional mutation being a guanine).

Optionally the vertebrate, cell or population comprises a plurality ofJH6 gene segments,, wherein the plurality comprises a human JH6 gene variant of SEQ ID NO: 2, wherein the variant comprises a W0 2013/041844 PCT/G82012/052296 cytosine at a position corresponding to position 106,329,434 on human chromosome 14; and optionally no further mutation from the sequence of SEQ ID NO: 2.

Optionally the variant comprises additionally a mutation at a position corresponding to (i) on 106,329,414 on human chromosome 14 nally the additional mutation being a guanine); and/or (ii) on 106,329,435 on human chromosome 14 (optionally the additional mutation being 3 guanine).

Optionally the vertebrate, cell or population comprises a plurality ofJH6 gene segments, wherein the plurality comprises a human JH6 gene variant of SEQ ID NO: 2, wherein the variant comprises a thymine at a position corresponding to position 106,329,411 on human chromosome 14; and optionally no further mutation from the ce of SEQ ID NO: 2. ally the variant comprises additionally a mutation at a on corresponding to position 106,329,435 on human chromosome 14 (optionally the additional on being a guanine).

Optionally the vertebrate, cell or population ses a plurality ofJH6 gene segments, wherein the ity comprises a human JH6 gene variant that is an antisense sequence of a variant described above.

Optionally the genome comprises SEQ ID NO:2; optionally cis at the same lg locus as one, two or more of the JH6 variants.

An embodiment provides a vertebrate, cell or population of the invention whose genome comprises a plurality of JH2 gene segments, wherein the ity comprises a human JHZ gene variant ofSEQ ID NO: 3, wherein the variant comprises a nucleotide on at one or more positions corresponding to positions 106,331,455 106,331,453, and 106,331,409 WO 41844 PCT/GBlOlZ/052296 on human chromosome 14.

Optionally the rate, cell or population comprises said plurality ofJHZ gene segments, wherein the plurality comprises a human JHZ gene variant of SEQ ID NO: 3, wherein the variant ses a guanine at a position ponding to position 106,331,455 on human chromosome 14; and ally no further mutation from the sequence of SEQ ID NO: 3.

Optionally the variant comprises additionally a mutation at a position corresponding to (i) position 106,331,453 on human chromosome 14 (optionally the additional mutation being an adenine),- and/or (ii) position 106,331,409 on human chromosome 14 (optionally the additional mutation being an adenine); (iii) position 106,329,434 on human chromosome 14 (optionally the onal mutation being an adenine).

Optionally the vertebrate, cell or population comprises a plurality ofJHZ gene segments, wherein the plurality ses a human JHZ gene variant of SEQ 1D NO: 3, wherein the variant comprises an adenine at a on corresponding to position 106,331,453 on human chromosome 14; and optionally no r mutation from the sequence of SEQ ID NO: 3.

Optionally the variant comprises additionally a mutation at a position corresponding to position 1,409 on human some 14 (optionally the additional mutation being an adenine).

Optionally the vertebrate, cell or population comprises a plurality of JHZ gene segments, wherein the plurality comprises a human JH2 gene variant of SEQ ID NO: 3, wherein the variant ses an adenine at a position corresponding to position 106,331,409 on human chromosome 14; and optionally no further mutation from the sequence of SEQ ID NO: 3.

Optionally the vertebrate, cell or population comprises a plurality ofJHZ gene segments, wherein the plurality comprises a human JH2 gene t that is an antisense sequence of a variant described above.

Optionally the genome comprises SEQ ID NO:3; optionally cis at the same lg locus as one, two or more of the JHZ variants. -8 PCT/G82012/052296 Optionally the vertebrate, cell or population genome comprises two or more different JH gene segments ed from SEQ ID NOs: 1 to 3 and variants described above; optionally wherein said JH gene segments are cis at the same immunoglobulin lg locus.

Multiple Human D Gene Segment Variants A specific ation of this configuration is the provision of multiple human D gene segments as s (as set out in ed clauses, starting at clause number 154). 154. A non-human vertebrate {eg, a mouse or rat) or a non—human vertebrate cell (eg, an ES cell or a B-cell) having a genome comprising at least 3 human D gene segments of the same type (eg, DZ-Z gene segments), wherein at least two ofthe human D gene segments are variants that are not cal to each other (eg, D2-2ref and 02-23).

In an example of any aspect of the sixth configuration of the invention (V, D, J or C), one or more or all of the variants are naturally-occurring human gene segments.

In an example of any aspect of the sixth configuration of the invention (V, D, J or C), one or more of the ts may be a synthetic variant of a human gene t. 155. A man vertebrate (eg, a mouse or rat) or a non-human vertebrate cell (eg, an ES cell or a B-cell) having a genome comprising at least 2 different non-endogenous D gene segments of the same type type (eg, DZ-Zref and DZ—Za) cis at the same lg locus. 156. A non-human vertebrate (eg, a mouse or rat) or a non-human vertebrate cell (eg, an ES cell or a ) having a genome comprising at least 2 different human D gene segments of the same type (eg, DZ-Zref and D2-2a) trans at the same lg locus; and Optionally a third human D gene segment (eg, (eg, D2*2ref, 02-23 or D2-2b) of the same type, wherein the third D is cis with one of said 2 different D gene segments. 157. A population of non—human vertebrates (eg, mice or rats) comprising a repertoire of human D gene ts, wherein the plurality comprises at least 2 different human D gene segments of the same type (eg, D2-2 gene segments), a first of said different D gene segments (eg, DZ—Zref) is provided in the genome ofa first vertebrate of the population, and a second of said different D gene segment (eg, D2-2a) being provided in the genome of a second vertebrate of the W0 2013/041844 PCT/G82012/052296 population, wherein the genome of the first vertebrate does not se the second D gene segment. 158. A man vertebrate (eg, a mouse or rat) or a non-human vertebrate cell (eg, an ES cell or a B-cell) having a genome comprising at least 2 different non-endogenous D gene segments of the same type (eg, human D2-2 gene segments), wherein the D gene segments are derived from the genome sequence of different human individuals that are not genetically related over at least 3 generations. 159. A method of enhancing the human immunoglobulin gene diversity of a non-human vertebrate (eg, a mouse or rat), the method comprising providing the vertebrate with a genome comprising at least 3 human D gene segments of the same type (eg, 02-2 gene segments), wherein at least two of the human D gene segments are variants that are not identical to each other (eg, f and D2-2a). 160. A method of enhancing the immunoglobulin gene diversity of a non-human vertebrate (eg, a mouse or rat), the method comprising providing the vertebrate with a genome comprising at least 2 different non-endogenous D gene ts of the same type (eg, human 02-2 gene ts) cis at the same lg locus. 161. A method of ing the immunoglobulin gene diversity of a non-human vertebrate (eg, a mouse or rat), the method comprising providing the vertebrate with a genome comprising at least 2 different human D gene ts ofthe same type (eg, D2-2ref and DZ—Za) trans at the same lg locus; and optionally a third human D gene segment (eg, D2-2ref, D2-2a or D2-2b) of the same type, wherein the third D is cis with one of said 2 different D gene segments. 162. A method of providing an enhanced human immunoglobulin D gene segment repertoire, the method comprising providing a population of non—human vertebrates (eg, a mouse or rat) comprising a repertoire of human D gene segments, wherein the method comprises providing at least 2 different human D gene segments of the same type (eg, D2-2refand DZ-Za), wherein a first of said different D gene segments is ed in the genome of a first vertebrate of the population, and a second of said ent D gene segments is provided in the genome of a second vertebrate of the tion, wherein the genome of the first vertebrate does not 2012/052296 comprise the second D gene segment. 163. A method of ing the human globulin gene diversity of a non—human vertebrate (eg, a mouse or rat), the method comprising providing the rate with a genome comprising at least 2 different non-endogenous D gene segments of the same type (eg, DZ-Zref and 02-23), wherein the D gene segments are derived from the genome sequence of different human individuals that are not genetically related over at least 3 generations. 164. The vertebrate or cell of clause 154, 156 or 158, or the method of clause 159, 161 or 163, wherein at least 2 or 3 of said different gene segments are provided cis at the same lg locus in said genome. 165, The vertebrate or cell of clause 154, 155 or 156, or the method of any one of clauses 159 to 162 and 164, wherein the D gene segments are derived from the genome sequence of different human individuals that are not genetically related over at least 3 tions. 166. The vertebrate or cell of any one of clauses 154 to 157, or the method of any one of clauses 159 to 162 and 165, wherein the D gene segments are derived from the genome sequence of two or more different human individuals; optionally wherein the different human duals are from ent human populations. 167. The vertebrate, cell or method of clause 166, wherein the individuals are not genetically related. 168. The vertebrate, cell or method of any one of clauses 154 to 167, wherein at least one of the different D segments is a synthetic mutant of a human germline D gene segment. 169. A method of enhancing the human immunoglobulin gene ity ofa non—human vertebrate (eg, a mouse or rat), the method comprising providing the vertebrate with a genome comprising at least 2 human D gene segments of the same type (eg, DZ-Zref and 02—23), wherein the D gene segments are derived from the genome sequence of different human individuals that are not genetically related over at least 3 generations; optionally wherein at least 2 or 3 of said different gene segments are provided at the same lgH locus in said genome.

W0 20131041844 PCT/GBZOIZI‘052296 170. The vertebrate or cell of any one of clauses 154 to 158 and 164 to 168, wherein the genome comprises a substantially complete onal repertoire of human D gene segment types supplemented with one, two or more variant human D gene ts, wherein said substantially complete onal repertoire and the supplementary D gene segments are not found together in the germline genome of a human dual. 171. The population of clause 157, wherein the population comprises a substantially complete functional repertoire of human D gene segment types supplemented with one, two or more variant human D gene segments, wherein said ntially complete functional repertoire and the mentary D gene segments are not found together in the germline genome of a human individual. 172. A non-human vertebrate (eg, a mouse or rat) or a non—human cell (eg, an ES cell or a B-cell) having a genome comprising a substantially complete functional repertoire of human D gene segment types supplemented with one, two or more variant human D gene segments, wherein said substantially complete functional repertoire and the supplementary D gene segments are not found together in the germline genome of a human dual. 173. A population of non-human vertebrates (eg, mice or rats) sing a ntially complete functional repertoire of human JH gene segment types supplemented with one, two or more variant human D gene segments, wherein said substantially complete functional repertoire and the supplementary D gene segments are not found together in the germline genome of a human individual. 174. The vertebrate or cell of clause 172 or the population of clause 173, comprising first and second D gene segments selected from D2-2ref and DZ-Za; or DZ-21ref and D2-21a; or D3-10ref and DB-lOa; or D3—16ref and DB-lGa; or D2-8ref and D2-8a; or D3-3ref and D3-3a; or D4-23ref and D4-233; or D6—13ref and 06-133; or WO 41844 DS-Qref and D3-93; or D4-4ref and D4-4a; or ef and ; Optionally wherein the first and/or second D gene segment is present in two or more copies.

For example, there are provided two or three copies of the first gene segment, optionally with one, two or three copies of the second gene segment. Copies can be arranged in cis or trans. 175. The vertebrate, cell or population of clause 174, comprising human gene segments DZ-Zref and 02-23; and f and D3-3a; and optionally also 02-15.

In an example, the vertebrate, cell or population comprises one or more D segments selected from human D3—3, D2-15, 03-9; 04-17; 03-10; D2-2; D5-24; 06-19; 03—22; 06—13; 05-12; 01-26; 01-20; 05-18; 03-16; D2-21; 01-14; 07-27; 01-1; 06-25; 02-14 and 04-23 (eg, selected from DB- 9*01; D4-17*01; Ol; D2-2*02; 01; D6-19‘01; 03-22*01; D6-13*01; 05—12*01; D1- 26*01; 01-20‘01; DS-18*01; D3-16*02; 02-21*02;Dl-14*01; D7-27*02; 01-1‘01; DG-ZS‘OI; D2-15*01; and D4-23*01), together with the reference sequence(s) of said ed segment(s).

These were found in variable domains having a HCDR3 length of at least 20 amino acids (see examples herein). 176. A non-human vertebrate or vertebrate cell according to clause 155, comprising a genome that comprises VH, D and JH gene repertoires comprising human gene segments, the D gene repertoire comprising one or more of a plurality of 02-2 gene segments provided by at least 2 different 02-2 gene segments in cis at the same lg locus in said genome; a plurality of 02-21 gene segments provided by at least 2 different 02-21 gene segments in cis at the same lg locus in said genome; a plurality of 03-10 gene segments provided by at least 2 different D3~10 gene segments in cis at the same lg locus in said genome; a plurality of 03—16 gene segments provided by at least 2 different 03—16 gene segments in cis at the same lg locus in said genome; a plurality of D2-8 gene segments ed by at least 2 different D2-8 gene segments in cis at the same lg locus in said genome; PCT/GBZOIZ/052296 a plurality of 03-3 gene segments provided by at least 2 different D3-3 gene segments in cis at the same lg locus in said genome; a plurality of D4-23 gene segments provided by at least 2 different 04-23 gene segments in cis at the same lg locus in said genome; a plurality of D6-13 gene segments provided by at least 2 different 06-13 gene segments in cis at the same lg locus in said ; a plurality of D3-9 gene segments provided by at least 2 different D3-9 gene segments in cis at the same lg locus in said genome; a plurality of 04-4 gene segments ed by at least 2 different D4—4 gene segments in cis at the same lg locus in said genome; and a plurality of 07-27 gene segments provided by at least 2 different D7-27 gene segments in cis at the same lg locus in said genome; optionally wherein the D gene segments are derived from the genome sequence of two or more different human individuals. 177. A man vertebrate or vertebrate cell according to clause 155, comprising a genome that comprises VH, D and JH gene repertoires comprising human gene segments, the D gene repertoire comprising one or more of a plurality of D2-2 gene segments provided by at least 2 ent 02-2 gene segments in trans in said genome; a ity of D2-21 gene segments provided by at least 2 ent D2-21 gene segments in trans in said genome; a plurality of D3-10 gene segments provided by at least 2 different D3-1O gene ts in trans in said genome; a plurality of D3-l6 gene segments provided by at least 2 different D3-16 gene ts in trans in said genome; a plurality of 02—8 gene segments ed by at least 2 different 02-8 gene segments in trans in said genome; a plurality of 03—3 gene segments provided by at least 2 different 03-3 gene segments in trans in said genome; a plurality of 04-23 gene segments provided by at least 2 different D4—23 gene segments in trans in said genome; a plurality of D6-13 gene segments provided by at least 2 different D6-13 gene segments in trans in said genome; a plurality of 03-9 gene segments provided by at least 2 different 03-9 gene segments in trans in said genome; a plurality of D4—4 gene segments provided by at least 2 different D4-4 gene segments in trans in said genome; and a plurality of 07-27 gene segments provided by at least 2 different D7-27 gene segments in trans in said genome; optionally wherein the D gene segments are derived from the genome sequence of two or more different human individuals. 178. A non-human rate or vertebrate cell (optionally an ES cell or B-cell), according to clause 154, comprising a genome that comprises VH, D and JH gene repertoires sing human gene ts, the D gene repertoire sing one or more of a plurality of 02-2 gene segments provided by at least 3 ent 02-2 gene segments; a plurality of 02—21 gene segments provided by at least 3 different 02-21 gene segments,- a plurality of D3~10 gene ts provided by at least 3 different 03-10 gene segments; a plurality of D3-16 gene segments provided by at least 3 different 03—16 gene segments; a plurality of D2-8 gene segments ed by at least 3 different 02-8 gene segments; a plurality of D3-3 gene segments provided by at least 3 different 03-3 gene segments; a plurality of D4-23 gene segments provided by at least 3 different 04—23 gene segments; a plurality of D6-13 gene segments provided by at least 3 different D6-13 gene segments; a plurality of D3—9 gene segments ed by at least 3 different D3-9 gene segments; a plurality of 04-4 gene segments ed by at least 3 different D4-4 gene segments; and a ity of 07-27 gene segments provided by at least 3 different D7-27 gene segments; optionally wherein the D gene segments are derived from the genome sequence of two or three different human individuals; optionally wherein at least 2 or 3 of said ent gene segments are provided in cis at the same WO 41844 lg locus in said genome. 179. The vertebrate or cell of clause 176, 177 or 178, wherein the ent human individuals are from different human populations. 180. The vertebrate or cell of any one of clauses 176 to 179, wherein the individuals are not genetically related. 181. The vertebrate or cell of any one of clauses 176 to 180, wherein at least one of the different D segments is a synthetic mutant of a human germline D gene segment. 182. A man rate or vertebrate cell (optionally an ES cell or B-cell) according to clause 158, comprising a genome sing human VH, D and JH gene repertoires, the D gene repertoire comprising of one or more of a plurality of D2-2 gene segments provided by at least 2 different D2-2 gene; optionally in cis in said genome; a plurality of 02-21 gene segments provided by at least 2 different D2-21 gene ; optionally in cis in said genome; a plurality of 03-10 gene segments provided by at least 2 different D3-10 gene ; optionally in cis in said genome; a ity of D3-16 gene segments provided by at least 2 different D3-16 gene; optionally in cis in said ; a plurality of D2-8 gene segments provided by at least 2 different 02—8 gene; optionally in cis in said genome; a plurality of D3-3 gene segments provided by at least 2 different 03—3 gene ; optionally in cis in said genome; a plurality of 04-23 gene segments provided by at least 2 different 04-23 gene; optionally in cis in said genome; a plurality of 06-13 gene segments provided by at least 2 ent 06-13 gene; optionally in cis in said genome; a plurality of 03-9 gene segments provided by at least 2 different D3-9 gene; ally in cis in said genome; a plurality of D4-4 gene segments provided by at least 2 different 04-4 gene; optionally in cis in PCT/GBZO]2/052296 said genome; and a plurality of D7-27 gene segments provided by at least 2 different D7-27 gene ; optionally in cis in said ; wherein the D gene segments are derived from the genome sequence of different human individuals that are not genetically related over at least 3 generations. 183. The vertebrate or cell of clause 182, wherein the human duals are from different human populations. 184. The vertebrate, cell or population of any one of clauses 154 to 183, wherein one or more of the D gene segments is a variant of a human germline D gene segment, wherein the variant gene segment s an amino acid sequence that differs by 1, 2 or 3 amino acids from the corresponding amino acid sequence encoded by the human germline D gene segment, provided in that said amino acid sequence encoded by the variant does not e a stop codon when said corresponding amino acid sequence does not include a stop codon.

Optionally, the variant and germline D gene segments encode the respective amino acid sequences in reading frame 2 (IMGT numbering). See Briney et a! 2012. 185. The vertebrate, cell or population of clause 184, n said corresponding amino acid sequence encoded by the human germline D gene segment is a hydrophilic or hydrophobic sequence (according to] Mol Biol. 1997 Jul 25;270(4):587-97; Corbett SJ et al; Table 2). 186. The vertebrate, cell or population of clause 184 or 185, sing said t and said germline human D gene segments; optionally wherein the variant and germline human D gene segments are cis on the same chromosome. 187. The vertebrate, cell or population of any one of s 184 to 186, wherein germline human D gene segment is a DZ, D3, 05 or 06 family gene segment; ally a 02-2, D2-15, 03-3, D3—9, 03-10, 03-22, 05-5, 05-18, 06-6, 06-13, 06-19 gene segment.

These D segments are usable in all three reading frames.

Optionally a variant of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all of these human germline D gene segments is used. 188. The vertebrate, cell or population of any one of clauses 154 to 187, comprising a plurality of D2-2 gene segments, wherein the plurality comprises DZ-Z gene segments that vary from each other at one or more nucleotide positions corresponding to positions 106,382,687 and 106,382,711 on human chromosome 14. 189. The vertebrate, cell or population of clause 188, n the plurality ses a human D2-2 gene segment ((optionally two copies and/or in homozygous state) comprising a thymine at a position corresponding to position 106,382,687 on human chromosome 14; and optionally no further on from the sequence of D2-2ref. 190. The vertebrate, cell or tion of clause 188 or 189, wherein the ity comprises a human D2-2 gene segment comprising a cytosine at a position ponding to position 106,382,687 on human chromosome 14; and optionally no further mutation from the sequence of DZ-Za. 191. The vertebrate, cell or population of any one of clauses 188 to 190, wherein the ity comprises a human D2-2 gene segment comprising an adenine at a position corresponding to position 106,382,711 on human chromosome 14; and optionally no further mutation from the sequence of D2-2b. 192. The vertebrate, cell or population of any one of s 188 to 191, wherein the plurality comprises a human D2-2 gene segment comprising an thymine at a position corresponding to position 2,711 on human chromosome 14; and optionally no further mutation from the sequence of DZ-Zref. 193. The vertebrate, cell or population of any one of clauses 154 to 192, comprising a plurality of 0727 gene ts, wherein the plurality comprises D7-27 gene segments that vary from each other at a nucleotide position corresponding to position 106,331,767 on human chromosome 14. 194. The vertebrate, cell or tion of clause 193, wherein the plurality comprises a human D7-27 gene segment (optionally two copies and/or in homozygous state) sing a cytosine at a position corresponding to position 106,331,767 on human chromosome 14; and optionally no further mutation from the sequence of D7-27ref. 195. The rate, cell or population of clause 193 or 194, wherein the plurality comprises a human D7-27 gene segment comprising a guanine at a position corresponding to position 106,331,767 on human chromosome 14; and optionally no further mutation from the ce of . 196. The vertebrate, cell or population of any one of clauses 154 to 195, comprising a plurality of D4-23 gene segments, wherein the plurality comprises 04-23 gene ts that vary from each other at a nucleotide position corresponding to position 106,350,740 on human chromosome 197. The rate, cell or population of clause 196, wherein the plurality comprises a human D4-23 gene segment (optionally two copies and/or in homozygous state) comprising an e at a position corresponding to position 106,350,740 on human chromosome 14; and Optionally no further mutation from the sequence of ef. 198. The vertebrate, cell or population of clause 196 or 197, wherein the plurality comprises a human D4-23 gene segment (optionally two copies and/or in homozygous state) comprising an guanine at a position ponding to position 106,350,740 on human chromosome 14; and optionally no further mutation from the sequence of . 199. The vertebrate, cell or population of any one of clauses 154 to 197, comprising a plurality of D2—21 gene segments, wherein the plurality comprises 02-21 gene segments that vary from each other at a nucleotide position corresponding to position 106,354,418 on human chromosome 200. The vertebrate, cell or population of clause 199, wherein the plurality comprises a human 02-21 gene segment nally two copies and/or in homozygous state) comprising an adenine at a position corresponding to position 106,354,418 on human chromosome 14; and optionally no further mutation from the sequence of D2-21ref. 201. The vertebrate, cell or population of clause 199 or 200, wherein the plurality comprises a human D2-21 gene segment nally two copies and/or in homozygous state) comprising 3 guanine at a position ponding to on 106,354,418 on human chromosome 14; and optionally no further mutation from the sequence of D2-21a. 202. The vertebrate, cell or population of any one of clauses 154 to 201, comprising a plurality of D3-16 gene segments, wherein the plurality comprises 03-16 gene segments that vary from each other at a nucleotide position corresponding to position 106,354,418 on human chromosome 203. The vertebrate, cell or population of clause 202, wherein the plurality comprises a human D3-16 gene segment (optionally two copies and/or in homozygous state) comprising a e at a position corresponding to position 106,361,515 on human chromosome 14; and optionally no r mutation from the sequence of D3-16ref. 204. The vertebrate, cell or population of clause 202 or 203, wherein the plurality comprises a human D3—16 gene segment (optionally two copies and/or in homozygous state) comprising a cytosine at a on corresponding to position 106,361,515 on human chromosome 14; and optionally no further on from the sequence of D3-16a. 205. The rate, cell or population of any one of clauses 154 to 204, comprising a plurality of D6-13 gene segments, wherein the plurality comprises D6-13 gene segments that vary from each other at a nucleotide position corresponding to position 7,013 on human chromosome 206. The vertebrate, cell or tion of clause 205, wherein the plurality comprises a human D6-13 gene segment (optionally two copies and/or in homozygous state) comprising a thymine at a position corresponding to position 106,367,013 on human chromosome 14; and optionally no further mutation from the sequence of D6—13ref.

PCT/G82012/052296 207. The vertebrate, cell or population of clause 205 or 206, wherein the plurality comprises a human D6-13 gene segment (optionally two copies and/or in homozygous state) comprising a cytosine at a on corresponding to position 106,367,013 on human chromosome 14; and ally no further mutation from the sequence of D6-13a. 208. The vertebrate, cell or population of any one of clauses 154 to 207, comprising a plurality of D3-10 gene segments, wherein the plurality comprises D3-10 gene segments that vary from each other at one or more nucleotide positions corresponding to positions 106,370,370 and 106,370,371 on human chromosome 14. 209. The vertebrate, cell or tion of clause 208, wherein the plurality comprises a human D3-10 gene segment (optionally two copies and/or in homozygous state) sing a thymine at a position corresponding to position 106,370,370 on human chromosome 14; and optionally no further mutation from the sequence of D3-10ref. 210. The vertebrate, cell or tion of clause 208 or 209, wherein the plurality comprises a human D3-10 gene segment (optionally two copies and/or in homozygous state) comprising a cytosine at a on corresponding to position 106,370,370 on human chromosome 14; and optionally no further mutation from the sequence of D3-10a. 211. The vertebrate, cell or tion of clause 208, 209 or 210 wherein the plurality comprises a human D3-10 gene segment (optionally two copies and/or in homozygous state) comprising an adenine at a position corresponding to position 106,370,371 on human chromosome 14; and optionally no further on from the sequence of D3-10ref. 212. The vertebrate, cell or population ofany one of clauses 208 to 211, wherein the ity comprises a human D3-10 gene segment (optionally two copies and/or in homozygous state) sing 3 guanine at a on corresponding to position 106,370,371 on human chromosome 14; and optionally no further mutation from the sequence of D3-10b. 213. The vertebrate, cell or population of any one of clauses 154 to 212, sing a plurality of D3-9 gene ts, wherein the plurality comprises 03-9 gene segments that vary from each WO 41844 PCT/G82012/052296 other at a nucleotide position corresponding to position 106,370,567 on human chromosome 214. The vertebrate, cell or population of clause 213, wherein the ity ses a human D3—9 gene segment (optionally two copies and/or in homozygous state) comprising an adenine at a position corresponding to position 0,567 on human chromosome 14; and optionally no further mutation from the sequence of D3-9ref. 215. The vertebrate, cell or population of clause 213 or 214, wherein the plurality comprises a human D3-9 gene segment (optionally two copies and/or in homozygous state) sing a thymine at a position corresponding to position 106,370,567 on human some 14; and optionally no further mutation from the ce of D3-9a. 216. The vertebrate, cell or population of any one of clauses 154 to 215, comprising a plurality of D2-8 gene segments, wherein the plurality comprises D2-8 gene segments that vary from each other at one or more nucleotide positions corresponding to positions 106,373,085; 3,086 and 106,373,089 on human chromosome 14. 217. The vertebrate, cell or population of clause 216, wherein the plurality comprises a human D2-8 gene segment (optionally two copies and/or in homozygous state) comprising a cytosine at a position corresponding to position 106,373,085 on human chromosome 14. 218. The vertebrate, cell or population of clause 216 or 217, wherein the plurality ses a human D2-8 gene segment (optionally two copies and/or in homozygous state) comprising a thymine at a position corresponding to position 106,373,085 on human chromosome 14; and optionally no further mutation from the sequence of D2-8b. 219. The vertebrate, cell or population of clause 216, 217 or 218 wherein the plurality ses a human D2-8 gene segment nally two copies and/or in homozygous state) comprising a cytosine at a position corresponding to position 106,373,086 on human chromosome 14; and 2012/052296 optionally no further mutation from the sequence of D2—8ref. 220. The vertebrate, cell or tion of any one of clauses 216 to 219, wherein the plurality comprises a human D2-8 gene segment comprising a thymine at a position corresponding to position 106,373,086 on human chromosome 14; and optionally no further on from the sequence of D2-8ref. 221. The vertebrate, cell or population of any one of s 154 to 220, comprising a plurality of 04—4 gene segments, wherein the plurality comprises D4-4 gene segments that vary from each other at one or more nucleotide positions ponding to ons 106,379,086; and 106,379,089 on human chromosome 14. 222. The vertebrate, cell or population of clause 221, n the plurality comprises a D4-4 gene t (optionally two copies and/or in homozygous state) comprising a cytosine at a position corresponding to position 106,379,086 on human chromosome 14; and optionally no further on from the sequence of f. 223. The vertebrate, cell or population of clause 221 or 222, wherein the plurality comprises a human D4-4 gene segment (optionally two copies and/or in homozygous state) comprising a thymine at a position corresponding to position 106,379,086 on human chromosome 14; and ally no further mutation from the sequence of 04-43. 224. The vertebrate, cell or population of clause 221, 222 or 223 wherein the plurality comprises a human D4-4 gene segment (optionally two copies and/or in gous state) comprising a cytosine at a position corresponding to position 106,379,089 on human chromosome 14; and optionally no further mutation from the sequence of D4-4ref or a cytosine at a position corresponding to position 106,379,086 on human chromosome 14. 225. The vertebrate, cell or population of any one of clauses 221 to 224, wherein the plurality comprises a human D4-4 gene segment (optionally two copies and/or in homozygous state) comprising a thymine at a position corresponding to position 106,373,089 on human chromosome 14; and optionally no further mutation from the sequence of D4—4a. 226. The vertebrate, cell or population of any one of clauses 154 to 225, comprising a plurality of D3-3 gene segments, wherein the plurality comprises D3-3 gene segments that vary from each other at one or more nucleotide positions corresponding to positions 106,380,241; and 106,380,246 on human chromosome 14. 227. The vertebrate, cell or population of clause 226, wherein the plurality comprises a D3-3 gene segment (optionally two copies and/or in homozygous state) sing a thymine at a position corresponding to position 106,380,241 on human chromosome 14; and optionally no further mutation from the sequence of D3-3ref. 228. The vertebrate, cell or population of clause 226 or 227, wherein the plurality comprises a human D3—3 gene segment (optionally two copies and/or in homozygous state) comprising a cytosine at a position ponding to position 106,380,241 on human chromosome 14; and optionally no further mutation from the sequence of D3-33. 229. The vertebrate, cell or population of clause 226, 227 or 228 wherein the plurality comprises a human D3—3 gene segment nally two copies and/or in homozygous state) comprising an adenine at a on corresponding to position 106,380,246 on human chromosome 14; and optionally no further mutation from the sequence of D3-3ref. 230. The vertebrate, cell or population of any one of clauses 226 to 229, wherein the plurality comprises a human D3-3 gene segment (optionally two copies and/or in homozygous state) comprising a e at a position corresponding to position 106,380,246 on human chromosome 14; and optionally no r mutation from the ce of D3-3a. l I I . I H i i .

A specific application of this configuration is the provision of multiple human JL gene segments UK and/or M) as follows (as set out in ed aphs, starting at paragraph number 80).

PCT/G32012/052296 80. A man vertebrate (eg, a mouse or rat) or a non—human vertebrate cell (eg, an ES cell or a B-cell) having a genome comprising at least 3 human JL gene segments of the same type (eg, JKl), wherein at least two of the human JL gene segments are variants that are not identical to each other. 81. A non-human vertebrate (eg, a mouse or rat) or a non—human vertebrate cell (eg, an ES cell or a B-cell) having a genome comprising at least 2 different non-endogenous JL gene segments of the same type (eg, 1K1) cis at the same lg locus. 82. A non-human vertebrate (eg, a mouse or rat) or a non-human vertebrate cell (eg, an ES cell or a B-cell) having a genome comprising at least 2 different human JLgene segments of the same type (eg, 1K1) trans at the same lg locus; and optionally a third human JL gene segment of the same type, wherein the third JL is cis with one of said 2 different JL gene segments. 83. A population of non-human vertebrates (eg, mice or rats) comprising a oire of human JL gene segments, n the plurality comprises at least 2 different human JL gene segments of the same type (eg, 1K1), a first of said different JLgene segments is ed in the genome of a first vertebrate of the population, and a second of said different JL gene segments being provided in the genome of a second vertebrate of the population, wherein the genome of the first vertebrate does not comprise the second H. gene segment. 84. A non-human vertebrate (eg, a mouse or rat) or a non—human vertebrate cell (eg, an ES cell or a B-cell) having a genome comprising at least 2 different non-endogenous JL gene segments of the same type (eg, Jxl), wherein the JL gene segments are derived from the genome sequence of different human duals that are not genetically related over at least 3 generations. 85. A method of enhancing the human immunoglobulin gene diversity of a non-human rate (eg, a mouse or rat), the method comprising ing the vertebrate with a genome comprising at least 3 human JL gene segments of the same type (eg, 1K1), wherein at least two of the human JL gene ts are variants that are not identical to each other. 86. A method of enhancing the immunoglobulin gene diversity of a non~human vertebrate (eg, a mouse or rat), the method comprising providing the vertebrate with a genome sing at least 2 different non-endogenous JL gene segments of the same type (eg, 1K1) cis at the same lg WO 41844 locus. 87. A method of enhancing the immunoglobulin gene diversity of a non-human vertebrate (eg, a mouse or rat), the method comprising providing the rate with a genome sing at least 2 different human JLgene segments of the same type(eg, Ji<1) trans at the same lg locus; and optionally a third human JL gene segment of the same type, wherein the third JL is cis with one of said 2 differentJL gene segments. 88. A method of providing an enhanced human immunoglobulin JLgene segment repertoire, the method comprising providing a population of non-human vertebrates (eg, a mouse or rat) comprising a repertoire of human lL gene segments, wherein the method comprises providing at least 2 different human JL gene segments of the same type (eg, 1K1), wherein a first of said differentJL gene segments is provided in the genome of a first vertebrate of the population, and a second of said ent JL gene segments is provided in the genome of a second vertebrate of the population, n the genome of the first vertebrate does not comprise the second JL gene segment. 89. A method of enhancing the human immunoglobulin gene diversity of a non-human vertebrate (eg, a mouse or rat), the method comprising providing the vertebrate with a genome comprising at least 2 different non-endogenous JL gene segments of the same type (eg, 1K1), wherein the JL gene segments are d from the genome sequence of different human individuals that are not genetically related over at least 3 generations. 90. The vertebrate or cell of aph 80, 82 or 84, or the method of paragraph 85, 82 or 89, wherein at least 2 or 3 of said different gene ts are provided cis at the same lg locus in said . 91. The vertebrate or cell of paragraph 80, 81 or 82, or the method of paragraph 85, 86 or 87, wherein the JL gene segments are derived from the genome sequence of different human individuals that are not genetically related over at least 3 generations. 92. The vertebrate or cell of aph 80, 81 or 82, or the method of paragraph 85, 86 or 87, wherein the JL gene segments are derived from the genome sequence of two or more different human individuals; optionally wherein the different human individuals are from different human populations. 93. The vertebrate, cell or method of aph 92, wherein the individuals are not genetically related. 94. The vertebrate, cell or method of any one of paragraphs 80 to 93, wherein at least one of the different JL ts is a synthetic mutant of a human germline JL gene segment. 95. A method of enhancing the human immunoglobulin gene diversity of a non-human vertebrate (eg, a mouse or rat), the method comprising providing the vertebrate with a genome comprising at least 2 human JL gene segments of the same type (eg, 1K1), wherein the JL gene segments are derived from the genome ce of different human individuals that are not genetically related over at least 3 generations; optionally wherein at least 2 or 3 of said ent gene segments are ed at the same Igl. locus in said genome. 96. The vertebrate or cell of any one of paragraphs paragraph 80 to 82 and 84, wherein the genome comprises a substantially complete functional repertoire of human JK and/or M gene segment types supplemented with one, two or more human JK and/or JA gene segments respectively, wherein said substantially complete functional repertoire and the supplementary gene segments are not found together in the germline genome of a human individual. 97. The population of paragraph 83, wherein the population comprises a ntially complete functional repertoire of human JL gene segment types supplemented with one, two or more human in and/or JA gene segments respectively, wherein said substantially complete functional repertoire and the mentary gene segments are not found er in the germline genome of a human individual. 98. A non-human vertebrate (eg, a mouse or rat) or a non-human cell (eg, an ES cell or a B-cell) having a genome comprising a ntially complete functional repertoire of human JK and/or JA gene segment types mented with one, two or more human JK and/or JA gene segments respectively, wherein said substantially complete functional repertoire and the supplementary gene segments are not found er in the germline genome of a human individual.

W0 2013/041844 PCT/G82012l052296 99. A population of non-human vertebrates (eg, mice or rats) comprising a substantially complete functional repertoire of human JK and/or JA gene segment types supplemented with one, two or more human JK and/or J7t gene segments respectively, wherein said substantially complete functional repertoire and the supplementary gene segments are not found together in the ne genome of a human individual. 100. A non-human vertebrate or vertebrate cell according to paragraph 81, comprising a genome that ses VL and JL gene repertoires comprising human gene segments, the JL gene repertoire comprising a plurality of human Ji<1 gene segments provided by at least 2 different human JKl gene segments in cis at the same lg locus in said genome; a plurality of human JKZ gene segments provided by at least 2 different human JKl gene segments in cis at the same lg locus in said ; a plurality of human JK3 gene segments provided by at least 2 different human JKl gene segments in cis at the same lg locus in said genome; a plurality of human JK4 gene segments provided by at least 2 different human JKl gene segments in cis at the same lg locus in said genome; a plurality of human JKS gene segments provided by at least 2 ent human JKl gene segments in cis at the same lg locus in said genome; a plurality of human JAl gene segments provided by at least 2 different human JAl gene segments in cis at the same lg locus in said ; a ity of human 1A2 gene segments provided by at least 2 ent human 1A2 gene segments in cis at the same lg locus in said genome; a plurality of human JAB gene segments provided by at least 2 different human 1A3 gene segments in cis at the same lg locus in said genome; a plurality of human JM gene segments provided by at least 2 different human 1A4 gene segments in cis at the same lg locus in said genome; a plurality of human JAS gene segments provided by at least 2 different human JAS gene segments in cis at the same lg locus in said genome; a plurality of human 1A6 gene segments ed by at least 2 ent human 1A6 gene segments in cis at the same lg locus in said genome; or a ity of human M7 gene segments provided by at least 2 different human 1A7 gene segments in cis at the same lg locus in said genome; II-S ally wherein the JLgene segments are derived from the genome ce of two or more ent human individuals. 101. A non-human vertebrate or vertebrate cell (optionally an ES cell or B-cell), according to aph 80, comprising a genome that ses VL and JL gene repertoires comprising human gene segments, the JL gene oire sing a plurality of human JKl gene segments provided by at least 3 (eg, 3,4,5, 6, or 7) different human 1K1 gene segments; a plurality of human JK2 gene segments provided by at least 3 (eg, 3, 4, 5, 6, or 7) different human JKl gene segments; a plurality of human JK3 gene ts provided by at least 3 (eg, 3, 4, 5,6, or 7) different human JKl gene segments; a plurality of human Ji<4 gene segments provided by at least 3 (eg, 3, 4, 5, 6, or 7) different human Jxl gene segments; a plurality of human JK5 gene segments provided by at least 3 (eg, 3, 4, 5, 6, or 7) different human JKl gene segments; a plurality of human Jltl gene segments provided by at least 3 (eg, 3, 4, 5, 6, or 7) different human JAl gene segments; a plurality of human JAZ gene segments provided by at least 3 (eg, 3, 4, 5, 6, or 7) different human 1A2 gene segments; a plurality of human JAB gene segments provided by at least 3 (eg, 3, 4, 5, 6, or 7) different human 1A3 gene segments; a plurality of human JA4 gene segments provided by at least 3 (eg, 3, 4, 5, 6, or 7) different human JM gene segments; a plurality of human 1A5 gene segments provided by at least 3 (eg, 3, 4, 5, 6, or 7) different human 1A5 gene segments; a plurality of human M6 gene segments provided by at least 3 (eg, 3, 4, 5,6, or 7) different human JAG gene segments; or a plurality of human JA7 gene segments provided by at least 3 (eg, 3, 4, 5, 6, or 7) different human DJ gene segments; optionally wherein the JL gene segments are derived from the genome sequence of two or three WO 41844 PCT/G32012/052296 different human individuals; optionally wherein at least 2 or 3 of said different gene ts are provided in cis at the same lg locus in said genome. 102. The vertebrate or cell of paragraph 104 or 105, n the different human individuals are from different human populations. 103. The vertebrate or cell of any one of aphs 104 to 106, n the individuals are not genetically related. 104. The vertebrate or cell of any one of paragraphs 104 to 107, wherein at least one of the different JL segments is a tic mutant of a human germline JL gene t. 105. A non-human vertebrate or vertebrate cell (optionally an ES cell or B-cell) according to paragraph 84, comprising a genome comprising human VL and JL gene repertoires, the JL gene repertoire comprising a plurality of human JKl gene segments ed by at least 2 different human JKl gene segments, optionally in cis at the same lg locus in said genome; a plurality of human JK2 gene segments provided by at least 2 different human Ji<1 gene segments, optionally in cis at the same lg locus in said genome; a plurality of human Ji<3 gene segments provided by at least 2 different human JKl gene segments, optionally in cis at the same lg locus in said genome; a plurality of human Ji<4 gene segments provided by at least 2 different human JKl gene segments, optionally in cis at the same lg locus in said genome; a plurality of human JKS gene segments provided by at least 2 different human JKl gene segments, ally in cis at the same lg locus in said genome; a plurality of human JM gene segments provided by at least 2 different human JM gene segments, optionally in cis at the same lg locus in said genome; a plurality of human JAZ gene segments provided by at least 2 different human 1A2 gene segments, optionally in cis at the same lg locus in said genome; a plurality of human M3 gene segments provided by at least 2 different human J13 gene segments, optionally in cis at the same lg locus in said genome; a ity of human M4 gene segments provided by at least 2 different human JM gene segments, Optionally in cis at the same lg locus in said genome; a plurality of human JAS gene segments provided by at least 2 ent human 1A5 gene segments, optionally in cis at the same lg locus in said genome; a plurality of human M6 gene segments provided by at least 2 different human JAB gene segments, optionally in cis at the same lg locus in said genome; or a plurality of human 1A7 gene segments provided by at least 2 different human 1A7 gene segments, optionally in cis at the same lg locus in said genome; wherein the JL gene segments are derived from the genome ce of different human individuals that are not genetically related over at least 3 generations. 106. The vertebrate or cell of paragraph 109, wherein the human individuals are from different human populations.

The d person will realise that standard molecular biology ques can be used to provide vectors comprising synthetic ations of immunoglobulin gene segments (eg, V, D and/or J) for use in the invention, such that the vectors can be used to build a transgenic immunoglobulin locus (eg, using homologous recombination and/or recombinase mediated cassette exchange as known in the art, eg, see US7501552 (Medarex), US$939598 (Abgenix), 364 (Abgenix), W002/066630 (Regeneron), W02011004192 (Genome Research Limited), 076464, W02009143472 and W02010039900 (Ablexis), the disclosures of which are explicitly incorporated herein. For example, such synthetic combinations of gene segments can be made using standard recombineering techniques in E coli to construct BAC vectors harbouring the tic combination prior to insertion in embryonic stem cells using homologous recombination or RMCE (eg, using cre/on site-specific recombination). Details of ineering can be found at wwwgengbrigggscom and in EP1034260 and EP1204740 the disclosures of which are explicitly incorporated . in one embodiment, it is useful to bias the immune response of the vertebrate (and thus resultant lead antibodies) to a predetermined gene segment, eg, one known to be commonly used in natural human immune responses to antigens, such as antigens of infectious e pathogens. For example, VH1-69 is commonly used to produce antibodies in humans against Infulenza virus; it is possible, therefore, to include two or more polymorphic DNA versions of the VH segment VH1-69 in PCT/0820121052296 the locus of the invention. The examples below illustrate how such a transgenic locus can be constructed in which diversity is extended by extending the VH1-69 gene segment repertoire based on naturally-occurring VH1-69 polymorphic variants.

In one embodiment in any configuration of the invention, the genome has been ed to prevent or reduce the expression of fully-endogenous antibody. Examples of suitable techniques for doing this can be found in PCT/GBZOlO/051122, US7501552, U56673986, U56130364, W02009/076464, EP1399559 and U56586251, the disclosures of which are incorporated herein by reference. In one embodiment, the non-human rate V0] region of the endogenous heavy chain immunoglobulin locus, and optionally VJ region of the endogenous light chain immunoglobulin loci a and/or kappa loci), have been inactivated. For example, all or part of the non-human vertebrate VDJ region is vated by inversion in the nous heavy chain immunoglobulin locus of the mammal, optionally with the ed region being moved upstream or downstream of the nous lg locus (see, eg, w02011004192, the disclosure of which is incorporated herein by nce). For example, all or part of the non-human vertebrate VJ region is inactivated by inversion in the nous kappa chain immunoglobulin locus of the mammal, optionally with the inverted region being moved upstream or downstream of the endogenous lg locus. For example, all or part of the non-human rate VJ region is inactivated by inversion in the endogenous lambda chain immunoglobulin locus of the mammal, optionally with the inverted region being moved upstream or downstream of the endogenous lg locus. In one ment the endogenous heavy chain locus is inactivated in this way as is one or both of the endogenous kappa and lambda loci.

Additionally or alternatively, the vertebrate has been generated in a genetic background which prevents the production of mature host B and T lymphocytes, optionally a RAG-l-deficient and/or RAG-2 deficient background. See US$859301 for techniques of generating RAG—1 deficient animals.

Thus, in one embodiment of any configuration or aspect of the invention herein, endogenous heavy and light chain expression has been inactivated.

W0 20131041844 PCT/G32012/052296 In one embodiment each said locus constant region is a heavy chain endogenous non-human vertebrate (optionally host mouse or rat) constant region.

In one embodiment each said locus constant region is a light chain nous man vertebrate (optionally host mouse or rat) constant region.

The invention provides a monoclonal or polyclonal antibody composition prepared by immunisation of at least one vertebrate (eg, mouse or rat) according to the ion, optionally wherein the antigen is an antigen of an infectious e pathogen (eg, a bacterial or viral pathogen antigen), optionally wherein the same antigen is used to immunise all the vertebrates; optionally wherein the antibody or antibodies are IgG-type (eg, lgGl).

The invention also provides a monoclonal or polyclonal antibody mixture produced by the method of the invention or a derivative antibody or mixture thereof, eg, where one or more constant region has been changed (eg, replaced with a different constant region such as a human constant region; or mutated to enhance or ablate Fc effector function). In an aspect of the invention, the monoclonal or polyclonal antibody mixture is provided for y and/or prophylaxis of a disease or condition in a human, eg, for the treatment and/or prevention of an ious disease, wherein optionally n each dy binds an antigen of an infectious disease pathogen, ably the same antigen.

In an aspect of the invention, there is provided the use of an isolated, monoclonal or polyclonal antibody according to the invention, or a mutant or derivative antibody thereof in the manufacture of a medicament for the treatment and/or prevention of a e or condition in a human, eg, an infectious disease, optionally wherein the infectious disease is a e caused by a bacterial or viral pathogen.

An e of a mutant antibody is one that bears up to 15 or 10 amino acid mutations in its variable regions relative to an isolated dy (eg, IgG-type, such as lgGl-type, antibody) obtainable or obtained by the method of the invention. An example of a derivative is one that has been modified to replace a constant region with a different constant region such as a human constant region; or mutated to enhance or ablate Fc effector function.

Examples of infectious diseases are diseases caused or ed by a bacterial or viral pathogen.

For example, the infectious disease is selected from the group consisting of a disease caused by a pathogen selected from the group ting of hilus influenza, E coli, Neisseria meningitidis, a herpes family virus, cytomegalovirus (CMV), HIV and influenza virus.

PCT/GB20 12/052296 The inventors realised that it would be desirable to provide for vertebrates, cells, methods etc for the production of therapeutic and/or prophylactic antibodies based on natural human immune responses to antigens, such as antigens of infectious disease pathogens. In this respect, the literature observes ntly used immunoglobulin gene segments to raise anti-infective responses in humans (Table 9).

In the various configurations, aspects, embodiments and examples above, the ion provides the skilled addressee with the possibility of ng immunoglobulin gene segments in a way that tailors or biases the repertoire for application to generating antibodies to treat and/or prevent infectious diseases. The inventors have categorised the following groups of gene segments for use in the invention according to the d application of resultant antibodies.

List A: (a) a VL gene segment ed from the group consisting ofa VAII gene family member, VAVII 4A, VAII 2.1, VAVII 4A, a VA1 gene family member, a Vx3gene family member, |GLV1SZ, Vx3-cM L70, lalh2, lalvl, la3h3, Kv325, a VKI gene family member, Kl-15A(KL012), VKII family member, a VKIII family member, a VKI gene family member, Kl-15A(KL012), Vgll A2 (optionally the A2a variant), VK A27 (Humkv3 25) and a gene segment at least 80% identical thereto. (b) a VA gene segment selected from a VAII gene family , VAVII 4A, Vxll 2.1, VAVII 4A, a Vxl gene family member, a VASgene family member, |GLV152, VA3-cML7O, lath, lalvl, |a3h3 and a gene segment at least 80% identical thereto. (c) a VK gene segment selected from Kv325, a VKI gene family , Kl—lSA ), VKII family member, a V,,l|| family member, a VKI gene family member, Kl-15A (KL012), VKII A2 (optionally the A2a t), VK A27 (Humkv325) and a gene segment at least 80% identical thereto. (d) a VH gene segment a VHIII gene family member (optionally, a VHIIIa or VHlllb family member), 3 WW gene family member, VHIII9.1(VH3-15),VHlIIVH26(VH3-23), VH3~21, , L5612.1, DP77 ), VH H11, VHIGRR, ha3h2, VHl-halcl, VHlll-VHZ-l, VH4.18, ha4h3, HleSl, 71-2, HvlflO, VH4.11, 71-4, VH251, VH1—69 and a gene t at least 80% identical thereto. (e) a J; gene segment selected from JAZ, JAB and a gene segment at least 80% identical thereto.

PCT/GBZOIZ/052296 (f) a D gene segment selected from Dkl, Dxp’l, Dn4r, DZr and a gene segment at least 80% identical o.

List A1: lmmuno Iobulin ' en se fr i tbind ana | ‘al Pathogen (a) a V) gene segment selected from a thl gene family member, VAVII 4A, Val! 2.1, VAVII 4A and a gene segment at least 80% identical thereto. (b) a VK gene segment selected from a Vxl gene family member, KI-15A(KL012),VK1| family member, a VKlll family member, a VKl gene family member, KI-15A(KL012), VKII A2 (optionally the AZa variant), VK A27 (Humkv325) and a gene t at least 80% identical thereto. (c) a VH gene segment a VH3 gene family member (optionally, a VHllla or VHlllb family member), VHIIl9.1(VH3-15), VHIII VH26 (VH3-23), VH3-21, LSGS.1, L8612.1, DP77 (V3~21), VH H11 and a gene segment at least 80% identical thereto. (d) a J) gene segment selected from JKZ, 1A3 and a gene segment at least 80% identical thereto. (e) a JH gene segment selected from JH2, 1H3, LA and a gene t at least 80% identical thereto. (a) a VA gene segment selected from a VAII gene family member, VAVII 4A, VAII 2.1, VAVII 4A and a gene segment at least 80% identical thereto. (b) a V.‘ gene segment selected from a V,” family member, a VKIII family member, a VKI gene family member, xI-15A ), VKII A2 (optionally the AZa variant), VK A27 325) and a gene segment at least 80% identical o. (c) a VH gene segment a VH3 gene family member (optionally, a VHlllb family member), VHIII 9.1 (VH3—15), VHIII VH26 (VHS-23), VHS-21, LSGB.1, 1, DP77 (V3-21) and a gene segment at least 80% identical thereto‘ (d) a J) gene segment selected from 1A2, JAB and a gene segment at least 80% cal thereto. ”a All; WO 41844 meningitidis (a) a VH gene segment a VH3 gene family member (optionally a VHllla or VHlllb ), WI” 9.1 (VH3-15), VH H11, VHlll VH26 (VH3-23) a gene segment at least 80% cal thereto, eg, VHIII 9.1 + JHB; or VH H11 + 1H4; or VHlll VH26 + 1H2. (b) a VK gene segment selected from a VKI gene family member, Kl-15A(KL012) and a gene t at least 80% identical thereto. (c) a VA gene segment ed from a V," gene family member, Vill 2.1 and a gene segment at least 80% identical thereto. (d) a JH gene segment selected from 1H2, JH3, 1H4 and a gene segment at least 80% identical thereto. (a) a VH gene segment selected from a VHIII gene family member, a VHIV gene family member, VHIII— VH26 (VH3—23), VHlGRR, ha3h2, VHl-halcl, VHllI-VHZ-l, VH4.18, ha4h3, HleSl, 71-2, HvlflO, VH4.11, 71-4, VH251, VH1-69 and a gene segment at least 80% identical thereto. (b) a V; gene segment selected from 3 WI gene family member, a Vlagene family member, |GLV1SZ, VA3-CML70, lalh2, lalvl, la3h3 and a gene segment at least 80% identical thereto. (0) a Vk gene segment selected from Kv325 and a gene segment at least 80% identical thereto. (d) a JH gene segment ed from 1H3, 1R5, JH6 and a gene segment at least 80% identical thereto. (e) a D gene segment selected from Dkl, Dxp’l, Dn4r, DZr and a gene segment at least 80% identical o. (f) a JA gene t selected from 1,2, hi! and a gene segment at least 80% identical thereto. (a) a VH gene segment selected from a VHIII gene family member, a VHIV gene family member, VHIII- VH26 (VH3-23), VHIGRR, ha3h2, VHl-halcl, VHlll-VH2-1,VH4.18, ha4h3, and a gene segment at least 80% identical thereto. (b) a VA gene segment selected from a Vxl gene family member, a e family member, lGLVlSZ, VAS-cML70, lalh2, lalvl, |a3h3 and a gene segment at least 80% identical thereto. (c) 3 JH gene segment selected from 1H3, JHS, 1,5 and a gene segment at least 80% identical thereto. (d) a D gene segment selected from Dkl, Dxp'l, Dn4r, Mr and a gene segment at least 80% cal thereto, (e) a J; gene segment ed from JAZ, J73 and a gene segment at least 80% identical thereto.

Imm l Ii i ' ' n n e mentsfora anti enex r b (a) a VH gene segment selected from Hv1051 and a gene segment at least 80% cal thereto. (b) a Vk gene t selected from Kv325 and a gene segment at least 80% identical thereto. (a) a VH gene segment selected from 71-2, Hv1f10, VH4.11, 71~4, VH251, VH 1-69 and a gene segment at least 80% identical thereto. (a) a VH gene segment selected from VH1-69 and a gene segment at least 80% identical o, Thus, Where one wishes to generate an dy or antibody mixture to treat and/or prevent an infectious disease, one or more V, D and/or or all J gene segments used in any configuration, aspect, method, example or ment of the invention can be selected from List A1. Thus, for example in (a) of the first configuration ofthe invention, the recited heavy chain V gene segment is selected from the VH gene segments in List A, optionally with a D in that list.

Where one wishes to generate an antibody or antibody mixture to treat and/or prevent an infectious disease caused or mediated by a bacterial pathogen, one or more or all V, D and/or] gene W0 2013/0418“ ZOI2/052296 segments used in any configuration, aSpect, method, example or embodiment of the invention can be selected from List A1.

Where one wishes to generate an antibody or antibody mixture to treat and/or prevent an infectious disease caused or ed by a viral pathogen, one or more or all V, D and/or] gene segments used in any uration, aspect, method, e or embodiment of the invention can be selected from List A2.

Where one wishes to generate an antibody or antibody mixture to treat and/or prevent an infectious disease caused or mediated by H influenza, one or more or all V, D and/orJ gene segments used in any configuration, aspect, method, example or ment of the invention can be selected from List A1.1.

Where one wishes to generate an antibody or antibody mixture to treat and/or prevent an infectious disease caused or ed by E Coli or Neisseria meningitidis, one or more or all V, D and/or] gene segments used in any uration, aspect, method, example or embodiment of the invention can be selected from List ALL Where one wishes to generate an antibody or antibody mixture to treat and/or prevent an infectious disease caused or mediated by Herpes Virus Family (eg, VZV or HSV), one or more or all V, D J gene segments used in any configuration, aspect, method, example or embodiment of the invention can be selected from List A2.1.

Where one wishes to generate an antibody or antibody mixture to treat and/or prevent an infectious disease caused or mediated by CMV, one or more or all V, D and/or 1 gene segments used in any configuration, aspect, method, e or embodiment of the invention can be selected from List A2.2.

Where one wishes to generate an antibody or antibody mixture to treat and/or prevent an infectious disease caused or mediated by HIV, one or more or all V, D and/or] gene ts used in any configuration, , method, example or embodiment of the invention can be selected from List A23.

Where one wishes to generate an dy or antibody mixture to treat and/or prevent an infectious disease caused or mediated by Influenza Virus, one or more or all V, D and/or] gene PCT/G32012/052296 segments used in any configuration, aspect, method, example or ment of the invention can be selected from List A2.4.

Optionally each VH segment in the locus of the invention is selected from List A1, A2, A11, A12, A21, A22, A23 or A2.4.

Optionally each VL segment in the locus of the invention is selected from List A1, A2, A1.1, A12, A2.1, A22, A23 or A2.4 Optionally each D segment in the locus of the invention is selected from List A1, A2, A1.1, A12, A21, A22, A23 or A2.4.

Optionally each JL segment in the locus of the inventiOn is selected from List A1, A2, A11, A12, A2.1, A22, A23 or A2.4.

‘II'EE EEIl'lE‘ Ei'fi The inventors, having undertaken the extensive Bioinformatics analysis exercise described herein, realised that the output of that analysis has made it possible to identify specific gene segments that are useful to produce antibody- and VH domain—based drugs that are tailored specifically to a patient’s ancestry (ie, genotype). That is, antibodies can be selected on the basis that they are made in vivo in a transgenic non-human rate (eg, mouse or rat with transgenic lgH loci) and ularly derived from gene segments that are relatively prevalent in members ofthe patient’s population, ie, from duals of the same human ancestry. Since variant distributions differ across different populations (see Table 13), this ably reflects the s of evolution, adaptation and conservation of useful variant gene types in those populations. Thus, by tailoring the antibody- based drugs according to the invention, it is possible to match the drug to the population gene , thus with the aim of making better drugs for that specific population of humans. Better can, for example, mean more efficacious, better neutralising, higher target antigen affinity, less immunogenic, less patient reactions to the drug etc. This can be ined empirically, as is standard in drug research and development processes.

Thus, the invention es the following embodiments (numbered from clause 345 onwards):- 345. An isolated antibody for administration to a Chinese patient, the antibody comprising a human heavy chain, the heavy chain comprising a le domain that is specific for an antigen and a constant region, wherein the constant region is a human nt region selected from a constant region (eg, an IGHG constant region) inTable 13 found in a Chinese tion and with a tive frequency of at least 1 or S%,-, and n (i) the variable domain is derived from the recombination of said human gene segments in a non-human vertebrate (eg, in a mouse or a rat); and/or ( ii) the variable domain ses non- human vertebrate (eg, mouse or rat) AID-pattern mutations and non-human vertebrate (eg, mouse or rat) terminal ucleotidyl transferase (TdT)-pattern mutations.

In another embodiment, the invention provides An isolated antibody for administration to a e patient, the antibody comprising a human heavy chain, the heavy chain comprising a variable domain that is specific for an antigen and a nt region, wherein the constant region is a human constant region selected from a constant region (eg, an IGHG constant region) present in a Chinese population with a cumulative frequency of at least 5%;, and wherein (i) the variable domain is derived from the recombination of said human gene segments in a non-human vertebrate (eg, in a mouse or a rat);and/ or( ii) the variable domain comprises non- human vertebrate (eg, mouse or rat) AID-pattern mutations and non-human vertebrate (eg, mouse or rat) terminal deoxynucleotidyl transferase (TdT)-pattern mutations In an example, the constant region is found in the 1000 Genomes se. In an example, the constant region is found in Table 13. 346‘ The antibody of clause 345 wherein the constant region is a IGHGIa, lGHG2a, IGHGaa, IGHGBb or lGHG4a constant region. 347. The antibody of clause 345 or 346, wherein the variable domain is derived from the recombination of a human VH gene segment with a human D gene segment and a human JH gene segment, the VH gene segment being selected from a VH in Table 13 found in a Chinese population and with a cumulative frequency of at least 5%.

In another embodiment, the ion provides The dy of clause 345 or 346, wherein the variable domain is derived from the ination of a human VH gene segment with a human D gene segment and a human JH gene segment the VH gene segment being selected from a VH present in a Chinese population with a cumulative frequency of at least 5%.

In an e, the gene segment is found in the 1000 Genomes database. In an example, the gene segment is found in Table 13. 348. The antibody of clause 345, 346 or 347, wherein the le domain is derived from the recombination of a human VH gene segment with a human D gene segment and a human JH gene segment the D gene segment being selected from 3 Din Table 13 found in a Chinese population and with a cumulative frequency of at least 5%.

In another embodiment, the invention provides The antibody of clause 345, 346 or 347, wherein the variable domain is derived from the recombination of a human VH gene segment with a human D gene t and a human JH gene segment, the D gene segment being selected from a D t in a Chinese population with a cumulative frequency of at least 5%.

In an example, the gene segment is found in the 1000 Genomes database. In an e, the gene segment is found in Table 13. 349. The antibody of clause 345, 346, 347 or 348 n the variable domain is derived from the recombination of a human VH gene segment with a human D gene segment and a human JH gene segment , the JH gene segment being selected from a JH in Table 13 found in a Chinese population and with a cumulative frequency of at least 5%.

In r ment, the invention provides The antibody of clause 345, 346, 347 or 348 n the variable domain is derived from the recombination of a human VH gene segment with a human D gene segment and a human JH gene segment, the JH gene segment being selected from a JH present in a e population with a cumulative frequency of at least 5%.

In an example, the gene segment is found in the 1000 Genomes database. In an example, the gene segment is found in Table 13. 350. An isolated VH domain identical to a variable domain as recited in any one of clauses 347 to 349, ally fused at its C-terminus to a polypeptide (eg, an antibody Fc).

In an embodiment, there is provided an isolated VH domain identical to a variable domain as recited in any one of clauses 347 to 349 which is part of a conjugate, conjugated with a label (eg, for imaging in the patient) or a toxin (eg, a radioactive toxic payload, such as for cancer treatment in the patient} or a half—life-extending moiety (eg, PEG of human serum albumin). 351. A pharmaceutical composition comprising the antibody or variable domain of any one of clauses 345 to 350 together with a pharmaceutically-acceptable excipient, diluent or a medicament (eg, a further n-specific variable domain, antibody chain or antibody). 352. An isolated antibody for administration to a Chinese patient, the dy comprising a human heavy chain, the heavy chain comprising a variable domain that is specific for an antigen PCT/G82012/052296 and a constant region, wherein the le domain is derived from the recombination of a human VH gene segment with a human D gene segment and a human JH gene segment the VH gene segment being selected from a VH in Table 13 found in a Chinese population and with a cumulative frequency of at least 5%; and n (i) the variable domain is derived from the recombination of said human gene segments in a non-human vertebrate (eg, in a mouse or a rat); and/or( ii) the variable domain comprises non- human vertebrate (eg, mouse or rat) AID—pattern mutations and non-human vertebrate (eg, mouse or rat) terminal ucleotidyl transferase (TdT)-pattern ons.

In another embodiment, the invention provides An isolated antibody for administration to a Chinese patient, the antibody comprising a human heavy chain, the heavy chain comprising a variable domain that is specific for an antigen and a constant region, wherein the variable domain is derived from the recombination of a human VH gene segment with a human D gene segment and a human JH gene segment , the VH gene segment being selected from a VH present in a Chinese population with a cumulative frequency of at least 5%; and n (i) the variable domain is derived from the ination of said human gene segments in a non-human vertebrate (eg, in a mouse or a rat); and/or( ii) the variable domain comprises non- human vertebrate (eg, mouse or rat) AID-pattern mutations and non-human vertebrate (eg, mouse or rat) terminal deoxynucleotidyl transferase (TdT)-pattern mutations. 353. The antibody of clause 352, wherein the variable domain is derived from the ination of a human VH gene segment with a human D gene segment and a human JH gene segment, the D gene t being selected from a D in Table 13 found in a Chinese population and with a cumulative ncy of at least 5%.

In another ment, the invention provides The antibody of clause 352, wherein the variable domain is derived from the recombination of a human VH gene segment with a human D gene t and a human JH gene segment the D gene segment being selected from a D present in a Chinese population with a tive frequency of at least 5%.

In an example, the gene segment is found in the 1000 Genomes database. In an example, the gene segment is found in Table 13. 354. The antibody of clause 352 or 353, wherein the variable domain is d from the recombination ofa human VH gene segment with a human D gene segment and a human JH gene segment the JH gene segment being selected from a JH in Table 13 found in a Chinese population and with a tive frequency of at least 5%.

PCT/032012/052296 In another embodiment, the invention provides The antibody of clause 352 or 353, wherein the variable domain is derived from the recombination of a human VH gene segment with a human D gene segment and a human JH gene t the JH gene segment being selected from 3 JH present in a Chinese population with a cumulative frequency of at least 5%.

In an example, the gene segment is found in the 1000 Genomes se. In an example, the gene segment is found in Table 13. 355. An isolated VH domain identical to a variable domain as recited in any one of clauses 352 to 354, optionally fused at its C-terminus to a polypeptide (eg, an antibody PC). in an ment, there is ed a VH domain identical to a variable domain as recited in any one of clauses 352 to 354 which is part of a conjugate, conjugated with a label (eg, for imaging in the patient) or a toxin (eg, a radioactive toxic payload, such as for cancer treatment in the patient) or a half-life-extending moiety (eg, PEG of human serum albumin). 356. A pharmaceutical composition comprising the antibody or variable domain of any one of s 352 to 355 together with a pharmaceutically-acceptable excipient, diluent or a medicament (eg, a further antigen-specific variable domain, antibody chain or antibody). 357. An antibody heavy chain or VH domain (eg, provided as part of an antibody) for y and/or laxis of a disease or medical condition in a Chinese patient, wherein the heavy chain is a heavy chain produced by the following steps (or is a copy of such a heavy chain):- (a) Selection of an antigen-specific antibody heavy chain or VH domain from a non-human rate (eg, a mouse or a rat), wherein the heavy chain or VH domain is derived from the recombination of a human VH gene segment with a human D gene segment and a human JH gene segment, the VH gene segment being selected from a VH in Table 13 found in a Chinese population and with a cumulative frequency of at least 5%; (b) Optional humanisation of the heavy chain by combining the variable domain of the heavy chain with a human constant region; or al humanisation of the selected VH domain by combining with a human constant region.

In r embodiment, the invention provides An antibody heavy chain or VH domain (eg, provided as part of an antibody) for therapy and/or prophylaxis of a disease or medical condition in a Chinese t, wherein the heavy chain is a heavy chain produced by the following steps (or is a copy of such a heavy chain):- (a) Selection of an antigen-specific antibody heavy chain or VH domain from a non-human vertebrate (eg, a mouse or a rat), n the heavy chain or VH domain is derived from the 2012/052296 recombination of a human VH gene segment with a human D gene segment and a human JH gene segment the VH gene segment being selected from a VH present in a Chinese population with a cumulative frequency of at least 5%; (b) Optional humanisation of the heavy chain by combining the variable domain of the heavy chain with a human constant region; or optional humanisation of the selected VH domain by combining with a human constant region.

In an e, the VH gene segment is found in the 1000 Genomes database. In an example, the gene segment is found in Table 13. 358. The antibody heavy chain or VH domain of clause 357, wherein the human constant region is as recited in clause 345 or 346. 359. An antibody heavy chain or VH domain as d in clause 357 or 358 for use in a medicament for therapy and/or prophylaxis of a disease or medical condition in a Chinese patient. 360. A method of treating and/or preventing a disease or medical condition in a Chinese t, the method comprising stering to the patient a therapeutically or prophylactically- effective amount of the antibody heavy chain or VH domain as recited in clause 357 or 358. 361. An isolated antibody for stration to a patient of European, East Asian, West African, South Asian or Americas ancestry, the dy comprising a human heavy chain, the heavy chain comprising a le domain that is specific for an antigen and a constant region, wherein the constant region is a human constant region selected from a constant region (eg, an IGHG constant region) in Table 13 found in a population of European, East Asian, West African, South Asian or Americas ry respectively and with a cumulative frequency of at least 1 or 5%;, and wherein (i) the variable domain is derived from the recombination of said human gene segments in a non-human vertebrate (eg, in a mouse or a rat); or( ii) the variable domain comprises non- human vertebrate (eg, mouse or rat) AID-pattern mutations and non—human vertebrate (eg, mouse or rat) terminal deoxynucleotidyl transferase (TdT)-pattern mutations. in another embodiment, the ion provides An isolated antibody for administration to a t of European, East Asian, West African, South Asian or Americas ry, the antibody comprising a human heavy chain, the heavy chain comprising a le domain that is specific for an antigen and a constant region, wherein the constant region is a human constant region selected from a constant region (eg, an IGHG constant region) present in a population of European, East Asian, West African, South Asian or Americas ry respectively with a cumulative frequency of at least 1 or 5%;, and wherein (i) the variable domain is derived from the recombination of said human gene segments in a W0 2013/0418“ PCT/G82012/052296 non-human vertebrate (eg, in a mouse or a rat); or ( ii) the variable domain comprises non- human vertebrate (eg, mouse or rat) AID-pattern mutations and non-human vertebrate (eg, mouse or rat) terminal deoxynucleotidyl transferase (TdTl-pattern mutations.

In an example, the constant region is found in the 1000 Genomes database. In an example, the constant region is found in Table 13. 362. The antibody of clause 361 n the constant region is a IGHGla, IGHGZa, IGHG3a, |GHG3b or IGHG4a constant region and the patient is of European ancestry. 363. The antibody of clause 361 or 362, wherein the le domain is derived from the recombination of a human VH gene t with a human D gene segment and a human JH gene segment, the VH gene segment being selected from a VH in Table 13 found in said population and with a cumulative ncy of at least 1 or 5%.

In another embodiment, the ion es The antibody of clause 361 or 362, n the variable domain is derived from the recombination of a human VH gene segment with a human D gene segment and a human JH gene segment the VH gene segment being selected from a VH present in a Chinese tion with a cumulative frequency of at least 5%.

In an example, the gene segment is found in the 1000 Genomes database. In an example, the gene segment is found in Table 13. 364. The antibody of clause 361,362 or 363, wherein the le domain is derived from the recombination of a human VH gene segment with a human D gene segment and a human JH gene segment, the D gene segment being selected from a D in Table 13 found in said population and with a cumulative ncy of at least 1 or 5%.

In another embodiment, the invention provides The antibody of clause 361, 362 or 363, wherein the variable domain is derived from the recombination of a human VH gene segment with a human D gene segment and a human JH gene segment , the 0 gene segment being selected from a D present in a Chinese population with a tive frequency of at least 5%.

In an example, the gene segment is found in the 1000 Genomes database. In an example, the gene t is found in Table 13. 365. The antibody of clause 361, 362, 363 or 364 wherein the variable domain is derived from the recombination of a human VH gene segment with a human D gene segment and a human JH gene segment the JH gene segment being selected from 3 JH in Table 13 found in said population and with a cumulative frequency of at least 1 or 5%.

PCT/GBZOIZ/052296 In another ment, the invention provides The antibody of clause 361, 362, 363 or 364 wherein the variable domain is derived from the recombination of a human VH gene segment with a human D gene segment and a human JH gene segment the JH gene segment being selected from a JH t in a Chinese population with a cumulative frequency of at least 5%.

In an example, the gene segment is found in the 1000 Genomes se. In an example, the gene t is found in Table 13. 366. An isolated VH domain identical to a variable domain as recited in any one of clauses 363 to 365, optionally fused at its C-terminus to a polypeptide (eg, an dy EC). 367. A pharmaceutical composition sing the antibody or variable domain of any one of clauses 361 to 366 together with a pharmaceutically—acceptable excipient, diluent or a medicament (eg, a further antigen-specific variable domain, antibody chain or antibody). 368. An isolated antibody for administration to a patient of European, East Asian, West n or Americas ancestry, the antibody comprising a human heavy chain, the heavy chain comprising a variable domain that is specific for an antigen and a constant region, wherein the variable domain is derived from the recombination of a human VH gene segment with a human D gene segment and a human JH gene t , the VH gene segment being selected from a VH in Table 13 found in a population of European, East Asian, West African, South Asian or Americas ancestry tively and with a cumulative frequency of at least 1 or 5%; and wherein (i) the variable domain is derived from the recombination of said human gene segments in a non-human vertebrate (eg, in a mouse or a rat); or ( ii) the variable domain ses non- human rate (eg, mouse or rat) AID-pattern mutations and non-human vertebrate (eg, mouse or rat) terminal deoxynucleotidyl transferase (TdT)-pattern mutations. in another embodiment the invnention provides:- An isolated antibody for administration to a patient of European, East Asian, West African or Americas ancestry, the antibody comprising a human heavy chain, the heavy chain comprising a variable domain that is specific for an antigen and a constant region, wherein the variable domain is derived from the recombination ofa human VH gene segment with a human D gene segment and a human JH gene segment, the VH gene segment being selected from a VH present in a population of an, East Asian, West African, South Asian or Americas ancestry respectively with a cumulative ncy of at least 1 or 5%; and wherein (i) the variable domain is derived from the recombination of said human gene segments in a non-human rate (eg, in a mouse or a rat); or ( ii) the variable domain comprises non- human vertebrate (eg, mouse or rat) AID-pattern mutations and non—human vertebrate (eg, PCT/G32012/052296 mouse or rat) terminal deoxynucleotidyl transferase (TdT)-pattern mutations.

In an example, the VH gene segment is found in the 1000 Genomes se. in an example, the gene segment is found in Table 13. 369. The antibody of clause 368, wherein the variable domain is derived from the recombination of a human VH gene segment with a human D gene segment and a human JH gene segment , the D gene segment being selected from a D in Table 13 found in said population and with a tive frequency of at least 1 or 5%. in another example there is provided The antibody of clause 368, wherein the variable domain is derived from the ination of a human VH gene segment with a human D gene segment and a human JH gene segment, the D gene segment being selected from a D t in said population with a cumulative frequency of at least 1 or 5%.

In an example, the D gene segment is found in the 1000 Genomes se. In an example, the gene segment is found in Table 13. 370. The antibody of clause 368 or 369, wherein the variable domain is derived from the recombination of a human VH gene segment with a human D gene segment and a human JH gene segment , the JH gene segment being selected from 3 JH in Table 13 found in said population and with a cumulative frequency of at least 1 or 5%.

In another e there is provided The antibody of clause 368 or 369, wherein the variable domain is derived from the recombination of a human VH gene segment with a human D gene segment and a human JH gene segment the JH gene segment being selected from a JH present in said tion and with a cumulative frequency of at least 1 or 5%.

In an e, the JH gene segment is found in the 1000 Genomes database. In an example, the gene segment is found in Table 13. 371. An isolated VH domain identical to a variable domain as recited in any one of clauses 368 to 370, optionally fused at its C-terminus to a polypeptide (eg, an antibody PC). 372. A ceutical composition comprising the dy or variable domain of any one of clauses 368 to 371 together with a pharmaceuticaIIy-acceptable excipient, diluent or a medicament (eg, a further antigen-specific variable domain, antibody chain or antibody). 373. An antibody heavy chain or VH domain (eg, provided as part of an antibody) for y and/or prophylaxis of a disease or medical condition in a patient of European, East Asian, West African, South Asian or Americas ancestry, wherein the heavy chain is a heavy chain produced by PCT/G32012/052296 the following steps (or is a copy of such a heavy chain):- (a) Selection of an antigen-specific antibody heavy chain or VH domain from a non-human vertebrate (eg, a mouse or a rat), wherein the heavy chain or VH domain is derived from the recombination of a human VH gene segment with a human D gene segment and a human JH gene t, the VH gene t being selected from a VH in Table 13 found in said population and with a cumulative frequency of at least 1 or 5%; (b) Optional humanisation of the heavy chain by combining the variable domain of the heavy chain with a human constant ; or optional humanisation ofthe selected VH domain by combining with a human nt region.

In another embodiment, there is ed:- An antibody heavy chain or VH domain (eg, provided as part of an antibody} for therapy and/or prophylaxis of a disease or l condition in a patient of European, East Asian, West African, South Asian or Americas ry, wherein the heavy chain is a heavy chain produced by the following steps (or is a copy of such a heavy chain):- (a) Selection of an antigen-specific antibody heavy chain or VH domain from a non~human vertebrate (eg, a mouse or a rat), wherein the heavy chain or VH domain is derived from the recombination of a human VH gene segment with a human D gene segment and a human JH gene segment, the VH gene segment being selected from a VH present in said population with a tive frequency of at least 1 or 5%; (b) Optional humanisation of the heavy chain by combining the variable domain of the heavy chain with a human constant region; or optional humanisation of the selected VH domain by combining with a human constant region.

In an example, the VH gene segment is found in the 1000 Genomes database. In an example, the gene segment is found in Table 13. 374. The antibody heavy chain or VH domain of clause 373, wherein the human constant region is as recited in clause 361 or 362. 375. An antibody heavy chain or VH domain as recited in clause 373 or 374 for use in a medicament for y and/or prophylaxis ofa disease or medical ion in a patient of said ancestry. 376. A method of treating and/or preventing a disease or medical condition in a patient of an, East Asian, West African, South Asian or Americas ancestry, the method comprising administering to the patient a therapeutically or prophylactically-effective amount of the antibody heavy chain or VH domain as recited in clause 373 or 374.

PCT/G32012/052296 In embodiments herein, a Chinese patient can be a Han Chinese patient.

In embodiments herein, a patient of European ancestry can be a patient of Northern or n an ancestry, Italian ancestry, British or Scottish ry, Finnish ancestry or Iberian ancestry.

In embodiments herein, a patient of East Asian ancestry can be a patient of Han e ancestry, Japanese ancestry Chinese Dai ancestry, Vietnamese ancestry or Kinh ancestry.

In embodiments herein, a patient of West African ancestry can be a patient of Yoruba ancestry, Luhya ancestry, Gambian ry or Malawian ancestry.

In embodiments , a patient ofAmericas ancestry can be a patient of African American ancestry, African Caribbean ancestry, n ancestry, Puerto Rican ancestry, Colombian ancestry or Peruvian ancestry.

In embodiments herein, a patient of South Asian ancestry can be a patient of Ahom ancestry, Kayadtha ancestry, Reddy ancestry, a ancestry, or Punjabi ancestry.

In an example of any aspect, the cumulative frequency is at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, , 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95%.

It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the ion. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine study, numerous lents to the specific procedures bed herein. Such lents are considered to be within the scope of this invention and are covered by the claims. All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent ation was specifically and dually indicated to be incorporated by reference. The use of the word "a“ or "an“ when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one.“ The use PCT/G32012/052296 of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the atives are mutually exclusive, although the disclosure supports a tion that refers to only alternatives and “and/or." hout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as “comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or “containing" (and any form of containing, such as ins" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps The term "or ations thereof" as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or ations thereof is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

Any part of this sure may be read in combination with any other part of the disclosure, unless otherwise apparent from the context.

All of the itions and/or methods disclosed and claimed herein can be made and ed without undue experimentation in light ofthe present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that ions may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the t, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as d by the appended claims.

The present invention is described in more detail in the following non limiting prophetic Examples, mums.

EXAMPLE 1 "Recombineered BAC Vectors to add Polymorphic V-regions to the Mouse Genome" Figure 1 h 3 depict recombineering methods (see references above) that can be used to uce polymorphic V-gene regions into genomic DNA. In one embodiment, a genomic fragment from the human heavy chain region is inserted into a bacterial artificial chromosome (BAC) vector by standard techniques. ably, such a BAC, which can range in size from 20-kb to ZOO-kb or more, can be isolated from libraries of BACs by standard techniques ing sequence searches of commercially ble libraries or by hybridization to bacterial colonies containing BACs to identify those with a BAC of interest.

A BAC is chosen that has several VH gene segments; in Figure 1, these are generically identified as VH[a] through VH[z] for example. One skilled in the art will readily identify appropriate genomic fragments, for example, an approximately 120-kb fragment from human VHS-78 through VH1-68 which includes 5 endogenous active VH gene segments and 7 VH psuedogenes. Using recombineering techniques, the endogenous Vl-l gene segments can be replaced by rphic VH or VL gene segments. In this example, two steps are required. The first step replaces the V-region coding exon of an endogenous VH gene segment with a positive-negative selection operon, in this example, an operon encoding an ampicillin resistance gene (Amp) and a streptomycin-sensitizing ribosomal protein (rpsL). Certain strains of ia can be ed for the absence of the rpsL gene by ance to streptomycin. Short stretches of DNA homologous to sequences flanking the PCT/GBZOl2/052296 endogenous VH gene exon are placed 5’ and 3' of the rpsL-Amp operon. In the presence of riate recombination factors per standard recombineering ques (see references above) recombination between the operon fragment and the BAC will result in replacement of the endogenous VH gene exon with the operon (Figure 1a) which are selected by resistance to llin. The second step uses the same homologous sequences in order to replace the inserted operon with a desired polymorphic VH gene segment. In this example, a human VH1-69 gene is inserted (Figure 1b and 1c). In particular the *02 t of VH1-69 is used [ref IMGT and Figure 5].

Successful integrations of the polymorphic VH gene segment are selected in bacteria that become resistant to streptomycin due to the loss of the operon, specifically the rpsL n.

In this example, the two step process as described can be repeated for each of the nous VH gene segments or for as many endogenous gene segments that one wishes to replace with polymorphic V gene segments (Figure 1d).

As is apparent, any polymorphic V gene segment can be inserted in this manner and any endogenous V gene t can act as a target, including pseudogenes. V gene segments in each of the heavy chain and two light chain loci can be replaced using this technique with appropriate genomic fragments available as BAC inserts.

Figure 2 depicts another method for creating a genomic nt encoding polymorphic V gene segments. In this example, polymorphic V gene segments are ed into a region of c DNA devoid of other genes, control elements or other functions. Such ’desert‘ regions can be selected based on sequence analysis and corresponding DNA fragments cloned into BACs or identified in existing BAC libraries. Starting with such a genomic fragment, ineering techniques can be used to insert polymorphic V gene segments at intervals of, for example, 10-kb. In this example, a 150-kb genomic fragment might odate insertion of up to 15 polymorphic V gene segments.

Insertion of the segments is a two-step process. The first recombineering step inserts the rpsL-Amp operon at a specific site. Sequences homologous to a specific site are used to flank the operon.

These are used by the recombineering system to insert the element ically into the BAC genomic fragment and positive events are selected by resistance to ampicillin (Figure 2a). The second step replaces the operon in the genomic fragment with a polymorphic V gene segment by a r recombineering step using the same sequence homology (Figure 2b). In this example, both exons and promoter element of a polymorphic VH gene segment are inserted, resulting in replacement of the rpsL-Amp operon and therefore resistance to streptomycin (Figure 2c).

The two step technique for inserting polymorphic V gene ts into a specific site on the genomic fragment can be repeated multiple times resulting in a BAC genomic fragment with several polymorphic gene ts, including their promoter elements. It is apparent that the examples shown in Figures 1 and 2 can be combined wherein the technique for ion can be used to add extra polymorphic V gene segments to a BAC genomic fragment as depicted in Figure 1. One might choose to add these extra segments to an IG genomic fragment since such a fragment would be more amenable to proper IG gene expression once inserted into a non-human mammal’s genome. It is known that a genomic fragment can have elements such as enhancers or elements that contribute to certain tin conformations, both important in ype gene sion.

Figure 3 depicts an additional method to create genomic fragments with polymorphic V gene segments. This method depends upon the efficiency with which short (around 50 to 150 bases, preferably 100 bases) single stranded DNA fragments recombine with a homologous sequence using recombineering (Nat Rev Genet. 2001 Oct;2(10):769—79; Recombineering: a powerful new tool for mouse functional genomics; Copeland NG, Jenkins NA, Court DL). The recombinases used in recombineering preferentially bind and use such short single—stranded fragments of DNA as a substrate for initiating homologous recombination. The efficiency can be as high as 10-2, that is, a positive event can be found in approximately 100 randomly picked (not ed) clones resulting from recombineering. A positive event in this example occurring when one or more single nucleotide changes introduced into the single—stranded fragment get transferred to the BAC insert containing V gene segments and nding genomic DNA, said nucleotide change or s occurring at a homologous sequence on the BAC.

Polymorphic V gene segments can differ from endogenous V gene segments by only 1 or 2, or up to or 15 nucleotide changes, for example. An example of such nucleotide polymorphisms are depicted in Figure 5. Short single stranded regions that encompass the rphic nucleotide changes can be chemically sized using standard techniques. The ing single stranded DNA fragments are introduced into bacteria and via recombineering techniques approximately 1 in 100 BAC fragments will have incorporated the polymorphic nucleotides w‘a gous incorporation of the single stranded fragment (Figure 3a). BACs with the desired nucleotide change can be identified by screening for example several hundred individual clones by polymerase chain reaction (PCR) amplification and sequencing, both by standard techniques. In the example, two nucleotide changes will convert a *01 gene segment into a VH1-69*02 gene segment (Figure 3b).

It is clear that this s can be repeated for multiple endogenous V gene segments contained on a single BAC genomic fragment. In addition, the techniques depicted in Figure 2 can be used to add additional polymorphic V gene segments by insertion into regions n existing V gene segments. As would be t to one d in the art, a combination of these techniques can be used to create numerous variations of both polymorphic and endogenous human V gene ts.

And it would be evident that several different genomic nts with engineered polymorphic V gene ts and endogenous human V gene segments can be combined to create even more variations.

EXAMPLE 2 ”Adding Polymorphic ons to the Genome using SRMCE of Modified BACs" Modified BACs with polymorphic V gene segments created using the methods described in e 1 can be used to alter the genome of non—human mammals. These alterations can result in an intact IG locus in which normal immunoglobin region recombination results in VDJ or VJ combinations which includes the human V gene segments. An example of how such an animal can be created is by altering the genome of, for example, mouse embryonic stem (ES) cells using the strategy outlined in Figure 4.

One technique to integrate modified BACs with polymorphic V gene segments into a genome is sequential recombinase mediated cassette exchange (SRMCE). The technique is described in W02011004192 e Research Limited), which is incorporated here in its entirety by reference.

PCT/GBZOIZ/052296 SRMCE es for a locus ed with a ’landing pad’ inserted at a specific location. This insertion can either be de novo via homologous recombination or as a consequence of a us BAC insertion. In this example, the landing pad is ed in the mouse IGH locus n the most 3' J gene segment and the Cu gene segment and a previous BAC insertion via SRMCE ques have resulted in the addition of 5 human V gene segments and 2 V region pseudogenes. The landing pad has elements as shown in Figure 4 that will allow the selection of correct insertion of a second targeting BAC fragment. The specificity of this insertion is provided by cre recombinase-mediated exchange between permissive on sites. A on site is permissive for recombination only with a compatible lox site. In this example, the loxP site will only recombine with ioxP and on2272 will only recombine with on2272. This es directionality to the ion of the BAC fragment as depicted in Figure 4b and 4c.

ES cell clones with correct insertions are selected from a pool of clones without insertions or with non-productive insertions by resistance to puromycin. ance to puromycin results from the juxtaposition of an active promoter element, PGK, with the puroTK coding region. Correct insertions are verified by rd techniques including PCR of junctions, PCR of internal elements, Southern blotting, ative genomic hybridization (CGH), sequencing and etc. In the example, correct I0x2272—lox2272 and loxP-loxP recombination also results in two intact sets of piggyBac elements that did not exist prior to insertion. An intact piggyBac element is comprised of a set of inverted repeats which are depicted in the figure by "PBS’" and ”P33”. An appropriated ed set of piggyBac elements are the substrate of piggyBac transposase which can catalyse recombination between the elements, resulting in deletion of intervening sequences as well as both elements. The DNA remaining after a piggyBac transposition is left intact and is lacking any remnant of the piggyBac element. In the example, ES cell clones with successful piggyBac transposition are selected by loss of the active puroTK element which renders the cells resistant to the drug FIAU (Figure 4c and 4d).

The final product of the SRMCE method in this example is a IGH locus with several polymorphic V gene segments inserted along with a set of endogenous unmodified VH gene segments between sequences of the mouse genome on the 5’ side and the mouse IGH constant region gene segments on the 3‘ side. The polymorphic V gene ts are positioned such that they can ipate in the recombination events associated with B cell maturation yielding VDJ gene segments. These gene PCT/GBZOIZ/052296 segments can then be transcribed and spliced to the mouse nt region. ation of these ripts will result in the production of an antibody heavy chain encoded by the polymorphic V gene segment, a human DH gene segment, a human JH gene segment and a mouse constant heavy chain gene segment.

As is well known to those skilled in the art, an ES cell clone can be used to create a line of genetically modified mice via injection of said cells into a mouse blastocyst embryo, erring the injected embryo to a suitable recipient and breeding the chimeric offspring that result. The modified gene locus can be propagated through breeding and made either zygous or homozygous depending on the genetic cross.

It is evident from the structure of the IGH locus provided in this e and by knowledge of the mechanisms involved in B cell receptor (BCR) and antibody gene rearrangements that a large set of different combinations of polymorphic V gene segments with various DH and JH gene segments will result and these can contribute to a large repertoire of functional antibody genes in a population of B cells in genetically ed animals. In this example, several different human VH 1-69 polymorphs are incorporated to provide superhuman VH diversity. This ular VH gene segment is known to be ent in antibodies that bind infectious disease pathogens (such as influenza virus) and therefore the antibody repertoire of a mouse with the genetic modification of this example would be ed to produce antibodies with a bias in favour of those that bind infectious disease pathogens. The repertoire, in other words, would have a larger subset of antibodies with superior ties for pathogen antigens. Examples of such pathogens include influenza virus, hepatitis C virus {HCV} and human immunodeficiency virus—1 ) (see also table above).

EXAMPLE 3 ”Alignment of 13 VH1—69 Alleles” Building a more diverse antibody oire by incorporating additional V gene segment polymorphs requires availability of polymorphic variants of V gene segments. One source of such variants include sequence databases. In this example, 13 distinct variants of the VH1-69 gene segment are provided.

PCT/GB2012l052296 These variant sequences and comparisons are drawn from the "IMmunoGeneTics" IMGT Information System (www.imgt.com) database. Figure 5 is a diagram of the alignment of variants ‘02 through *13 with the *01 variant. The VH1»69*01 nucleotide and amino acid sequence is provided at the top of the figure. Where the remaining variants are identical to the *01 variant sequence a dash is inserted below the sequence. Nucleotide ences are noted alongside the riate t and if the sequence change results in a protein coding change, the amino acid change is ted above the triplet.

Figure 5 s between 1 and 4 amino acid changes for each variant in comparison to the *01 variant. All of the amino acid changes occur in the part of the heavy chain protein encoding the mentarity determining regions (CDRs). These s are responsible for antigen specificity and the affinity of the antibody for the antigen. It is evident that providing additional polymorphic CDRs in a repertoire of antibodies will increase the likelihood of there being an antibody with superior g characteristics for various antigens. In several reports, it has been observed that the VH1encoded variable region ofthe heavy chain is often found in antibodies that bind influenza virus, HCV and HlV-l antigens (see table above). Therefore incorporating the polymorphic V gene segments of this e into a transgenic animal model using the methods of Examples 1 and 2 would likely result in an antibody repertoire in said enic animal with more dies that bind to antigens associated with these and other ens. And as is known in the art, a larger repertoire increases the probability of finding monoclonal antibodies using, for example, hybridoma technology, that bind with high affinity and specificity to a desired antigen.

This disclosure therefore describes in these examples a transgenic mouse model which can be immunized with pathogen or other antigens. Plasma B cells from such an immunized mouse can be used to make a hybridoma library that can be ed for production of dies that bind the pathogen antigens. This library will be superior to libraries from traditional transgenic mice for finding such antibodies given the addition of polymorphic VH1-69 gene segments to the IGH locus in said enic mouse.

These examples are not limiting to the human polymorphic V gene segments that can be chosen or to the methods used to introduce them into an animal model. The method can be used to construct a transgenic locus with immunoglobulin D and/or] segments. The V, 0,] segments can be from a plurality of human sources (optionally more than one human ethnic population).

PCT/G82012/052296 EXAMPLE 4 Data is presented for human 1H2, 5 and 6 variants. In Tables 10A, 11A and 12A samples from humans from various populations are listed where the ce analysis of the ors has revealed the presence of polymorphisms in one or both IgH JH alleles. The population codes are explained in Table 8 above. The polymorphisms are nucleotide variants from Hz, 5 and 6 reference sequences (SEQ ID NOS: 1, 2 and 3 respectively; see below). All references are sequences taken from the Ensembl database (www.ensembl.org). The JHS reference is human IgH 15-001 disclosed in that database. The JH6 reference is human IgH 16—001 disclosed in that database. The JHZ nce is human 001 disclosed in that database.

The reference nucleotide and encoded amino acid sequences are shown on the next page.

Alignments with encoded amino acid sequences are also provided, including the corresponding position numbers on human chromosome 14. t Frequencies are shown in Tables 10A, 11A and 12A and these relate to the frequency of the variants in the 1000 Genomes Database se current at October 2011).

Tables 108, 113 and IZB show the nonymous nucleotide polymorphisms in the human JH variants, as sorted by the present inventors from the 1000 Genomes database. Position s corresponding to tide positions on human chromosome 14 are shown for variant positions (chromosome 14 being the chromosome bearing the IgH locus in humans). Thus, for example, the first entry in Table 118 is ”14:106330027zA/C" which refers to a position in a variant JH5 sequence wherein the position corre5ponds to position 106,330,027 on human chromosome 14, such position being A (adenine) in the reference sequence. The "C" indicates that the present inventors observed a mutation to cytosine at this position in the variants found in the 1000 Genomes data base. This change leads to a change at the amino acid level of the encoded sequence (ie, a ynonymous" change), in this case a change from a serine (found in the reference) to an alanine in the variant.

Example 5: The genomic coding region coordinates for each target gene for variant analysis were fied from the Ensembl WWW site (www.ensembl.orgi using coordinates from the GRCh.p8 Human Genome assembly (yaw.my.nlm.nih.ggx‘grgiggslgenomelassemblx‘gm. Using the collected gene location coordinates, variant data was extracted from the public ftp site of the 1000 s Project using the Perl ’Variant Pattern Finder’ (VPF —WW finder-agi—documentationl.

Data extracted by VPF was post processed using software to extract all non—synonymous (NSS) variants with their associated genotype calls. Genotypes calls were assembled to form unique haplotypes, representing groups of N55 variants associated with 1000 Genome population groups and frequency of occurrence within those populations.

The output of the analysis results in tables such as in Table 13. The main body of the table describes each haplotype in turn giving a unique ID for that gene (in the range a-z,aa-zz), the population frequencies and ence in individuals and unique population groups; one or more subsequent columns describe the DNA base calls at each location that form the haplotype giving both the base from the reference sequence or the variant base call.

Table 13 was constructed in this manner. The table can be read as follows: The first four columns (left to right) consist of (1) the ype ID letter (’ref’ indicates reference — the DNA base call at each genomic on from the GRCh37 Human Reference ly) (2) the observed tive frequency of the haplotype among the different populations (3) the number of individuals in which a specific haplotype was ed (4} the number of unique population groups that the identified individuals belong to (the actual population group identifiers are displayed as a string of ID’s in the most right hand column for each haplotype. For example haplotype ‘a’ has a population ID string of ’3,4,9,13’).

The populations are numbered as follows ation labels being according to 1000 Genomes Project nomenclature) 1= ASW; 2: CEU; 3=CHB; 4=CHS; =CLM; 6=F|N; 7=GBR; 8=|BS; PCT/G82012/052296 9:] PT; =LWK; 11=MXL; 12=PUR; 13=TSl; 14=YR|.

Subsequent columns detail a single point t and have the following format (top to bottom) (1) the human genomic location of the variant (format [chromosome number]: [location] e.g. '14:106204113’); (2) The identifier for the point variant as defined in DbSNP (www.ncbi.n|m.nih.gov(gro]ects{SNPll; (3) One or additional rows show the amino acid change as result of the variant for a ic transcript (denoted by the Ensembl transcript ID in the most right- hand column for each row), the format is the amino acid in the reference sequence followed by ’->’ and the amino acid caused by the substitution of the variant in the reference sequence (e.g. 'Gly- >Arg’ means a that the translated nce sequence would result in a glycine at that location, whereas the substitution of the identified variant would result in ated protein containing arginine) using the IUPAC three letter amino acid codes (mtg-“gag.iggag.9rg(gublicationsmacmgfllgzzmgflflm§figglmfl uent rows (one per haplotype) show the DNA base at each location, bases matching the reference sequence are shown in black on white back ground, bases varying from the reference are shown as white text on a black ound.

The most right-hand column contains the Ensembl transcript ID's (e.g. ’ENST00000390542’) for each ofthe gene ript and relates to the amino acid changes to the left of this column. e the ripts are differing lengths each variant position may or may not have an associated amino acid change at the that position.

A functional human gene segment repertoire (from VH2-26 to 1H6, see the IMGT database for the structure of the human lgH locus; uman&grou2=lGK ) was sectored by the inventors to produce two different transgenic heavy chain alleles (denoted 52F and 53F) and corresponding mice. The transgenic alleles were expressed in the mice and the heavy chain repertoires were assessed at the RNA transcript level. Deep sequence W0 20131041844 PCT/G82012/052296 is was carried out using Bioinformatics methods to assess V, D and JH gene usage, including in variable domain sequences having a HCDR3 length of at least 20 amino acids. Endogenous, mouse variable region gene segments were vated by inversion (as per the method described in W02011004192, this disclosure being incorporated herein by reference). 'fica ‘ f ‘ r' DNA s from 9 anonymised consenting human donors were obtained by taking cheek swabs.

The samples were processed and the DNA Samples were extracted follow the protocol of QlAamp DNA Mini Kit (Cat.No.51304, Qiagen).

PCR reactions were set up to amplify the JH6 region and PCR products were sequenced (PCR Oligos sequence: Fwd. CCAGCAGAGGGTTCCATG-3’ (SEQ ID NO: 444), Rev. 5'- GGCTCCCAGATCCTCAAGGCAC-3’ (SEQ ID NO: 445)).

Sequence analysis was d out by comparing to the JH6 reference sequence from lMGT annotated se (http://www.imgt.org/), and this identified that all 9 donor genomes contained the human JH6*02 variant, with this variant being in the homozygous state in 7 out of the 9 .

The inventors also consulted the genomic sequences publicly available for Jim Watson and Craig Venter at Ensembl human genome database [http://www.ensembl.org/]. These too contained the human JH6*02 variant. This confirmed to the inventors that human JH6*02 is a common, conserved variant in humans, and thus a good candidate for construction of a transgenic lgH locus as per the invention H T l fi.|l|l “55 HE; A series of human bacterial cial chromosome (BAC) clones were identified from Ensemble (httgzflwwwensgmmgrgfindex.html) or UCSC (httgzflggngmeucsceduz) human database searches based on gene name (16H) or location (chromosome 14: 106026574-107346185). Seven human RP11 BAC clones (see an extract of the UCSC databse in Figure 10, identified BACs being circled) were selected, RP11-1065N8 BAC carrying human JH6*02. In total, the following BACs were identified as sources of human IgH locus DNA: RP11—1065N8, RP11—659819, RP11-141W, RP-112H5, RP11-101624, RP11-12F16 and RP11-47P23.

With a similar approach, different BAC ciones (eg, different RP11 clone IDs or different sources from R911) or genetically engineered BACs can be selected for insertion into the mouse IGH locus to provide different sets of human repertoires in the transgenic mouse.

Construction of Transgenic IgH Loci Insertion of human heavy gene segments from a lst IGH BAC (RP11-1065MB) into the IGH locus of mouse ABZ.1 ES cells (Baylor College of Medicine) was performed to create a heavy chain allele denoted the 51 allele. The inserted human sequence corresponds to the sequence of human chromosome 14 from position 106494908 to on 106328951 and comprises functional heavy gene ts VH2-5, VH71, VH4-4, VH1—3, VH1—2, VHS-1, D1-1, 02-2, D3-9, , 04-11, 05-12, D6-13, D1-14, 02-15, D3-16, 04-17, 05-18, 06—19, 01-20, 02-21, D3-22, D4-23, 05-24, D6-25, D1-26, D7-27, 1H1, 1H2, 1H3, 1H4, 1,5 and JH6 (in 5’ to 3’ order), wherein the JH6 was chosen to be the human JH6*02 variant. The insertion was made between ons 114666435 and 114666436 on mouse chromosome 12, which is upstream of the mouse Cu region. The mouse V“, D and JH gene segments were ed in the locus, immediately upstream of (5’ of) the ed human heavy chain DNA‘ A second allele, 52 was constructed in which more human functional VH gene segments were inserted upstream (5’) of the 5‘-most VH inserted in the $1 allele by the sequential insertion of human DNA from a second BAC (BACZ). The inserted human sequence from BACZ ponds to the sequence of human chromosome 14 from position 106601551 to position 106494909 and comprises functional heavy chain gene segments VH3-13, , VH3-9, VH1—8, VH3-7. The mouse V“, D and JH gene segments were retained in the locus, immediately upstream of (5’ of) the ed human heavy chain DNA. In a subsequent step, these were inverted to inactivate them, thereby producing 52F mice in which only the human heavy chain variable region gene segments are active.

A third allele, S3 was ucted in which more human onal V“ gene segments were inserted upstream (5') of the 5’-most VH inserted in the $2 allele by the sequential insertion of human DNA ZOIZ/052296 from a third BAC . The inserted sequence corresponds to the sequence of human chromosome 14 from position 106759988 to position 106609301, and comprises functional heavy chain gene segments, VH2-26, VH1-24, VH3-23, VH3—21, , VH1-18, and VHS-15. The mouse VH, D and JH gene segments were ed in the locus, immediately upstream of (5’ of) the inserted human heavy chain DNA. In a uent step, these were inverted to inactivate them, thereby producing 53F mice in which only the human heavy chain variable region gene segments are active.

Mice bearing either the 52F or 33F insertion into an endogenous heavy chain locus were generated from the ES cells using standard procedures. The other endogenous heavy chain locus was inactivated in the mice by insertion of an vating sequence comprising neoR into the mouse JH- Cu intron (to produce the "HA” allele).

Immunisation procedure enic mice of the 52F or 83F genotype were primed with 20-40ug recombinant proteins obtained commercially or produced in house with n 1 (OVA (Sigma A7641),- Antigen 2 (a human infectious disease pathogen antigen) and Antigen 3 (a human antigen) via the ip route in complete Freunds adjuvant {Sigma F 5881) and 10ug/animal CpG (CpG oligo; Invivogen, San Diego, California, USA) and then boosted twice in about two weekly intervals with about half the amount of antigen in incomplete Freunds adjuvant (Sigma F 5506) and 10ug/animal CpG. Final boosts were administered two weeks later iv without any adjuvant and contained 5-10 ug protein in PBS.

I'l [' I Spleens were taken 3 days after the final boost and spleenocytes were treated with CpG (25 um final concentration) for and left until the following day. Cells were then fused with SPO/2 Ag14 myeloma cells (H PA Cultures Cat No 85072401) using a BTX ECM2001 electrofusion instrument. Fused cells were left to recover for 20 minutes then seeded in a T75 flask until next morning. Then the cells were spun down and plated out by dilution series on l culture plates and left for about 10 days before ing. Media was changed 1-3 times during this period. ;! gains W0 2013/041844 PCT/GBZOIZ/052296 Screening Culture supernatants of the hybridoma wells above were screened using homogenious time ed fluorescence assay (htrf) using um cryptate labelled anti-mouse IgG (Cisbio anti-mouse lg Europium Cryptate) and a biotin tagged target antigen with a commercially available streptavidin conjucated donor (Cisbio; streptaviding conjugated D2) or by lgG-specific 384 well ELISA. Positive wells identified by htrf were scaled to 24-well plates or immediately counterscreened using an lgG- specific detection ELISA method. Positives identified by primary ELISA screen were immediately expanded to 24-well plates. Once cultures were expanded to 24-well stage and reached eny, supernatants were ted using htrf or IgG-specific ELISA to m g to target antigen.

Supernatant of such confirmed es were then also analysed by surface plasmon resonance using a BioRad ProteOn XPR36 instrument. For this, antibody expressed in the hybridoma cultures was captured on a biosensor GLM chip (BioRad 176-512) which had an anti-mouse IgG (GE care BR38) )covalently coupled the biosensor chip e. The antigen was then used as the analyte and passed over the captured hybridoma antibody surface. For Antigen 2 and Antigen 3, concentrations of 256nM, 64nM, 16nM, 4nM and 1nM were typically used, for Antigen 1, concentrations of 1028nM, 256nM, 64nM, 16nM and 4nM were typically used, binding curves were double referenced using a OnM injection (i.e. buffer alone). Kinetics and overall affinities were determined using the 121 model inherent to the BioRad ProteOn XPR36 analysis re.

Any clones with med binding activity were used for preparing total RNA and followed by PCR to recover the heavy chain variable region sequences. Standard 5’-RACE was d out to analyse RNA transcripts from the transgenic heavy chain loci in the 52F and 53F mice. Additionally, deep sequence is of almost 2000 sequences produced by the mice was carried out.

Bionformatics Analysis Sequences for analysis were obtained from two different methods: 0 The first is from RNA extracted from the spleen: first cDNA strand was synthesized using an oligo based on the Cmu region of the mouse IGH locus as a PCR template. PCR was med using this oligo with an oligo dT—anchor primer. Then PCR product was cloned into pDrive vector (Qiagen) and then sequenced.

PCT/GBZOIZ/052296 - The second is from hybridomas generated through o-fusion: total RNA was extracted from hybridoma lines of st using standard Trizol methods and frozen at -80 °C for long term storage. cDNA was generated from 100ng total RNA using rd Superscript Ill reverse transcriptase and a pecific reverse primer binding to all mouse IgG isotypes for heavy chain and a mouse kappa constant region primer for the light chain amplification. 23 ul of cDNA were then used as template in a PCR reaction using Pfu DNA polymerase and a panel of degenerate forward s annealing to the leader sequence of the human immunoglobulin variable domain as well as one mouse pan-lgG e primer. PCR products were run out of a 1% agarose gel and bands of approximately 350-450 basepairs extracted and purified. DNA was then sequenced.

The sequences from the first method can either be from lgM from Naive mice or lgG from immunised mice. The samples from the second method are all from IgG from immunised mice, and specific to the immunising antigen. Almost 2000 sequences were analysed.

The sequences were obtained as a pair of forward and reverse reads. These were first trimmed to remove low-quality base calls from the ends of the reads (trimmed from both ends until a 19 nucleotide window had an average quality score of 25 or more). The reads were combined together by taking the reverse complement of the reverse read, and aligning it against the forward read. The alignment scoring was 5 for a match, —4 for a mismatch, a gap open penalty of 10 and a gap extension penalty of 1. A consensus ce was then ed by stepping through the alignment and comparing bases. When there was a disagreement the base with the highest y value from sequencing was used.

The BLAST+ (Basic Local Alignment Search Tool) (Camacho C., Coulouris G., n V., Ma N., Papadopoulos J., Bealer K., & Madden T. L. (2008) "BLAST+: architecture and applications." BMC Bioinformatics 10:421 /www.ncbi.n|m.nih.gov/pubmed/20003500) program ’blastn’ was then used to find the germlineJ and V segments used in each sequence. A wordsize of 30 was used for V matching, and 15 forJ matching. The database searched against was constructed from the N65 sequencing of the BACs which were used to generate the Kymouse.

If a sequence d both a V and a J segment, the sequence between the two was then compared to a database of germline D segments in the mouse using ’blastn’ with a wordsize of 4 and the options ’blastn-short’ and 'ungapped’. This was used to assign a D segment, if possible. The CDR3 was identified by ing for the conserved “TA‘ITACTGT” sequence in the V segment, and the “CTGGGG” in theJ segment. If these motifs were not found, then up to 4 ches were d.

The IMGT definition of CDR3 was used, so the CDR3 length is calculated from after the 'TGT” in the V to before the ”TGG” in the J. Sequences with an out of frame junction (those which do not have a CDR3 nucleotide length divisible by 3) or which contained a stop codon ("TAA”, “TAG” or "TC-EA”) were excluded.

The identity of the matching V, J and D segments as well as the CDR3 length from this ment were then saved as a table for downstream analysis. The ratio of IGHJ6*02 used increased from the naive to immunised mice, as well as being enriched in the sub-population of sequences with a long HCDR3 (defined as consisting of 20 or more amino acids): All HCDR3>20 \ \' JH6*02% \ s TotalCount JH6*02% 5T0talC0unt %HCDR3>20 22.31% x x 91.11% Immunised 37.50% § x 66.67% Hybridoma 36.13% x x 63.64% This shows that the JH6*02 gene segment is selected for by sation, as the proportion of JH6*02 usage increases after immunisation. JH6*02 is also used in the majority of antibodies with a long HCDR3 length, which is desirable for targets which are ically bound by long HCDR3 length antibodies. 109 980-039 D— < 00(1) l-OQEEM (30(1) L09 €€®8€ > O (D 00}— <f—> l09 Emmi? -| (D 0 L09 ” L— 109 EEO‘dbG '- < N0:6) L-<o ID 00 |ine=SEQ Bottom N025, -SEQ line: Reference L09 980-099 [L <u 0 Middle L09 £96099 NO:1, L09 99mg JHS l09 98M 3 L9.— < ID < i— 1 SEQ 00 NO: L~o L3 nment: L09 92mg 2 ID Ali line L09 £90053 L—u <: JHS (top 5H rsom <p—o— b-ULD o u < LOQEZQMQX hzas p-UO 00 00:3 < r— 0 00 UH‘: 0‘93 .- r— w <n-> .1 o < Loaezm «Duo L09€zQ? LL "U0 o (I—E ,_ < £09 9363348 < U 0 g toe tam 109 tzagw a loatzqmz> o @un— g < 2, IOQSZW E L098?!fig? 11 v-(L'J E (9 B r—(>- g (r— l09€3®t6 <0< n: (30 '_ r-<(>- c9 O (I— < V.‘ 00 o r— u u .E i—<>~ 0 <f— o w E U E E 00 < 0 0 2 messages a: ‘ r—<>- ‘a-J '3 0 0: O 4:»— ‘ID LDr—I— : o —\ 00 E a (Tf ’— OJ Lu E p—<>_ r—uo ‘fi) 0 2 C Q 5’. g LOBEEW C finrsom <uo u E :2 0 W0 2013/041844 PCT/GBZOIZ/052296 L09 sung? r— < G 0w NO:10) G line=SEQ C Bottom CGGTCAC NO:9, line:- GAAGCTAGAGACCCCGGCACCGTGGGACCAGTGACAGAGGA G G nce Middle L09€€l§6> GGGACCA < ,_ NO: mama? JH2 o o; < r— NO: nment: SEQ o o :— <2:> ID Ali line Lossmm JH2 (top ISH rsoot W0 2013/0418“ PCT/G82012/052296 26:23:. c8833 :2: mm; 32%: cmaofi :? 8:30: mm a 9: 83?; 29:7. SE 8:838 m_ 238 3 9; :32”: 385cm acmtm> .239 .6 :9: 29238 m 8* 2 E Ho“.n gm m:_>>o__2 3313.3 or: 5.82 0... 9: venues 5 8:33 34% “Eat—g 05 E395 a: u...— vmumBEouE 3 m_ mmcmfi @3952: can; “9: 08 2m 3.542. “Em 0: :0: Ba): m 2 ommmmx yo $5865 338:2 .EEm; c0530: .oz umwums _...mE 8382 mE_m_u 9: 5320: Squam 9: a. 3%: Lo .333 c2383.

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W0 2013/0418“ PCT/G82012/052296 Below is a summary of the ethnic population origin of samples that the 1000 Genomes Project sequences.

Population European ancestry Utah residents (CEPH) with Northern and Western European ancestry (CEU) Toscani in Italia (TSI) British from England and Scotland (GBR) Finnish from d (FIN) Iberian populations in Spain (IBS) East Asian ancestry Han Chinese in Beijing, China (CHB) Japanese in Toyko, Japan (JPT) Han Chinese South (CH5) e Dai in Xishuangbanna (CDX) Kinh in Ho Chi Minh City, Vietnam (KHV) Chinese in Denver, Colorado (Cl-ID) (pilot 3 only) West African ancestry Yoruba in lbadan, Nigeria (YRI) Luhya in Webuye, Kenya (LWK) n in Western Division, The Gambia (GWD) Malawian in Blantyre, Malawi (MAB) K00004-1 W0 PCT/G82012/052296 Population West African Population (TBD) Americas African Ancestry in est US (ASW) African American in n, MS (AJM) African Caribbean in Barbados (ACB) Mexican Ancestry in Los Angeles, CA (MXL) Puerto Rican in Puerto Rico (PUR) Colombian in Medellin, Colombia (CLM) Peruvian in Lima, Peru (PEL) South Asian ancestry Ahom in the State of Assam, India Kayadtha in Calcutta, India Reddy in Hyderabad, India Maratha in , India Punjabi in Lahore, Pakistan W0 2013/041844 “82 “Emma “mmma mag 83 “$2 82 Emma “meow 33:. “32.. £305 :5 “$2.. csEE_ 5.9:. :__u c388.

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”E. ogwmwmh _m>A.m_< mermh 2%- ofivr @2430 vmmmwmwofifir wmmwwmmvwm E D‘mwwmwowu‘ wwwwm £er E 5N CNN w v _. we. # indvs - w>:0_ mvnmx. oww 03 .o To o mgood 95006 mVoood 099. 29m... h.W.D.e (EDUDQ PCT/G82012/052296 H ill! h H H as. h Hi Egg-mg g H! p H o HIIHII 0 0 a éééééééé IIIIIIIIIIIIII Hiiill HI'IIII'IIII IIIIIIIIIIIIII IIIIIIIIII. HIIIIIII HH HHHHIIHH HHHHIIHII HHHHIIHII HHHIIHH HI 0 mfiommoriag .u 0 0 ééfififll u il I'll IIHH IIHH IIH. IE! HH'IHIIIIH Eli! S n m v N N m F F F F F r F r F mgr—$10. . -250 $8.0 88.0 mmmood 38.0 88.0 88.0 88.0 3086 9.83 3086 383 mgood 358.0 $08.0 380.0 958.0 - , Fmd.‘ min... h WW .9 a n. u v v. a : m c _ * _ E _ a 0 2012/052296 mF.~F.m.F.m.F ¢F,FF_oF_N OFF 26 w. 0 I'll Illl Hi H! E! H! H! II EH ii 0 F H! ill ll £3. F F F 703 II I I o. . . 52A . lg!4 HI o I . mtmmmmmeF an HI! K0 8 Jr. m . 822 amiafi IIIIIII< I'IIIH IIIIIH I'll lllll'll IIIIIIII HIHH IHHH H!!! Ii! I'll! Illii HIE! IIIIIH iii! 0 mF F m v N N N F F F F F F F F F o7 A «>10. 25.0 «wood $8: 383 88.9 88° 88.0 88.0 383 maood $58.0 $08.0 $58.0 $58.0 383 $80.0 $80.9 035... h w..W 3. a a o n x 0 c .F m F. F _ E _ a o WO 41844 503396 .. $8me aa o aa HHII II II IIH IIH p HH H gmom. - 2888:: mmwmmomze a.. . Io III0 I0 o . IIo I g lag HHII II I II IIHH II H H gE HH II II II I'll F H H H E H II m: mg we 2 9 m N F F F F r mm.F>IO_ - 8;: 83.0 38.0 .o mwmmod good 88.0 as: 88.0 383 983 383 383 383 NW». 2an m a 0 3 m h m x z ._ _ _ PCT/GBZOIZ/052296 3.3.94 3.9.9; mmoommooooohmzw fiofimdflddéfidé fiofimfiKddéfiNé vim—xmfiofimé N,_,_'‘—.0).Ln.‘1m 3.2.05 min 9 . Nmow n. 8E w__A-_m> 585:3 33?; @2420 05:: ea _m>A.m_< w: mom m9. m_. mr m N F _. _. F F ._0_ - ed momvd 3.36 mmmmod mwood wwood mNNood moood 56006 mvoood mvoood vaood @3006 N99. cum.freq 29¢... hlype (“.0013 (Du. U)._x.C <2 I § g (D (I) 000000 000 R 4 m a E fl 93 I— 1—}— l—F—i—l—I—l—i—l—k—l—i—l—l—F—l—I—P— 8 i il 142107170011 4|:9691 rs Ala->Thr IIIIF I II I‘— ll}— 670994 r51 Lys->Arg I ll 11111111 142107169995 64565 r51 Ser->Thr a 831 ' r51 Thr->Arg m+—-:-—-:_—11111111 42107169948 ‘— 13190613018 Val->Leu II"000 mNooco Nh-IOLD N NNN Ni-FN — . (00’)!— 69 #Indvs. moumwoovmmmmmsmmwmmmmmommmmmmmm 0:00:5ngocncooorxNNNw—mmoooooooovvvvxxco urn Or'mNOLOONNOV-FOOOOOOOOOOOD m‘f‘fQQo’QODDOQOOQOOQQQQQQQQOOOO Yoooo oQQQQoQQoQQooooooooQQOC’. o 000:: 00 DD 0000 3.33 cum.freq Table “" h.type emnooum_b.c~__E_.:z/>xmmuruhcfimcmonmm>~0.: o 'c I: mxcn WO 41844 PCT/032012/052296 11111111111 In". 1111111 11111111111 111111111111 1111111“mm—u—m-e- “mu—u—“_ “mm—m—“— “mm—m—m— --_G 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 IGHV1-69 0.00045 0.00045 0.00045 0.00045 0.00045 0.00045 5 0.00045 0.00045 0.00045 0.00045 0.00045 0.00045 0.00045 5 0.00045 0.00045 0.00045 0.00045 0.00045 3.33 Table az a} aa aq V al ab ak ap N EIV au am at aw aY ar as ae ai 2012/052296 14:1071701 64 rs1064 l’-I|'_m 170091 ' ‘210 rs18901 Leu->Pro HIHIHIHHHiiiHiiiHiHiiiHiHiW """l"0 14110717007 rs11845244 P-F- Ili‘r— F-I— Hit— h—HI I",- ._ PI '0062 . 1010 000" 170056 "214708 illlilllil ’— 14210 rs ililil r58009570 I”I 14:107170034 A A A (I CE <2 170028 ':10 Wmsmsmgs “mu—.- -_—-_—“-_- ml1« 1« ‘ ”NmOFV’NleDCDLDl-nvm NNNNNNu—x—V—F NV—x—NMNNFx—Q— ”('3‘— lGHV1-69 #indvs . mmmx-ooveommmmvxtnmcom mmmmcnmmmmm txcomoochV-romwoorxwmmrmmmowmncotoowNOw 0000000000003v<rg00 O’T‘TQQO'QOOOOQOOQO 900.990.0000 ”.0000 090.90.090.09 00000053900. 0 0000 00 0 0000 13.33 Table "- h.type anoo>a>_u.c~_._E._:wxmtugmfim:monmm>0.: o 'c C (0x0) 2012/052296 '— I'll Il— ._ II“ 11111111!!! "II” 1— l- l»— }-— II— 0 o lo 0 0 It!) “0 i IIIIIO I I II I I II HHHHHHHHHHHHHHHH '0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 lGHV1-69 :3mmm2$£“£mmmgaeesgeseeemoogggooogogggooooooooooo3oooooooooooooooooooooooooQQQQQQQQQQQQQQQQQQQQQQQQO.ooooooooooooooooooooooooo 13.33 Table N...rucr _.0x a. > JEHB 1. 1/10.- “SCUM“!>NNNUNNBNLXNGMMNMQNNN PCT/GBZOIZ/052296 ":8 000 00000000<900 _§ 0000 0 0000 000 (—II 23 9+— P—i—I—D—l—i—I—I— r—1—I—r—I—r—r—I—r—v— rum 0.

DCD A 1100 .L :5 .5 .500 0% 3“<<<<<<< <<<<<<<<<< B A ‘7.“ b 39 .C 0'30 v—CD :3 g 000 000 0000 a; 2‘ 215:) g 3e 0) m n. no A 3 c. a ,3 t. E r— 0 0 0 0 OI—I—OO r- v— 0 A ‘7- d: E Z it?) 0 0 .000 ’1‘— -~ ‘— ‘u‘ PF:- 10 mwcoco hmmwmmvmmwww NNN NPI—N r'x—F (00‘- 69 I Locncovoovoomtotnualxuamoommmcnmmmmcncntommln moogogu—omcowrxwmww—mmooooooooggvvno u': Ou-CDNQDLDONNOw—VOOOOOOOO oo m‘T‘TQQdQOOOOQDOC’.OOQQQQQQQQOOOO Yoooo QQQQQOQQOQQOQOOOOOOQQOQ o 0000 oo 00 0000 13.33 Table *5 0: 0....‘0 : mxc: hmnomm_‘o_:..._E_‘:wamwumcum:mo.omm>. 11111111 “m c c HIIMIom<I“—mm mm—“m—“m—-e-u—m- T <“< A —“m—---- mm—m T Ollo T T C T I mm— G G G “m—“m—m— “m—mm—T 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 IGHV1-69 mmmmmmmgggugmmgggmmmmmmng3333333gooooggooogggggggoooooooooooooooooooooooooooQQQQQQQQQQQQQQQQQQQQQQQQOooooooooooooooooooooooooo 13.33 Table ._.mc- _.o c. > :E...3>~h man- mmmw>mmmcm~3mhxmmmmmmammm PCT/G32012/052296 8088088me 3.2.9;fiofimdxdwéwN; 3.9.3;totm.w.$.m.w.m.N.F 3.2.3»:.or.m.N.w.m.v.m_N.F 903; 3N3; 3.993. 3.9; thfiidtNdédNéHéi: 3:53.33 Ntotmwfid 3% 3'E9?. EHlumilm' Hlilml III?! Hlflvfllll Hg! Hgii Hg!" Hg! Hg!" Hg!" F F F mNm N; m: 2 ; a NN 5 m: 3 m m m v m N N N N N N N N N F F F F F>IO_ -332 38.0 NEE 58.0 28.0 :3 85.0 889.0 880.0 383 208.0 Rood mNNood mNNood $86 353 358 88.0 885 88.0 88.0 88.0 886 88.0 88.0 $806 $08.0 383 $80.0 min... a a o o a _ o g * _ E ._ : w x um cm a cm a E c 5 o 8 xm mm > W0 20131041844 O FO V 031'? O ('3 FNNmt—x—V—va—w—v—VQNNFMNF ('0‘— S 2 : ll II E IIH Ii HHHHHHH HHHHHHHo I(<< lumill gilo' gillv ' III gill. Infill gill. gill Igill III IE, '5 I! l?! II! I! giH. II! I'ol I! 82II I%H_ IEiH F F F F F F F F F F F F F F F F F F F F F F F F F 91.2.6... $500.0 $600.0 9500.0 mvoood 0300.0 9500.0 9500.0 9500.0 0300.0 9500.0 0300.0 $500.0 vaood mvoood mvoood mgood mgood mvoood mvoood mgood 9600.0 9500.0 mvoood mvoood mwoood 2an .3 .8 mm cm > E nm xm U am N 3 >m . x 3m Em 6 3m xm .m a mm mm _m W0 2013/041844 PCT/G32012/052296 1216 (5'.

Th Inlll 649961 r818 r573371428 '— Ul II 160 8 6 4 3 IGHV1-8 - 0.8844 0.1016 0.00405 0.0032 0.0018 0.00135 0.00135 0.0009 0.00045 0.00045 000045 3.34 Table ._.C'J._ WO 41844 PCT/G32012/052296 mmmommOOO.O Z Vim—umfiwfiofiqfim; 9.2 NtNFidfiN E; :N :K 3 N 3 2 @M!! m N o N N N N F F r owe m 0 v m m N r _ F w. I —.>_._0_ 55....:n- o wee—mo 8306 N83 $8.0 880.0 850.0 886 383 380.0 038.9 min... 0.. m o n u m N 1 ._ m _ PCT/G82012/052296 :wommoooookmzw viz o. a «.../7.5% ‘ 833va on.... . ; oN-N>IO_ _\ @Nr mr h N F F _. - . mnfir mvmm mm o 50.0 Nwood Nmood @0906 mvoood @3090 95006 wasp. EL (0203—00543 Nwmmowomrmh 34:3 mwmmw 3 :9 at vmmo m . wmwmmwmwm‘. 0.34:? o 0 o o I o o o o o k. lilol o IIIIIIIIII EN". ovofithmF a '0' F II F H II II h II II . NmmmwoFE % HHE F HEHH E Es“. lama Em Ii II. II. II '0' o II o ill o m-~>_._0_ - mF v N F F F F F F F andv 25.0 805.0 88.0 88.0 308.0 383 380.0 380.0 380.9 380.0 $80.0 29w... hN..D.e .2 a c o x m g _ a F _ W0 2013/041844 ZO]2/052296 _..o_..m.w.~,o_m.v.m.m.v 339nm Snow; 3.0V miv PCT/0132012/052296 13976 r31 IIIIO III on— r— r— l— p->Tyr 44976346 rs Tyr->Asp '8~86 HV2-70 .- IG l - a 31 £303aaaaagggssmmmmmmmmmesm‘— 13.37 O 0 0.16595 coat: oooooooooooooooooooooo m. o.{dqQQQQQOO'OQQQQQQQQQQQQQO o 00 000000 oooooooooooooo Table "5 u. ang) _m 8' _x o. .anomu—_>~Nm£mwmm _:xmmu >mmUm WO 41844 PCT/G82012/052296 11111111111 IGHV2-70 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 13.37 00045 mammmmmgggggmtgm303333300000303ooooooooooooooo90.952539999999990.0 ooooooooooooooo Table 0 DE 1.) L— 11).: thxmtv_:mEmo.mmmo PCT/082012/052296 r861734 Met Thr-> rs187704106 Leu->Phe ""l". -| rs18229294 Val->Gly IIIIIII Arg->Lys III— I- l— l- I— l— II— I— r—— "+— ""I.

Gly->Ser rs148856487 GIu->Asp 0|“ r52073669 Leu->Arg HU U 0 :0 U 0 rs1648 II 7-“.

IGHV2-70 ‘_ .- ,. 584 353 132 94 70 - 31 13.37 0 016595 006035 004295 0.0319 0.00405 0.00225 000135 0.00135 5 0.00135 0.0009 0.0009 0.0009 0.00045 0.00045 0.00045 0.00045 5 0.00045 0.00045 5 0.00045 0.00045 0.00045 0.00045 00045 0 0.00045 Table a b d c e f 1 2 af h 0') S ab ah 39 1 U k 81 aa t aq v al ak 8p PCT/G32012/052296 IG I IIIIIIIIIII Ikhlkkilkpl IIIIIIIIIII IIIIIHHHHII ““— '0 IO U U "U U U EFFFFFV—T‘r‘r‘f—Y—Fv—v—FV‘ Immmmmmmgmmmglgmexg gaggggggogggoogoo oocoooooooo nooqooooooqqqqqqo goooooooooooooooo In? o '0 E I-EmhxmmmcwEiammmou q)_:: PCT/G82012/052296 votmdfiqmewN;I; 35E;totmgdmdgl; Vt O V ‘— 1— 05 V ‘_ . ‘_: O .m._. m O) .N . O F. p. ‘— (D on.N>I0_ F gm mmm «9 5 E mmmmmmNNNFFFFI—I—V—I—Fu—FT—rr hmdv -883 :6 833 308.0 mmmvod 28.0 833 380.0 880.0 380.0 860.0 928.0 88.0 88.0 88.0 308.0 3.83 308.0 383 $58.0 $08.0 $80.0 380.0 $80.0 $08.0 $80.0 @380 958.0 958.0 flow... MDUOQ)». >~N hm ll) am am am W mm cm _m xm am (‘7 T‘FNV Nw <0 ‘— V'LDF F? 0) E.

U 2'F" 9IFl 9Il—U on-~>10_- ‘_ V‘fiFWFt—Fvvw—i—w—t—w—F .? mvoood mvoood mVoood mvoood mvoood mvoood mvoood mvoood mvoood mvoood mvoood mvoood mvoood 9300.0 9500.0 @3090 03m... Om um Em 00 5m mm F. Em PCT/GBZO]2/052296 Fowomwoooookmzw m.v.m mam m moor“: gEH o H II nmwm 9<A-m>._ o mworné g 0249.. I I I on N _‘ —. # mdn - _ twmd 8990 88.0 308.0 wmf. and m mvEa... h w. D.e m a 0 W0 2013/041844 PCT/G32012/052296 mwmwwmwo m_ F A..._ ;:liio :0 0 II0 '0”«F .vm:merlllllll- "I._0 vmmow we. 8:998 Aiww IIo .852: Nwmvma. mmmowmoo; Vela g5 mm: ’— 58 I’— I_ II— ”'— wa. w $8835:GIF II H $83039 F I'll F II! 3" II: I IIIIII iii ii! ii! H I I iii Hi! 0 HHi FH F.m>IO_ u QFN Fm mm NI—x—Fx—u—w—FF mmnmwd mem F mwawod mo mNNood m mm . Fo and F F.o 5.0 Food Food @0006 .o 5500.0 mvoood mvoood mvoood mvoood 9600.0 mvoood 03w... Fm: mnuow.-U>-._x.c ._E E_ 0 2012/052296 8.8.5.0333va 3.2.9553ng Séflidtmqnwg 3.362. 3.0: o: 9.2 N 3 3 3 m S mommw, w 0 0 < wornv Fwwnmhm rm; H II atI H HH FH H HH wmvowmwr Fm; I'll 'III: H ”—1510. mNN now mm m m w m N _‘ r _‘ w w _. r . mvwm mmdr mNnmwd _‘ 59.0 mmmwod merod mNNood wwood mmgqo moood .o mvoood mvoood mvoood 9500.0 9500.0 mvoood mgood flank MQOU®._.U)-._x.c CE PCT/G32012/052296 .8858; R808 2868; fig 8888; 383:8 $358; wfi mmmommmmre mrnm>I@_ 3:. om P _‘ _\ - mmvwmd unwed mowed mvoood mvoood mvoood 29m... m n. U w o Vomommooooohwzm 333NFI0' :dwNFé—uo—uhfié vwéfiofir Nmrmmwoor I 3 $8899 I a2:8va I I'll IIo Nmowmmoo r“: 5.3% @3420 Illo'r OON NNV E o .

Tm>Im= mmowd msoe _.o.o _..o mmonod mwmood mmrood flash Fm: 32-8 3.2.3.?teddéwinqF 2N:teddéwédqF 3.2.3.3 3.2m :_m S N 3 F OI...- wwmmmmowvfl'flflg H Ell illH o w .

I I I womomm.‘ u ill IE! HIE! iii! HHIF «9 HII I I vwmrmnmmh HIIIHI'II I IHIIII I Hi! I I IHHH H H o: 5N 8 m N F F F F cN-m>I0_ -880 mg: mom; mag; 353 88.0 380.0 382 383 $08.9 29¢... m a u . v a m c _ W0 2013/041844 PCT/G32012/052296 Egg éé...x. Io I I 0 I'll ll! mvwwmw.:r IIHIIHHII IIIIIIIHH IIIIIIIIHI'III IIIIIIIH IIIIIIIIHHIII IIHHHHH I!!! gas h .w Ragga o 0 IlHHiHII IlHHHH HHHIH HHII H! «3m..wm§§ I'll Ell o I. o I. o I IIIHIIHHII IIHHHHII m v m r N r r r r r r w v r vN.m>_._0_ .am.0 -wamd mmmood 38.0 823 88.0 88.0 308.0 mvoood mvoood 3086 mvoood 968.0 mvoood 9586 mvoood win... m!c P E. m n o n : v. m m w c E _ _ _ PCT/GBZOIZ/052296 ooofimZm 32:? 1...: :2 N n N v 3 m : momwmowop. .07 v 832%.. E IIIIII Flill Hi! PH Hi! I II m|mwrmo©or3w vmfinwo—‘mwm _m>A-m_< I I I vi! mmwrmwmofi ommowo 204m? o o o 0 v wwwh wwmkmwvm I I I I E” ”:45IMVIIIIIIM' IIIIIIII QFHHHII Hi II!!! H H!!! H!!! H II LDVl‘Nu—Nu—I-I—t-ru—FQ—F Fuum>10_ # indvs - wamd mNNood m50.0 @0006 950.0 moood 9600.0 mvoood mwoood $500.0 $500.0 @3006 mvoood mvoood mvoOod mvdv 295... h.type KU'CODL ._._~zc)ms._._v:E_ PCT/082012/052296 142106725433 M 1111111111 142106725400 M 142106725397 ly 725357 Glu—>Asp 11 0 142106725346 Ala->Val 142106725329 Gly->Ser 142106725328 Gly->Asp 142106725320 r5189958807 M “mmumu—n “mmmm—m IGHV3-23 190 58 29 18 10 5 5 5 4 2 2 1 1 1 1 1 1 # indvs - 13.44 0.83265 0.09825 0.02695 0.01365 0.00865 0.00455 0.00225 0.00225 0.00225 020018 0.0009 0.0009 0.00045 00045 00045 0. 0. 5 0.00045 0.00045 Table cum.freq ref a b c d e f k g n h i U t V S q l’ W0 2013/041844 PCT/G82012/052296 3..NF.NF.FF.oF.w.F.m_m.m.N.F FF.NF.FF.oF.m.m.F..m.F 3.FF.oF.m_w.3.m.F 3.0%; F,F 9.: 3.8.3; FFdF 3.th 3 93 VF 2 w S 3 3 a o S N N é? ééggg H HHHIH Hail!!! HHIIIHHH ll'lllllll IIHIIIH HHHIHH HHIIIH IIHIIIE H! HHHIH oF HHHIH IIo a o HHHI Egg o o o o o 0 o 0 8va 839% H F 83F HFHHHIHHH HHHIHHH IIHIIIHHHH . IIHIIIIIHH. HF HHHIHHHF HHHF FF HHIHHHF EF IIIIIHHF HHF HHHIHHHF HHHIHHHF HHHIHHH mw-m>IO_ 8F mm mN NF OF m m m v N N F F F F F F F F F F F 3.6—. -9880 mNmmod mmmNod 890.0 $83 333 383 mNNood mNNood 88.0 88.0 88.0 958.0 $08.0 38.0 380.0 #88 38.0 38.0 38.0 $08.0 #83 938.9 032. m a 0 u m F x a : c _ a F > m a F _ e _ Q 0 WO 41844 on.n>10_ - mwmnm «V5.0 mmood 0 mmEod mmPOQd mwoood mvdr win... (5.01300 PCT/GBZOIZ/052296 .o_.w.~.w.m.v.~.v 3.2.8.: .Sdidwd; 3.25 3.9.03.2 Ed: 3.9.N 03.. no or S — 32.00vath mwwvmwmmrw. E Nn—Vmwmrm‘. Egg wvamtrFB 2%: 522 II F. IUHIOUI J. . 0. F mvm ‘_ OOCDVO'DNFPF N ‘— 9.6—. -mommwd mad mwmood Nmood NN000 300.0 $200.0 @0000 mvoood 0300.0 0300.0 03a... hm; m DQQUSV._U).— WO 41844 PCT/GBZOIZ/052296 Nm¢mvmmotqw mrwmmomisé “rememootwr mm.

NNN 00—. m _. mmum>I0_ # mdvs . mwomwd mot—no mammod good mvoood 22m... I1 type 3.. QDU‘U ZOIZ/052296 ewommooocolrmzw vw.mr.mr.:.o_.......mm\.©mvmn..mr 3.. 2...: 3.9.3.9; mfifim I‘m—.wmww 2m wwh 4:3 IE II H bun-Ono. $00 wNo 0 wm. II III 0 II II mgl..mwwnw Io o II I . . .. wmr mm ww m_. wm-m>10_ #indvs - mwmw o mwhod mrmmod mwhood wooed cum.freq «v.2. 032. m a o U PCT/G82012/052296 “a é HHIHHI'IH II} I'll III'II I'll HHIHHI'I iii Hi H II H II rég ; I I .. H! H I “ Hi EH é o o o 0 o 0 0 HIII' o o 0 . IIHIH IIHIHH Hal!!! o Hi! o h .F HHII Hi! ..... gééé 0 0 % II H'Illl IIIIIII HHIIIIII F HHIH. Hi!0H IHHHIH Q o 0 HHI! I! u o II IIH HI! HHHIII HHHIII H «E x: 8 ow on NN 9 S w m .0. n N v F F v mYm>Imx .mqtmd mmvg 880.0 88.0 $8.0 330.0 880.0 88.0 88.0 $80.0 383 928.0 380.0 88.0 958.0 $08.0 $08.0 $80.0 2an m a 0 m v m 3 _ w v. E 9 a : ,_ _ 0 oEmwooooEmzm 3.2.9;totqmgdgda.F mr.§.:.m.~.w.m.v.m; .ow.m.w.m.v.m; 3.3 :.S.m.w.v.m.m 9.3.3 3.3.2.: 3.2; 9.3% :9 2.2 2; 2 S S m Eve roommt E o mus—L: 36E:38.35.10?II II ._.

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Each .nw..pe mouo~.cnwc_xmo-_._:E_Qo W0 2013/0418“ PCT/GB20 12/052296 g mFFFoFm.F.omF.mNF| SNEmFmE wwomoomeg I ...... IIIIII I!!! o o 0 ©2252 IIIIIIII o III 'I'IIIH I'll! Hill! III! II 0 H! .85. ow 0 0 I O r9 1.51m? $33. zoiflow ll II o a H. H HH 3.4540. 8 w v v m F F F . 2. - r memd £899 883 28.0 38.0 350.0 383 308.0 $80.0 0351.. Fm: a u v m . m g _ PCT/GBZOIZ/052296 .rv.o_..m.w.m.w.m.v.m.u._‘ 3.9.9 Edie: 3.9.3 v.20: 3.? Ed 34 2‘ S 3%; Egg éég IIIII ill Ell H.F HII Hi Hi oH 0 w 0 o 0 Elli 0 0 is. v omvrwwh 0 I oIoo 934% 33% a 1. H II.I I'll EIII' IIIIII IIIIII I H!lolllll o I H 57 Elli. r>40_ - 8 mm m v m N F F wrv.n r -mmfiwo 88.0 808d 830.0 286 38.9 88° 958.0 $08.0 032. h mi E a 0 u m h 0 g _ 2012/052296 Shvmmofie mwmvwvwfi IF o .— II II 0.5 mm m v an. . r>4m= Nmmnd mmmmfio moved mwmood good wrunm Bank h E w..D.e ._ m a 0 U W0 418“ PCT/G82012/052296 3.2.92 ommomMOOOQOszm F _..o_..w.m.w.m.m._‘ N...m.m #6 $3899 Stovfieg Hi 0 HE! 0 .5: E 8E IIIIIIII o o 392N892 HHHIIII H I .u I .... lllilllllllul HEIIIII». £80 l I :3 IIHHIIIIIIII HIIIIHIIII I'll II all!!! Elli. Hill! 54540. Fww (DVQ'NPV—r‘ Nmnwd h: mmmood m m . v.0 50.0 wooed mgr—32‘ 50.0 mvoood mvoocd @3006 Oink NDUOOOLH PCT/G82012/052296 mrwwwg ovaNwme vownmwmwrw mNNooomm—‘wh wuw>4mz NNNN“ fin mwnmd Nmood Nmood moood moood $000.0 $000.0 v mkood mmgod mNNood 0300.0 03m... X: anDm-—®u—._£ W0 2013/041844 ma < emmnmvw Fw‘. _m>A-;_o Emwwe $31.56. IIIO "0 0 l0 '0 lo ""0"“0 avvaSQ a242o lr—r-v—r— II—"r— I—Ir—Ir—I—m—“r—I— mwmmm 3.. F029;? ‘0.- mwmwwm E EwAéO I}. H“. II- II. mmwvorww r2 57-5w 5000.500 Fm: 5245 I- II.— IIIMF2%-:3 .— I— I)— H ‘- ”’— ""ib 09‘. v0 mm Fm (O‘OIDV‘TV'NNNFFI—rrx-q— v—..«>._0_ F F mmwomd mmmwvd mmwmod V0000 mmmwod V050 mmmood 0000.0 0000.0 0000.0 mw000 nwood mwwood 0050.0 800.0 900.0 0000.0 0000.0 0000.0 0300.0 0300.0 9000.0 3000.0 $500.0 mvoood 9300.0 030... monomwcu-._8c._x..cro.o>.::u>w3x 2012/052296 «5888855 3.9a:F,or.m.m.$.m.v,m.m.r 3.9.3;totmdgdwéadg 3.3.3.2.m66i3 S.S.m.o.m....m_~; 35 :.o_..m 023% 3.2.99.3 ~23.“ 9.8.9.3 0366;” N23 3 9.:56 3 S 2.“ a 2 o 0 F v m m gE5IIII p F ii p h II II ._. H I II iH II H II i IIi H H H 527.3% mmogwmw : Fm. E omw vmr mmw E NN 9 or N_, m (OLDKDVVVNNNV—Vx-x-FFF VTN>._0_ -mmwwmd mwmwvd mmwmod wowed nonwod 3306 Q5006 wooed mmood wmood hwood nwood mNNood @2090 300.0 300.0 @0006 moood moood 9300.9 @3090 mvoood 9600.0 @3006 mvoood mgood ¢... mcabmmz..__._.Ec ._x._cro.o>.:u>w§>< “ 2012/052296 3.923;._‘.o_um.w.n.w.m.v.m.w._‘II EdwKéfié 890;; w mom P-N>._0_ - mNNN mwm. o 500.0 was... h.W“Pe w a PCT/G82012/052296 GIu->Asp :648111 n—>Glu r51: GI I'll!!! 49282 r5- Ser->Arg H 192358 rs1' GIy->Arg liiilflli Ser->Cys HHIHHHHH Val->Glu VaI-> HHIHHHIIHH rs1216625 Thr—>Asn I: IGLV2-23 I a.) 0.34655 memafiggmemmmms|_‘_gzmmmwoosoaaggo. OOOOOOODO «2 c5 C! Q C? Cat? c5 <5 <2 <2 C? C? :2 Cal: 0 09000 0000000 13.120 Table anoum».a:xo....c._:E_o.

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WO 41844 PCT/G82012/052296 we V N mun-«>40. - mmmwmd mtood 5006 @0090 FNFJUV 035... a a 0 ZOIZ/052296 w.N>4m: - vawd $3.0 ovood mrmood hwood wrood mvoood «afar Each NDUUQH W0 2013/041844 PCT/G32012/052296 gag; Hi 0 H!!! Emma»: F eéggg IIIIIIHIIIIHIIH IIIIIIH'I I'III'HIIIIHI'H‘ I'll IIIIIIIIIIII II IoI o IIIIIIIIII Elli: IIIH. oIIIIF I IIHHH IiF oF HHHHF B Fv o F F F F Or-n>40_ - mwmmmd mvod mowed 96006 95006 9500.0 9500.9 mvoood mafimr 035k Fm: NDO‘OC’IOh PCT/G82012/052296 ooookmzm N adeéE II 2. . km iv m F _. F w Tn>dmz . mvmmmd mgd wowed mwmood nvoood mvoood mgood 0300.0 nN—un 2an h w.. «0 D.e L NDUUU‘IQ’u— W0 2013/04 1844 2012/052296 $$~5Leé SSBNQ 9 II mammooow. II m r r r Nvun>u_0_ # . mow CNN mmr Indvs . mvvwvd mrnwmd 9. v.0 finned marood mvoood mvoood mvoood VN—nm cum freq. flaw... hw.. pe 3.. NDUUU’QJw— :mommoooooszm mé vw.mr.m_‘.o_._m.n.m.wé 3.5.ordé 3.9.? Nwoww rm; 504:3 am<n-_m> wwevwmmvwmh cm<A¢3 a H§A4m> rwfiomo—rmh Ilo Ilo ""0.mwfifia Fomo rm; llo3,995 Ilo ""0 F H p n. 2% £8252: FE HI I'll all HI I'll HI HI II I w—...n>._0_ - Na 9‘ or v m e V _. r # Indvs . mNrdr mmmmd mvaod 50vo 960.0 $006 mmgod @3006 36006 mvoood mvoood Saw... h.type .9. MDOLUGQV— PCT/G32012/052296 rs18849 6281239 rs Ser->Asn rs Ser->Asn lu HI 8459 r->Ala Th !!!!!!! rs193091 !!!!!!!HI [5144678284 Gln->Pro !!!!!!! lGLV3-19 ocommm NN F‘- mmmmmmmmm LDLO mroowmmooo - mONNx-FOOO 33 3.126 wovoooooo oo Q‘TQQQQddd 0.9. 000000 00 Table ...

Emno'o._..x 39.3 momommoooofimzm rréoémem? :iei? C) ‘- 9 3 a 3 m. Q 9 9?. ,— .F 9< «H E II «II! 0 20420 IIIII Hal I HHH 05 1.0rm. 33.5 vHHHI I mmmmm 0 o w I'lllmlllll; llih‘9.vloIHIuoIIII mHHHII HHHHI IE!!! II. HHHHH. Hiill HHIIII _ all. I mvnm>4mz - 09. mm IDO‘JMNNNNNT-FI—Fw-v-v-v- # indvs - . mmmww. mated mNNood morood mmwood moood moood wooed wooed $090.0 wN—unv o 962.6 $600.0 $500.0 3600.0 9600.0 9500.0 mvoood mvoood mgood 63m... NDQ'D_ ._.x .C._ amochcho PCT/G82012/052296 Gly->Aro 36453 r561 Pro->Ala 86574 {e l' Thr->l “m“ Lem: G L H L— Eva-m ->A|aGly->Ala GI mu—m Ann—“mmmm 4““flmmmmm mm—“mmmm mm—“um “n—m—n “m—mn «mu—“m mm—mm 1-.--“- 'mmnm O w-u— n xmmmmmmmmmmmmmmmmmazmtnmm > LDIOOWMONNNOOv'V-FFFFw-OOOOOOOcocoa—‘—Nmmmvrmmmmmmmooooocoggg _| Fodooqoooqqoooooooqqqqqqqooo Q 0.9 QQOQQQOQQQQQQQQOOOOOOOQQQ oo 0:: 000 9000000 000 fi " I— 31130220a)...>~._._cm>me_QmU._mNmm:xmtn .1: ‘tv ._ “m “m—mmmmm “m—mmmm“ 1 1 1 1 1 1 FVF‘. 1 F‘_.‘_‘_‘_ 1 1 1 (Ammunmmnnunmuzmm 333333oooooooocoo OQQQQQQQQQQC’. Toooooooocoo000045 0.00045 0.00045 0.00045 0.00045 0.00045 0.00045 0.00045 r—ggfir‘éfic%zg.fi au am at ar as ai an ZOIZ/052296 .- I: 1111 § ’— E2 “m “ l Iiii! s->Thr L iH” <0" N u 0') GI n. Lys-> HH HHHH ->Arg G GI H: V- 55097 Lysw>||e A r5191 IH H 9 ->Glu rs‘l: Lys H H! W 1rs183495‘ s Ser—>C H zz 0 1' r5189 Thr->Pro H -““““ 7 6 5 5 5 4 4 # indvs IGLV3-1 - 0.9255 0.00315 0.0027 0.00225 0.00225 0.00225 0.0018 0.0018 0.00135 0.00135 0.00135 0.00135 0.00135 0.00135 0.00135 0.0009 0.0009 0.0009 0.0009 0.0009 0.0009 0.0009 0.00045 0.00045 0.00045 13.127 Table .3' Q‘ 1:m UDSOQ’ u C ah W0 2013/041844 2012/052296 II I ””HHHHF < < “mum“ 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 asefiamsmemmmmeeeeamm ODOOOOOOCOOOOODOOOD OQQQQQQQQQQQQQQQQQQO TOODOOOOOCOCOOOOOOOO “nachos: a c “DE... .w._¢: l—mmmmmcmamhmmmmEmmmm W0 2013/041844 W681 731 r561 Met->Leu HHHHHHHHHHHHHHHHHHHH HHHHHHH W Ser->Asn = 30449 Ser->Gly ""0 {8.3164939 Gln->G|u C HIHHHHI Val—>Net G HI!!! to r561731687 Pro->Ala HIE! r361731688 ->Arg Lys H!!! W494 r5186— >Aro Tr- II': s->Trp ) C III!!! 'm—“mm “—“mm -“—“m In—““ “—mm “—“m“ “nun-s- “—“mu 'm—m—m m—“mm ‘--“--‘- lm—mmm .n—“m-e-mmm 40 13 7 7 6 1 1 1 # indvs IGLV3-1 5 mmmmh—mmmwwmmmmmmmcncncncacaozcnlntnnncoo-ru-NNNNx—rmmmmmmmooooooovgvOOWWONNNOOFi-I—I—FI—i—OOOOOOOC o OO’OOQOOOQQOOOOOOOQQQQQQQOOO 0. 0.0.OQQQQQQQQQQQQOOOOOCOQQO o oo coo 0000000 000 27 cum.fre Table v.5 S) .C U _ hmonsowu>.__c >wxm_QmU_mNmm:xm PCT/G32012/052296 Hi. i- I O HII{Hunts Illo HIIIIO 0'0 0 II0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ‘l 1 1 1 1 5mmmmmmmm£m9m3mu€$mmlg>33333335c3o3o30o339 JOOOOOOOOCOODOOOOODO OQQOOOQOOCOQQQQQQQQO TOOOOOOOOOODOOOOOOOO hmgmmmctuSmhfimmmEfimmm(U _D¥ D. O 3 E u U)._C PCT/G82012l052296 Exmeré Fmommooooohwzm e.ow.m.w.n.©.m.v.m.w Edvdfimfiuwdfi 3.9.3.2666; vie—umdidé 3.0—3.6” Enorfidé Viofimdfi mfidfié Sdfifiw 3.9.5; Edged mfimd wwdfim ofimfi 3.0;” mfiw N man. 36 aim N Nfiw var Cd 3.9. 3‘ ‘— 1-0! OEEBSQ 57.3: ""lll"!I'— iIEII IIH II 53:. 05450 . 35 ”Semis ”34.3.IHHHU< 308889 “Hm/«33 ._. 3m. 304m? 0 IIHHHIII HHHHIH IIHHHIH aR858 0 DIOVVMWMMMMMNNNNNNNFFF # indvs 94540. - mmwmd mow—‘06 mmmood mood mrmood mrmood nwood mNNood mNNood mmmood wrood wrood mmrood moroo‘o mum—bod mmvood A“um—bod 0900.0 @2006 moood @0006 @0006 wooed moood aoccd moood mvoood mvoood mvoood thdr cumf"m 2an h.type U) E .C NOD:O‘D~>«._ C (15>an _D.Q'D.—.U)N E3 3.! _m PCT/GBZO]2/052296 LOEEuD-LOFNIOFCONMF D‘— 10 4 2 1 "0infllm -mm—mmm 1 1 1 1 1 1 1 1 1 1 1 1 1 IGLV3-1 0.00045 0.00045 0.00045 0.00045 0.00045 0.00045 5 0.00045 0.00045 0.00045 0.00045 0.00045 0.00045 13.127 Table aa aq al ab ak 8P a0 am at ar as ai an PCT/GBZOIZI052296 WW 4943 Emma Pro->Leu C 6145459232 Tyr—>Asp T illill C III-I III-III. c rs1008910 GIn->L Thr->Ala A rs190621299 {5115492210 556 338 296 IGLV3-21 5 0.178 0.1613 13.128 Table ref mnohumw:_._m~x.._:>Ucan EQNFFSSSSSEIIIIII «5335;33va 3.2a:_.e.m.m.%.3.m§ Eatwtofiwflqwé $65,936.13 3.9.38; 2.5.3 .w.v.w 3.23 9.3 9.0.3 a; NE S 0: N 2 w w w 2 2 8:463, p 598%; EH :3 HE! 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Snoré ofivd vvdr mic—um or v. «v.9. 3.; 3.0—. 2‘ VF -3 N m w m m N m F F N N N w r NN.N>40_ pops - mNm m COtDEQVV‘mmNNNNFF:0 ‘— #Indvs - - mm mm mN—umr mmood Nwmmd mvmwod mwwood mmood nmood wrood 300.0 50.0 50.0 moood moood moood moood 9500.0 3500.0 03F“. .9 (0.9001: cu:....-_chx_ rs188941 rs182651541 r5443 J' rs12628782 367 41 9 3 2 2 1 1 1 1 1 1 1 1 1 1 1 1 IGLV3-25 0.19965 0.02105 0.00405 0.00135 0.0009 0.0009 0.00045 0.00045 0.00045 5 0.00045 5 0.00045 0.00045 0.00045 0.00045 0.00045 0.00045 13.130 cum.freq Table :- Q 'Um 007_.__!WUL h.._CEQ.0 PCT/G32012/052296 874336 1 a->Thr rs AI rs1895 HHIHI [3181030260 Ala->Ser rs1847734- 3145801212 rs192490624 367 41 9 3 2 2 1 1 1 1 1 1 1 1 1 1 IGLV3-25 # indvs 0.19965 0.02105 0.00405 0.00135 0.0009 0.0009 0.00045 5 0.00045 0.00045 0.00045 0.00045 Lnln-nmvoto383333000000QQQQQQoooooo Table .3«21:m —u—XWUI. 5..-:an PCT/G82012/052296 monomMOOQOOHme 3.2.9....flordfifidfifiddé 3.9.3;r.ow.m.w.h.m.m,v.m.m.w Fr.m.m.v.m.w oedddé «fimN N m.m o m or 3. 3 w m w — N F FFFV-v-erv—Fu— nwm 3. 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NAME.

IGLJ 7-a ggagzs PCT/G82012/052296 —'GW -—- ‘ IGLV 46-a 0.9166 9m. W WM n W MMT NAME. wecv mm IGU IGU7-a 0.93225 .13. 1 :NA’I’VE REPE ___— __-_ __-657% WO 41844 lGHJ3*02 \ IGHJ4*02 IGHJ5"02 ——ll_- _——EE- —IGHDI-26*01 ———_ ———E- —_—l_ —GHD4—23*01 PCT/GBZ012/052296 - ant \ IGHJ6*02 IGHD6-19*01 _——- 1———- _——- ———I'.- ———I- TABLE lfiggz All Na'l've — V Q J 2] *03 IGHJ6*02 5 _-I_l- —_-_ —_—l- ---34 IGHJ6*02—_ —_—E_ _——_ IGHV3-15*01 PCT/GBZOIZ/052296 __—- __—I- —_—lm 2*02 IGHJ6‘02 IGHV71*01 IGHJ6‘02 IGHJ6*02 IGHV1v18*01 IGHJ6*02 1 IGHV4-4*02 IGHJ6*02 0 - IGHV3-9*0] IGHJ6*02 - IGHV3-23*04 GHJ6*02 I- 16: IMM NI ED HEAW H __—— _-_I_ PCT/G32012/052296 TABLE 168; All Immunisgd —J Q D Qsagg -__mLenth . _—I_I_ ———_ IGHD6-I9*01 IGHD6-I3*01 IGHJ6*02 IGHD3-9‘0] —_ IGHJ6*02 ——I_ --nm' ‘l'tl 0. t IGHJ6*02 IGHD3-10*01 IGH16*02 IGHD6-l9*01—_ H1902 IGHD1-26*01—I— _-MM IGI-IV3-7*0] IGHJ6*02 _- IGHV]-8*01 02 —_—_ __I_— ———_ IGHJ6*02 —_ W0 2013/041844 \m_-— --I-a-V Lnth o t ----7*01 IGHJ6*02 21 4 TABLE 17: N-SPE lFlC HEAW HAIN REPERT IRE TABLE 12A: All Anti n- ific— IGI-IJI *01 68% GHJ3 *02 .36% IGHJ4*02 3.78% IGHJ5*02 .04% ——_— ———— -MM_c,It'n tI. .

IGHJ4*02 22 9.09% IGHJ5*02 21 27.27% 2012/052296 TABLE 128: All Antiggnfingcifig —g Q D -_L anLn_th o t -——I_ _——I_ IGHJ6*02 IGHD1«26*01 IGHJ6*02 lGHD3-16*02 —_ IGHJ6*02 ]GHD5-18*01__ mm——_ HCDR3 n _- . ., IGHJ6*02 IGHD3-10*0] —_l_l_ IGHJ6*02 I__ ——-_ IGH16*02—_ IGHJ6*02—_ ———— V J Len-th nt IGHV4-4*02 WO 41844 PCT/082012/052296 ___— _——I- ____ JH2 reference (nucleotide sequence) WDESCRIPTION: w JHZ reference 1 JHS reference (amino acid otide ce) sequence) H p.- IGHV1-2*01 JH6*03=JH6 reference [—1 N |GHV1-3*01 (nucleotide sequence) JHZ reference p.» U) lGHVl—8’01 (nucleotide sequence) JH6*02 (nucleotide sequence) .-IGHV1-24*01 IGHV1-4S*01 JHS reference (nucleotide .- sequence) JHS reference (amino acid --|GHV1-46*01 H lGHVl-58*01 sequence) JH6*03=JH6 .- nce l—‘ C!) IGHV1-69*01 (nucleotide sequence) I—‘ to |GHV1-C*01 JH6*O3=JH6 reference (amino acid sequence) ZOI2/052296 NO |GHV1-f*01 L» U1 |GHV33*01 N p IGHV2-5*Ol U) cn |GHV3-33*Ol NN IGHV2-26*01 U) \J |GHV3-35*01 NW IGHV2-70‘01 U) 00 lGHV3-38*01 N1:. lGHV3-7*01 0.) k0 lGHV3-43‘01 N U‘ lGHV3-9*01 |GHV3-47*01 IGHV3-11*01 b 1-4 |GHV3~48*01 .-lGHV3-13*01 .-lGHV3-49*01 N00 |GHV3~15*01 AU) |GHV3-53*01 N\D |GHV3-16*01 |GHV3-64*01 IGHV3-l9*01 .5 U1 IGHV3-66*Ol W H IGHV3-20*01 4:. 01 72*01 WN |GHV3-21*01 A\l |GHV3-73*01 U.) W lGHV3-23*01 74*01 u)b |GHV3—30*01 Ak0 lGHV3-d*01 U'I |GHV3—h*01 lGHV6—1*01 Mp 4*01 03U1 |GHV71"‘01 lGHV4-28*01 |GHV7-81*01 U‘ u) lGHV42*01 03 7 |GHDl-1*01 lGHDl-7*01 U19 lGHV44*01 |GHDl—14*01 |GHV4-31*01 \lO IGHD1-20*01 \l 1 IGHD1-26*01 IGHV4—34*01 \1 N IGHD2-2*01 3 lGHD2-8*01 57 -|GHV4-39*01 \l4 IGHD2-15*Ol \l5 |GHDZ-21*01 U'i (X) lGHV4-55*01 \l6 |GHD3-3*01 U1\D IGHV4-59*01 \l7 |GHD3-9*01 \l8 |GHDB—10*01 61*01 \l9 lGHDS-16*01 IGH03-22*01 IGHV4-b*01 00 IGH D4-4*01 00 2 lGHD4-11*01 lGHV5-51*Ol 003 |GHD4-17*01 0‘! U) a*01 IGHD4-23*01 00 U1 IGHDS-S*01 IGHDS—12*01 WO 41844 PCT/G82012/052296 |GKV1-16*01 IGKVID-16*Ol m— IGKV1-17*01 IGKV1D—17*01 EI- |GKV1~27*01 9S JH2*01 IGKV1-33*01 JH3*01 97 JH4*01 IG 3*01 JH5*01 JH6*01 |GKV1-37*01 100 |GKV1—S*01 IGKVID-37*Ol |GKV1-6*01 IGKV1-39*01 JGKV1—8*01 |GKV1D-39 lGKVlD—8*01 lGKVlD—42*01 |GKV1-9*01 |GKV1D-43*01 |GKV1-12*01 |GKV2-24*01 IGKV1~13*01 IGKVZD-24*01 PCT/G82012/052296 |GKV2-28*01 |GKV3-20*01 IGKVZD—28*Ol |GKV3D-20*01 |GKV2-29*01 lGKV4-1*01 -29*01 |GKV5v2"‘01 IGKV2-30*01 21*01 lGKVZD-30*Ol lGKV6D-21*01 IGKV2-40*Ol IGKV60-4l*01 |GKV2D~40*01 IGLV1-36*01 |GKV3-7*01 |GLV1-40*01 |GKV3-7*03 IGLV1-4l‘01 -7*01 |GLV1-44*01 lGKV3-11*Ol |GLV1-47*01 |GKV3D~11*01 IGLV1~SO*01 |GKV3w15*01 IGLV1-51*01 lGKVBD-15*01 |GLV2-8*01 PCT/GBZOIZ/052296 IGLV2-11*01 |GLV4-3*01 IGLV2-14*01 |GLV4-60*01 IGLV2-18*01 lGLV4-69*01 23*01 lGLV5-37*Ol IGLV2-33*01 |GLVS-45*01 lGLV3-1*01 iGLV3-10*01 |GLV5~48*01 lGLV3—12*01 IGLV5~52*01 16*01 lGLV6-57‘01 |GLV3-19*01 IG LV7-43*01 |GLV3-21*01 |GLV7-46*01 |GLV3~22*01 |GLV8—61*01 *01 |GLV9-49*01 |GLV3-27*01 lGLV3-32*01 |GLV10-54*Ol W0 20131041844 PCT/0820 12/052296 |GLV11-55*01 |GLV11-55*01 |GLV2-14*01 18*01 |GLV2-23*01 -—185 186 |GLJ1*01 IGLV2u8*01 187 IGLl2*01 188 |GLJ3*01 |GLV3-1*01 189 IGU4*01 lGLV3-12‘01 190 IGL|5*01 191 |GLJ6*01 19*01 192 IGIJ7*01 193 IGKVZD-26*01 IGLV3-21*01 |GLV1-36*01 |GLV3—22*01 |GLV1-47*01 |GLV3-25*01 |GLV1-50*01 |GLV4-60*01 51*01 |GLV5-37*01 |GLV10-54*01 IGLV5-45*01 W0 41844 PCT/GBZOI2/052296 |GLVS-48"'01 lGLV6-57*01 |GLV7-46*01 IGLV8-61*01 W0 2013/0418“ PCT/(382012/052296 (nudeofide sequence) JH6*01 8L JH6*02(amino acid sequence) Human RSS-JH6*02 Human RSS IGHjl ref 2012/052296 240 IG HJ 2 ref IGHV3-16 ref 241 IGHJ3 ref IGHV3-20 ref 242 IG H14 ref 243 IGHJS ref lGHV3-21 ref 244 IGHJS ref 245 |GHV1v18 ref lGHV3-23 ref 246 IGHVl-Z ref lGHV3-30 ref 247 IGHV1-24 ref 248 IGHV1—3 ref |GHV3~33 ref 249 IGHV1-45 ref IGHV3-35 ref 250 IGHV1-46 ref lGHV3-38 ref 58 ref IGHV3-43 ref IGHV1-69 ref IGHV1-8 ref 48 ref IGHV2-26 ref IGHV3-49 ref IGHVZ-S ref IGHV2-7O ref IGHV3-53 ref IGHV3—11 ref IGHV3-64 ref IGHV3-13 ref IGHV3-66 ref IGHV3-1S ref IGHV3-7 ref lGHV3-72 ref 292 IGKJS ref 293 lGKVl—16 ref lGHV3-73 ref 294 IGKV1-17 ref 295 lGKVl-S ref 277 lGHV3-74 ref 6 ref 278 lGHV3-9 ref IGKV1-8 ref 279 lGHV4-28 ref IGKVl-9 ref 280 31 ref 281 IG HV4-34 ref 299 lGKVlD—lZ ref 282 lGHV4-39 ref 300 IGKVlD-lB ref 283 lGHV4-4 ref 301 lGKVlD-l7 ref 284 lGHV4-59 ref 302 IGKVlD-42 ref 303 |GKV1D-43 ref 285 IGKVlD-8 “if lGHV4-61 ref 286 Sl ref IGKV2-24 ”91‘ 287 lGHV6-1 ref 'GKV2'30 I'Ef 288 lGHV7-81 ref 289 IGKJZ ref 290 IGKJ3 ref lGKVZD—24 ref 291 |GKJ4 ref IG KVZD-ZG ref 323 IGUZ ref 324 |GLI3 ref 325 IGUS ref IG KVZD-29 ref 326 IGLl6 ref 327 |GU7 ref 328 lGLVlO—54 ref 329 IGLVll-SS ref 330 IGLV1—36 ref 33 1 IGLV1-4O ref 16 LV1-44 ref 333 IGLV1—47 ref i IG LV 1-50 ref lGLVl—Sl ref 1 ref lGLV2-11 ref |GKV5~2 ref 337 l4 ref IGKV6-21 ref 338 IGLV2-18 ref 339 IGLV2-23 ref IGKVGD-Zl ref 340 IGLV2-33 ref IGKVSD-4l ref lGLJl ref PCT/G82012/052296 8 ref lGLV4-69 ref 342 lGLV3-l ref 56 7 ref 357 lGLVS—45 ref iiIG LV3-10 ref lGLV5-48 ref lGLV3-12 ref lGLV5-52 ref lGLV3-16 ref lGLV6-57 ref lGLV3-19 ref lGLV7-43 ref lGLV7-46 ref lGLV3—21 ref lGLV8-61 ref lGLV9-49 ref IGLV3-22 ref [6 LV3-25 ref lGLV3-27 ref lGLV3-32 ref lGLV3-9 ref lGLV4-3 ref 354 lGLV4-60 ref IGHGl ref GENOMIC IGHGZ ref - GENOMIC IGHGZ ref - CDS (ensembl ript ENST00000390545) lGHGZ-a CDS IGHGZ-a GENOMIC IGHGB ref - GENOMIC IGHGl ref - CDS (ensembl transcript ENST00000390542) IGHGl ref - CDS (Ensembl transcript IGHG3 ref — CDS ID (ensembl transcript ENST00000390549) ID ENST00000390551) IGHGl ref - CDS (ensembl transcript a CDS E NST00000390548) W0 2013/041844 PCT/0320] 96 lGHG3-a GENOMIC lGHG4-a GENOMIC IGHAl ref GENOMIC lGHG3-b CDS lGHAl ref- CDS lGHGB-b GENOMIC (ensembf transcript ENST00000390547) IGHAl-a GENOMIC lGHG4 ref- GENOMIC a CDS IGHAZ ref GENOMIC |GHG4 ref - CDS (ensembl transcript ENST00000390543) lGHG4—a CDS IGHAZ ref- CDS (ensembl transcript ENST00000390539) IGHAZ-a GENOMIC IGHD ref GENOMIC lGHAZ-a CDS IGHAZ-b GENOMIC b CDS IGHD ref- CDS (ensem bl transcript ENST00000390556) PCT/GBZOIZ/052296 IGHE ref GENOMIC IGHM-a GENOMIC IGHM-a CDS |GHM«b GENOMIC IGHE ref- CDS (ensembl transcript ENST00000390541) IGHE rEf- CDS (ensembl transcript ENST00000576077) IG HE ref- CDS 'IGHM-b CDS (ensem bl transcript ENST00000577108) lGH D2-15 GENOMIC IGHM ref GENOMIC 407 IGHDB 16 C IGH D6—6 ref GENOMIC lGHDS-l8 ref GENOMIC IGHDZ-Z ref IGHM ref- CDS GENOMIC (ensem bl transcript ID lGHD4-11 ref 000390559) GENOMIC lGHDS-lZ ref PCT/GBZOl2/052296 GENOMIC IGHD3-22 ref GENOMIC IGHD3-3 ref GENOMIC IGHDS-S ref GENOMIC IGHDZ-8 ref C 19 ref GENOMIC lGHDd-4 ref GENOMIC lGHDl-7 ref GENOMIC IGHD4-23 ref GENOMIC Rabbit JH6 (amino acid) IGHDl-14 ref GENOMIC Rabbit JH6 (nucleotide) ref GENOMIC Sheep JH6 (amino acid) IGHD1-26 ref GENOMIC SheepJH6 (nucleotide) lGHDS—9 ref GENOMIC Bovine JH6 (amino acid) IGHDl-l ref GENOMIC Bovine JH6 (nucleotide) IGHDS-ZS ref GENOMIC 438 Dog JH3 (amino acid} IGHDS-24 ref 439 Dog JH3 (nucleotide) GENOMIC 440 Human JH6*02 IGHDZ-Zl ref (amino acid) GENOMIC Human JH6*02 ZO ref (nucleotide) GENOMIC IGH06-13 ref (nucleotide) GENOMIC amino acid IGHD4-17 ref GENOMIC ment ion Classical pathway Altemattv e pathway Fc receptor recognition FcyRI FeyRHa, 131R/R FcyRJIa, 131H.’H Fc-yRIIb Fc R111 IGKv IGKVID-s-b 0.01785 1.79%—— IGHV IGHV3a 0.01695 1.70%——010-1 -IGHV IGHVS-Sl-a 0.0164 1.64% HV GHVZ-S-b 0.01055 1.06% V LV2_ 0.010 1 04% HV7b 0.0100014:. 1.01% < KVlDb 5 1.01% GKV3~7-b .0o._. 1.00% iHV1d 0.00995 1.00% GLV2e 0.00995 1.00% Bééé7:<<< LV9a 0.00865 0.87% LV1-44—b 0.00775 0.78% LV2a 5 0.78% KV3d 0.007U) 0.73% GKVlDb 0.00685 0.69% GHV3—38-d 0.0064 0 64% I—‘t—ll-II—II—ll-‘HNHNNNI-IUJ IGLV IGLVSc 12 -IGHv4i -—-——_ IGHV IGHVl—24-c 0.00415 0.42%_— IGHV S-c 000405 0.41%_i—NU-I IGLV IGLVld 000405 0.41%——u.) -_-——_ IGHv sa-h ——— IGHV IGHv2c -IGU6-b IGLV IGLV9-49—b --_——_ IGLV I6Lv10f -———_— IGHv IGHv1mm law _—_—_a IGLv IGLv10im -_————=.I -_—-—_ IGHJ IGHJZ-b _—__ IGHV IGHv1ap _——_ IGHv1c WO 41844 PCT/G82012/052296 __——— -_———— 5I< GHv3e —_——www 55555 xIIII<<<<< G) Hv3f 0-00225__—u; HV3g 0.00225 0.23% 5 HV4p 0.00225 0.23% GHV4—31 5 0.23% KV1d 0.00225 0.23% 555 xxx<<< _iiiKV3a 5 0.23% KV3c 0.00225 0 23% GKV3d 0.00225 0.23% C< KV3D-1l—b 0.00225 0.23% UB-a 0.00225__— IGLV GLv11g _——— -—_——_UJUJN 0.00225 0.23% -IGLV I GLV2f 0.00225 0. 23% IGLV GLV3C 0.00225 0.23% GLVSC 0.00225 0. 23% LVSk 0.00225__— < —i 5__l—NUJ HJI-a 0.0018__— HVI- 0.0018__— Hv1y 0.0018__—www 55555 IIIII <<<<< —iiii5HV1ar 0.0018 0 18% HV1ai 0.0018 0.18% HV1s 0.00100 0.18% GHV1d 0.001 0.18% 55 II<< iHVZ-S-d 0.00100 0.18% GHV3-13 ' 0.001 0.18% 5I< 5HV3d 0.001 0.18% 5I< 5HV3n 0.001 0.18% 5I< E<H”U)H“ o. 0.001 0.18% 5I< 5HV3e 0.001 0.18% 5I< 5HV3. 0.001 0.18% I< 5HV4y 0.00100 0.18% 5555 7:11: <<<‘— 5HV4n 0.001 0.18% 5HV4o 0.001on 018% 5HV7—81—c 0.001 0.18% 5KJZ-e 0.001 0.18% GKVZDc 0.00100 0.18% E!55 7:7: iiKV3Dg 0.0018 0.18% KV3Df 0.001on 0.18% GKV4w 0.0018 0.18% I: ’T‘m 0.0018 0.18% PCT/G32012/052296 iiI—l—< ill V GLV10k 0.18%.— N LV11e IllIllllllllllllllllllllllllllllllllllllllllll00018 00018 018%.—U.) '—< GLV1e 0.0018——0.18% 4 555555 f_l_l_l_l_f_ <<<<<< LV1f 0.0018_-0.18% LV1d 0.0018_—I0.18% LV1e 0.001 —_l0.18% -g 0.001 8% ,_< ’1‘U: C.’ 0. 0.0018—_l0.18% 55 ,.< ’T"U1 ’T‘ Q. 0.0018—_I0.18% 55 r-r-<< LV1-51—c 0.0018_—I0.18% GLV2r 0.0018_-I0.18% §§§§<<<< —b 0.0018——0.18% LV2-8~e 0.0018——0.18% LV3h 0.0018—_I0.18% LV3-21 ’ 0.001oo__I0 18% Q< LV3g 0.001 —_I0.18% 99<< -h 0.001 __l0.18% 5LV5-48 ' »_‘ 0.001oo—_l0.18% 5I‘— HJ4-d 0.0013U'i_—0.14% 5555 IIII <<<< HV1—18—d 0. 0013 U!_—0.14% HV1—18-e 0.00135 0 14% HV1h 0.00135——0.14% HV1f 0.00135——0.14% 5I< GHVlaa 0.00135——0.14% 5555 IIII <<<< HV1—3—ak 0.00135——0.14% HV1~3~W 0.00135 0 14% HV1at 0. 00135_—0.14% HV1-69—k 0.00135__0.14% 5I< GHV1ac 0.00135——0.14% 5I< 8-e 0.00135——0.14% 5I< f 0.00135_—0.14% 5I< GHV2y 0.0013 U'l——0.14% 5555 IIII <<<< HV2z 0.00135——0.14% HV2af 5——0.14% HV2h 0.00135——0.14% HV3 5——0.14% 5I< GHV3—16-e 0.0013U1——0.14% 5555 III: <<<< HV3f 0.00135——0.14% -d 0.00135 0 14% HV3C 0.0013 (J!——0.14% HV3h 0.00135—_0.14% GHV3k 0.00135——0.14% 55 III <<< ii—HV3m 0.0013U"——0.14% HV3n 0.0013U1—_0.14% GHV3—53—m 0.00135_—0.14% iHV3f 0.00135__0.14% WO 41844 PCT/GBZOIZ/052296 IGHV IGHV3c 0.00135 0.14%_—U.) IGHV IGHv3h 000135 0-14%——|—‘ 55555 IIIII IGHV4h 0.00135 0.14% HV4—39-u 0.00135 0.14% HV4t 0.0013 0.14% HV4q 0.0013 0.14% HV4ao 0.0013U1 0.14% 5I< GHV4ac 0.00135 0.14% E!5I HV4—4—ae 0.00135 0.14% 5I iiHVS-SI-g 0.00135 0 14% 3 aI< GHV7-81—_e 0.00135__—H 5555 5555 KJZ-f _———NKJ3—d ' J4-s 35__—U.)KJS-c _0.00135 0 14% 55 5KJS-b —0.00135 0.14% 55 XX<< KVl—S-c 35 0.14% KV1-9 ' —0.00135 0.14% KV1D—17—c _0.0013mm 0.14% 5555 XXXX <<<< KVlD-17vb _0.0013 0.14% KV1D~42-e 0.0013 0 14% 2-d 0.0013 0.14% 4-b -0.00135 0.14% 7:< GKVZDc _0.0013U1 0.14% 5555 xxxx <<<< KV3-20—d 3U1 0.14% KV3f _0.00135 0.14% KV3D-11—d _0.0013U1 0.14% r —0.00135 0.14% 7:< GKV4-1—x _0.00135 0.14% 55 X7:<< iiKV4e _0.00135 0.14% KV4—1-o _0.00135 0 14% a I: 5U6-c _0.00135 0.14% 5C iU7-b _0.0013U1 0.14% GLVlO’S4-_ _0.0013U1 0.14% 55l—l_<< iiLVll—SS—h -0.00135 0.14% LV11f —0.00135 0 14% GLV1-4O-1 _0.0013U'I 0.14% (7)555 I—I—I—I—<<<< Lv1f _0.00135 0.14% LV1~47-f _0.00135 0 14% LV2—23—e 0.0013 0.14% LV3d 0.0013 0.14% GLV3d _0.00135 0.14% 55 r-r-<< iiLV3—19-d _0.00135 0.14% LV3—19-l _0.0013U'I 0.14% GLVS-l-ag _0.00135 0.14% GLV3-1—y -0.00135 0.14% PCT/G32012/052296 IGLV IGLVB—l-s 000135 014%_—UJ IGLV IGLVS-l—ad IGLV IGLVB-ZZ-i IGLVSs ) -———__N -—————5.: -—————N --———_N -—————[NJ IGHV IGHVI—IS-f IGHv IGHv IGHv1z IGHV IGHv1ae IGHv IGHv1k IGHv 58-- 0.0009 IGHv IGHv1ad 0.0009 0.0009 IGHv IGHv1o 0.0009 IGHv1h PCT/GBZOIZ/052296 55 :2:<< HV2f 0.0009 0.09% HVZ-S-c 0.0009 0.09% 5I< HV2-70—g 0.0009 0.09% 5555 IIII <<<< Hv2-7o-s 0.0009 0.09% Hv2—7o-ab 0.0009 0.09% -b 0.0009 0.09% HV3f 0.0009 0 09% 5555 IIII <<<< —ii_GHV3-20—d 0.0009 0.09% -c 0.0009 0.09% HV3h 0.0009 0.09% GHV3-23 ' 0.0009 0.09% 5555 IIII <<<< HV3h 0.0009 0.09% HV3f 0.000{D O 09% HV3p 0.0009 0.09% HV3h 0.000to 0 09% 5I< G HV3—66—h 0.000u.) 0.09% IGHV G HV3—74—C 0.0009 0.09% IGHV IGHV3o 0.0009 0 09% 009% 0.09% 0.09% 0.09% 0.09% IGHV IG HV4-34—d 0.0009 0.09% 5I HV4—34-g 0.0009 0.09% 5I HV4b 0.0009 0.09% 5I HV4aa 0.0009 0.09% 5I HV4s 0.000no 0 09% 5I< GHV4x 0.000 0.09% 5I< iilllllllllllllHV4 I 0.000 0.09% 5I< HV4aa 0.000to 0.09% 5I< GHV4af 0.000 0.09% 55555 11223: <<<<< HV4~4~X 0.000 0.09% HV4—4-ad 0.000 0.09% HV4ai one 0.09% HV5-51~d 0.000£0 0.09% GHVS-Sl-k 0.0000 0.09% 5555 III: <<<< -c 0.000 0.09% HV6a 0.000 0 09% HV6c 0.0000'0 0.09% HV7d 0.000Lo 0.09% 55 55r." : 0.000 0.09% HH55 iKJ3-b 0.000 0.09% — GKJ41_ 0.0009 0.09% 5E"F‘ (‘3 0.000LO 0.09% GKJ4-k 0.0009 0.09% PCT/0820 l 2/052296 55<5 iiKJ4-h 0.0009 O. 09% KV1d 0.000LO O 09% 7i< GKV1e 0.000LO 0.09% 5555 XXXX <<<< d 0.000LO 0.09% KV1—8-h 0.0009 0.09% KV1i 0.0009 0.09% KVID—lG-c 0.0009 0 09% 2 GKV1Db 0.0009 0.09% 55 XX<< KVIDg 0.0009_—N GKVlDf 0.0009__— IGKv GKv10e _——— -—_——— HNi—‘l-I -—__—— -—_———NN -_—__— -————_ -—_——— --__——NHI—IN --————M 0.09% -IGKV iiG KV3e 9 0.09% KV3C _0.0009 0.09% IGKV ll-h —0.0009 0.09% __—KV3D-11—i 0.0009 0.09% _-KV3Dd 0.0009 0.09% -—-_KV4 0.0009 0.09% GKV4-1 _0.0009 0.09% 5x< KV4t _0.0009 0.09% iiXR<< KV4i _0.0009 0.09% _0.0009 0.09% 5x< 5x<J? ’7‘ 0.0009 0.09% 5c: 51:: wk.) “0 0.000 0.09% 5I: 5 0.0009—0.09% 5L: 5US—b 0.000 0.09% < 5LV10n 0.000 0.09% _ GLV10' 0.000 0.09% Qééé<<<< LV1h 0.000 0.09% LV1-40*e 0.000 O 09% LV1e 0.000 0.09% LVl-Sl-_ 0.000to 0 09% GLV2' 0.000ED 0.09% §§<< 11-f 0.000 0.09% LV2i 0.000U3 0.09% GLV2-1 1-e 0.0009 0. 09% GLV2p 0.0009 0. 09% PCT/GBZO]2/052296 IGLV IGLv2q 0.0009 0.09% IGLV IGLVZ-M-o 0.0009 0.09% IGLV IGLVZ-B—k 0-0009 009% -_————N -——_——H -—————N IGLV IGLVB-l-ah -IGLV3i -IGL\I3~1’2 -—————N -—————N -——_——N ——_——_I—I -——_—_N -—-————g.) -—————N -—————N IGLv IGLvs—zz—o IGLv IGLv3a IGLv IGLV4—60-g IGLV IGLV IGLVS -————_l IGLV sz—b IGLv7c IGLv IGLV9-49~c _—_———WU) _—_———W -_—_-_U) -—_———0-) -—__——U) ____——w _—_——— IGLv1—44-b 0.0064 IGU6—a 0.00595 0.0055 0.00545 IGHV4—31-i 0.0054 IGLv1c 0.00455 5I< lGHVS-Sl-a 0.0164 52-8 WO 41844 PCT/G82012/052296 PCT/G82012/052296 .5 m w ...». taum mwwavmon-momvamofi¢H EU mmmwmoHéhwwmvafiVH mmnonéfiammnwofivH ommahvonbvmanvoofivH mmmmmmmofiémmmmmwofiwa mmmwmmoOHAmvonwmmOfiVH hREhOhOfimnmwnonofiE” :oxuumEmz mNmHmvaH¢mo 32:3; 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Nat Biotechnol. 2005 Sep;23(9):1117-25; Human antibodies from transgenic animals; Lonberg N. 2. J Clin Invest. 1992 Mar;89(3):729-38; lmmunoglobulin light chain variable region gene sequences for human antibodies to Haemophilus influenzae type b capsular polysaccharide are dominated by a limited number of V kappa and V lambda segments and VJ combinations; Adderson EE, Shackelford PG, Insel RA, Quinn A, Wilson PM, Carroll WL. 3. J Immunol. 1993 Oct 15;151(8):4352-61; Clonal characterization ofthe human IgG dy repertoire to hilus influenzae type b polysaccharide. V. In vivo expression of individual antibody clones is dependent on lg CH haplotypes and the categories of antigen; Chung GH, Scott MG, Kim KH, Kearney J, Siber GR, Ambrosino DM, Nahm MH. 4. J Immunol. 1998 Dec 1;161(11):6068-73; Decreased frequency of rearrangement due to the synergistic effect of nucleotide changes in the heptamer and nonamer ofthe recombination signal sequence of the V kappa gene A2b, which is associated with increased susceptibility of Navajos to Haemophilus influenzae type b e; Nadel B, Tang A, Lugo G, Love V, Escuro G, Feeney AJ.

. J Clin Invest. 1996 May 15;97{10);2277-82; A defective Vkappa A2 allele in Navajos which may play a role in increased susceptibility to haemophilus influenzae type in disease; Feeney AJ, Atkinson MJ, Cowan MJ, Escuro G, Lugo G. 6. infect Immun. 1994 Sep;62(9):3873-80; le region sequences of a protective human monoclonal antibody specific for the Haemophilus influenzae type b capsular polysaccharide; Lucas AH, Larrick JW, Reason DC. 7. J Clin . 1993 Jun;91(6):2734-43; Restricted immunoglobulin VH usage and VDJ combinations in the human response to Haemophilus influenzae type b capsular polysaccharide. Nucleotide sequences of monospecific anti-Haemophilus antibodies and polyspecific antibodies reacting with self antigens; Adderson EE, Shackelford PG, Quinn A, Wilson PM, gham MW, Insel RA, Carroll WL. 8. J Clin Invest. 1993 Mar;91(3):788-96; le region expression in the antibody responses of infants vaccinated with Haemophilus influenzae type b polysaccharide-protein conjugates. ption of a new lambda light chain—associated idiotype and the on between idiotype expression, avidity, and vaccine formulation. The Collaborative Vaccine Study Group; Granoff DM, Shackelford PG, Holmes SJ, Lucas AH. 9. infect Immun. 1994 May;62(5):1776-86; Variable region sequences and pic expression of a protective human immunoglobulin M antibody to capsular polysaccharides of ria meningitidis group B and Escherichia coli K1; Azmi FH, Lucas AH, Raff HV, Granoff DM.

. J Clin Invest. 1992 Dec;90(6):2197-208; Sequence analyses of three immunoglobulin G anti- virus antibodies reveal their utilization of autoantibody-related immunoglobulin Vh genes, but not V lambda genes; Huang DF, Olee T, Masuho Y, Matsumoto Y, Carson DA, Chen PP. 11. Science. 2011 Aug 12;333(6044):834-S, mistry. Catching a moving target, Wang TI”, Palese P 12. Science. 2009 Apr 10;324(5924):246-51. Epub 2009 Feb 26; Antibody recognition of a highly conserved influenza virus epitope; Ekiert DC, Bhabha G, Elsliger MA, Friesen RH, Jongeneelen M, Throsby M, Goudsmit J, Wilson IA. 13. PLoS One. 2008;3(12):e3942. Epub 2008 Dec 16; Heterosubtypic neutralizing monoclonal antibodies cross-protective against H5N1 and H1N1 recovered from human lgM+ memory 8 cells; Throsby M, van den Brink E, Jongeneelen M, Poon LL, Alard P, Cornelissen L, Bakker A, Cox F, van Deventer E, Guan Y, Cinatl J, ter Meulen J, Lasters I, Carsetti R, Peiris M, de Kruifl, Goudsmit J. 14. Nat Struct Mol Biol. 2009 Mar;16(3):265-73. Epub 2009 Feb 22,Structura| and functional bases for spectrum neutralization of avian and human nza A viruses, Sui J, Hwang WC, Perez 5, Wei G, Aird D, Chen LM, Santelli E, Stec B, l 6, Ali M, Wan H, Murakami A, Yammanuru A, Han T, Cox NJ, Bankston LA, Donis RO, Liddington RC, Marasco . Science. 2011 Aug 12;333(6044):843-50. Epub 2011 Jul 7, A highly conserved neutralizing epitope on group 2 nza A viruses, Ekiert DC, Friesen RH, Bhabha G, Kwaks T, Jongeneelen M, Yu W, Ophorst C, Cox F, Korse HJ, nburg B, Vogels R, BrakenhoffJP, Kompier R, Koldijk MH, Cornelissen LA, Poon LL, Peiris M, Koudstaal W, Wilson lA, Goudsmit *1le ‘JUu

Claims (2)

CLAIMS : 1. A non-human vertebrate or non-human vertebrate cell whose genome comprises an immunoglobulin heavy chain locus comprising unrearranged human gene segment JH6*02, one or more VH gene segments and one or more D gene segments am of a nt region; wherein the gene segments in the heavy chain locus are operably linked to the constant region thereof so that the vertebrate is capable of producing an antibody heavy chain produced by recombination of the human JH6*02 with a human D segment and a human VH segment, wherein the JH6*02 gene segment comprises the following nucleotide sequence: ATTACTA CTACTACTAC GGTATGGACG TCTGGGGCCA CACG GTCACCGTCT CCTCAG, wherein the vertebrate has been immunised with a target antigen and es an antibody heavy chain specific for the target antigen, wherein the target n is an antigen of an infectious disease pathogen, and wherein the variable domain of the heavy chain is the product of recombination between a human VH, D and JH6*02 and wherein the HCDR3 length is at least 20 amino acids. 2. An isolated antibody-producing cell that is a progeny of a non-human vertebrate cell whose genome ses an immunoglobulin heavy chain locus comprising human gene segment JH6*02, one or more human VH gene segments and one or more human D gene segments upstream of a nt region; wherein the gene segments in the heavy chain locus are operably linked to the constant region thereof for producing an antibody heavy chain produced by recombination of the human JH6*02 with a D t and a VH segment, wherein the JH6*02 gene segment comprises the following nucleotide sequence: A CTAC GGTATGGACG TCTGGGGCCA AGGGACCACG GTCACCGTCT CCTCAG wherein the antibody-producing cell comprises a heavy chain locus comprising a rearranged variable region produced by ination of human JH6*02 with a human D segment and a human VH segment, wherein the antibody-producing cell produces an antibody heavy chain specific for a target antigen, wherein the target antigen is an antigen of an infectious disease pathogen, and 7797479_1 (GHMatters) P96492.NZ 27-May-16 wherein the variable domain of the heavy chain is the product of recombination between a human VH, human D and human JH6*02 and wherein the HCDR3 length is at least 20 amino acids. 3. The cell of claim 2, wherein the antibody-producing cell is a B-cell. 4. The cell of claim 2 or claim 3, which is a B-cell or oma that expresses a target antigenspecific antibody comprising a heavy chain that comprises a rearranged variable region produced by recombination of human JH6*02 with a human D segment and a human VH segment selected from: VH3-11 and D3-9; VH3-20 and D3-10; VH4-4 and D3-10; VH3-9 and D3-10; or VH1-8 and D310. 5. The cell of claim 4, wherein the human VH 3-11 segment is a VH3-11*01 segment. 6. The cell of claim 4 or claim 5, wherein the VH3-20 segment is a VH3-20*01 t. 7. The cell of any one of claims 4 to 6, wherein the VH4-4 segment is a VH4-4*02 segment. 8. The cell of any one of claims 4 to 7, wherein the VH3-9 t is a VH3-9*01 segment. 9. The cell of any one of claims 4 to 8, wherein the VH1-8 segment is a 01 segment. 10. The vertebrate or cell of any one of claims 1 to 9, wherein the target n is a bacterial or viral pathogen antigen. 11. The vertebrate or cell of any one of the preceding claims, wherein the HCDR3 length is at least 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids. 12. The vertebrate or cell of any one of the preceding claims, wherein the HCDR3 length is no more than 35, 34, 33, 32 or 31 amino acids. 13. The rate or cell of any one of claims 1 to 11, wherein the HCDR3 length is 20, 21, 22, 23 or 24 amino acids. 7797479_1 (GHMatters) P96492.NZ 27-May-16 14. The vertebrate or cell of any one of the preceding claims, wherein the heavy chain locus comprises a human JH6*02 recombination signal sequence (RSS) operably connected 5' to the JH6*02 gene segment. 15. The vertebrate or cell of claim 14, wherein the RSS is SEQ ID NO: 238 or a sequence having an identical 9mer and 7mer sequence flanking a sequence that is at least 70% identical to the 22mer ce of SEQ ID NO: 238. 16. The vertebrate or cell of claim 15, wherein the RSS and JH6*02 are provided as SEQ ID NO: 237. 17. The vertebrate or cell of claim 1, wherein the locus comprises one, more or all human D gene segments D3-9, D4-17, D3-10, D2-2, D5-24, D6-19, D3-22, D6-13, D5-12, D1- 26, D1-20, D5- 18, D3-16, D2-21, D1-14, D7-27, D1-1, D6-25, D2-14, and D4-23. 18. The vertebrate or cell of claim 17, wherein the locus comprises one, more or all human D gene segments D3-9, D3-10, D6-19, D4-17, D6-13, D3-22, D2-2, D2-25, and D3-3. 19. The vertebrate or cell of any one of the preceding , wherein i) the JH6*02 is the only JH6-type gene segment in the genome or ii) the JH6*02 is the closest JH gene t to the constant region in the locus. 20. The vertebrate or cell of claim 1, wherein the locus ses a ity of human D gene segments and the JH6*02 is in human ne configuration with respect to the 3'-most human D gene segment. 21. The vertebrate or cell of any one of the preceding claims, wherein the locus comprises one, more or all of IGHV gene segments selected from V3-21, V3-13, V3-7, V6-1, V1-8, V1-2, V7- 4-1, V1-3, V1- 18, V4-4, V3-9, V3-23, V3-11, V3-20, D3-9*01, D3-10*01, D6-19*01, D6-13*01, D1-26*01, IGHV1-8*01, IGHV4-61*01, IGHV6-1*01, 4*02, IGHV1-3*01, IGHV3-66*03, IGHV3-7*01 and IGHV3-9*01. 22. The vertebrate or cell of any one of the preceding claims, wherein the antibody heavy chain is a product of the recombination of JH6*02 with a human VH gene segment selected from V3- 21, V3-13, V3-7, V6-1, V1-8, V1-2, V71, V1-3, V1- 18, V4-4, V3-9, V3-23, V3-11, V3-20, D3-9*01, D3-10*01, D6-19*01, D6-13*01, D1-26*01, IGHV1-8*01, IGHV4-61*01, IGHV6-1*01, 4*02, IGHV1-3*01, 66*03, IGHV3-7*01 and IGHV3-9*01 and/or a D gene segment selected from D3-9, D4-17, D3-10, D2-2, D5-24, D6-19, D3-22, D6-13, D5-12, D1- 7797479_1 (GHMatters) P96492.NZ 27-May-16 26, D1-20, D5-18, D3-16, D2-21, D1-14, D7-27, D1-1, D6-25, D2-14, and D4-23 or selected from D3-9, D3-10, D6-19, D4-17, D6-13, D3-22, D2-2, D2-25 and D3-3. 23. The vertebrate or cell of any one of the preceding , wherein all nous non-human vertebrate heavy chain variable region gene segments have been vated in the genome and/or wherein the genome is homozygous for said heavy chain locus. 24. The vertebrate or cell of any one of claims 1 to 23, wherein the constant region is a human, mouse or rat constant region. 25. The vertebrate or cell of claim 24, wherein the constant region is an nous constant region of said rate or cell. 26. The vertebrate or cell of any one of the preceding claims, wherein the vertebrate is a mouse or a rat, or the cell is a mouse cell or a rat cell. 27. The vertebrate of claim 26, which is a mouse comprising functional heavy gene segments VH2-5, VH71, VH4-4, VH1-3, VH1-2, VH6-1, D1-1, D2-2, D3-9, D3-10, D4-11, D5-12, D6- 13, D1-14, D2-15, D3-16, D4-17, D5-18, D6-19, D1-20, D2-21, D3-22, D4-23, D5-24, D6-25, D1-26, D7-27, JH1, JH2, JH3, JH4, JH5 and JH6, in 5' to 3' order, wherein the JH6 is the human JH6*02 variant, wherein the insertion of human DNA is made between positions 114666435 and 114666436 on mouse chromosome 12, wherein human functional heavy chain gene segments VH3-13, VH3-11, VH3-9, VH1-8, VH3- 7 are inserted upstream of VH2-5, wherein the mouse VH, D and JH gene segments are retained in the locus, ately upstream of (5' of) the ed human heavy chain DNA, and wherein the mouse VH, D and J segments are ed to inactivate them, thereby producing mice in which only the human heavy chain variable region gene segments are active. 28. The vertebrate of claim 27 additionally comprising VH2-26, VH1-24, VH3-23, VH3-21, VH3- 20, VH1-18, and VH3–15 inserted upstream (5') of the 5'-most VH. 7797479_1 (GHMatters) P96492.NZ 27-May-16 29. A method for producing a heavy chain, a VH domain, or an antibody specific to a target antigen, the method comprising isolating from the vertebrate of any one of claims 1 or 10 to 28, the heavy chain, VH domain or antibody specific to the target antigen or a cell ing the heavy chain, VH domain or antibody, wherein the heavy chain, VH domain or an antibody ses a HCDR3 that is derived from the recombination of human JH6*02 with a human VH gene segment and a human D gene segment, wherein the JH6*02 gene segment comprises the following nucleotide sequence: ATTACTA CTACTACTAC GGTATGGACG TCTGGGGCCA AGGGACCACG GTCACCGTCT CCTCAG, wherein the vertebrate produces said antibody heavy chain ic for the target antigen, n the le domain of the heavy chain is the product of recombination between a human VH, human D and JH6*02, and wherein the HCDR3 length is at least 20 amino acids. 30. The method of claim 29, n the constant region of the locus is a non-human vertebrate, nt region, and the non-human constant region of the isolated heavy chain or antibody is replaced with a human constant region. 31. The method of claim 30, wherein the constant region is a mouse or a rat constant region. 32. The method of any one of claims 29 to 31, comprising isolating a heavy chain, VH domain or an antibody, wherein the HCDR3 length is at least 20 amino acids. 33. The method of any one of claims 29 to 32, comprising isolating a B-cell or hybridoma expressing a heavy chain VH domain that is cal to the VH domain of the heavy chain of claim 32. 34. The method of any one of claims 29 to 31, comprising removing B lymphocytes from the vertebrate and ing one or more B lymphocytes expressing antibodies that bind to the antigen, optionally immortalising said selected B lymphocytes or progeny thereof, optionally by producing hybridomas therefrom, and ing an antibody expressed by the B lymphocytes. 35. The method of claim 34, further comprising isolating from said B lymphocytes nucleic acid encoding said antibody. 7797479_1 (GHMatters) P96492.NZ 27-May-16 36. The method of claim 35, further sing exchanging the heavy chain constant region nucleotide sequence of the antibody with a nucleotide sequence encoding a human or humanised heavy chain constant region, and optionally affinity maturing the variable region of said antibody. 37. The method of claim 35 or claim 36, further comprising inserting said nucleic acid into an expression vector and optionally a host cell. 38. The method of claim 37, comprising expressing the heavy chain, VH domain or antibody from the host cell and providing an isolated heavy chain, VH domain or antibody. 39. A heavy chain, a VH domain, or an antibody produced by the method of any one of claims 29 to 38. 40. A heavy chain or an antibody produced by the method of any one of claims 29 to 38, wherein the HCDR3 length is at least 20 amino acids and the nt region is mouse or rat. 41. Use of human JH6*02 for generating a HCDR3 of at least 20 amino acids in a man rate whose genome comprises an immunoglobulin heavy chain locus comprising human gene segment JH6*02, one or more human VH gene segments and one or more human D gene ts upstream of a constant region; wherein the gene segments in the heavy chain locus are operably linked to the constant region thereof so that the vertebrate is capable of producing an antibody heavy chain produced by recombination of the human JH6*02 with a human D segment and a human VH segment and wherein the JH6*02 gene segment comprises the following nucleotide sequence: ATTACTA CTACTACTAC GGTATGGACG TCTGGGGCCA AGGGACCACG GTCACCGTCT CCTCAG, wherein the antibody heavy chain specifically binds a target antigen of an infectious e pathogen. 42. The use of claim 41, wherein the target n is a bacterial or viral en antigen. 43. The use of claim 41, wherein the vertebrate is the vertebrate any one of claims 1 or 10 to 28. 44. The use of claim 41, wherein the vertebrate is sed with the target antigen. 45. The use of claim 41, wherein the vertebrate comprises a mouse Eµ and Sµ in the heavy chain locus between the JH6*02 and the constant region. 46. The use of claim 45 wherein the Eµ and Sµ are Eµ and Sµ of a mouse 129-derived genome or of a mouse C57BL/6-derived genome. 9_1 (GHMatters) P96492.NZ 27-May-16 47. A heavy chain, a VH domain, or an antibody comprising the HCDR3 generated by the use of claim 41. 48. An antibody comprising the heavy chain of claim 47. 49. A pharmaceutical composition comprising the heavy chain, VH domain, or antibody of claim 39 or claim 47 or claim 48, together with pharmaceutically-acceptable excipient, diluent or a medicament. 50. The vertebrate of claim 1 or the cell of claim 2, substantially as hereinbefore described with reference to the examples and figures. 51. 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