NZ623745B2 - Manipulation of immunoglobulin gene diversity and multi-antibody therapeutics - Google Patents

Abstract

The invention provides improved non-human vertebrates and non-vertebrate cells capable of expressing antibodies comprising human variable region sequences. The present invention is directed to the provision of long HCDR3s from non-human vertebrates and cells. The present invention is also directed to the provision of novel V, D and J pairings in immunoglobulin heavy and light chain loci. Novel, biased antibody diversities and potentially expanded diversities are provided. The invention also provides for novel and potentially expanded diversity or diversity that is biased towards variable gene usage common to antibodies useful for treating and/or preventing certain diseases or conditions, such as infectious diseases. The invention also provides methods of generating antibodies using such vertebrates, as well as the antibodies per se, therapeutic compositions thereof and uses. o the provision of novel V, D and J pairings in immunoglobulin heavy and light chain loci. Novel, biased antibody diversities and potentially expanded diversities are provided. The invention also provides for novel and potentially expanded diversity or diversity that is biased towards variable gene usage common to antibodies useful for treating and/or preventing certain diseases or conditions, such as infectious diseases. The invention also provides methods of generating antibodies using such vertebrates, as well as the antibodies per se, therapeutic compositions thereof and uses.

Description

PCT/G82012/052298 MANIPULATION OF IMMUNOGLOBULIN GENE DIVERSITY AND MULTI-ANTIBODY THERAPEUTICS FIELD OF THE INVENTION The present ion relates to the provision of antibodies with long HCDR3 lengths. The present invention is also directed to the provision of novel V, D and J pairings in immunoglobulin heavy and light chain loci. Novel, biased antibody diversities and potentially ed diversities are provided.

The invention also provides for novel and potentially ed diversity or diversity that is biased towards variable gene usage common to antibodies useful for ng and/or preventing certain diseases or conditions, such as infectious es. This ability to bias the antibody repertoire also provides methods of simplifying the tion of antibody mixtures, such as polyclonal antibody therapeutics useful for the treatment and/or prevention of infectious diseases where a polyclonal approach to target multiple pathogen antigens is ble. To this end, the t invention also provides bispecific antibodies that are e of binding to more than one antigen (eg, multiple infectious antigens expressed by the same pathogen), thus providing advantages (such as manufacturing, dosing and stration ages) not le with polyclonal antibody mixtures.

The present invention provides vertebrates and cells, such as transgenic mice or rats or transgenic mouse or rat cells. Furthermore, the invention relates to methods of using the vertebrates to isolate antibodies or nucleotide sequences encoding antibodies. Antibodies, nucleotide sequences, pharmaceutical compositions and uses are also provided by the invention.

BACKGROUN D The state of the art provides non-human vertebrates (eg, mice and rats) and cells comprising transgenic globulin loci, such loci comprising human variable (V), diversity (D) and/orjoining (J) segments, and optionally human constant regions. Alternatively, endogenous constant regions of the host vertebrate (eg, mouse or rat constant regions) are provided in the transgenic loci. Methods of constructing such transgenic vertebrates and use of these to te antibodies and nucleic acids thereof ing antigen immunisation are known in the art, eg, see US7501552 (Medarex), US5939598 ix), US6130364 ix), WO02/066630 (Regeneron), WO2011004192 e Research Limited), WO2009076464, WO2009143472 and WO2010039900 (Ablexis), the disclosures of which are itly incorporated herein. Such transgenic loci in the art include varying amounts of the human V(D) J repertoire.

Existing enic immunoglobulin loci tend to be based on a single human DNA source. The potential diversity of human antibody variable regions in non-human rates bearing such transgenic loci is thus confined by the repertoire used.

It would be desirable to provide for novel and potentially expanded repertoire and ity of human variable regions in transgenic immunoglobulin loci and non-human vertebrates ring these, as well as in antibodies produced following immunisation of such s.

SUMMARY OF THE INVENTION The present inventors have discovered, by way of construction of transgenic non-human vertebrates, immunisation, antibody heavy chain collection and deep bioinformatics analysis, how to rationally design for VH domains, heavy chains and antibodies having long HCDR3s. These are useful for addressing antigens (such as infectious disease pathogen antigens, receptors and enzyme clefts) where a longer CDR better addresses the target.

The present inventors also ed the possibility of providing combinations of V, D and J gene segments in new ways to provide synthetic gene segment combinations in immunoglobulin loci that are not found in nature or in state-of-the-art loci. The inventors realised the importance of this to provide for novel and ially expanded repertoire and ity of human variable regions in transgenic immunoglobulin loci and non-human vertebrates harbouring these, as well as in antibodies produced following immunisation of such animals. The ors realised that it would be desirable to bias the novel repertoire 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, provides for antibodies to epitopes that have hitherto been intractable by prior transgenic immunoglobulin loci or difficult to address. For example, the ors envisaged a specific ation to bias the novel repertoire for the production of antibodies useful in the therapy and/or prevention of ious disease.

A first aspect of the invention provides a method of producing an antibody with an HCDR3 sequence of at least 20 amino acids in length, wherein the antibody binds to an antigen of an infectious disease pathogen, the method comprising: (a) providing a transgenic mouse or rat whose genome comprises an immunoglobulin heavy chain locus comprising one or more human VH gene segments selected from the group consisting of: a VHIII gene family member nally, a 7592861_1 (GHMatters) P96496.NZ 5-Apr-16 VHIIIa or VHIIIb family member), a VHIV gene family member, VHIII 9.1 (VH3-15), VHIII VH26 (VH3-23), VH3-21, LSG6.1, LSG12.1, DP77 (V3-21), VH H11, VH1GRR, ha3h2, VHI- ha1c1, VHIII-VH2-1, , ha4h3, Hv1051, 71-2, , VH4.11, 71-4, VH251, VH1-69 and a gene segment at least 80% identical; one or more human D gene segments and one or more human JH gene segments upstream of a constant region; wherein the heavy chain locus comprises VH and/or D gene segments capable of yielding HCDR3s of 20 or more amino acids in , comprising VH gene segments IGHV1- 2*02, IGHV1-18*01, IGHV3-7*01, 1*01, IGHV3-9*01, IGHV2-5*10, IGHV71*01, IGHV1-3*01, IGHV4-4*02, IGHV3-13*01, IGHV3-23*04, IGHV1-8*01, IGHV3-21*03 and/or IGHV3-11*01, and/or comprising D segments IGHD2-2*02, IGHD3-9*01, IGHD3-10*01, IGHD6-13*01, IGHD4-17*01, IGHD6-19*01, 22*01 and/or IGHD1-26*01, and an globulin light chain locus comprising one or more human V gene segments and one or more human J gene segments upstream of a constant region, wherein the gene segments in the heavy chain locus are operably linked to the constant region thereof, and the gene segments in the light chain locus are operably linked to the constant region thereof, so that upon immunisation the mouse or rat is capable of ing an antibody comprising heavy chains produced by recombination of the heavy chain locus and light chains produced by recombination of the light chain locus; (b) immunising said mouse or rat with said antigen; (c) removing B lymphocytes from the mouse or rat and selecting one or more B lymphocytes expressing antibodies that bind to the antigen; (d) optionally immortalising said ed B lymphocytes or progeny thereof, optionally by producing hybridomas therefrom; and (e) isolating an antibody sed by the B cytes.

A second aspect of the invention provides a method of producing a polyclonal antibody mixture, the method sing carrying out a method ing to any one of claims 1 to 3 by separately immunising first and second mice or rats with antigen and combining the anti-antigen antibodies isolated from each mouse or rat, or s or derivatives of said antibodies, to produce a polyclonal antibody mixture; optionally wherein the following (i), (ii) or (iii) apply separately or the following (i) and (ii) or (i) and (iii) apply in combination: (i) the mice or rats are immunised with the same antigen or different ns, optionally wherein the different ns are expressed by the same pathogenic organism or a family member thereof; (ii) prior to immunisation the light chain loci of the mice or rats contain the identical VH gene repertoire, optionally a single VH gene, and optionally the identical J repertoire; optionally the light chain loci of the mice or rats are identical prior to immunisation; (iii) prior to immunisation the light chain loci of the mice or rats contain the identical rearranged VJ or VDJ repertoire, optionally a single VJ or VDJ; optionally the light chain loci of the mice or rats are identical prior to immunisation. 7592861_1 (GHMatters) .NZ 5-Apr-16 A third aspect of the ion provides a method of producing a onal antibody mixture, the method comprising ng out a method according to the first or second aspect by immunising one or a plurality of mice or rats with first and second antigens and combining the anti-antigen dies isolated from each mouse or rat, or mutants or derivatives of said antibodies, to produce a polyclonal antibody mixture; optionally wherein the following (i), (ii) or (iii) apply separately or the following (i) and (ii) or (i) and (iii) apply in combination: (i) the antigens are expressed by the same pathogenic organism, or a family member thereof; (ii) prior to immunisation the light chain loci of the mice or rats contain the identical VH gene repertoire, optionally a single VH gene, and optionally the identical J repertoire; optionally the light chain loci of the mice or rats are identical prior to immunisation; (iii) prior to immunisation the light chain loci of the mice or rats contain the identical rearranged VJ or VDJ repertoire, optionally a single VJ or VDJ; optionally the light chain loci of the mice or rats are identical prior to immunisation.

A fourth aspect of the invention provides a method of producing host cells capable of expressing a polyclonal antibody e, the method comprising, in a method according to the first aspect: (a) immunising one or a plurality of mice or rats with first and second ns, optionally wherein the different antigens are expressed by the same pathogenic organism or a family member thereof; (b) isolating nucleic acid encoding first and second ntigen antibodies from B cytes from said mice or rats; (c) determining the nucleotide sequences of the heavy and light chain variable regions of the first antibody; (d) determining the nucleotide ce of the heavy variable region and optionally the light chain variable region of the second antibody; (e) inserting the heavy chain variable region coding sequence of each antibody into a heavy chain expression vector; optionally wherein the constant region coding sequence of each heavy chain is exchanged for a nucleotide sequence that encodes a human or humanised constant region; (f) inserting the light chain variable region coding sequence of the first antibody into a light chain expression vector; optionally wherein the constant region coding sequence of the light chain of the first antibody is exchanged for a nucleotide sequence that encodes a human or humanised nt region; (g) optionally inserting the light chain variable region coding sequence of the second antibody into a light chain expression vector; optionally wherein the constant region coding sequence of the light chain of the second antibody is ged for a nucleotide sequence that encodes a human or humanised constant region; and (h) introducing each expression vector into a host cell and co-expressing antibody chains in a mixture of said host cells to produce antibodies, each antibody comprising one or both of said heavy chain variable regions and a light chain; ally wherein the sion s are introduced 7592861_1 (GHMatters) P96496.NZ 5-Apr-16 together into the same host cell, optionally a CHO cell or HEK293 cell, so that the cell is capable of sing antibody light chains and heavy chains, such that the cell or a plurality of the host cells express antibodies, each comprising one or both of said heavy chain variable regions and a light chain; (i) optionally: prior to immunisation the light chain loci of the mice or rats contain the identical VH gene repertoire, optionally a single VH gene segment, and optionally the identical J repertoire, optionally a single J gene segment; optionally the light chain loci of the mice or rats are identical prior to immunisation; or prior to immunisation the light chain loci of the mice or rats contain the identical rearranged VJ or VDJ repertoire, optionally a single VJ or VDJ; optionally the light chain loci of the mice or rats are identical prior to immunisation.

A fifth aspect of the invention provides a method of producing a monoclonal or polyclonal antibody mixture, the method comprising ng out a method according to the fourth aspect and expressing a monoclonal antibody or polyclonal mixture of said antibodies; optionally followed by ing an antibody sing the heavy chain le region of the first and/or second antibodies.

A sixth aspect of the invention provides use of a transgenic mouse or rat for producing an antibody with an HCDR3 sequence of at least 20 amino acids in length, wherein the antibody binds to an antigen of an ious disease pathogen, n the mouse or rat genome comprises human VH, D and J gene segments provided in a heavy chain locus upstream of the endogenous mouse or rat constant region, wherein the VH gene segments comprise IGHV1-2*02, IGHV1-18*01, 7*01, IGHV6-1*01, IGHV3-9*01, IGHV2-5*10, IGHV71*01, IGHV1-3*01, IGHV4-4*02, 13*01, 23*04, IGHV1-8*01, 21*03 and/or 11*01, and wherein the D segments se IGHD2-2*02, IGHD3-9*01, IGHD3-10*01, IGHD6-13*01, IGHD4- 17*01, IGHD6-19*01, IGHD3-22*01 and/or IGHD1-26*01.

A seventh aspect of the invention es an isolated antibody that specifically binds an antigen of an infectious disease en, the antibody comprising human heavy chain variable regions and non-human constant regions, wherein the variable regions are derived from the recombination in a non-human vertebrate of (i) a human VH gene segment selected from IGHV1-2*02, IGHV1-18*01, IGHV3-7*01, IGHV6-1*01, IGHV3-9*01, IGHV2-5*10, IGHV71*01, IGHV1-3*01, IGHV4-4*02, IGHV3-13*01, IGHV3-23*04, IGHV1-8*01, 21*03 and/or IGHV3-11*01 with (ii) a human D gene segment selected from IGHD2-2*02, IGHD3-9*01, IGHD3-10*01, IGHD6-13*01, IGHD4-17*01, IGHD6-19*01, IGHD3-22*01 and/or 26*01 and with (iii) a human JH gene segment, optionally JH6; wherein the antibody has an HCDR3 sequence of at least 20 amino acids in length; and non-human 40 vertebrate AID-pattern somatic hypermutations, optionally mouse or rat AID-pattern mutations, when 7759020_1 (GHMatters) P96496.NZ 18-May-16 compared to corresponding human germline V, D and J sequences and/or non-human, optionally mouse or rat, al deoxynucleotidyl transferase (TdT)- pattern junctional mutations when compared to corresponding human germline V, D and J sequences.

An eighth aspect of the invention provides use of a onal or polyclonal antibody produced by the method of any one of the first to third or fifth aspects or an antibody according to the seventh aspect in the manufacture of a medicament for treating and/or preventing an infectious disease.

Also disclosed is a non-human vertebrate (optionally a mouse or a rat) or vertebrate cell whose genome comprises: (a) An immunoglobulin heavy chain locus sing one or more human V gene segments, one or more human D gene segments and one or more human J gene segments upstream of a constant region; ally wherein the heavy chain locus is according to any configuration of the ion described below; and (b) An immunoglobulin light chain locus sing : (i) one or more human VH gene segments and one or more human J gene segments upstream of a constant region (optionally a rearranged VHJLCL or VHJACL, wherein the CL is CA or CK); or (ii) one or more human VL gene segments, one or more human D gene segments and one or more human JH gene segments upstream of a constant region (optionally a nged VLDJHCL or VADJHCL, wherein the CL is CA or CK); or (iii) one or more human VL gene segments selected from the group consisting of: a VAII gene family member, VAVII 4A, VAII 2.1, VAVII 4A, a VA1 gene family member, a VA3gene family member, 15 IGLV1S2, L70, lalh2, lalvl, la3h3, Kv325, a VKI gene family member, KI-15A (KL012), VKII family member, a VKIII family member, a VKI gene family member, KI-15A (KL012), VKII A2 (optionally the A2a allele), VK A27 (Humkv325) and a gene segment at least 80% identical thereto, and one or more human JL gene ts upstream of a constant region; Wherein the gene segments in the heavy chain locus are operably linked to the constant region thereof, and the gene segments in the light chain locus are ly linked to the constant region thereof, so that upon immunisation the mouse is capable of producing an antibody comprising heavy chains produced by recombination of the heavy chain locus and light chains produced by recombination of the light chain locus. 7592861_1 (GHMatters) P96496.NZ 5-Apr-16 In one embodiment, in (b)(i) the V gene segment oire of the light chain locus comprises or consists of one or more VH gene segments selected from the group consisting of: a VHIII gene family member (optionally, a 7592861_1 (GHMatters) P96496.NZ 5-Apr-16 VHIIIa or VHIIIb family member), a VHIV gene family member, VHIII 9.1 (VHS-15), VHIII VH26 (VH3- 23), VHS-21, LSGB.1, LSG1241, DP77 ), VH H11, VHlGRR, ha3h2, VHl-halct, VHIII-VH2-1, VH4.18, ha4h3, Hv1051, 71-2, , VH4.11, 71-4, VH251, VH1-69 and a gene segment at least 80% identical thereto; or in (b)(iii) the light chain locus V gene segment repertoire ts of one VL gene segment type (optionally and one or mutants thereof), wherein the VL gene segment is selected from said group of VL gene segments.

Also disclosed is non-human vertebrate (optionally a mouse or a rat) or vertebrate cell whose genome comprises: (a) An immunoglobulin heavy chain locus comprising one or more human V gene segments, one or more human D gene segments and one or more human J gene segments am of a constant region; and (i) An unrearranged immunoglobulin light chain locus comprising one or more human VH gene segments and one or more human J gene segments upstream of a constant region, wherein each human VH gene segment is a human gene segment identical to (or mutant of) a human VH gene segment used to produce a rearranged VDJ encoding a heavy chain variable region of a human antibody from an antibodyexpressing cell wherein said antibody binds to an antigen of an infectious disease en (optionally the variable s of said antibody being identical to an antibody from a human individual suffering, tible to, or recovered from, a disease or condition caused or mediated by an organism harbouring or secreting said antigen; or from a human individual harbouring said organism); or (ii) An immunoglobulin light chain locus sing a nged VJ region or VDJ region upstream of a constant region, wherein the nucleotide sequence of the recombined region is identical to a nucleotide sequence produced by the recombination of a human J gene segment and optionally a human D gene segment with a human VH gene segment that is identical to (or mutant of) the human VH gene segment used to produce a rearranged VDJ encoding a heavy chain variable region of a human antibody from an antibody-expressing cell n said antibody binds to an antigen of an infectious e pathogen (optionally the variable regions of said antibody being identical to an antibody from a human individual suffering, tible to, or recovered from, a e or condition caused or mediated by an organism harbouring or ing said antigen; or from a human individual harbouring said organism); 6804761_1 (GHMallers) POMDG NZ LEOWNR 18-Aug—l5 (c) Wherein the gene segments in the heavy chain locus are operably linked to the constant region thereof, and the gene ts or VJ or VDJ in the light chain locus are operably linked to the constant region thereof, so that upon immunisation the mouse is e of producing an antibody sing heavy chains produced by ination of the heavy chain locus and tight chains derived from the light chain locus; (d) Optionally when (b)(i) applies, each said VH gene segment in the light chain locus is selected from the group consisting of: a VHIII gene family member (optionally, a VHllla or VHIIlb family member), a VHIV gene family member, VHIII 9.1 (VH3-15), VHIII VH26 3), VHS-21, LSG6.1, LSG12.1, DP77 (VS-21), VH H11, VH1GRR, ha3h2, VHI-ha1c1, VHIll-VH2-1, 1O , ha4h3, Hv1051, 10 712, Hv1f10, VH4,11, 71-4, VH251, VH1-69 and a gene segment at least 80% identical thereto; (e) Optionally when (b)(ii) applies, the nucleotide sequence of the recombined region is identical to a tide sequence produced by the recombination of a human J gene segment and ally a human D gene segment with a human VH gene segment selected from the group consisting of: a VHIII gene family member (optionally, a VHllla or VHlllb family member), a VHlV gene family , VHIII 9.1 (VH3—15), VHIII VH26 (VH3-23), VH3-21, LSG6.1, LSG12.1, DP77 (VB-21), VH H11, VH1GRR, hash2, VHl-ha1c1, VHlll-VH2-1, VH4.18, ha4h3, Hv1051, 71—2, Hv1f10, VH4.11, 71-4, VH251, VH1-69 and a gene segment at least 80% identical thereto.

In one embodiment, the V gene segment oire of the light chain locus comprises or consists of one human VH gene segment; optionally germline VH and one or more polymorphic variants thereof, eg, where each polymorphic variant differs from the germline VH nucleotide sequence by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 positions. In one . the V gene segment repertoire of the light chain locus comprises or consists of human VH1-69 gene segment; optionally germline VH1-69 and one or more polymorphic variants thereof, eg, where each polymorphic variant differs from the germline VH1-69 25 nucleotide sequence by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ons. An example of ucting an immunoglobulin locus comprising VH1-69 and polymorphic variants is given below. By using a particuiar gene segment (eg, one commonly found in human antibodies raised in humans against an infection or other condition) and one or more polymorphic variants thereof, it is possible to provide a repertoire of genes and yet still bias the antibody gene repertoire to a gene segment that is relevant to the disease (eg, an infectious disease, such as a bacterial or viral disease, eg, influenza). This provides a useful pool of genes from which to ultimately generate and isolate a lead antibody for a therapeutic/prophylactic t the disease in on. In an example, the polymorphic variants are l variants seen in human beings or human populations. The skilled person will know of sources of human antibody gene sequences, such as IMGT (wwwimgtorg), GenBank (www.ncbi.nlm.nih.gov/genbank) and the 1000 Genomes databases (www.10009enomes.org). 680‘761_1 (GHMallers) P96496.NZ LEO\NNR 19-Augv15 ormatics tools for database manipulation are also readily available and known to the skilled , eg, as publicly available from the 1000 Genomes Project/EBI (www.10009enomes.org) In another ment, the genome of said rate or cell is homozygous for light chain locus (b)(i) or (ii); optionally wherein: - the V gene t repertoire of the light chain loci consists of one or more human VH gene segments selected from the group consisting of: a VHIII gene family member (optionally, a VHllla or VHlllb family member), a VHIV gene family member. VHIII 9.1 (VH3-15), VHIII VH26 (VH3~23), VH3- 21, LSG6.1, LSG12.1, DP77 ), VH H11. VH1GRR, ha3h2, VHI- ha1c1, VHIII-VH2-1, VH4.18, ha4h3, Hv1051, 71-2, Hv1f10, VH4.11, 71-4, VH251, VH1-69 and a gene segment at least 80% identical thereto; or - the recombined VJ or VDJ repertoire of the light chain loci consists of sequences identical to one or more nucleotide sequences produced by the recombination of a human VH gene segment selected from the group consisting of: a VHIII gene family member (optionally‘ a VHllla or VHlllb family member)‘ a VHIV gene family member, VHlll 9.1 (VHS-15). VHIII VH26 (VH3-23). , LSG6.1, LSG12.1, DP77 (V3-21), VH H11, VH1GRR. ha3h2, VHI- ha1c1, VHIII—VH2—1, VH4.18, ha4h3, Hv1051, 20 71-2, Hv1f10, , 71-4, VH251, VH1- 69 and a gene segment at least 80% identical thereto, with a human J gene segment and ally a human D gene segment.

In another embodiment, each immunoglobulin light chain locus of said vertebrate or cell is according to (b)(i) and comprises only a single human VH gene segment selected from the group ting of: a VHIII gene family member (optionally, a VHIIIa or VHIllb family member), a VHIV gene family member, VHlll 9.1 (VH3-15), VHIII VH26 (VH3—23), VH3-21, LSG6.1, L8612.1. DP77 (VS-21), VH H11,VHiGRR,ha3h2.VHl-ha1c1.VHlll-VH2-1,VH4.18,ha4h3, Hv1051, 71-2. Hv1f10, VH4.11, 71- 4, VH251. VH1-69 and a gene segment at least 80% identical thereto, optionally wherein the genome of the vertebrate or cell is homozygous for said light chain so that all light chain loci comprise the same, single human VH gene segment.

Also disclosed is a first method of isolating an antibody that binds a predetermined antigen, the method comprising: (a) providing a vertebrate (optionally a mouse or rat) according to any preceding configuration or aspect; (b) immunising said vertebrate with said antigen (optionally wherein the antigen is an antigen of an infectious disease en); (c) removing B lymphocytes from the vertebrate and ing one or more B lymphocytes expressing antibodies that bind to the antigen; 8W761_1 (GHMallars) PWWNZ LEOWNR 16-Aua-l5 (d) optionaiiy immortalising said selected B lymphocytes or progeny thereof, optionally by producing hybridomas therefrom; and (e) isolating an dy (eg, and IgG-type antibody) expressed by the B lymphocytes.

A second method comprises carrying out the first method and the step of isolating from said B lymphocytes c 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 sion vector and optionally a host.

An embodiment provides method of producing a polyclonal antibody mixture, the method sing carrying out the first method by tely immunising first and second vertebrates (optionally first and second mice or first and second rats) with n and combining the anti-antigen antibodies isolated from each rate (or mutants or derivatives of said antibodies) to produce a polyclonal antibody mixture; optionally wherein the ing apply separately or in combination ((i) and (ii); or (i) and (iii)): (i) the rates are immunised with the same antigen or different antigens (optionally wherein the different antigens are sed by the same pathogenic organism (or a family member thereof»; (ii) prior to immunisation the light chain loci of the vertebrates contain the cal VH gene repertoire (optionally a single VH gene) and optionally the identical J oire; optionally the light chain loci of the mammals are identical prior to immunisation; (iii) prior to immunisation the light chain loci of the vertebrates contain the identical rearranged VJ or VDJ repertoire (optionally a single VJ or VDJ); optionally the light chain loci of the vertebrates are identical prior to sation.

An embodiment provides a method of producing a polyclonal antibody mixture, the method comprising carrying out the first method by immunising one or a plurality of vertebrates (optionally mice or rats) with first and second antigens and combining the anti-antigen antibodies isolated from each vertebrate (or mutants or derivatives of said antibodies) to produce a polyclonal antibody e; optionally wherein the following apply separately or in combination ((i) and (ii); or (i) and (iii)): (i) the antigens are expressed by the same enic organism (or a family member thereof)); 8804781_| (GHMuliers) PWW NZ LEOVVNR 18-Auq-IS (ii) prior to immunisation the light chain loci of the vertebrates contain the identical Vl-l gene repertoire (optionally a single VH gene) and optionally the cal J repertoire; optionally the light chain loci of the mammals are identical prior to immunisation; (iii) prior to immunisation the light chain loci of the vertebrates contain the identical rearranged VJ or VDJ repertoire (optionally a single VJ or VDJ); optionally the light chain loci of the vertebrates are identical prior to immunisation.

An embodiment provides method of producing host cells capable of expressing a polyclonal dy mixture, the method comprising, in the second :- (a) immunising one or a plurality of vertebrates (optionally mice or rats) with first and second antigens (optionally wherein the different antigens are expressed by the same pathogenic organism (or a family member thereof»; ing nucleic acid encoding first and second ntigen antibodies from B lymphocytes from said vertebrates; ining the nucleotide sequences of the heavy and light chain variable regions of the first antibody; determining the nucleotide ce of the heavy variable region and optionally the light chain variable region of the second antibody; inserting the heavy chain variable region coding sequence of each dy into a heavy chain sion vector; optionally wherein the constant region coding sequence of each heavy chain is exchanged for a nucleotide sequence that s a human or humanised constant region; inserting the light chain variable region coding sequence of the first antibody into a light chain expression vector; optionally wherein the constant region coding sequence of the light chain of the first antibody is exchanged for a nucleotide sequence that encodes a human or humanised constant region; (g) optionally inserting the light chain variable region coding sequence of the second dy into a light chain expression vector; optionally wherein the constant region coding sequence of the light chain of the second antibody is exchanged for a nucleotide sequence that encodes a human or humanised constant region; and (h) introducing each sion vector into a host cell and co-expressing antibody chains in a mixture of said host cells to produce antibodies, each antibody comprising one or both of said heavy chain variable regions and a light chain; optionally n the expression vectors are $8M781_1 (GHMalleB) 1396498 NZ LEOVVNR IS-Auqu uced together into the same host cell (eg, a CHO or HEK293 cell) so that the cell is capable of expressing antibody light chains and heavy , such that the cell or a ity of the host cells express antibodies, each comprising one or both of said heavy chain variable regions and a light chain; (i) optionally: prior to immunisation the light chain loci of the vertebrates contain the identical Vl-l gene repertoire (optionally a single VH gene segment) and optionally the identical J repertoire (optionally a single J gene segment); optionally the light chain loci of the vertebrates are identical prior to sation; or prior to immunisation the light chain loci of the vertebrates contain the identical rearranged VJ or VDJ repertoire (optionally a single VJ or VDJ); optionally the light chain loci of the vertebrates are identical prior to immunisation.

Also disclosed is a method of producing a monoclonal or polyclonal antibody mixture, optionally for use in medicine, optionally for the treatment and/or prevention of an infectious disease, wherein optionally wherein each antibody binds an antigen of an infectious e pathogen, preferably the same antigen The invention also provides the use of an isolated, monoclonal or polyclonal antibody. or a mutant or derivative antibody thereof in the cture of a ment for the treatment and/or prevention of an infectious disease, optionally wherein the infectious disease is a disease caused by a bacterial or viral pathogen.

Also disclosed is an isolated antibody (eg, lgG-type antibody) obtainable or obtained by a method of the invention, or a mutant or derivative antibody thereof n (i) the isolated antibody comprises two copies of the heavy chain variable region of said first antibody paired with two copies of the light chain variable region of said first antibody; or (ii) the isolated antibody comprises two copies of the heavy chain variable region of said second antibody paired with two copies of the light chain variable region of said first antibody; or (iii) the isolated antibody is a bispecific antibody comprising one copy of the heavy chain variable region of said first antibody paired with a copy of the light chain variable region of the first antibody, and one copy of the heavy chain variable region of said the antibody paired with a copy of the light chain variable region of the first antibody, optionally n the bispecific antibody binds to said first and second antigens are expressed by the same pathogenic organism (or a family member thereof; optionally for use in medicine, optionally for the ent and/or prevention of an infectious disease.

In an embodiment, there is provided a nucleotide sequence ng an antibody of the invention, optionally wherein the nucleotide sequence is part of a vector. 6804761_l (GHMaiwrs) P96496 NZ LEOWNR icAuws In an embodiment, there is provided a pharmaceutical composition comprising the antibody or antibodies of the invention and a diluent. excipient or r.

Also disclosed is non-human vertebrate nally a mouse or a rat) or rate cell whose genome comprises: (a) An globulin heavy chain locus comprising either:- (i) one or more human VL gene segments, one or more human D gene segments and one or more human J gene segments upstream of a constant region (optionally a rearranged H or H); or (ii) one or more human VH gene segments selected from the group consisting of: a VHIII gene family member (optionally, a VHllla or VHlllb family member), a VHIV gene family member, VHIII 9.1 (VH3-15), VHIII VH26 (VH3-23), VH3-21, LSGB.1, LSG12.1, DP77 (VB-21), VH H11, VH1GRR, ha3h2, VHI- halci, VHIII-VH2-1, VH4.18, ha4h3, Hv1051, 71-2, Hv1f10, . 71-4, VH251, VH1-69 and a gene segment at least 80% identical; one or more human D gene segments and one or more human JH gene segments upstream of a constant region; and (b) An immunoglobulin light chain locus comprising one or more human V gene ts and one or more human J gene segments upstream of a nt region, optionally wherein the light chain locus is according to (b)(i) or (b)(ii) of the first configuration of the invention; Wherein the gene segments in the heavy chain locus are operably linked to the constant region thereof, and the gene segments in the light chain locus are operably linked to the constant region thereof, so that upon immunisation the mouse is e of producing an antibody comprising heavy chains produced by recombination of the heavy chain locus and light chains produced by recombination of the light chain locus.

Also disclosed is non-human vertebrate (optionally a mouse or a rat) or vertebrate cell whose genome comprises: (i) An unrearranged immunoglobulin heavy chain locus comprising one or more human VL gene segments, one or more human D gene ts and one or more JH gene segments upstream of a constant region, wherein each human VL gene segment is a human gene segment identical to (or mutant of) a human VL gene segment used to produce a rearranged VJ encoding a light chain variable region of a human antibody from an antibody-expressing cell wherein said antibody binds to an antigen of an infectious disease pathogen (optionally the variable regions of said antibody being 8804701_l IOIS) P96‘95NZ LEOWNR 18-Aug-15 identical to an antibody from a human individual suffering, susceptible to, or recovered from, a disease or condition caused or mediated by an organism harbouring or secreting said antigen; or from a human individual harbouring said organism); or (ii) An immunoglobulin heavy chain locus comprising a rearranged VJ region or VDJ region upstream of a constant region, wherein the nucleotide sequence of the recombined region is identical to a nucleotide sequence ed by the recombination of a human J gene segment and optionally a human D gene segment with a human VL gene segment that is cal to (or mutant of) the human VL gene segment used to produce a rearranged VJ encoding a light chain le region of a human dy from an antibody-expressing cell wherein said antibody binds to an antigen of an infectious e pathogen (optionally the variable regions of said antibody being identical to an antibody from a human dual suffering, susceptible to, or recovered from, a disease or condition caused or mediated by an organism harbouring or secreting said antigen; or from a human individual ring said organism); An immunoglobulin light chain locus comprising one or more human V gene segments and one or more human J gene segments upstream of a constant region; and Wherein the gene ts in the light chain locus are operably linked to the constant region thereof, and the gene segments or VJ or VDJ in the heavy chain locus are operably linked to the constant region thereof, so that upon immunisation the mouse is capable of ing an antibody comprising light chains produced by recombination of the light chain locus and heavy chains derived from the heavy chain locus; (d) Optionally when (a)(i) applies, each said VL gene segment in the heavy chain locus is selected from the group consisting of a VL gene segment selected from the group consisting of a VAII gene family member, VAVlt 4A, VAII 2.1, VAVII 4A, a VA1 gene family member, a VA3gene family member, |GLV182, VA3-cML70, lalh2, lalvl, Ia3h3, Kv325, a VKI gene family member. KI-15A (KLO12), 20 VKII family member, a VKlll family member, a VKI gene family member, Kl-15A (KL012), VKll A2 (optionally the A2a allele), VK A27 (Humkv325) and a 3O gene segment at least 80% identical o; ally when (a)(ii) applies, the tide sequence of the recombined region is identical to a nucleotide sequence produced by the recombination of a human J gene segment and optionally a human D gene segment with a human VL gene segment selected from the group consisting of a VL gene t selected from the group consisting of a VAII gene family member, VAVII 4A, VAII 2.1, VAVII 4A, a VA1 gene family member, a VASgene family member, 2, VA3- cML70, lalh2, lalvl, la3h3, Kv325, a VKI gene family member, Kl- saa4m_i (GHMallars) mosnz LEOWNR 18—Aug-15 15A (KL012), VKII family member, a VKIII family member, a VKI gene family member. K|a15A ), VKII A2 (optionally the A2a allele). VK A27 (Humkv325) and a gene segment at least 80% cal o.

In one embodiment, the genome of said vertebrate or cell is homozygous for heavy chain locus (a)(i) or (ii); optionally wherein: - the V gene segment repertoire of the heavy chain loci consists of one or more human VL gene segments selected from the group consisting of a VL gene t selected from the group consisting of a VAII gene family member, VAVII 4A, VAII 2,1, VAVII 4A, a VA1 gene family , a VA3gene family member, lGLVlSZ, VA3~cML70, Ialh2, lalvl, Ia3h3, Kv325, a VKI gene family member, Kl-15A (KL012), VKII family member, a VKIII family member, a VKI gene family member, Kl-15A (KL012), VKII A2 (optionally the A2a allele), VK A27 (Humkv325) and a gene segment at least 80% identical thereto; or - the ined VJ or VDJ oire of the heavy chain loci consists of sequences identical to one or more nucleotide sequences produced by the recombination of a human VL gene segment selected from the group consisting of a VL gene segment selected from the group consisting of a VAII gene family member, VAVII 4A, VAII 2.1, VAVII 4A, a VAl gene family , a VA3gene family member, IGLV182, VA3-cML70, lalh2, lalvl, la3h3, Kv325, a VKI gene family member, KI-15A (KL012), VKII family member, a VKlll family member, a VKI gene family member, KI-15A (KL012), VKII A2 (optionally the A2a allele), VK A27 (Humkv325) and a gene segment at least 80% identical thereto with a human J gene segment and optionally a human D gene segment.

Also disclosed is a monoclonal or polyclonal antibody composition prepared by sation of at least one vertebrate (eg, mouse or rat) according to any preceding configuration or aspect with an antigen, optionally wherein the antigen is an antigen of an infectious disease pathogen, optionally wherein the same antigen is used to immunise all the vertebrates; optionally wherein the dy or antibodies are lgG-type.

Also disclosed is an isolated chimaeric antibody for treating and/or preventing an infectious disease or condition, the dy comprising a non-human vertebrate (optionally a mouse or rat) heavy chain constant regions and human variable regions that bind an antigen of an infectious disease pathogen. wherein the antibody is obtainable or obtained in a method comprising sation of a non-human vertebrate of the invention with said antigen.

Also disclosed is an isolated human antibody for treating and/or preventing an infectious disease or condition, the antibody comprising human heavy chain nt s and human variable s that bind an antigen of an ious disease pathogen, wherein the antibody is obtainable or obtained in a method comprising affinity maturation of antibody variable regions in vivo in a transgenic GUDWNJ (GHMBHWS) PWSG NZ LEOWNR 1&Aur15 non-human vertebrate (eg, mouse or rat) when said variable s are operably linked to heavy chain constant regions of said vertebrate (eg, mouse or rat heavy chain constant regions) by (a) immunisation of a vertebrate of the invention with said antigen, (b) isolation of nucleic acid encoding a chimaeric antibody according to the invention, (c) replacing the nucleotide sequences of the nucleic acid that encode the non-human vertebrate heavy chain constant regions with nucleotide ce encoding human heavy chain constant regions to e nucleic acid encoding a human antibody; (d) expressing the human antibody in vitro (optionally from CHO or HEK293 cells harbouring the human nucleic acid) and (e) isolating the human antibody (optionally with further ty maturation of the antibody and/or producing a derivative thereof).

An ment provides a mixture of first and second human antibodies, each dy being capable of binding to an antigen of an infectious disease pathogen (optionally wherein the first antibody binds a first n and the second antibody binds a second n, said antigens being from the same pathogen; or wherein the antigens are the same). In an embodiment, a common light chain is used which enables simplified manufacture of the antibody mixture. Thus, there is provided in the mixture. the light chain amino acid sequence of the first antibody that is identical to the light chain amino acid sequence of the second antibody, or has up to amino acid changes therefrom.

Also disclosed is a host cell comprising one or more expression s encoding 3 or more first and second antibody heavy and light chains, Also disclosed is synthetic immunoglobulin locus comprising one or more variable and J gene segments (and optionally one or more D gene segments) operably linked 5' of a constant region, wherein the locus comprises a 5' to 3' V(D)J arrangement selected from the group consisting of immunoglobulin locus can be ucted with one or more of the following arrangements (5' to 3'):- (a) [V (heavy, lambda or kappa)] - [two-turn RSS] — urn RSS] - [D] - [JH], wherein said RSSs are in an opposite ation; (b) [VH] - [D] - [two-turn RSS] — [one-turn RSS1 - [J ], wherein said RSSs are in an opposite orientation; (c) [VH] - [D] - [one-turn RSS] — [two-turn RSS] - [J kappa], wherein said RSSs are in an opposite orientation; (d) [VH or V kappa] - [two-turn RSS] — [one-turn RSS] - [J lambda], n said RSSs are in an opposite orientation; (e) [V kappa] - [one-turn RSS] —— [two-turn RSS] - [JH or J lambda], wherein said RSSs are in an opposite ation; 6304701_l (SHMnllors) 996496 NZ LEO‘NNR 18-Aug-15 (f) [V , lambda or kappa)] - [one-turn R88] — [two-turn R88] - [D] - [JH], wherein said R888 10 are in an opposite ation; (9) [VH] - [D] - [one-turn R88] — [two-turn R88] - [J lambda], wherein said R885 are in an opposite orientation; (h) [VH] - [D] - [two-turn RSS] — [one-turn RSS] - [J kappa] wherein said RSSs are in an opposite orientation; (i) [VH or V kappa] - [one-turn R88] — [two-turn R88] - [J lambda], wherein said R885 are in an opposite orientation; (j) [V kappa] - [two-turn R88] —— urn R88] - [JH or J lambda], wherein said RSSs are in an opposite orientation.

Also disclosed are means for generating VH domains. heavy chains and antibodies having a long HCDR3 . In this context. the disclosure provides:- A non-human rate (eg. a mouse or a rat) or a non-human rate cell (eg, a mouse cell or a rat cell) whose genome comprises a human immunoglobulin D gene segment repertoire that is biased to the human D2 and/or D3 family or biased to one, more or all human D gene segments selected from the group D1-26, 02-2, 03-9, 03-10, D3-22, 04-17, D6-13 and 06-19 A non-human vertebrate (eg, a mouse or a rat) or a non-human rate cell (eg. a mouse cell or a rat cell), optionally according to any preceding claim, whose genome comprises a human immunoglobulin VH gene segment repertoire that is biased to one, more or all of gene segments selected from the group VH1-2, VH1-3, VH1-8, VH1-‘l8, VH5-51, VH1-69. VH2-5, VH3-7, VHS-9, VH3— 11, VH3-13, VH3-20. VH3-21. VH3’23, VH4~4, VH6-1 and VH71. 860476Ll (GHMBllersJ P96496142 LEOWNR 18~Au045 W0 20131041846 PCT/G82012/052298 A non-human vertebrate (eg, a mouse or a rat) or a man vertebrate cell (eg, a mouse cell or a rat cell) whose genome comprises a human immunoglobulin JH gene segment repertoire that is biased to human 1H6.

A monoclonal or polyclonal antibody ition or a population of antibody—producing cells for producing such composition, wherein the composition or population is prepared by immunising at least one vertebrate according to any preceding claim with an antigen, wherein the antibody or antibodies have human heavy chain variable regions comprising non-human vertebrate AID-pattern somatic utations, (eg, mouse or rat AID-pattern mutations) when compared to corresponding human germline V, D and J sequences and/or non-human (eg, mouse or rat) terminal deoxynucleotidyl transferase (TdT)- pattern junctional mutations when compared to corresponding human germline V, D and] sequences; wherein the composition comprises at least one antigen- ic antibody having a HCDR3 length of at least 20 amino acids (according to IMGT).

A repertoire of antibody heavy chains (eg, provided by antibodies) comprising one or more heavy chains whose variable domain HCDR3 has a length of at least 20 amino acids (according to IMGT) and derived from the recombination of a human VH, D and JH, wherein the VH is selected from the group VH1-2*02, VH1-3*01, VH1-8*01, VH1-18*01, VH2-5*10, 01, VH3-9*01, VH3-11*01, VH3- 13*01, VH3-21*03, VH3-23*04, VH4-4*02, VH6—1*Ol and VH71*01 and the D is selected from the group 02-2‘02, DEB-9‘01, 03-10*01 and 03-22*01, or DZ—2*02, 1 and D3~10*01, or 03-901 and 03-10*01, or 01—26, 02—2, 03-9, 03-10, 03-22, 04-17, 06-13 and 06-19, or 01—26*01, DZ-2*02, 1, 03-10*01, 01, 01,06-13*01 and D6-19*01, or 02-2, 03-9, D3-10, 03-22, D4-17, D6-13 and 06-19, or D2-2*02, D3-9*01, D3-10*01, D3-22*01, D4- 17*01, D6-13*01 and 06-19‘01, or 0126, 02—2, 03—10 and 06-19, or 02~2, 03-9 and D3—10; and optionally the JH is JH6 (eg, JH6*02); W0 2013/0418-‘16 PCT/G32012/052298 Wherein (a) the heavy chain variable domain has been produced in vivo in a non-human vertebrate (eg, a mouse or a rat); and/or (b) the heavy chain variable domain ses non-human rate AID-pattern somatic hypermutations, (eg, mouse or rat AID—pattern mutations) when compared to corresponding human germline V, D and J sequences and/or non—human (eg, mouse or rat) al deoxynucleotidyl transferase (TdT)- pattern junctional mutations when compared to corresponding human germline V, D andJ sequences.

BRIEF DESCRIPTION OF THE FIGURES Figures 1 to 3: Schematic illustrating 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 segments to a mouse genome using sequential recombinase mediated cassette ge (sRMCE); Figure 5 (in 4 parts): Alignment of 13 IGHV1-69 alleles showing the variable (V) coding region only.

Nucleotides that differ from VHl—69 allele *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 the corresponding triplet. Boxed s correspond to CDRl, CDRZ and CDR3 as indicated; and Figure 6: RSS structure and ination schematic.

DETAILED DESCRIPTION OF THE IQN A source for human V, D and J gene segments is ial Artificial Chromosomes (RPClll BACs) obtained from Roswell Park Cancer Institute /|nvitrogen. See httpflbacgac.chori.org[hma|e11.htm, which describes the BACs as follows:- ”RPC/ - 11 Human Male BAC Library The RFC/~11 Human Male BAC Library (Osoegawa et 0]., 2001 J was constructed using improved P’nninn tt’r‘hrfimle‘s (Osoegawa et a/., 1998) developed by Kazutoyo Osoegawa. The library was wo 2013/041846 zoizl05229s generated by Kazutoyo Osoegawa. Construction wasfunded by a grantfrom the al Human Genome ch Institute (NHGRI, NIH) (#1R01R601165-03). This library was generated ing to the new NHGRl/DOE "Guidance on Human Subjects in Large-Scale DNA Sequencing...

”Ma/e blood was obtained via a double-blind ion 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, Catanese J], de Jong PJ; Genome Res. 2001 Mar;11(3):483-96; ”A bacterial artificial some library for cing the complete human genome"; 0 Osoegawa, K., Woon, P.Y., Zhao, 8., Frengen, E., Tateno, M., Catanese, JJ, and de Jong, PJ. (1998); ”An Improved Approach for Construction of Bacterial Artificial Chromosome Libraries”,- Genomics 52, 1-8.

As a source of antibody gene segment sequences, the skilled person will also be aware of the following available databases and ces (including updates thereof):- 1.1. The Kabat Database (G. Johnson and T. T.Wu, 2002; httngzwww.kabatdatabase.coml.

Created by E. A. Kabat and T. T. Wu in 1966, the Kabat database publishes aligned sequences of antibodies, T-cell receptors, major histocompatibility x (MHC) class I and II molecules, and other proteins of immunological interest. A searchable interface is provided by the Seqhuntll tool, and a range of utilities is available for sequence alignment, sequence up 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/mmunological Interest, 5th ed., NIH Publication No. 91- 3242,Bethesda, 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; http:l[www.bioinf.org.uk[abs[simkab.html). This is a web interface to make simple queries to the Kabat sequence database. wo 2013/041846 PCT/GBzoiz/05229s 1.3. IMGT, the ational ImMunoGeneTics Information System”; M.—P. Lefranc, 2002,- hug-(gimgtcinesjrl. IMGT is an integrated ation system that specializes in dies, T cell receptors, and MHC les of all vertebrate species. It provides a common portal to standardized data that include nucleotide and protein sequences, oligonucleotide primers, gene maps, genetic polymorphisms, icities, and two-dimensional (2D) and three-dimensional (3D) structures. IMGT includes three sequence databases (lMGT/LlGM—DB, lMGT/MHC—DB, IMGT/PRIMERDB), one genome database (IMGT/GENE—DB), one 30 structure database (lMGT/3Dstructure-DB), and a range of web resources (”IMGT Marie—Paule page”) and interactive tools. 1.4. V-BASE (i. M. Tom/Inson, ttp://www.mrc-cpe.cam.ac.uk/vbase). V-BASE is a comprehensive directory of all human antibody germline variable region sequences compiled from more than one thousand published sequences. It includes a version of the alignment software DNAPLOT (developed by Hans-Helmar s and Werner Milller) that allows the assignment of rearranged antibody V genes to their closest germline gene segments. 1.5. Antibodies—Structure and Sequence(A. C. R. , 2002; httgsawwwbioinf.org.uk(absl.

This page summarizes useful information on antibody structure and sequence. It provides a query interface to the Kabat antibody sequence data, general information on dies, crystal structures, and links to other antibody-related information. It also distributes an ted summary of all antibody structures deposited in the Protein Databank (PDB). Of particular interest is a thorough description and comparison of the various numbering schemes for antibody variable regions. 1.6. AAAAA—AHo’s Amazing Atlas body Anatomy (A. Honegger, 2001,- httgzgwww.unizh.ch(~antibodxl. This resource includes tools for structural is, modeling, and engineering. It adopts a unifying scheme for comprehensive structural alignment of antibody and T- celI-receptor sequences, and includes Excel macros for antibody analysis and graphical representation. 1.7. WAM—Web Antibody ng (N. egg and A. R. Rees, 2001,- htth'gantibodgbath.acukl. Hosted by the Centre for Protein Analysis and Design at the University wo 2013/041846 of Bath, United Kingdom. Based on the AbM package (formerly marketed by Oxford Molecular) to construct 3D models of antibody Fv sequences using a combination of established tical methods, this site also includes the latest antibody structural information. 1.8. Mike’s lmmunog/obulin Structure/Function Page (M. R. Clark, 2001; www.gathcantac.ukg~rnrc7gmikeimageshtin/i These pages e educational materials on immunoglobulin structure and function, and are illustrated by many colour images, models, and animations. Additional information is available on antibody humanization and Mike Clark’s eutic Antibody Human gy Project, which aims to ate clinical efficacy and anti- globulin responses with variable region sequences of therapeutic antibodies. 1.9. The Antibody Resource Page (The Antibody Resource Page, 2000; httgfiwww.onn'bodzresourcecoml. This site describes itself as the ”complete guide to dy research and suppliers.” Links to amino acid sequencing tools, nucleotide antibody sequencing tools, and hybridoma/cell-culture ses are provided. 1.9. Humanization bY Design (J. Saldanha, 2000; httgzmeoglecgist.bbk.ac.ukg~ubcg0 75). This resource provides an ew on antibody humanization technology. The most useful feature is a searchable database (by sequence and text) of more than 40 hed humanized antibodies including information on design issues, framework choice, framework back-mutations, and binding affinity of the humanized constructs.

See also Antibody Engineering Methods and Protocols, Ed. Benny K C Lo, Methods in Molecular Biologym, Human Press. Also at httQ:awwwblogsua.commdeantibody-engineering-methods-and- grotocolsantibody—engineering-methods—and-grotocols.gdf In one embodiment throughout the present text, "germline" refers to the canonical germline gene segment sequence. wo 2013/041846 PCT/G82012/052298 The present invention is directed to the provision of novel V, D and J pairings in immunoglobulin heavy and light chain loci. Novel, biased antibody diversities and potentially ed diversities are provided. One aspect of the invention exploits the natural pairing of compatible ination signal sequences (RSSs) during antibody V(D)J recombination in vivo, and this aspect of the invention provides new, synthetic combinations of V, D and J gene segments using the observation of RSS compatibility.

Another aspect of the invention is based on the observation of V, D andJ usage bias in naturallyv occurring human dies raised against infectious disease pathogens. The invention is useful for lating the antibody gene diversity in transgenic non—human s, thus providing for novel and potentially expanded diversity or ity that is biased towards variable gene usage common to dies useful for treating and/or preventing certain diseases or ions, such as infectious diseases. This ability to bias the antibody repertoire also provides methods of simplifying the production of antibody mixtures, such as polyclonal antibody therapeutics useful for the treatment and/or prevention of infectious diseases where a onal approach to target multiple pathogen antigens is desirable. To this end, the present invention also provides bispecific dies that are capable of binding to more than one antigen (eg, multiple infectious antigens expressed by the same en), thus providing advantages (such as manufacturing, dosing and administration advantages) not possible with polyclonal antibody es.

The present invention provides vertebrates and cells, such as transgenic mice or rats or transgenic mouse or rat cells. Furthermore, the invention relates to methods of using the vertebrates to e antibodies or nucleotide sequences encoding antibodies. Antibodies, nucleotide sequences, pharmaceutical compositions and uses are also provided by the invention.

To this endI the present invention grovidesl in a first configuration A nonwhuman vertebrate (optionally a mouse or a rat) or vertebrate cell whose genome comprises: wo 2013/041846 PCT/GBzoiz/05229s (a) An immunoglobulin heavy chain locus sing one or more human V gene segments (optionally a ity of VH), one or more human D gene segments and one or more human J gene segments upstream of a nt region; optionally wherein the heavy chain locus is according to (a) of the second configuration described below; and (b) An immunoglobulin light chain locus comprising either (i) one or more human VH gene segments and one or more human J gene segments upstream of a constant region (optionally a rearranged VHJ,CL or VHJACI, wherein the CL is CA or CK); or (ii) one or more human VL gene segments, one or more human 0 gene segments and one or more human JH gene segments upstream of a constant region (optionally a rearranged VLDJHCL or VKDJHCL, wherein the CL is CA or CK); or (iii) one or more human VL gene segments selected from the group consisting of: a Vill gene family member, ViVll 4A, Vill 2.1, VAVII 4A, a Vxl gene family member, a Vx3gene family member, IGLVlSZ, VA3-CML70, lalh2, lalvl, |a3h3, Kv325, a VKI gene family , KI-lSA (KL012), VKII family member, a VKIII family member, a VKI gene family member, Kl-lSA (KL012), VKII A2 (optionally the A23 allele), V.< A27 (Humkv325) and a gene segment at least 80% identical o, and one or more human JL gene segments upstream of a constant region; optionally the one or more VL gene segments are selected from List A1, A2, A1.1, A12, A2.1, A2.2, A243 or A2.4 below.

Wherein the gene segments in the heavy chain locus are operably linked to the nt region thereof, and the gene segments in the light chain locus are operably linked to the constant region thereof, so that upon immunisation the mouse is capable of producing an antibody comprising heavy chains produced by recombination of the heavy chain locus and light chains produced by recombination of the light chain locus.

This configuration of the invention, thus, provides for the possibility of novel, tic antibody and gene repertoires in a transgenic non-human vertebrate, such as a mouse or rat. Such new repertoires are desirable, since they provide for the possibility of a novel pool of antibodies from which lead antibodies can be selected following sation of the vertebrate with a predetermined n. This, therefore, provides for a pool from which antibodies with desirable teristics can be isolated, for example, dies with relatively high ty for specific target antigen binding. It is desirable to isolate high affinity antibodies directly from the immunised vertebrate, since this can provide for an antibody lead that is potentially useful as a therapeutic PCT/G82012/052298 and/or prophylactic medicament without the need for further extensive affinity maturation (eg, by in vitro antibody display such as ribosome display or phage display). Modification of the effector portions of the antibody can be made as desired (eg, humanisation of the constant region), without the need to late the ces of the variable regions. Alternatively, or additionally, the pool of antibodies may allow for selection of a lead antibody with desirable bi0physica| teristics and/or epitope specificity. The latter may be important for finding lead antibodies against epitopes that have not previously raised therapeutic and/or prophylactic antibodies or epitopes that are difficult to reach by antibodies generated by antibody gene diversities generated by prior non-human vertebrates bearing transgenic immunoglobulin loci, eg, those based on the single human genome represented by the RPCI—ll BACs.

The cells of the ion (according to any aspect or configuration) is, for example, a B-cell, hybridoma or a stem cell, optionally an embryonic stem cell or haematopoietic stem cell. In one aspect the ES cell is derived from the mouse C57BL/6N, C57BL/6J, 12955 or 129$v strain. In one aspect the man vertebrate is a rodent, ly a mouse, and cells of the invention, are rodent cells or ES cells, suitably mouse ES cells. The ES cells of the present invention can be used to generate animals using techniques well known in the art, which comprise injection of the ES cell into a blastocyst followed by implantation of ric blastocystys into females to produce offspring which can be bred and selected for homozygous recombinants having the ed insertion. In one aspect the ion relates to a transgenic animal comprised of ES cell-derived tissue and host embryo derived tissue. In one aspect the invention relates to genetically-altered subsequent generation animals, which e animals having a homozygous recombinants for the V0] and/or VJ regions.

Vertebrates bearing one or more light chain loci according to (b)(i) and (ii) provide for novel and potentially ed antibody and gene repertoires by ting synthetic, non—naturallyvoccurring, combinations of immunoglobulin gene segments (V, D, J, C). In this respect, the present inventors have realised the desirability and possibility of providing for antibody and gene repertoires that mix heavy chain gene segments with those of light chain loci. This is based on observations of the inventors: Firstly, nature suggests the possibility of functional antibodies having VH—VH or VL-VL pairings (as opposed to more classical VH-VL pairings). For example, reference is made to heavy chain antibodies of Came/idae which produce antibodies with paired VH domains and is devoid of wo 2013/041846 PCT/G82012/052298 light chain VL domains (eg, see Nature. 1993 Jun 3;363(6428):446-8; Naturally occurring antibodies devoid of light chains; Hamers-Casterman C, uch T, Muyldermans S, Robinson 6, Hamers C, Songa EB, Bendahman N, Hamers R). These antibodies function to specifically bind antigen, such antibodies being found in the blood of such Came/idae (eg, llamas, camels, alpacas). Such dies with VH pairs can also be synthetically produced to provide therapeutic and prophylactic medicaments (eg, see W01994004678, W02004041862, W02004041863). Transgenic mice also can produce such heavy chain dies and the in vivo production of the antibodies allows the mouse’s immune system to select for VH-VH pairings, sometimes selecting for such pairings in which ons have been introduced in vivo by the mouse to accommodate the pairing (W02010109165A2). Thus, the inventors realised that the on of an in vivo antibody production system (rather than an in vitro system such as phage or ribosome display of antibodies) is desirable to accommodate the synthetic immunoglobulin gene segment combinations that are now contemplated by the present invention, A second observation of the present inventors lies in the architecture of naturally-occurring immunoglobulin loci, and in particular the arrangement of recombination signal sequences (RSSs) that mediate V(D)J recombination in viva (see, eg, Cell. 2002 Apr;109 Supplzs45-55.

The mechanism and regulation of chromosomal V(D)l recombination; Bassing CH, Swat W, Alt FW, the sure of which is incorporated herein by reference). As illustrated in Fig. 6, two types of RSS element have been identified: a one-turn RSS (12-RSS) and a two-turn RSS (23—RSS). In natural VJ ination in the lambda light chain locus, recombination if effected between a two-turn RSS that lies 3’ of a V lambda and a one—turn RSS that lies 5’ of a J , the R555 being in opposite orientation. In natural VJ ination in the kappa light chain locus, recombination if effected between a one-turn RSS that lies 3’ of a V kappa and a two-turn RSS that lies 5’ of a J kappa, the R855 being in opposite orientation. In natural VD ination in the heavy chain locus, recombination if effected between a two-turn RSS that lies 3' of a VH and a one-turn RSS that lies 5’ of a D, the R555 being in opposite ation. In natural DJ recombination in the heavy chain locus, recombination if effected between a one-turn RSS that lies 3‘ of a D and a rn RSS that lies 5’ of a JH, the R555 being in opposite orientation. Thus, lly a two-turn RSS is compatible with a one-turn RSS in the te orientation. The ors realised that they could use this observation in constructing transgenic immunoglobulin loci such that a 5’ gene segment can recombine with a 3’ gene segment (eg, a V with a J; or a V with a D) when there is provided a two-turn RSS and a one-turn RSS in the opposite orientation, with each RSS adjacent a respective one of the gene segments. Thus, the wo 2013/041846 PCT/G82012/052298 inventors realised in one embodiment that an immunoglobulin locus can be constructed with one or more of the following arrangements (5' to 3’):- (k) [V (heavy, lambda or kappa)] — [two-turn RSS] --- [one-turn RSS] ~ [D] ~ [JH], wherein said R555 are in an opposite orientation; (I) [VH] — [D] — [two-turn RSS] [one-turn RSS] — [J lambda], n said RSSs are in an opposite orientation; (m) [VH] — [D] — [one-turn RSS] [two-turn RSS] - [J kappa], wherein said RSSs are in an opposite ation; (n) [VH or V kappa] - [two-turn RSS] [one-turn RSS] — [J lambda], wherein said RSSs are in an opposite orientation; (0) [V kappa] - [one-turn RSS] [two-turn RSS] — [JH orJ lambda], wherein said RSSs are in an opposite orientation; (p) [V (heavy, lambda or kappa)] — urn RSS] [two-turn RSS] — [D] « [JH], wherein said RSSs are in an opposite orientation; (q) [VH] ~ [D] ~ [one-turn RSS] [two-turn RSS] ~ [J lambda], wherein said RSSs are in an opposite orientation; (r) [VH] — [D] — [two—turn RSS] [one—turn RSS] — [J kappa], wherein said RSSs are in an opposite orientation; (5) [VH or V kappa] - [one—turn RSS] [two-turn RSS] — [J lambda], wherein said RSSs are in an opposite orientation; (t) [V kappa] - [two-turn RSS] [one-turn RSS] — [JH orJ lambda], wherein said RSSs are in an te orientation.

The skilled person will realise that rd molecular biology techniques can be used to provide vectors comprising synthetic combinations of RSS with V, D orJ for use in this aspect of the ion, such that the s can be used to build a transgenic immunoglobulin locus (eg, using gous ination and/or recombinase mediated cassette exchange as known in the art, eg, see US7501552 (Medarex), US$939598 (Abgenix), 364 (Abgenix), W002/066630 eron), W02011004192 (Genome Research d), W02009076464, W02009143472 and W02010039900 (Ablexis), the disclosures of which are explicitly incorporated herein. For example, such synthetic combinations with RSS and gene segments can be made using standard mmmhineerina techniques in E coli to construct BAC vectors harbouring the synthetic combination wo 41846 2012/052298 prior to insertion in embryonic stem cells using homologous recombination or RMCE (eg, using cre/lox pecific recombination). Details of recombineering can be found at www.genebridges.com and in EP1034260 and EP1204740 the disclosures of which are explicitly incorporated herein.

In one embodiment of (b)(i), all of the light chain locus V gene segments are human VH gene segments (optionally with one or more human V lambda gene segments).

In one embodiment of (b)(i), the nt region is a mouse, rat or human CL, eg, CA. In one embodiment, theJ and constant regions are provided by one or more human JACK.

Although having utility lly to any antigen and disease setting, rates bearing one or more light chain loci according to (b)(iii) are useful, in particular, for generating antibody leads against infectious disease pathogens. In this respect, the present inventors have realised the desirability and possibility of providing for antibody and gene repertoires that are biased to immunoglobulin gene segments commonly found in natural antibody reactions of humans to infectious disease pathogens. The inventors realised that it would be ble to provide for vertebrates, cells, methods etc for the production of eutic and/or prophylactic antibodies based on natural human immune responses to ns, such as antigens of infectious disease pathogens. In this respect, the literature observes frequently used immunoglobulin gene segments to raise anti- infective responses in humans (Table I). wo 41846 PCT/G82012/052298 PCT/GBZOIZ/052298 N$3 “wag .mmmH isfiééamséwa $3 mama “$3 wvmmfi Hmeow “32. mama .8355 ~32: 502: EC _oc:EE_ csEE_ 52: 532m EU SEE: 7228 3m _L_m 7:8 a 3m 685 “62 6 .38 3m um 885 59.222 E 6 \_m 32 32 32 ._m :28 2 am E um 05mm 93:3 5:55 $83; 955 6an >963 mmui :85; c0952)» £82: 56133686 $83,: _E~< 52:2: 88.9.5 3232:. 825 g! mecwzcE max mZm m:_EQoEwmI mm tensed. 001.53 58:: ._<_m m.._.U<m n ma? 9.: an. 2:5 mm~cmszfi £5 :50. mszcaoEmmI wncmfiuazoa 955:: "H new -_v_ ES Amcmw 2an 9:. 58 mm + mcmm AN< .va N< H.~<> 3: mc: E B mcmm wf rwnEmE .8552 285E 9 mmzmo mcmm 8 Ebm >=E£ 3sz >=E£ > mc__E.mm >=E£ _mu_ucw_u_ .mmmiEJIv mcwm 382 __> >__E£ (his. <> «35.8 msoonEo: 05> __ __ __ x> va _v.> x> m <3 (Om—23 58 $50 96 ,3 $3 Tmoooox SEE. __CD 2:38 mutchSzOm mcmm 8me ___I> I> 5-9V B 31> ©NI> 2306 mm SomeoEo: 2: E5 9 ___I> ___:> “Scam“ a? a: dw mucmEmwm new 30:82 <¢ E ~< NHS; 253680: Néwéémm ANGS __>‘> E>+dm __g>+ =K>$NI> ___I> (v 53; mmeO *8 8mm +3 fim “ad O>> _=I> + fim __>4> =< I> I> 3: €83 do. ___x> ___x> ___I> 51> 5:) H-mOOOOV_ :02: 3m 7:23 mama: 583: 95.6 ”39.: A>N>v mfibfiEcmE m2; m3; m3; 382 xwagm N->mI 338 mZmGOICQn. 380: 392.. NH.>mI m__mu:m> 52358338 ._<x_> £30583» 268382» .muuouomc_cm_2 2ESam:_>umumAw¢~vE>_om 92% 9-03 £2 Bum 63 >N> >mI B + 5:3 LwQEmE NmH>._D_ is gNQ mg? ._o 2:3 8&2 + >=Emw LwnEwE SEQ m_.:. o 0536 + >_T_> + $13 m<> MI_. Nfi m<> .30 + ._O + 02, :_I> 53> LO N»: deI> 2.) ©NI> 31> 9.: +xz0HI> NmH>._0_ O Tmoooox WO 20131041846 ”SS. «Nam: :8 meow .05 :25. 52.. 856m 88 «:0 .22 3.8 EU :6 mo: as; a 858 853 ._m 3m 32$ ‘_m .6 E 52 ‘5 E 3 w E 3 mama: 953 :03 38:: . a 5m $3 .8 .8 .2 .NH .2 .3 .2 H < 959 9490 .0 .NzNI anI .mm 3:235 masoammsogu .o .mES N 955 4sz NZAI 2:3 Sconce. \mm .0 5E5 $.23 N78... 353:5 . . 23.; EcsammmEmmI £5,313 5E 5%, :13 $13 2%; 32 2%; $2 .o an? 53 52 :3 £3 AHK>:>E_ 5E 53 :5 02, >N> >N> 8“. 22 L8 mama. 38>: $3. Ni 02:: 2.3:, #2 Hm~1> moi; H-moooox 2012/052298 KOOOOS-l 2012/052298 References: 1. Nat Biotechnol. 2005 Sep;23(9):1117-25; Human antibodies from transgenic animals; g 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. Jlmmunol. 1993 Oct 15;151(8):43SZ-61; Clonal characterization of the human IgG antibody repertoire to Haemophilus influenzae type b polysaccharide. V. In vivo expression of individual dy clones is dependent on lg CH haplotypes and the categories of antigen; Chung GH, Scott MG, Kim KH, Kearney], Siber GR, ino DM, Nahm MH. 4. J l. 1998 Dec 11):6068-73; Decreased frequency of rearrangement due to the synergistic effect of nucleotide changes in the heptamer and nonamer of the recombination signal sequence of the V kappa gene AZb, which is ated with increased susceptibility of Navajos to Haemophilus influenzae type b disease; Nadel B, Tang A, Lugo G, Love V, Escuro G, Feeney AJ, . JClin Invest. 1996 May 15;97(10):2277~82; A defective Vkappa A2 allele in Navajos which may play a role in increased susceptibility to hilus influenzae type b disease; Feeney AJ, Atkinson MJ, Cowan MJ, Escuro G, Lugo G. 6. Infect Immun. 1994 Sep;62(9):3873-80; Variable 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 Invest. 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 cross-reacting with self ns; on EE, Shackelford PG, Quinn A, Wilson PM, Cunningham MW, Insel RA, Carroll WL. wo 2013/041846 PCT/GBZU]2I052298 8. J Clin Invest. 1993 Mar;91(3):788-96; Variable region expression in the antibody responses of infants vaccinated with Haemophilus nzae type b polysaccharide-protein conjugates.

Description of a new lambda light chain-associated idiotype and the relation between idiotype expression, avidity, and vaccine formulation. The Collaborative Vaccine Study Group; Granoff DM, Shackelford PG, Holmes SJ, Lucas AH.

Infect Immun. 1994 May;62(5):1776-86; Variable region sequences and idiotypic 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 . 1992 Dec;90(6):2197-208; Sequence analyses of three immunoglobulin G anti- virus antibodies reveal their ation 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}:834-5, Biochemistry. Catching a moving target, Wang ‘l'l', Palese P 12. Science. 2009 Apr 10;324(5924):246-51. Epub 2009 Feb 26; Antibody recognition of a highly ved influenza virus epitope; Ekiert DC, Bhabha G, Elsliger MA, Friesen RH, eelen M, Throsby M, Goudsmit J, Wilson IA. 13. PLoS One. 2008;3(12):e3942. Epub 2008 Dec 16; Heterosubtypic lizing monoclonal dies cross—protective against H5N1 and H1N1 recovered from human IgM+ 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 KruifJ, it J. 14. Nat Struct Mol Biol. 2009 Mar;16(3):265-73. Epub 2009 Feb 22,5tructural and functional bases for broad-spectrum neutralization of avian and human influenza A viruses, Sui J, Hwang WC, Perez S, Wei G, Aird D, Chen LM, Santelli E, Stec B, Cadwell G, Ali M, Wan H, Murakami A, Yammanuru A, Han T, Cox NJ, Bankston LA, Donis R0, Liddington RC, Marasco wo 2013/041846 PCT/GBZO]2I052298 . Science. 2011 Aug 12;333(6044):843—50. Epub 20111u| 7, A highly conserved neutralizing epitope on group 2 influenza A viruses, Ekiert DC, Friesen RH, Bhabha G, Kwaks T, Jongeneelen M, Yu W, Ophorst C, Cox F, Korse HJ, Brandenburg B, Vogels R, BrakenhoffJP, Kompier R, Koldijk MH, Cornelissen LA, Poon LL, Peiris M, Koudstaal W, Wilson lA, Goudsmit In one embodiment, in (b)(i) the J gene segments of the light chain locus are it gene segments and optionally the constant region of the light chain locus is a lambda constant region; or in ) the VL is a VA and optionally the constant region of the light chain locus is a lambda constant region.

Alternatively, the constant region is C kappa.

In one embodiment, in (b)(i) the V gene segment repertoire of the light chain locus comprises or consists of one or more VH gene segments selected from the group consisting of: a VHIII gene family member (optionally, a VHIIla or VHIIIb family member), a VHIV gene family member, VHIII 9.1 (VH3- ), VHIII VH26 (VH3-23), VHS-21, LSG6.1, LSGlZ.1, DP77 (VS-21), VH H11, VHlGRR, ha3h2, VHI-halcl, VHlll-VHZ-l, VH4.18, ha4h3, Hv1051, 71-2, Hv1f10, VH4.11, 71-4, VH251, VH1-69 and a gene segment at least 80% cal thereto. These gene segments are useful because they expand the oire in vivo to VH gene ts that are found in l human immune responses to antigens, such as antigens of infectious e pathogens. This is useful, for example, when the vertebrate is immunised with an antigen of an ious disease pathogen, for generation and isolation of an antibody for treating and/or preventing a disease or condition mediated by said pathogen. In one example, in (b)(i) the V gene segment repertoire of the light chain locus comprises or consists of only VH gene segment selected from the group consisting of: a VHIII gene family member (optionally, a VHIIIa or VHIIIb family member), a VHIV gene family , VHIII 9.1(VH3— ), Will VH26 (VH3-23), VH3-21, LSGG.1, LSGlZ.1, DP77 (V321), VH H11, VHlGRR, ha3h2,VHl-ha1c1, VHlll-VH2-1,VH4,18,ha4h3, Hv1051, 71-2, , , 71-4, VH251, VH1-69 and a gene segment at least 80% identical thereto. This 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 ly used in natural human immune responses to antigens, such as antigens of infectious disease pathogens. For example, VH1-69 is commonly used to produce antibodies in humans t lnfulenza virus (see Table 1); it is possible, therefore, to e the single VH segment to VH1-69 in wo 2013/041846 ment (b)(i) of the invention.

In one ment, in (b)(iii) the light chain locus V gene segment repertoire ts of only one (optionally only two, three or four) VL gene segment type (optionally and one or s thereof), wherein the VL gene segment is selected from said group of VL gene segments. This 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 disease pathogens.

In one embodiment, in (a) said constant region is a heavy chain endogenous non-human vertebrate (optionally host mouse or rat) constant region and/or in (b) said constant region is a light chain endogenous non-human vertebrate (optionally host mouse or rat) nt .

In one embodiment in any configuration of the invention, the genome has been modified 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, 986, US6130364, W02009/076464, EP1399559 and U56586251, the disclosures of which are incorporated herein by reference. In one embodiment, the non—human vertebrate VDJ region of the endogenous heavy chain immunoglobulin locus, and optionally VJ region of the endogenous light chain immunoglobulin loci (lambda and/or kappa loci), have been inactivated. For example, all or part of the non-human vertebrate VDJ region is inactivated by inversion in the nous heavy chain immunoglobulin locus of the mammal, ally with the inverted region being moved upstream or downstream of the endogenous lg locus (see, eg, 004192, the disclosure of which is incorporated herein by reference). For example, all or part of the non-human vertebrate VJ region is inactivated by inversion in the endogenous 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 vertebrate VJ region is inactivated by inversion in the endogenous lambda chain immunoglobulin locus of the , optionally with the inverted region being moved upstream or downstream of the nous lg locus. In one embodiment the endogenous heavy chain locus is inactivated in this way as is one or both of the endogenous kappa and lambda loci. wo 2013/041846 PCT/G82012/052298 Additionally or alternatively, the rate has been generated in a genetic ound which prevents the tion of mature host B and T lymphocytes, optionally a RAG-l-deficient and/or RAG-2 ent background. See U55859301 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.

In a second configuration of the inventionI there is provided A non‘human vertebrate (optionally a mouse or a rat) or vertebrate cell whose genome comprises: (a) An immunoglobulin heavy chain locus comprising one or more human V gene ts (eg, a plurality of VH), one or more human D gene segments and one or more human J gene segments upstream of a constant region; and (b) (i) An unrearranged immunoglobulin light chain locus comprising one or more human VH gene segments and one or more human J gene segments upstream of a constant region, wherein each human VH gene segment is a human gene segment identical to (or mutant of, eg, having up to 15 or 10 nucleotide changes from the human gene segment) a human VH gene segment (eg, a germline VH gene segment; eg, a gene segment selected from List A1, A2, A11, A1.2, A2.1, A22, A23 or A2.4 below.) used to produce a rearranged VDJ ng a heavy chain variable region of a human antibody from an antibody—expressing cell n said antibody binds to an antigen of an ious disease pathogen (optionally the variable regions of said antibody being identical to an dy from a human individual suffering, susceptible to, or recovered from, a disease or condition caused or mediated by an organism harbouring or secreting said antigen; or from a human dual harbouring said organism); (ii) An immunoglobulin light chain locus comprising a rearranged VJ region or VDJ region upstream of a constant region, wherein the nucleotide sequence of the recombined region is identical to a nucleotide sequence produced by the recombination of a human J gene segment and optionally a human D gene segment with a human VH gene segment that is identical to (or mutant of; eg, having up to 15 or 10 nucleotide changes from the human wo 2013/041846 PCT/G82012/052298 gene segment” the human VH gene segment (eg, germline VH gene segment; eg, a gene segment selected from List A1, A2, A1.1, A1.2, A2.1, A2.2, A2.3 or A2.4 below.) used to produce a rearranged VDJ encoding a heavy chain variable region of a human antibody from an dy-expressing cell wherein said antibody binds to an antigen of an infectious disease pathogen (optionally the variable regions of said antibody being identical to an antibody from a human individual suffering, susceptible to, or red from, a disease or condition caused or mediated by an organism harbouring or secreting said antigen; or from a human individual harbouring said organism); (C) Wherein the gene ts in the heavy chain locus are operably linked to the constant region thereof, and the gene segments or VJ or VDJ in the light chain locus are opera bly linked to the constant region thereof, so that upon immunisation the mouse is capable of producing an antibody comprising heavy chains produced by recombination of the heavy chain locus and light chains derived from the light chain locus; (d) Optionally when (b)(i) applies, each said VH gene segment in the light chain locus is selected from the group consisting of: a VHIII gene family member (optionally, a VHllla or VHlllb family member), a VHIV gene family member, VHIII9.1(VH3-15),VHlllVH26(VH3—23),VH3- 21, , LSGlZ.1, DP77 (V3-21), VH H11, VHlGRR, ha3h2, lcl, VHIII-VHZ-l, , ha4h3, HleSl, 71-2, Hvlf10,VH4.1l, 71-4, VH251, VH1-69 and a gene t at least 80% identical thereto; optionally each VH gene segment is selected from List A1, A2, A1.1, A1.2, A2.1, A22, A2.3 or A2.4 below.

Optionally when (b)(ii) applies, the nucleotide sequence of the recombined region is identical to a nucleotide sequence produced by the recombination of a human J gene segment and optionally a human D gene segment with a human VH gene segment ed fr0m the group consisting of: a VHIII gene family member (optionally, a VHIIIa or VHIIIb family member), a VHlV gene family member, VHIII 9.1(VH3-15),VHII|VH26(VH3—23),VH3- 21, LSGGJ, LSGlZ.1, DP77 (VS-21), VH H11, VHlGRR, VHI-halc1, VHIII-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; ally each VH gene segment is selected from List A1, A2, A11, A12, A2.1, A22, A23 or A2.4 below. wo 2013/041846 2012/052298 In one embodiment, the antigen is an antigen expressed by a bacterial or viral infectious disease pathogen, eg, any of the pathogens listed in Table 1. For e, the n is an antigen selected from the antigens listed in Table 1.

In one embodiment of any aspect, configuration or embodiment of the invention herein, the "human individual harbouring said organism" is a patient that has natural resistance to the pathogen and produces antibodies that bind to the pathogen or an antigen expressed thereby.

In one embodiment of the second configuration, the) gene segments of the light chain locus are JA gene ts and optionally the constant region of the light chain locus is a lambda constant region. Alternatively, the constant region is C kappa.

In one embodiment of the second configuration, the V gene segment repertoire of the light chain iocus comprises or consists of one or more VH gene segments selected from the group consisting of: a VHlll gene family member (optionally, a VHllla or VHlllb family member), a VHIV gene family member, VHIII 3-15),VHIIIVH26(VH3-23), VH3-21, LSG6.1, LSGlZ.1, DP77 (VS-21), VH H11, VHlGRR, ha3h2, VHI—halcl, VHIII—VHZ-l, VH4.18, ha4h3, Hv1051, 71-2, Hv1f10, VH4.11, 721-4, VH251, VH1—69 and a gene segment at least 80% identical thereto. These gene segments are useful because they expand the repertoire in vivo to VH gene segments that are found in l human immune responses to antigens, such as antigens of infectious disease ens. This is useful, for example, when the vertebrate is immunised with an antigen of an infectious disease pathogen, for generation and isolation of an antibody for ng and/or preventing a disease or ion ed by said pathogen. In one example, in (b)(i) the V gene segment repertoire of the light chain locus comprises or consists of only VH gene segment selected from the group consisting of: a VHlll gene family member (optionally, a VHIIIa or VHIllb family member), a VHIV gene family member, VHIII 9.1(VH3- ),VH1|| VH26 (VH3—23), VH3-21, , 1, DP77 (VB-21), VH H11, VHlGRR, ha3h2,VHl—ha1c1, VHlll-VH2-1,VH4.18, ha4h3, Hv1051, 71-2, Hv1f10, VH4.11, 71-4, VH251, VH1—69 and a gene t at least 80% identical thereto. This is useful to bias the immune response of the vertebrate (and thus resultant lead antibodies) to a ermined gene segment, eg, one known to be commonly used in natural human immune responses to antigens, such as antigens of infectious disease pathogens. For example, VH1-69 is commonly used to produce antibodies in humans against lnfulenza virus (see Table 1); it is possible, therefore, to confine the single VH segment to VH 1-69 in embodiment (b)(i) of the invention.

In one embodiment of the second configuration, in (a) said constant region is a heavy chain nous non-human vertebrate nally host mouse or rat) constant region.

In one ment of the second configuration, in (b) said constant region is a light chain endogenous non-human vertebrate (optionally host mouse or rat) constant region.

In one embodiment of the second uration, the genome of said vertebrate or cell is homozygous for light chain locus (b)(i) or (ii); optionally wherein: - the V gene segment repertoire of the light chain loci consists of one or more human VH gene segments selected from the group consisting of: a VHIII gene family member (optionally, a VHllla or VHIIIb family member), a VHIV gene family member, VHIII 9.1(VH3-15),VHIIIVH26(VH3-23), VH3- 21, LSGG.1, LSGlZ.1, DP77 (V3-21), VH H11, VHlGRR, ha3h2, lcl, VHIII-VH2-1, VH4.18, ha4h3, Hv1051, 71-2, Hv1f10, VH4.11, 71-4, VH251, VH1-69 and a gene t at least 80% identical thereto; or — the recombined VJ or VDJ repertoire of the light chain loci consists of sequences identical to one or more nucleotide sequences ed by the recombination of a human VH gene segment selected from the group consisting of: a VHIII gene family member (optionally, a VHIIIa or VHIIib family member), a VHIV gene family member, VHIII9.1(VH3-15), VHlll VH26 (VH3-23), , , LSGlZ.1, DP77 (V3-21), VH H11, VHlGRR, ha3h2, VHl—halcl, VHlll—VHZ-l, VH4.18, ha4h3, Hv1051, 71-2, Hv1f10, VH4.11, 71-4, VH251, VH1-69 and a gene segment at least 80% cal thereto, with a humanJ gene segment and optionally a human D gene segment. In one embodiment, all of the light chain locus V gene segments are from this group.

In one embodiment of the second configuration, endogenous heavy and light chain expression has been inactivated, and wherein light chain loci according to the second configuration are the only wo 41846 PCT/GBZO]2/052298 functional light chain loci in the genome of the vertebrate or cell.

In one embodiment of the second uration, each immunoglobulin light chain locus of said vertebrate or cell is ing to (b)(i) and comprises only a single human VH gene segment ed from the group consisting of: a VHIII gene family member (optionally, a VHllla or VHlllb family member), a VHIV gene family member, VHIII9.1(VH3-15), VHIII VH26 3), VH3-21, LSGB.1, LSGlZ.1, DP77 ), VH H11, VHlGRR,ha3h2,VHI-ha1c1,VHIll-VH2-1,VH4.18, ha4h3, Hv1051, 71-2, Hv1f10,VH4.11, 71-4, VH251, VH1—69 and a gene segment at least 80% identical thereto, optionally wherein the genome of the vertebrate or cell is homozygous for said light chain so that all light chain loci comprise the same, single human VH gene segment. In this ment (and lly in other embodiments, configurations and aspects of the invention), confinement of heavy and/or light chain locus architecture is useful for biasing or controlling the antibody and gene oire, eg, to mirror human immune responses as mentioned above. Provision of a single light or heavy chain variable (and optionally D and/or J) gene segment (or only this with closely related mutants thereof) — or confinement in embodiments below to a single rearranged V(D)J region or single heavy or light chain — is advantageous for simplifying the expression and production of eutic/prophylactic antibodies since this restricts the number of antibody species produced during downstream manufacturer A common heavy or light chain is advantageous to enable co- expression of a plurality (eg, two, three or more) different antibodies in the same expression medium, for example from the same host cell. See, eg, EP1523496 (Merus BV) and W02011097603 (Regeneron Pharmaceuticals, Inc). in one embodiment of the second configuration, each globulin light chain locus of said vertebrate or cell is according to (b)(ii) and comprises only a single rearranged VJ or VDJ region, optionally wherein the genome of the vertebrate or cell is homozygous for said light chain so that all light chain loci comprise the same, single rearranged VJ or VDJ region. in one embodiment of the second configuration, each immunoglobulin light chain locus further comprises a VH gene segment or rearranged region that is a mutant (eg, having up to 15 or 10 nucleotide changes from the VH gene segment) respectively of said selected human VH gene segment or rearranged region, optionally wherein the genome of the vertebrate or cell is homozygous for said light chain mutant VH gene segment or rearranged region.

PCT/G82012/052298 In one embodiment of the second configuration, each immunoglobulin light chain locus ses only two or three human VH gene segments selected from said group, optionally wherein the genome of the rate or cell is homozygous for said two or three light chain human VH gene segments.

In one embodiment of the second configuration, each immunoglobulin light chain locus comprises only two or three of said rearranged VJ or VDJ regions, optionally wherein the genome of the vertebrate or cell is homozygous for said two or three light chain rearranged VJ or VDJ regions.

The invention provides a onal or polyclonal antibody composition prepared by immunisation of at least one vertebrate (eg, mouse or rat) according to any configuration, aspect or embodiment of the invention, optionally wherein the antigen is an antigen of an infectious disease pathogen (eg, a bacterial or viral pathogen antigen or an antigen listed in Table 1), optionally wherein the same antigen is used to immunise all the vertebrates; optionally wherein the antibody or antibodies are lgG—type (eg, lgG 1).

The invention provides a first method of isolating an antibody that binds a predetermined antigen (eg, a bacterial or viral pathogen n or an n listed in Table 1), the method comprising (a) providing a vertebrate (optionally a mouse or rat) according to according to any configuration, aspect or embodiment of the invention; (b) immunising (eg, using a rd prime-boost method) said vertebrate with said antigen (optionally wherein the antigen is an n 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 n; (d) optionally immortalising said selected B lymphocytes or progeny thereof, optionally by producing hybridomas therefrom; and (e) ing an antibody (eg, and lgG-type antibody) expressed by the B lymphocytes.

In a first embodiment of the first method of the ion, the method ses the step of isolating from said B lymphocytes nucleic acid encoding said antibody that binds said n; optionally exchanging the heavy chain nt region nucleotide sequence of the antibody with a wo 2013/041846 PCT/G82012/052298 nucleotide ce encoding a human or humanised heavy chain constant region and optionally affinity maturing the variable region of said antibody; and ally inserting said nucleic acid into an expression vector and ally a host. The skilled person will be aware of rd molecular biology techniques to do this. For example, see Harlow, E. & Lane, D. 1998, 5th edition, Antibodies: A tory 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 ed by techniques readily available in the art, Such as using conventional recombinant DNA and RNA technology as will be apparent to the skilled person. See e,g. Sambrook, J and Russell, D. (2001, 3’d edition) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, Plainview, NY).

In one embodiment of the first method of the invention, the method sesfurther ma king a mutant or derivative of the antibody.

A method of producing a polyclonal antibody mixture is provided, the method comprising carrying out the first method of the invention by separately immunising first and second vertebrates (optionally first and second mice or first and second rats) with antigen and combining the anti~ antigen antibodies isolated from each vertebrate (or mutants or derivatives of said antibodies) to produce a polyclonal antibody mixture; ally wherein the following apply separately or in combination ((i) and (ii); or (i) and (iii)): (i) the vertebrates are immunised with the same antigen or different antigens (optionally wherein the different antigens are expressed by the same pathogenic organism (or by family members or different strains of the sm)); (ii) prior to immunisation the light chain loci of the vertebrates n the identical VH gene repertoire (optionally a single VH gene) and optionally the identical J repertoire; ally the light chain loci of the s are identical prior to immunisation,- (iii) prior to immunisation the light chain loci of the vertebrates contain the identical rearranged VJ or VDJ repertoire (optionally a single VJ or VDJ); optionally the light chain loci ofthe vertebrates are identical prior to immunisation.

PCT/G82012/052298 A method of ing a polyclonal antibody e is provided, the method comprising carrying out the first method of the ion by immunising one or a plurality of vertebrates (optionally mice or rats) with first and second antigens and combining the anti-antigen antibodies isolated from each vertebrate (or mutants or derivatives of said antibodies) to produce a polyclonal antibody mixture; optionally wherein the following apply separately or in combination ((i) and (ii); or (i) and (iii)): (i) the antigens are expressed by the same pathogenic sm (or by family members or different strains of the organism)); (ii) prior to immunisation the light chain loci of the vertebrates contain the identical VH gene repertoire (optionally a single VH gene) and ally the identical J repertoire; optionally the light chain loci of the mammals are identical prior to immunisation; (iii) prior to immunisation the light chain loci of the vertebrates n the identical nged VJ or VDJ repertoire (optionally a single VJ or VDJ); optionally the light chain loci of the vertebrates are identical prior to immunisation.

The invention provides a second method: A method of producing host cells (eg, Chinese Hamster Ovary (CHO) or HEK293 cells) capable of expressing a polyclonal antibody e is provided, the method comprising, in a method according to said first embodiment of the first method of the ion:- (a) immunising one or a plurality of vertebrates (optionally mice or rats) with first and second antigens (optionally wherein the different antigens are expressed by the same enic organism (or a family member thereof)); (b) isolating nucleic acid encoding first and second anti-antigen antibodies from B lymphocytes from said vertebrates; (c) determining the tide sequences of the heavy and light chain variable regions (optionally the entire heavy and/or light chain sequences) of the first antibody; (d) determining the nucleotide sequence of the heavy variable region and optionally the light chain variable region of the second antibody; 2012/052298 (e) inserting the heavy chain variable region coding sequence of each antibody into a heavy chain expression vector; optionally wherein the constant region coding sequence of each heavy chain is exchanged for a nucleotide sequence that encodes a human or sed constant region; (f) inserting the light chain variable region coding ce of the first antibody into alight chain expression vector; optionally wherein the constant region coding sequence of the light chain of the first antibody is exchanged for a nucleotide sequence that s a human or humanised constant region; (g) optionally inserting the light chain variable region coding sequence of the second antibody into a light chain sion vector; optionally wherein the constant region coding sequence of the light chain of the second antibody is exchanged for a nucleotide sequence that s a human or humanised constant region; and (h) introducing each expression vector into a host cell and co—expressing dy chains in a mixture of said host cells to produce antibodies, each antibody comprising one or both of said heavy chain variable regions and a light chain; optionally wherein the expression vectors are introduced together into the same host cell (eg, a CHO or HEK293 cell) so that the cell is capable of expressing antibody light chains and heavy chains, such that the cell or a plurality of the host cells express antibodies (eg, two, three, four or more different dies), each comprising one or both of said heavy chain variable regions and a light chain; (i) ally: prior to immunisation the light chain loci of the vertebrates contain the identical VH gene repertoire (optionally a single VH gene segment) and optionally the identical J repertoire (optionally a single 1 gene segment); optionally the light chain loci of the vertebrates are identical prior to immunisation; or prior to immunisation the light chain loci of the vertebrates contain the identical rearranged V! or VDJ repertoire (optionally a singie VJ or VOJ); optionally the light chain loci of the vertebrates are identical prior to immunisation. (j) optionally: producing a monoclonal or polyclonal dy mixture, by expressing a monoclonal antibody or polyclonal mixture of said dies; optionally followed by isolating an dy comprising the heavy chain variable region of the first and/or second antibodies.

The invention also provides a monoclonal or polyclonal antibody mixture 50 produced or a derivative antibody or mixture thereof, eg, where one or more constant region has been changed (eg, replaced wo 2013/041846 PCT/G82012/052298 with a ent constant region such as a human constant region; or mutated to enhance or ablate Fc effector function). (optionally the entire heavy and/or light chain sequences) In any of the methods of the invention, optionally each vertebrate used for immunisation is provided (a) isolating from a human blood or tissue (eg, B lymphocytes (PBLs), peripheral blood mononuclear cells (PBMCs), bone marrow, , tonsil or lymph node) sample a B lymphocyte that expresses an antibody that binds a predetermined antigen (eg, an antigen expressed by an infectious disease pathogen; ally wherein said serum or tissue was from a human individual suffering, susceptible to, or red from, a disease or condition caused or mediated by an organism harbouring or secreting said antigen; or from a human individual harbouring said organism); (b) determining which human germline VH gene segment was recombined in the human to produce the tide ce of said B cyte that s the heavy chain variable region of the antibody; (c) constructing a transgenic vertebrate wherein said human ne VH gene segment is provided in a light chain locus thereof according the first or second configuration of the invention; (d) providing said transgenic vertebrate for immunisation in the first method of the invention.

The term "Human blood” herein includes a human blood product minus one or more non-B lymphocyte cellular populations, provided that the product retains antibody-producing cells, eg, PBLS.

In an ment of the first method of the invention, each vertebrate used for immunisation is provided by (a) isolating from a human blood or tissue (eg, B cytes, PBMCS, bone marrow, spleen, tonsil or lymph node) sample a B lymphocyte that expresses an antibody that binds a predetermined antigen (eg, an antigen expressed by an infectious disease pathogen; optionally wherein said serum or tissue was from a human individual ing, susceptible to, or recovered from, a disease or condition caused or mediated by an organism harbouring or secreting said antigen; or from a human individual harbouring said organism); (b) determining a nucleotide sequence of said B lymphocyte that encodes a rearranged VDJ or wo 2013/041846 PCT/G82012/052298 VJ region of the antibody; (c) constructing a transgenic vertebrate wherein said rearranged VDJ or VJ region is provided in a light chain locus thereof according to the first or second configuration of the invention; and (d) providing said transgenic vertebrate for immunisation in the first method of the invention.

Common Li ht Chain Antibodies & Bis ecifics e to two atho en anti ens for infectious diseases The invention provides an isolated dy (eg, IgG-type, such as lgGl-type, antibody) obtainable or obtained by the second method of the invention (including step (j), or a mutant or derivative antibody thereof wherein (i) the isolated antibody comprises two copies of the heavy chain le region of said first antibody paired with two copies of the light chain variable region of said first antibody; or (ii) the isolated antibody comprises two copies of the heavy chain le region of said second antibody paired with two capies of the light chain variable region of said first antibody; or (iii) the isolated antibody is a bispecific antibody comprising one copy of the heavy chain variable region of said first antibody paired with a copy of the light chain variable region of the first antibody, and one copy of the heavy chain variable region of said the antibody paired with a copy of the light chain variable region of the first antibody, optionally wherein the bispecific antibody binds to said first and second antigens recited in claim 24; optionally for use in medicine, ally for the treatment and/or prevention of an infectious disease.

In an aspect of the invention, there is ed a monoclonal or polyclonal antibody mixture (eg, IgG-type antibody or antibodies), wherein the monoclonal antibody or mixture is according to any configuration, , embodiment or example of the invention, or a mutant or derivative antibody thereof optionally for use in medicine, optionally for the treatment and/or prevention of an infectious disease, wherein optionally wherein each antibody binds an n of an infectious disease pathogen, preferably the same n In an aSpect of the invention, there is provided the use of an ed, onal or onal antibody according to any configuration, , embodiment or example of the invention, or a mutant or derivative dy thereof in the manufacture of a medicament for the treatment and/or prevention of an infectious disease, optionally wherein the infectious disease is a disease caused by a bacterial or viral pathogen. wo 2013/041846 ‘3 PCT/082012/052298 An example of a mutant antibody is one that bears up to 15 or 10 amino acid mutations in its variable regions relative to an isolated antibody (eg, lgG-type, such as lgGl-type, antibody) obtainable or obtained by the second method of the invention (including step (j). An e 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 or function.

Examples of ious diseases are es caused or mediated by a bacterial or viral pathogen, eg, a pathogen listed in Table 1. Examples of antigens are those listed in Table 1.

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

The invention further provides a nucleotide sequence encoding an antibody according to any uration, aspect, embodiment or example of the invention, ally wherein the nucleotide sequence is part of a .

The invention further es a pharmaceutical composition comprising the antibody or antibodies of any configuration, aspect, embodiment or example of the invention and a diluent, exclpient or carrier.

In a third configuration of the inventionI there is provided A non-human vertebrate (optionally a mouse or a rat) or vertebrate cell whose genome comprises: (a) An immunoglobulin heavy chain locus comprising either:— (i) one or more human VL gene segments, one or more human D gene segments and one or more human J gene segments upstream of a constant region (optionally a rearranged VLDJHCH or VADJHCH); or (iii one or more human VH gene segments selected from the group consisting of: a VHlll wo 2013/041846 PCT/G32012/052298 gene family member (optionally, a VHIIIa or VHlllb family member), a VHIV gene family member, VHIII9.1(VH3—15), VHlll VH26 (VH3-23), VH3-21, LSGS.1, LSGlZ.1, DP77 (VB-21), VH H11, VHlGRR, h83h2, VHl-halcl, VHIII-VHZ-l, VH4.18, ha4h3, Hv1051, 71-2, HvlfIO, VH4.11, 71-4, VH251, VH1-69 and a gene segment at least 80% identical; one or more human D gene segments and one or more human JH gene segments upstream of a constant region; optionally each VH gene segment (and optionally each D) is selected from List A1, A2, A1.1, A1.2, A2.1, A2.2, A2.3 or A2.4 below; and (b) An immunoglobulin light chain locus comprising one or more human V gene segments (eg, a ity of VL) and one or more human 1 gene segments am of a constant region, optionally wherein the light chain locus is according to (b)(i) or (b)(ii) of the first configuration of the invention; Wherein the gene segments in the heavy chain locus are operably linked to the constant region thereof, and the gene segments in the light chain locus are operably linked to the constant region thereof, so that upon immunisation the mouse is e of producing an antibody comprising heavy chains produced by recombination of the heavy chain locus and light chains produced by recombination of the light chain locus. in one e, in (a)(i) all of the heavy chain locus V gene segments are human VL gene segments.

In one embodiment of the third configuration, the V gene segment repertoire of the light chain locus comprises or consists of one or more VL gene segments selected from the group consisting of a VAII gene family , VAVII 4A, VAII 2.1, VAVlI 4A, a Vxl gene family member, a angene family , lGLVlSZ, L70, lalh2, lalvl, la3h3, Kv325, a Vxl gene family member, Kl—lSA (KLOlZ), VKII family member, a Vklll family member, a VKI gene family member, Kl-15A (KL012), Vkll A2 (optionally the A2a allele), VK A27 (Humkv325) and a gene segment at least 80% identical o; optionally each VL gene segment is ed from List A1, A2, A1.1, A1.2, A2.1, A2.2, A2.3 or A2.4 below; and/or wherein or in (a)(ii) the heavy chain locus V gene segment repertoire consists of only one (or two, three or four) VH gene t type (optionally and one or s thereof), wherein the VH gene segment is selected from said group of VH gene segments. This 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 disease pathogens.

In one nr‘nlmrlirwent of the third configuration, in (a) said constant region is a heavy chain WO 41846 PCT/G32012/052298 endogenous non-human vertebrate (optionally host mouse or rat) constant region and/or in (b) said constant region is a light chain endogenous non—human vertebrate (optionally host mouse or rat) constant region.

In one embodiment of the third configuration, endogenous heavy and light chain expression has been inactivated.

A fourth configuration of the present invention provides A non-human vertebrate (optionally a mouse or a rat) or vertebrate cell whose genome comprises: (a) (i) An unrearranged globulin heavy chain locus comprising one or more human VL gene segments, one or more human D gene segments and one or more 1.. gene segments upstream of a constant region, wherein each human VL gene segment is a human gene segment cal to (or mutant of, eg, having up to 15 or 10 nucleotide changes from the human gene segment) a human VL gene segment (eg, a germline VL gene segment; eg, a VL gene t selected from List A1, A2, A1.1, A1.2, A2.1, A2.2, A23 or A2.4 below) used to produce a rearranged VJ encoding a light chain variable region of a human antibody from an antibody-expressing cell wherein said antibody binds to an antigen of an infectious disease en (optionally the variable regions of said antibody being identical to an dy from a human dual suffering, susceptible to, or recovered from, a disease or condition caused or mediated by an organism harbouring or secreting said antigen; or from a human individual harbouring said organism); or (ii) An immunoglobulin heavy chain locus comprising a rearranged VJ region or V0] region upstream of a constant region, wherein the nucleotide ce of the recombined region is identical to a tide sequence produced by the recombination of a human J gene segment and optionally a human D gene segment with a human gene segment identical to (or mutant of, eg, having up to 15 or 10 nucleotide changes from the human gene segment) a human VL gene segment (eg, a germline VL gene segment; eg, a VL gene segment selected from List A1, A2, A1.1, A1.2, A2.1, A2.2, A2.3 or A2.4 below) used to produce a rearranged VJ encoding a light chain le region of a human antibody from an antibody-expressing cell wherein said antibody binds to an antigen of an infectious e pathogen (optionally the variable regions of said antibody being cal to an antibody from a human individual suffering, susceptible to, or recovered from, a disease or condition WO 41846 PCT/G32012/052298 caused or mediated by an organism harbouring or secreting said n; or from a human individual ring said organism); (b) An immunoglobulin light chain locus comprising one or more human V gene segments (eg, a plurality of VL) and one or more human J gene segments upstream of a constant region; and (C) Wherein the gene segments in the light chain locus are operably linked to the constant region thereof, and the gene ts or VJ or VDJ in the heavy chain locus are operably linked to the nt region thereof, so that upon immunisation the mouse is capable of producing an antibody comprising light chains produced by recombination ofthe light chain locus and heavy chains derived from the heavy chain locus; (d) Optionally when (a)(i) applies, each said VL gene segment in the heavy chain locus is selected from the group consisting of a VL gene segment selected from the group consisting of a VAII gene family member, VAVlI 4A, VAII 2.1, VAVII 4A, a VM gene family member, a VABgene family member, lGLVlSZ, VA3-cML70, |a|h2, lalvl, la3h3, Kv325, a VKI gene family , Kl-15A (KL012), VKIl family member, a VKI” family member, a VKI gene family member, Kl—15A(KL012), VKII A2 (optionally the A2a allele), VK A27 (Humkv325) and a gene segment at least 80% identical thereto; optionally each VL gene segment is selected from List A1, A2, A1.1, A12, A2.1, A22, A23 or A2.4 below; Optionally when (a)(ii) applies, the nucleotide sequence of the recombined region is cal to a tide sequence produced by the recombination of a human J gene t and optionally a human D gene segment with a human VL gene segment ed from the group consisting of a VAII gene family member, VAVII 4A, VAII 2.1, VAVIl 4A, a Vltl gene family member, a Wtagene family member, IGLVlSZ, Vks-cM L70, |a|h2, lalvl, la3h3, Kv325, a VKI gene family member, Kl-15A (KL012), VKII family member, a VKIII family member, a VKl gene family member, Kl-lSA (KL012), VK“ A2 (optionally the A2a allele), VK A27 (Humkv325) and a gene segment at least 80% identical thereto.

The group of VL gene segments 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 l human immune responses to antigens, such as antigens of infectious disease pathogens.

In an embodiment of the fourth configuration, the VL gene segments of the heavy chain locus are VA gene segments. wo 2013/041846 PCT/G32012/052298 In an embodiment of the fourth configuration, in (a) said constant region is a heavy chain endogenous non-human vertebrate (optionally host mouse or rat) nt region.

In an embodiment of the fourth configuration, in (b) said constant region is a light chain endogenous non-human vertebrate (optionally host mouse or rat) nt region.

In an embodiment of the fourth configuration, the genome of said vertebrate or cell is homozygous for heavy chain locus (a)(i) or (ii); ally wherein: - the V gene segment repertoire of the heavy chain loci consists of one or more (or consists only of) human VL gene segments ed from the group consisting of a VL gene segment selected from the group consisting of a VAII gene family member, VAVII 4A, WI 2.1, VAVII 4A, a Val gene family , a VABgene family member, IGLV1SZ, Via-cML70, lath, lalvl, la3h3, Kv325, a VKl gene family member, Kl-15A (KL012), VKII family member, a Vxlll family member, a VKI gene family member, Kl— 15A (KL012), VKII A2 (optionally the A2a allele), VK A27 (Humkv325) and a gene segment at least 80% identical thereto; or - the recombined VJ or VDJ oire of the heavy chain loci consists of sequences identical to one or more nucleotide sequences produced by the recombination of a human VL gene segment selected from the group consisting of a VL gene segment ed from the group consisting of a VAII gene family member, VAVII 4A, VAll 2.1, VAVIl 4A, a VM gene family member, a VA3gene family member, iGLVlSZ, VA3—cML70, lalh2, lalvl, la3h3, Kv325, a VKI gene family member, Kl-15A(KL012), VKII family member, a Will family member, a VKI gene family member, (KL012), VKli A2 nally the A2a allele), VK A27 (Humkv325) and a gene segment at least 80% identical thereto with a humanJ gene segment and ally a human D gene segment.

In an embodiment of the fourth configuration, endogenous heavy and light chain expression has been inactivated, and wherein heavy chain loci according to the fourth configuration are the only functional heavy chain loci in the genome of the vertebrate or cell.

In an embodiment of the fourth configuration, each immunoglobulin heavy chain locus of said vertebrate or cell is according to (a)(i) and comprises only a single human VL gene segment selected wo 2013/041846 PCT/GBZOIZ/052298 from the group ting of a VL gene t selected from the group consisting of a VAI! gene family member, VAVII 4A, VMI 2.1, VAVII 4A, a VM gene family member, a VA3gene family member, iGLVlSZ, VA3-cML70, lalh2, Ialvl, |a3h3, Kv325, a VKI gene family member, KI-15A (KL012), VKII family member, a VKIII family member, a VKI gene family member, Kl*15A(KL012), VKII A2 (optionally the A23 aliele), VK A27 (Humkv325) and a gene t at least 80% identical thereto, optionally wherein the genome of the vertebrate or cell is homozygous for said heavy chain so that all heavy chain loci comprise the same, single human VL gene segment.

In an embodiment of the fourth uration, each immunoglobulin heavy chain locus of said vertebrate or cell is according to ) and ses only a single rearranged VJ or VDJ region, optionally wherein the genome ofthe vertebrate or cell is homozygous for said heavy chain so that all heavy chain loci comprise the same, single rearranged VJ or VDJ region.

In an embodiment of the fourth configuration, each immunoglobulin heavy chain locus r comprises a VL gene segment or rearranged region that is a mutant respectively of said ed human VL gene segment or rearranged region, optionally wherein the genome of the vertebrate or cell is homozygous for said light chain mutant VL gene segment or rearranged region.

In all configurations, aspects, examples and ments of the invention, where a ”mutant" is mentioned, this includes a mutant sequence that is identical to a reference sequence (eg, reference VH, VL, VJ or VDJ) but with 1, 2, 3,4,5, 6,7, 8, 9 or 10 nucleotide or amino acid changes therefrom.

In an embodiment of the fourth configuration, each immunoglobulin heavy chain locus comprises only two or three human VL gene segments selected from said group, Optionally wherein the genome of the vertebrate or cell is homozygous for said two or three heavy chain human VL gene segments.

In an embodiment of the fourth configuration, each immunoglobulin heavy chain locus comprises only two or three of said rearranged VJ or VDJ regions, ally wherein the genome of the vertebrate or cell is homozygous for said two or three heavy chain rearranged VJ or VDJ regions.

The invention provides a monoclonal or polyclonal antibody composition prepared by sation of at least one vertebrate (eg, mouse or rat) according to the third or fourth embodiment of the invention with an antigen, optionally wherein the antigen is an antigen of an infectious disease pathogen, optionally n the same n is used to immunise all the vertebrates; ally wherein the antibody or antibodies are lgG-type.

The invention provides a third method: A method of isolating an antibody (eg, lgG-type, such as lgGl) that binds a predetermined antigen, the method comprising (a) providing a vertebrate (optionally a mouse or rat) according to the third or fourth embodiment of the ion; (b) immunising (eg, using standard prime-boost) said vertebrate with said antigen (optionally wherein the antigen is an antigen of an infectiOus disease pathogen); (c) removing B lymphocytes from the rate and selecting one or more B lymphocytes expressing dies that bind to the n; (d) ally immortalising said selected B lymphocytes or progeny thereof, optionally by producing hybridomas therefrom; and (e) isolating an antibody (eg, and IgG-type antibody) expressed by the B cytes; (f) Optionally, the third method comprises the step of isolating from said B cytes 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.

Optionally, the third method further comprises making a mutant or derivative of the antibody.

The invention provides a fourth method: A method of producing a polyclonal antibody mixture, the method comprising carrying out the third method by separately immunising first and second vertebrates (optionally first and second mice or first and second rats) with antigen (eg, any antigen sed herein) and combining the anti-antigen antibodies isolated from each vertebrate (or mutants or derivatives of said dies) to produce a polyclonal antibody mixture; optionally wo 2013/041846 wherein the following apply separately or in ation ((i) and (ii); or (i) and (iii)): (i) the rates are immunised with the same antigen or different ns (optionally wherein the different antigens are sed by the same pathogenic organism (or different family members thereof or ent strains of the organism”; (ii) prior to immunisation the heavy chain loci of the vertebrates contain the identical VL gene repertoire (optionally a single VL gene) and ally the identical D and/or] repertoire; optionally the heavy chain loci of the mammals are identical prior to immunisation; (iii) prior to immunisation the heavy chain loci of the vertebrates contain the cal rearranged VJ or VDJ repertoire (optionally a single VJ or VDJ); optionally the heavy chain loci of the vertebrates are identical prior to immunisation.

The ion provides a fifth method: A method of producing a polyclonal antibody mixture, the method comprising carrying out the third method by immunising one or a plurality of vertebrates (optionally mice or rats) with first and second antigens and combining the anti-antigen antibodies isolated from each vertebrate (or mutants or derivatives of said antibodies) to produce a polyclonal antibody mixture; optionally n the following apply separately or in combination ((i) and (ii); or (i) and (no): (i) the antigens are expressed by the same pathogenic sm (or different family members thereof or different strains of the organism); (ii) prior to immunisation the heavy chain loci ofthe vertebrates n the identical VL gene repertoire (optionally a single VL gene) and Optionally the identical 0 and/or] repertoire; optionally the heavy chain loci of the mammals are identical prior to immunisation; (iii) prior to immunisation the heavy chain loci of the vertebrates contain the identical rearranged VJ or VDJ repertoire (optionally a single VJ or VDJ); optionally the heavy chain loci of the vertebrates are identical prior to immunisation. wo 2013/041846 The invention provides a sixth method: A method of ing host cells e of expressing a polyclonal antibody mixture, the method comprising, in the third method n step (f) is carried out:— (a) immunising one or a plurality of vertebrates (optionally mice or rats) with first and second antigens (optionally wherein the different antigens are expressed by the same pathogenic organism (or a family member thereof)); (b) isolating c acid encoding first and second anti-antigen antibodies from B lymphocytes from said vertebrates; (c) determining the nucleotide sequences of the heavy and light chain variable regions (optionally the entire heavy and/or light chain ces) of the first antibody; ((1) determining the nucleotide sequence of the light le region and optionally the heavy chain variable region of the second antibody; (e) inserting the light chain variable region coding sequence of each antibody into alight chain expression vector; optionally wherein the nt region coding ce of each light chain is exchanged for a nucleotide sequence that encodes a human or humanised constant region; (f) inserting the heavy chain variable region coding ce of the first antibody into a heavy chain sion vector; optionally wherein the constant region coding sequence of the heavy chain of the first antibody is exchanged for a nucleotide ce that encodes a human or humanised constant region; (g) optionally inserting the heavy chain variable region coding sequence of the second antibody into a heavy chain expression vector; optionally wherein the constant region coding sequence of the heavy chain of the second dy is exchanged for a nucleotide sequence that encodes a human or humanised constant region; and (h) introducing each expression vector into a host cell and co-expressing antibody chains in a mixture of said host cells to produce antibodies, each antibody comprising one or both of said light chain variable regions and a heavy chain; optionally wherein the expression vectors are introduced together into the same host cell (eg, 3 CH0 or HEK293 cell) so that the cell is capable of expressing antibody light chains and heavy , such that the cell or a plurality of the host cells express antibodies (eg, two, three or four different antibodies), each comprising one or both of said light chain variable regions and a heavy chain; (i) optionally: prior to immunisation the heavy chain loci of the vertebrates contain the identical VL gene wo 2013/041846 PCT/GBZO]2/052298 repertoire (optionally a single VL gene segment) and optionally the identical D and/or] repertoire (optionally a single D and] gene segment); optionally the heavy chain loci of the vertebrates are identical prior to immunisation; or prior to immunisation the heavy chain loci of the vertebrates contain the identical rearranged VJ or VDJ repertoire (optionally a single VJ or VDJ); optionally the heavy chain loci of the vertebrates are identical prior to sation.

The invention also provides a monoclonal or polyclonal antibody mixture so produced 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 nt region; or mutated to enhance or ablate Fc or function).

The invention provides a seventh : A method of producing a monoclonal antibody or polyclonal antibody mixture, the method comprising carrying out the sixth method and expressing a monoclonal dy or polyclonal mixture of said antibodies; ally ed by isolating an antibody sing the light chain variable region of the first and/or second antibodies.

Optionally, each vertebrate used for sation is provided by (a) isolating from a human blood or tissue (eg, B lymphocytes, PBMCs, bone marrow, spleen, tonsil or lymph node) sample a B lymphocyte that expresses an antibody that binds a predetermined n (eg, an antigen expressed by an infectious disease pathogen; optionally wherein said serum or tissue was from a human individual ing, susceptible to, or recovered from, a e or condition caused or mediated by an organism harbouring or secreting said antigen; or from a human individual harbouring said organism); (b) determining which human germline Vt gene segment was recombined in the human to produce the nucleotide sequence of said B lymphocyte that encodes the light chain variable region of the antibody; (c) constructing a transgenic vertebrate wherein said human germline VL gene segment is provided in a heavy chain locus thereof according to the third or fourth configuration of the invention; and (d) providing said transgenic vertebrate for immunisation in the fourth, fifth or sixth method of PCT/082012/052298 the invention.

In another embodiment, each vertebrate used for immunisation is provided by (a) isolating from a human blood or tissue (eg, B lymphocytes, PBMCs, bone marrow, spleen, tonsil or lymph node) sample a B lymphocyte that expresses an antibody that binds a predetermined antigen (eg, an antigen sed by an ious disease pathogen; optionally wherein said serum or tissue was from a human individual ing, susceptible to, or recovered from, a e or condition caused or mediated by an organism ring or secreting said antigen; or from a human individual harbouring said organism); (b) determining a nucleotide sequence of said B lymphocyte that encodes a rearranged VDJ or VJ region of the dy; (c) constructing a enic vertebrate wherein said rearranged VDJ or VJ region is provided in a heavy chain locus thereof according the third or fourth configuration of the invention; and (d) providing said transgenic vertebrate for immunisation in the method of the fourth, fifth or sixth method of the invention.

Common Heav Chain Antibodies&Bis ecifics e to two atho en anti ens forinfectious diseases The invention provides an isolated antibody (eg, IgG-type antibody) obtainable or obtained by the seventh method, or a mutant or derivative dy thereof wherein (i) the ed antibody comprises two copies of the heavy chain variable region of said first antibody paired with two capies of the light chain variable region of said first antibody; or (ii) the isolated antibody comprises two copies of the heavy chain variable region of said second antibody paired with two copies of the light chain variable region of said first antibody; or (iii) the isolated antibody is a bispecific antibody comprising one copy of the heavy chain variable region of said first antibody paired with a copy of the light chain variable region of the first antibody, and one copy of the heavy chain variable region of said the antibody paired with a copy of the light chain variable region of the first antibody, optionally wherein the bispecific antibody binds to said first and second antigens described above; optionally for use in medicine, optionally for the treatment and/or tion of an ious disease. wo 2013/041846 PCT/GBZOIZ/052298 The invention provides a monoclonal or polyclonal antibody mixture (eg, pe antibody or antibodies), wherein the monoclonal antibody or mixture comprises or consists of antibodies produced by the fourth, fifth, sixth or seventh method, or a mutant or derivative dy thereof ally for use in medicine, optionally for the treatment and/or prevention of an infectious disease, wherein ally wherein each antibody binds an antigen of an infectious disease pathogen, preferably the same antigen.

The following ments relate to antibodies, host cells, nucleic acids and compositions and apply to such elements obtained or obtainable by any previous configuration or method of the invention:- The invention provides an ed chimaeric antibody for treating and/or preventing an infectious disease or condition, the antibody comprising a non-human vertebrate (optionally a mouse or rat) heavy chain constant regions and human variable regions that bind an antigen of an infectious disease en, wherein the antibody is obtainable or obtained in a method comprising immunisation of a vertebrate according to of any one of the first to seventh methods of the invention with said antigen. The n is, for e, any antigen mentioned above. The disease or condition is, for example, any disease or condition ned above.

The invention provides an isolated human antibody for treating and/or preventing an infectious e or condition, the antibody comprising human heavy chain constant regions and human variable regions that bind an antigen of an infectious disease pathogen, wherein the antibody is obtainable or obtained in a method comprising ty maturation of antibody variable regions in vivo in a transgenic non—human vertebrate (eg, mouse or rat) when said variable regions are operably linked to heavy chain constant regions of said rate (eg, mouse or rat heavy chain constant regions) by (a) immunisation of a vertebrate of any configuration of the invention with said antigen, (b) isolation of nucleic acid encoding a chimaeric antibody as bed above, (c) replacing the nucleotide sequences of the nucleic acid that encode the non-human vertebrate heavy chain constant regions with nucleotide sequence encoding human heavy chain nt regions to produce nucleic acid encoding a human antibody; (d) expressing the human antibody in vitro nally from CHO 0r HEK293 cells harbouring the human nucleic acid) and (e) isolating the human antibody (optionally with further ty maturation of the antibody and/or producing a PCT/G82012/052298 derivative thereof). The ion provides a mixture of first and second such human antibodies (an optionally also third and optionally fourth dies), each antibody being capable of binding to an antigen of an infectious disease pathogen (optionally wherein the first antibody binds a first antigen and the second antibody binds a second antigen, said antigens being from the same pathogen; or wherein the antigens are the same). Optionally, the light chain amino acid sequence of the first dy is identical to the light chain amino acid sequence of the second antibody, or has up to 15 amino acid s therefrom. The advantages of such a common (or closely—related) chain are explained above, and e ve ease of manufacture.

The antigen is, for e, any antigen ned above. The disease or condition is, for example, any disease or condition mentioned above. The pathogen is, for e, any pathogen mentioned above.

The invention provides an antibody comprising human variable domains that bind a predetermined antigen (eg, an antigen expressed by a bacterial or viral pathogen), wherein the variable domain ces are encoded by rearranged VDJ and VJ regions, each of the VDJ and/or VJ being a hybrid region produced by the in vivo rearrangement of human heavy and light chain variable region gene segments (V and J and Optionally D segments); optionally wherein the antibody comprises human constant regions.

The invention provides a method of producing an isolated human antibody for treating and/or ting an infectious disease or condition, the antibody comprising human heavy chain constant regions and human le regions that bind an antigen of an infectious disease pathogen, wherein the method ses affinity maturing antibody variable regions in vivo in a enic non—human vertebrate (eg, mouse or rat) when said variable regions are operably linked to heavy chain constant regions of said vertebrate (eg, mouse or rat heavy chain constant s) by (a) immunisation of a vertebrate of any configuration of the invention with said antigen, (b) isolation of nucleic acid encoding a chimaeric antibody as described above, (c) replacing the nucleotide sequences of the nucleic acid that encode the non-human vertebrate heavy chain constant regions with nucleotide sequence encoding human heavy chain constant regions to produce nucleic acid encoding a human antibody; (d) expressing the human antibody in vitro (optionally from CHO or HEK293 cells harbouring the human nucleic acid) and (e) isolating the human antibody (optionally with further PCT/G82012/052298 affinity maturation of the dy and/or producing a derivative thereof). The antigen is, for example, any antigen mentioned above. The disease or condition is, for example, any disease or condition mentioned above. The pathogen is, for example, any pathogen mentioned above.

The invention provides the use of any isolated, onal or polyclonal antibody or e of the invention as described above, in the manufacture of a medicament for the treatment and/or tion of an infectious disease, ally wherein the infectious disease is a disease caused by a bacterial or viral pathogen. The e or condition is, for example, any disease or condition mentioned above. The pathogen is, for example, any pathogen mentioned above. For example, the infectious e is selected from the group consisting of a disease caused by a pathogen selected from the group consisting of Haemophilus influenza, E coli, Neisseria itidis, a herpes family virus, cytomegalovirus (CMV), HIV and influenza virus.

The invention provides first and second nucleotide sequences (eg , DNA, RNA, mRNA, cDNA) encoding the heavy and light chains of an antibody according to any configuration, aspect, example or embodiment of the invention or at least the le regions thereof, optionally wherein each nucleotide sequence is part of a vector.

The invention provides a host cell comprising one or more expression vectors encoding the heavy chains of the first and second dies mentioned above, and the light chain of the first antibody mentioned above (and optionally also the light chain of the second antibody). Again, reference is made to the discussion above about the advantages of having a common antibody chain for the production of antibody mixtures.

The invention provides a pharmaceutical composition comprising the antibody or antibodies of any configuration, aspect, example or embodiment 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 skilled person will know standard diluents, excipients and rs le for pharmaceutical application.

Throughout this description, where it is mentioned ”at least 80% identical”, there is contemplated in the alternative one of the following identities: at least 85%, 90, 95, 96, 97, 98 or 99 cal and the disclosure herein contemplates that one or more of these identities may be recited in a claim herein in place of “at least 80% identica In in V Dllncor oration Into lmmuno lobin Loci ForThe Generati not Anti i A in t Infectious Disease In the various configurations, aspects, embodiments and examples above, the invention provides the skilled addressee with the possibility of choosing immunoglobulin gene segments in a way that tailors or biases the repertoire for application to generating antibodies to treat and/or prevent infectious es. The inventors have categorised the following groups of gene segments for use in the invention according to the desired ation of resultant antibodies.

List A: lmmunoglobulin gene segments for antibodies that bind an antigen expressed by a en (a) a VL gene segment ed from the group consisting of a Vill gene family member, VAVII 4A, VAII 2.1, VRVII 4A, a Vil gene family member, a Vi3gene family member, lGLVlSZ, L7O, lalh2, lalvl, l33h3, Kv325, a VKl gene family member, Kl-lSA (KL012), VKII family member, a V.‘||| family member, a Vxl gene family member, Kl-lSA (KL012), VKII A2 (optionally the A2a ), VK A27 (Humkv325) and a gene segment at least 80% identical o. (b) a VA gene segment selected from a VAII gene family member, VAVII 4A, VAII 2.1, VAVII 4A, a VA1 gene family member, a e family member, IGLVlSZ, VA3-cML7O, Ialh2, lalvl, |a3h3 and a gene segment at least 80% identical thereto. (c) a V,< gene segment selected from Kv325, a VKI gene family member, Kl-15A (KL012), VKII family member, a VKIII family member, a Vxl gene family member, Kl-15A (KL012), Vxll A2 (optionally the A2a allele), VK A27 (Humkv325) and a gene segment at least 80% identical thereto. (d) a VH gene segment a VHIII gene family member (optionally, a VHllla or VHIIIb family ), a VHIV gene family member, VH|||9.1(VH3-15), VHIII VH26 (VH3-23), VHS-21, LSGG.1, LSGlZ.1, wo 2013/041846 PCT/082012/05229s DP77 (VB-21), VK H11, VHlGRR, h33h2, VHI-halcl, VHlll-VH2-1,VH4.18, ha4h3, HleSl, 71—2, Hvlf10,VH4.11, 71-4, VH251, VH1-69 and a gene segment at least 80% identical thereto, (e) a J) gene segment selected from 1A2, #3 and a gene segment at least 80% identical thereto. (f) a D gene t selected from Dkl, Dxp’l, Dn4r, D2r and a gene segment at least 80% identical thereto.

List A1: lmmunoglobulin gene segments for antibodies that bind an antigen sed by_ a Bacterial PLhogLn (a) a V) gene segment selected from a VAII gene family member, VAVII 4A, VAII 2.1, VAVII 4A and a gene segment at least 80% cal thereto. (b) a VK gene segment selected from a VKI gene family member, Ki-15A (KL012), V‘II family , a VKIII family member, a Vxl gene family member, KI-15A(KL012), VKII A2 (optionally the AZa allele), VK A27 (Humkv325) and a gene segment at least 80% identical thereto. (c) a VH gene segment a VH3 gene family member (optionally, a VHIIIa or VHIIIb family member), VHIII 9.1(VH3-15), VHIII VH26 (VH3-23), , LSG6.1, LSGlZ.1, DP77 (VB-21), VH H11 and a gene segment at least 80% identical o. (d) a1) gene segment selected from JAZ, JAS and a gene segment at least 80% identical o. (e) a JH gene segment selected from JHZ, 1.3, J..4 and a gene segment at least 80% identical thereto.

List All: Immunoglobulin gene segments for antibodies that bind an antigen expressed by H influenza WO 20131041846 PCT/GBZOIZ/052298 (a) a V, gene segment selected from a Vxll gene family member, VAVII 4A, VAII 2.1, VAVII 4A and a gene segment at least 80% identical thereto. (b) a VK gene segment selected from a VKII family member, a Vxlll family member, a VKI gene family member, Kl—lSA (KL012), VKII AZ (optionally the A2a allele), VK A27 (Humkv325) and a gene segment at least 80% identical thereto. (c) a VH gene t a VH3 gene family member (optionally, a VHlIIb family member), VHIII 9.1 (VH3-15), VHlll VH26 (VH3-23), VH3-21, LSG6.1, LSG]2.1, DP77 (VS—21) and a gene segment at least 80% identical o. (d) a1) gene segment selected from JAZ, Jx3 and a gene t at least 80% identical thereto.

List A1.22 lmmunoglobulin gene segments for antibodies that bind an antigen exgressed by E Coli or Neisseria meningitidis (a) a VH gene segment a VH3 gene family member (optionally a VHIIIa or VHlllb member), VHIII 9.1 (VH3-15), VH H11, VHIII VH26 (VH3-23) a gene segment at least 80% identical thereto, eg, WI“ 9.1 + 1.3; or VH H11 + 1H4; or VHIII VH26 +JH2. (b) a VK gene t selected from a Vxl gene family member, KI-15A (KL012) and a gene segment at least 80% identical thereto. (c) a VA gene segment selected from a VAII gene family , Vill 2.1 and a gene segment at least 80% identical thereto. (d) a JH gene segment selected from JHZ, 1H3, 1,14 and a gene segment at least 80% identical thereto. immunoglobulin gene segments for antibodies that bind an antigen exgressed by a viral Pathogen PCT/0820121052298 (a) a VH gene segment selected from a VHlll gene family member, a VHIV gene family , VHIII- VH26 (VH3-23), VHlGRR, haShZ, VH|~halc1, VHZ—l, VH4.18, ha4h3, HV1051, 71-2, HvlflO, VH4.11, 71-4, VH251, VH 1-69 and a gene segment at least 80% identical thereto. (b) a VA gene segment selected from a V11 gene family member, a VABgene family member, lGLVlSZ, VA3-cML7O, lath, lalvl, l33h3 and a gene segment at least 80% identical thereto. (c) a Vk gene segment selected from Kv325 and a gene segment at least 80% cal thereto. (d) 3 JH gene segment selected from JH3, 1H5, 1,6 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 thereto. (f) a JA gene segment selected from JAZ, JAB and a gene segment at least 80% identical thereto.

All: Immunoglobulin gene segments for antibodies that bind an antigen exgressed by Herges Virus Family (egI VZV or HSV] (a) a VH gene segment selected from a VHIII gene family , a VHIV gene family member, VHlll- VH26 (VH3-23), , ha3h2, VHl-halc1,VHIII-VH2-1, VH4.18, ha4h3, and a gene segment at least 80% identical thereto. (b) a V,\ gene segment selected from a VA1 gene family member, a e family member, IGLVlSZ, VA3-CM L70, lthZ, lalvl, |a$h3 and a gene segment at least 80% identical thereto. (c) 3 JH gene segment selected from JH3, JHS, 1H6 and a gene segment at least 80% identical thereto. (d) a D gene segment selected from Dkl, Dxp'l, Dn4r, DZr and a gene segment at least 80% identical thereto. (e) a it gene t ed from 1A2, 1A3 and a gene segment at least 80% identical thereto.

PCT/GBZO]2/052298 A22: lmmunoglobulin gene segments for antibodies that bind an antigen expressed by CMV (a) a VH gene segment ed from Hv1051 and a gene segment at least 80% identical thereto. (b) a Vk gene segment selected from Kv325 and a gene segment at least 80% identical thereto.

A2.3: Immunoglobulin gene segments for antibodies that bind an antigen expressed by HIV (a) a VH gene segment selected from 71-2, Hv1f10, VH4.11, 71-4, VH251, VH1-69 and a gene segment at least 80% identical thereto.

A2.4: Immunoglobulin gene segments for antibodies that bind an antigen expressed b! influenza Virus (3) a VH gene segment selected from VH1—69 and a gene segment at least 80% identical thereto.

Thus, Where one wishes to te an antibody or dy 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, , example or embodiment of the ion can be selected from List A1. Thus, for example in (a) of the first configuration of the 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 te an antibody or antibody e 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 wo 2013/041846 PCT/GBZOIZ/052298 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 mediated by a viral en, one or more or all V, D and/or] gene ts used in any configuration, aspect, method, example 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 t an infectious disease caused or mediated by H influenza, one or more or all V, 0 and/or J gene ts used in any configuration, aspect, method, e or embodiment of the invention can be selected from List A1.1.

Where one wishes to generate an antibody or antibody e to treat and/or prevent an infectious disease caused or mediated by E Coli or Neisseria meningitidis, 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 A1.2.

Where one wishes to generate an antibody or dy 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 and/orJ gene segments used in any configuration, aspect, , 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] gene segments used in any configuration, aspect, method, example or embodiment of the invention can be selected from List A2.2.

ZOIZ/052298 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 segments used in any configuration, aspect, method, example or embodiment of the invention can be selected from List A23».

Where one wishes to generate an antibody or antibody e to treat and/or prevent an infectious disease caused or mediated by Influenza Virus, one or more or all V, O and/or] gene segments used in any configuration, aspect, method, example or embodiment of the invention can be selected from List A24.

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

Optionally each VL segment in the locus of the ion is selected from List A1, A2, A11, Al.2, A2.1, A2.2, A23 or A2.4 Optionally each D segment in the locus of the invention is selected from List A1, A2, A1.1, A12, A21, A2.2, A23 or A24.

Optionally each JL segment in the locus of the ion is selected from List A1, A2, Al.1, A1.2, A2.l, A2.2, A23 or A24.

Long HCDR3 g site; & ing ngg §ggmgn§ t9 Pgthgggn; 8L Othgr Antigens This aspect of the invention relates to the development of rates, cells, methods and antibodies with relatively long HCDR3 binding sites. There is also provided embodiments in which genomes and antibodies are tailored in terms of their gene segments usage to address infectious e pathogen antigens or other antigens which are advantageously addressed with a longer HCDR3 length for binding or neutralisation. Antibodies may be raised in the vertebrates by wo 2013/041846 PCT/GBZO]2/052298 immunisation with a non-pathogen target n, eg, an antigen bearing an epitope in a cleft requiring along CDR for contact, or an antigen from a pathogen that causes or is implicated in harmful human disease or conditions Examples are bacterial or viral pathogens and the target antigen may be a bacterial cell-surface antigen or a viral surface-exposed antigen (eg, coat n).

Additionally or alternatively, the antigen may be an antigen that is released (eg, secreted) from a pathogenic bacterium or virus. The invention is not limited to sing pathogen antigens, but is also useful for addressing other antigens where along CDR3 would be useful for binding (eg, an enzyme active site or receptor cleft).

Antibodies with long HCDR3 (at least 20 amino acids according to IMGT nomenclature) have been shown to effectively lise a variety of pathogens including HIV, Influenza virus, malaria and Africa trypanosomes. nce is also made to lly-occurring d (eg, llama or camel) heavy chain-only antibodies which bear long HCDR3S for reaching relatively inaccessible epitopes (see, eg, 140). Long HCDRBS can form unique stable subdomains with extended loop structure that towers above the antibody surface to confer fine specificity. In some cases, the long HCDR3 itself is sufficient for epitope binding and neutralization (Liu, L et al; l 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 surface. In human peripheral blood, there is around 3.5% of naive B antibodies or 1.9% of memory B lgG antibodies containing the HCDR35 with lengths of more than 24 amino acids (Briney, BS et al, referenced given below) (Fig. 1 of Briney, BS et al). The usage analysis indicates that these antibodies have the preference to use human VH1-69, D2-2, 03-3, 02-15 and JH6 segments (Figs. 2- of Briney, BS et of). There are around 20% of all HCDR3 length antibodies using JH6. However, in those antibodies with more than 24 amino acids of HCDR3, there are 70% using JH6 (Fig.2 of , BS et al). Human VH5~51 is also commonly used for anti-HIV dies (see Gorny et al, PLoS One. 2011;6(12):eZ7780. Epub 2011 Dec 2.

Human 3 HlV-l monoclonal antibodies encoded by the VHS-Sl/Vl. lambda genes define a conserved antigenic structure, incorporated herein by reference).

Supplementing these observations, the inventors have found (see examples) that other ed human heavy chain variable region gene segments (V, D, J) recombine in transgenic non-human vertebrates to produce long HCDR3 (at least 20 amino acids). wo 2013/041846 PCT/GBZOIZ/052298 Thus, as ned further in the examples, the inventors constructed transgenic lgH loci in ES cells, wherein the loci purposely included ed human heavy chain variable region gene ts (V, D, J) that recombine to produce long HCDR3 (at least 20 amino acids). From the ES cells, the inventors generated transgenic non—human vertebrates (both naive and immunised with a range of different target antigen types — disease pathogen and human antigenic s), isolated antibodies and heavy chain sequences based on the ed gene segments as well as B-cells expressing these and made hybridomas expressing antigen-specific antibodies that are based on the selected gene segments.

There is a need in the art for genetically modified non—human animals that prefer to make human antibodies that have long HCDR3s, as well as antibodies that can be selected from such animals wherein the antibodies can address target epitopes more easily accessed by long HCDR3s. Long CDRH3 is also useful for penetrating highly glycan-covered epitope sites (eg, virus epitopes or any glycoprotein targets, eg, see Nature. 2011 Dec 14;480(7377):324—5. doi: 10.1038/480324a; Vaccinology: "A sweet cleft in HIV‘s armour", Sattentau QJ, incorporated herein by reference), and the target antigen can comprise such a target epitope.

The present invention provides vertebrates that can artificially simulate those naturally-occurring human long HCDR3 antibodies, and can e antibody, heavy chain and le domain repertoires from which can be selected an dy, heavy chain or variable domain having a long HCDR3 (eg, having a HCDR3 length of 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30 or more amino acids ding to IMGT). The invention provides for the combination of human VH, D and J gene oires am of non-human vertebrate (eg, mouse or rat, eg, endogenous mouse or rat) constant region in heavy chain loci comprised by the vertebrate genomes. This enables the recombination, maturation and selection of the human gene segments in the context of endogenous or other non-vertebrate nt regions which enhances the development of good sized antibody, heavy chain and variable domain repertoires from which to select long type binding sites.

Thus, in an example of any configuration of the invention, the human gene segments are provided in a heavy chain locus upstream of a non-human vertebrate (eg, endogenous) constant region.

Similarly any antibody of the invention comprises human variable domains and non-human wo 2013/041846 PCT/G82012/052298 vertebrate (eg, endogenous) s. The latter can be replaced by human constant domains after selection and isolation.

For example, the following antibodies of the invention are contemplated (eg, produced in a vertebrate of this aspect of the invention by a method sed herein) or a copy or derivative of an antibody so produced:- An isolated, synthetic or recombinant antibody comprising human heavy chain variable domains having a HCDR3 length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids (according to IMGT), the heavy chain le domains being derived from the recombination of a human VH gene segment selected from a VH group sed herein with a human D gene segment and a human JH gene segment (optionally a 1H6), wherein the antibody binds a target n; n the heavy chain variable domains have non-human vertebrate AID-pattern somatic hypermutations, (eg, mouse or rat AID-pattern mutations) when compared to ponding human germline V, D and J sequences and/or non-human (eg, mouse or rat) terminal deoxynucleotidyl erase (TdT)- pattern junctional mutations when ed to corresponding human germline V, D and J sequences. In an example, the antibody of the invention has a HCDR3 length of 20, 21, 22, 23, 24, , 26, 27, 28, 29 or 30 amino acids (according to IMGT). In an example, the antigen is an antigen of a pathogen that causes or is implicated in a human infectious disease or condition, eg, a pathogen listed in Table 1. In an example, the antibody specifically binds an active site or cleft of an n (eg, an enzyme active site or receptor cleft). This can be determined, eg, using standard X-ray crystallography of a complex of the antibody (or heavy chain or VH domain) with the cognate antigen, as is known to the skilled person.

Mouse AID-pattern somatic hypermutations and/or mouse de—pattern mutations can be provided, for example, wherein VH domain is produced in a mouse sing mouse AID and/or mouse TdT (eg, nous 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 associated discussion on AID hotspots in mouse); and Curr Opin lmmunol. 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 incorporated herein by reference. Such mice can be made using ponding mouse ES cell technology.

PCT/G82012/052298 In an example, the antibody specifically binds to a HIV antigen. Several naturally-occurring human antibodies are known to be neutralising of HIV and have rather long HCDR3 lengths (20 amino acids or more according to IMGT; see Breden et al, PLoS One. 2011 Mar ):e16857; "Comparison of antibody repertoires produced by HIV-1 infection, other chronic and acute infections, and systemic autoimmune disease" (incorporated herein by reference) - VH1-69 preferred for long HCDR3). See also PLoS One. (5):e36750. Epub 2012 May 9; "Human peripheral blood antibodies with long HCDRBS are ished primarily at original recombination using a limited subset of germline genes”; Briney BS 9 a/ (incorporated herein by reference). Thus, it is desirable to provide antibodies of the ion that have similarly long HCDR3 lengths. The antibody of the invention is, in one example, provided for treating and/or ting HIV infection, eg, c HIV infection, in a human. The invention also provides a method of treating and/or ting HIV infection, eg, chronic HIV infection, in a human, the method comprising stering a pharmaceutically acceptable dose of the antibody of the invention. The dose can be split into one or more administration aliquots, eg, stered over a time course according to a medically-determined regimen, as the skilled person will be able to determine.

In an example, the antibody specifically binds to Hemophilus influenza type b polysaccharide. The antibody of the invention is, in one example, provided for treating and/or preventing Hemophilus influenza ion, eg, chronic Hemophilus influenza infection, in a human. The invention also provides a method of treating and/or preventing Hemophilus influenza infection, eg, chronic Hemophilus influenza infection, in a human, the method comprising administering a ceutically able dose of the antibody of the invention. The dose can be split into one or more administration ts, eg, administered over a time course according to a medically- determined regimen, as the skilled person will be able to determine.

In an example, the antibody specifically binds to a rotavirus antigen (eg, protein 6 or 7). The antibody of the invention is, in one example, provided for treating and/or preventing rotavirus infection, eg, c rotavirus infection, in a human. The invention also provides a method of treating and/or preventing rotavirus infection, eg, chronic rotavirus infection, in a human, the method comprising stering a pharmaceutically acceptable dose of the antibody of the invention. The dose can be split into one or more administration aliquots, eg, administered over a wo 41846 ZOIZ/052298 time course according to a medically-determined regimen, as the skilled person will be able to determine.

In an example, the antibody specifically binds to a cytomegalovirus antigen (eg, cytomegalovirus gB antigen). The antibody of the ion is, in one example, provided for treating and/or preventing cytomegalovirus infection, eg, chronic cytomegalovirus infection, in a human. The invention also provides a method of treating and/or preventing cytomegalovirus infection, eg, chronic cytomegalovirus infection, in a human, the method comprising administering a pharmaceutically acceptable dose of the antibody of the invention. The dose can be split into one or more administration aliquots, eg, administered over a time course according to a medically-determined regimen, as the skilled person will be able to determine.

The invention also provides a vertebrate or cell for expressing such an antibody; thus the invention provides a non~human vertebrate (eg, a mouse or a rat) or a non—human vertebrate cell (eg, a mouse cell or a rat cell) whose genome comprises a human immunoglobulin VH gene segment repertoire that is biased to one, more or all human VH gene segments selected from a VH group disclosed herein.

The invention also provides a method of isolating an antibody that binds 3 HIV antigen, Hemophilus influenza type b polysaccharide, cytomegalovirus antigen or rotavirus antigen, the method comprising (a) providing the human VH biased vertebrate of the ion; (b) immunising said vertebrate with said HIV antigen, ilus influenza type b polysaccharide, cytomegalovirus n or rotavirus antigen; (c) ng B lymphocytes from the vertebrate and ing one or more B lymphocytes expressing antibodies that bind to the antigen,- (d) optionally immortalising said selected B lymphocytes or progeny thereof, optionally by producing hybridomas rom; and (e) isolating an antibody (eg, and lgG-type dy) expressed by the B lymphocytes, wherein the antibody has a HCDR3 length of 20 amino acids or more. wo 2013/041846 PCT/GBZO]2/052298 Optionally, the method further comprises the step of isolating from said B lymphocytes nucleic acid encoding said dy 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.

Optionally, the method further ses making a copy, mutant or derivative (eg, humanised version) of the antibody produced by the .

This aspect of the invention also provides A pharmaceutical composition comprising the anti-HIV antibody, for treating and/or preventing HIV in a human (eg, an infant .

A pharmaceutical composition comprising the anti-Hemophi/us influenza type b polysaccharide dy, for treating and/0r preventing Haemophiius nza in a human (eg, an infant human).

A pharmaceutical composition comprising the anti-rotavirus antibody, for treating and/or ting rotavirus in a human (eg, an infant human).

A pharmaceutical composition sing the anti- cytomegalovirus antibody, for treating and/or preventing cytomegalovirus in a human (eg, an infant .

The invention also provides a method of generating such an antibody (eg, any one of embodiments (i) et seq above) by immunising a vertebrate of the ion with the target antigen and isolating the antibody from the vertebrate, optionally also making a copy or derivative of the antibody. In a further step, a B-cell capable of expressing the antibody is isolated from the vertebrate. In a further step, a nucleic acid encoding the antibody (or a VH domain thereof) is isolated from the vertebrate (eg, a nucleic acid PCR cloned from a B-cell isolated from the vertebrate). wo 2013/041846 In an e, the antibody of the invention is a neutralising antibody. In an example, the antibody of the invention has a HCDR3 length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids (according to IMGT). In an example, the antibody of the invention has a HCDR3 length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids (according to IMGT). In an example, the antibody of the invention is isolated from a non-human vertebrate (eg, a mouse or a rat), for example a vertebrate of the invention; or the antibody is a copy or derivative (eg, humanised version) thereof. In an example, the antibody of the ion has non-human vertebrate constant regions (eg, mouse or rat constant regions); these may be replaced using standard recombinant DNA technology with human constant regions, so the invention also provides for human ns of the antibodies recited above, wherein the human antibody comprises human variable and constant regions, n the variable regions bind the antigen. In an example, the antibody of the has |ambda~type human light chain variable domains. In another example, the antibody of the invention has kappa-type human light chain variable domains.

Antibody competition can be determined, for example, by ELISA or surface plasmon resonance (SPR; eg, by ition Biacore‘" or Proteon) as is standard.

The ion also provides the following embodiments (recited below as numbered clauses):- D Bias 1. A man vertebrate (eg, a mouse or a rat) or a non—human vertebrate cell (eg, a mouse cell or a rat cell) whose genome comprises a human immunoglobulin D gene segment oire that is biased to the human DZ and/or D3 family or biased to one, more or all human D gene segments selected from the group D1-26, 02-2, 03-9, D3-10, D3-22, D4-17, 06—13 and 06-19.

For example, the repertoire consists of only human D gene ts from the D2 and/or 03 family.

Optionally the repertoire is biased to one or more of human 02—2, D2-15, D3-3, 03-9, 03-10 and D3- 22, or the repertoire consists of one, more or all of these D gene ts. These produce long wo 2013/041846 PCT/GBZOIZ/052298 HCDR3 lengths (eg, see Table 2 and references cited herein).

For example, the repertoire is biased to one or more of human of DZ—Z*02, 03-9*01, 03-10*01 and D3-22*01, or the oire consists of one, more or all of these 0 gene segments.

For example, the repertoire is biased to one or more of human DZ-2*02, D3-9*01 and D3-10*Ol, or the repertoire consists of one, more or all of these D gene segments.

For e, the repertoire is biased to 1 and 03-10*01, or consists of one, more or all of these D gene segments.

Optionally the repertoire consists of one, more or all of human 01-26, 02-2, 03-9, D3-10, D3-22, D4- 17, 06-13 and 06-19. These produce long HCDR3 lengths (eg, see Table 2). ally the oire is biased to one or more of human Dl—26*01, 02-2*02, DB-9*01, D3- ‘01, 03-22*01, D4-17*01, 06—13*01 and 06-19*01, or the repertoire consists of one, more or all of these D gene segments.

Optionally the repertoire is biased to one or more of human D2-2, 03-9, D3-10, D3-22, D4-17, D6-13 and D6-19, or the repertoire ts of one, more or all of these D gene segments. Optionally the repertoire is biased to one or more of human DZ-2*02, D3-9*01, D3-10*01, D3-22*01, D4-17*Ol, D6-13*01 and D6-19*01, or the repertoire consists of one, more or all of these D gene segments.

These produce long HCDR3 lengths in naive repertoires (eg, see Table 2). ally the repertoire is biased to one or more of human D1-26, 02-2, 03-10 and 06-19, or the repertoire ts of one, more or all of these D gene segments. Optionally the oire is biased to one or more of human D1-26*01, D2-2*02, 03-10*01 and D6-19‘01, or the repertoire consists of one, more or all of these D gene segments. These produce long HCDR3 lengths in immunised repertoires (eg, see Table 2).

Optionally the repertoire is biased to one or more of human 02—2, 039 and 03—10, or the repertoire consists of one, more or all of these D gene segments. Optionally the repertoire is biased to one or more of human DZ—2*02, D3-9‘01 and 03-10*Ol, or the repertoire ts of one, more or all of these D gene segments. These produce long HCDR3 lengths in antigen-specific repertoires (eg, see wo 41846 PCT/082012/052298 Table 2).

IMGT nomenclature is used for all gene segments.

Throughout this text, Gen bank is a reference to Genbank release number 185.0 or 191.0; the 1000 Genomes database is Phase 1, release v3, 16“1 March 2012; the Ensembl database is assembly GRCh37.p8 (10/04/2012); the IMGT database is available at www.imgt.org. The sequences of all VH gene segments explicitly mentioned herein are disclosed herein in their entirety (for possible inclusion in clauses in conjunction with any aspect of the invention as clauseed), such sequences being those in the IMGT and 1000 Genomes databases.

In one embodiment, the genome comprises an lgH locus sing a targeted insertion of said human 0 gene segments. In an example, the lgH locus comprises (in 5’ to 3’ order) one or more human VH gene segments, said D gene segment repertoire, one or more human JH gene segments and a constant region (eg, wherein the constant region is a human constant region or a non-human (eg, endogenous, eg, m0use or rat) constant region).

In another embodiment, the genome comprises said human D gene segments randomly inserted therein. This can be ed, eg, by incorporating human DNA borne by YACS into the genome of ES cells wed optionally by generation of a non-human vertebrate rom, as is standard).

Optionally, the human D gene segment oire further ses no more than 5 additional human D gene segments, for example, the repertoire es 1, 2, 3, 4 or 5 additional human D gene segments. 2. The vertebrate or cell of clause 1, wherein the D gene segment repertoire consists of or substantially consists of one, two or three human gene segments selected from the group 01-26, 02-2, D3-9, D3-10, 03-22, 04-17, 06-13 and D619. 3. The vertebrate or cell of clause 1 or 2, wherein the genome comprises an ranged globulin heavy chain locus comprising (in 5' to 3’ order) human VH, D and JH gene segments and said human 0 gene segments recited in clause 1 are spaced from the VH gene segment(s) by no more than four other D gene segments (eg, by no D gene segments). wo 41846 PCT/082012/052298 This provides for bias wherein proximal D gene segments (those more 3’, ie, closer to the constant region) are likely to be more frequently used than those segments from distal (ie, 5' or r away from the constant region).

The vertebrate or cell of any preceding clause, n the genome comprises an unrearranged immunoglobulin heavy chain locus sing said human D gene segments and there are no other D gene segments in the locus between said human D gene segments.

This is another way of biasing the repertoire of D gene segments. Thus, the desired D5 are provided in , aimed to promote use in recombination.

The vertebrate or cell of any preceding clause, wherein the genome comprises three or more copies of a human D gene segment selected from D1—26, 02—2, 02-15, 03-3, 03-9, 03-10, D3-22, D4—17, D6—13 and D6—19.

For example, the genome comprises three or more copies of a human D gene segment selected from 01-26, 02—2, D3—9, D3-10, D3-22, D4-17, 06-13 and D6-l9.

This is another way of biasing the repertoire of D gene segments.

The vertebrate or cell of clause 5, wherein the genome comprises first and second human D gene segments selected from 01-26, D2-2, 02-15, 03-3, 03-9, D3-10, DEB-22, 04-17, 06-13 and 06-19 when the first D gene segment is present as three or more copies and wherein the second D gene segment is present as three or more copies.

For example, the first and second gene segments are selected from 01-26, 02—2, D3—9, D3-10, D3-22, D4—17, D6—13 and D6—19.

The various gene segment biasing techniques described herein can be performed using conventional DNA manipulation in the construction of transgenic vertebrates or cells of the invention, which techniques (eg, recombineering and recombinant DNA technology) will be known to the skilled . For e, BACs can be constructed using these techniques in wo 2013/041846 PCT/G32012/052298 which the desired combination of human gene segments is provided, and these BACs can be introduced into ES cells for incorporation of the human gene segments into the genomes thereof (eg, by ed insertion into lg loci). The ES cells can be used to generate transgenic vertebrates as is standard and cells (eg, B-cells) can be isolated from these wherein the genome is as per the invention.

In one ment, the biased D gene segment(s) are selected from the IMGT database of variants or the 1000 Genomes database. 7. The rate or cell of any preceding clause, wherein the D gene segments are selected from D2—2*02, D3-9*01, 01 and D3-22*01, or selected from Dl—26*01, 02-2*02, 1, D3- *01, 03-22‘01, D4-17*01, 01 and D6-19‘01.

VH Bias 8. A non-human vertebrate (eg, a mouse or a rat) or a non-human vertebrate cell (eg, a mouse cell or a rat cell), optionally according to any preceding clause, whose genome comprises a human immunoglobulin VH gene segment repertoire that is biased to one, more or all of gene segments selected from the group VH1-2, VH 1-3, VH1-8, , VHS-51, VH1-69, VHZ—S, VH3—7, VH3-9, VH3-11, VHS-13, VH3-20, VH3-21, VH3-23, VH4-4, VH6-1 and VH7-4~1.

These produce long HCDR3 lengths (see Table 2 and references cited herein).

For example, the VH repertoire is biased to one, more or all of VH1—2, VH1—3, VH1-8, VH1—18, VH2—5, VH3-7, VH3—9, VH3—1l, , VH3—20, VH3—21, VH3~23, VH4-4, VH6-1 and VH71.

These produce long HCDR3 lengths (see Table 2), or the oire consists of one, more or all of these VH gene segments. For example, the VH repertoire is biased to one, more or all of VH1- 2*02, VH1-3‘01,VH1—8*01, VH1—18‘01,VH2—5*10, VH3-7‘01, VH3-9‘01,VH3-11*01,VH3- 13*01, *03, VH3-23‘04, VH4-4*02, VH6-1‘01 and VH71*01, or the repertoire consists of one, more or all of these VH gene segments. wo 41846 PCT/G82012/052298 For example, the VH repertoire is biased to one, more or all of VH1—2*02, VH1—8*01, VH1-18‘01, VH 1-3*01, VH2-5*10, VH3-7*Ol, VH3-9‘01, VH3~13*01, VH3—21‘03, VH3-23‘O4, VH4A4*02, VH6-1*01 and VH71*01, or the repertoire consists of one, more or all of these VH gene segments. These e long HCDR3 lengths in naive repertoires (see Table 2).

For example, the VH repertoire is biased to one, more or all of VH4-4*02, ‘01 and VH3- 7‘01, or the repertoire consists of one, more or all of these VH gene segments. These produce long HCDR3 lengths in immunised repertoires (see Table 2).

For example, the VH repertoire is biased to one, more or all of VH1-3*01, VHl-8*01,VH3-7*01, 01,VH3-11*01 and VH4-4*02, or the repertoire consists of one, more or all of these VH gene ts. These e long HCDR3 lengths in antigen—specific repertoires (see Table 2); Optionally, the human VH gene segment repertoire further comprises no more than 5 onal human VH gene segments, for example, the repertoire includes 1, 2,3, 4 or 5 additional human VH gene segments.

In one embodiment, the genome comprises an lgH locus comprising a targeted insertion of said human VH gene segments. In an e, the lgH locus comprises (in 5’ to 3‘ order) said VH gene segment repertoire, one or more human D gene segments, one or more human JH gene segments and a constant region (eg, wherein the constant region is a human nt region or a non-human (eg, endogenous, eg, mouse or rat) constant region).

In another embodiment, the genome comprises said human VH gene segments randomly inserted therein. This can be effected, eg, by incorporating human DNA borne by YACS into the genome of ES cells (followed optionally by generation of a nonwhuman vertebrate therefrom, as is standard). 9. The vertebrate or cell of clause 8, wherein the VH gene segment repertoire substantially consists of or substantially consists of one, two or three human gene segments selected from VH1-2, VH1-3, VH 1-8, VH1-18, , VH1—69, VH2-5, VHS—7, VH3-9, VH3-11, VH3-13, VH3-20, VH3—21, VH3-23, VH4-4, VH6-l and VH71. wo 2013/041846 PCT/032012/052298 For example, the VH gene segment repertoire substantially consists of or substantially consists of one, two or three human gene segments selected from the group consisting of VH1-2*02, VH1-3*01, 01,VH1~18*01,VH2~5*10,VH3—7*01,VH3—9*01,VH3—11*01,VH3-13*01, VH3-21‘03, ‘04, 02, VHS-1‘01 and VH71*01.

. The vertebrate or cell of clause 8 or 9, wherein the genome comprises an unrearranged immunoglobulin heavy chain locus comprising (in 5' to 3' order) human VH, D and JH gene segments and said human VH gene segments are spaced from the D gene t(s) by no more than four other VH gene segments (eg, by no VH gene segments).

This provides for bias wherein proximal VH gene segments (those more 3', ie, closer to the constant region) are likely to be more frequently used than those segments from distal (ie, 5' or further away from the constant region). 11. The vertebrate or cell of any one of clauses 8 to 10, wherein the genome comprises an unrearranged immunoglobulin heavy chain locus comprising said human VH gene segments and there are no other VH gene segments in the locus between said human VH gene segments.

This is another way of g the oire of VH gene segments. 12. The vertebrate or cell of any one of clauses 8 to 11, wherein the genome comprises three or more copies of a human VH gene segment selected from the group consisting of VH1-2, VH1-3, VH1-8, VH1-18, VHS—51, VH1-69, VH2~5, VHS—7, VH3-9, VH3-11, VH3-13, VHS-20, VHS—21, VH3- 23, VH4-4, VH6-1 and VH7-4—1.

For example, the genome comprises three or more copies of a human VH gene segment selected from the group consisting of 02, VH1—3*01, VH1-8*01, VH1-18*01, VH2-5*10, VH3—7*01, VH3-9*01, *01, VH3-13*01, VH3-21*03, *04, VH4-4*02, VH6-1*01 and VH7—4— 1*01.

This is another way of biasing the repertoire of VH gene segments. wo 2013/041846 PCT/G32012/052298 13, The vertebrate or cell of clause 12, wherein the genome comprises first and second human VH gene segments selected from the group consisting of VH1-2, VH 1-3, VH1-8, VH1-18, VHS-51, VH1—69, VH2—5, VH3-7, VHS-9, VH3-11, VH3-13, VH3-20, VH3-21, VH3-23, VH4-4, VHS-1 and VH71 when the first VH gene segment is present as three or more copies and wherein the second VH gene t is present as three or more copies.

For example, the genome comprises first and second human VH gene segments selected from the group consisting of VHl-2*02, VH1-3*01, VH1-8*01, VH1-18*01, VH2-5*10, 01, VH3— 9*01, VH3-11*01, VH3-13‘01, VH3-21*03, VH3-23*04, VH4-4*02, VH6-1*01 and VH7—4-1*01 when the first VH gene t is present as three or more copies and wherein the second VH gene segment is present as three or more copies.

In an embodiment, all or substantially all of VH gene segments are present as three or more copies each.

The various gene segment biasing techniques described herein can be performed using conventional DNA manipulation in the construction of transgenic rates or cells of the invention, which techniques (eg, recombineering and inant DNA logy) will be known to the skilled person. For example, BACs can be constructed using these techniques in which the desired combination of human gene segments is provided, and these BACs can be uced into ES cells for oration of the human gene segments into the genomes thereof (eg, by targeted insertion into lg loci). The ES cells can be used to generate transgenic vertebrates as is standard and cells (eg, B-cells) can be isolated from these wherein the genome is as per the invention.

In one embodiment, the biased D gene segment(s) are selected from the IMGT database of variants or the 1000 Genomes se. 14. The vertebrate or cell of any one of clauses 8 to 13, n the VH gene segments are selected from the group consisting of VH1-2*02, VH1-3‘01, VH1-8*01,VH1—18*01,VH2-5*10,VH3-7‘01, VHS-9‘01, *01, VH3-13*01, VH3-21*03, VH3—23*04, VH4-4*02, VH6-1*01 and VH7 1*01. wo 2013/041846 PCT/GBzoiz/05229s In an embodiment, the genome comprises a human immunoglobulin VH gene segment repertoire that is biased to VH1-69.

In an embodiment, the human immunoglobulin VH gene segment repertoire substantially consists of one or more human VH1-69 gene segments.

The gene segments are provided in one or more immunoglobulin loci. For example, the gene segment repertoire (D and/0r VH) is provided in both lgH loci (ie, in a homozygous state).

. The rate or cell of any one of clauses 8 to 16, comprising an immunoglobulin heavy chain locus comprising two or more copies of a VH gene segment selected from said group.

Thus, at least one of said copies is closer to the constant region of the locus than the germline distance in a human from a human nt region. The aim is to e by bias by providing more than one copy on the same locus. Also as at least one of the copies is closer (more proximal to) the constant region and J—C intron (which includes regulatory elements such as the Emu enhancer ), this may favour use of the gene segment, thus contributing to the desired bias.

Optionally, the genome is homozygous for the heavy chain locus.

Optionally the two or more copies of gene segments are identical (eg, all VH1-69‘01, using IMGT lature). In another example, copies are variants of each other, eg, naturally-occuring human variants. Alterntatively, synthetic variants may be used with or without a naturally— occuring variant.

In any embodiment of the invention, the vertebrate is naive or sed with a target antigen. 16. The vertebrate or cell of any clause, wherein the genome comprises a human JH gene segment repertoire consisting of one or more human JH6 gene ts.

This biases the JH repertoire for the production of long HCDR3, since this is the longest naturally- occurring human JH gene segment type and is commonly found in naturally-occurring human wo 2013/041846 PCT/G82012/052298 antibodies having long HCDR3.

For example, the repertoire comprises two or more different JH6 variants. In an example, the repertoire comprises two or more JH6*02 variants (IMGT nomenclature). 17. The vertebrate or cell of any preceding clause, wherein the genome comprises a human immunoglobulin JH gene segment repertoire that is biased to JH6, optionally JH6*02. 18. The vertebrate or cell of clause 17, wherein the JH gene segment repertoire ts or substantially consists of three or more human JH6 gene segments. 19. The vertebrate or cell of any ing clause, wherein the sequence of each of said human gene segments is a human germline gene t sequence.

. The vertebrate or cell of any preceding clause, wherein one, more or all of the selected gene segments are t in the genome as two or more copies, the copies being variants of each other.

Thus, one, more or all of the human V, D and JH gene segments of said genome is present in two or more variant versions, such as naturally—occurring human variants, eg, ts found in the 1000 Genomes database and/or IMGT database. In another example, one or more of the variants may be a synthetic variant. 21. The vertebrate or cell of any preceding , wherein said human gene ts are provided by homozygous immunoglobulin heavy chain loci.

In an example, no other (non-human) active heavy chain VH, D or JH gene segments are present in heavy chain loci of the genome. Additionally, in an example no active non-human light chain VL or JL gene segments are present in the genome.

This is useful for ensuring that endogenous (non-human) variable region expression is wo 2013/041846 PCT/GBZOIZ/052298 inactivated. Thus, all heavy chains produced by the vertebrate or cell will have human variable regions, which is useful for producing drugs for administration to humans. 22. A non-human vertebrate (eg, a mouse or a rat) or a non-human vertebrate cell (eg, a mouse cell or a rat cell) whose genome comprises a human immunoglobulin VH gene segment oire, one or more human D gene segments and one or more human JH gene segments, wherein the VH repertoire does not se one, more or all VH gene segments selected from the group VH1~2, VH1e3, VH1-8, VH1-18, VHS-51, VH 1-69, VHZ-S, VH3-7, VH3-9, VH3-11, VH3-13, VH3—20, VH3-21, VH3-23, VH4-4, VH6-1 and VH71. 23. A non-human vertebrate (eg, a mouse or a rat) or a non-human vertebrate cell (eg, a mouse cell or a rat cell) whose genome comprises a human immunoglobulin D gene segment oire, one or more human VH gene segments and one or more human JH gene segments, wherein the D repertoire does not comprise one, more or all D gene ts selected from the group D1~ 26, D2-2, 03-9, D3-10, D3-22, 04-17, 06-13 and D6-19.

In instances it has been ed in that the art that certain human gene usage may te the immune response to infectious disease pathogen antigens or other antigens. While this may yield many specific antibodies, typically these may not be neutralising and thus the immune se is relatively ineffective. This may happen, for example, where the antigen is a decoy antigen expressed by the pathogen. The present embodiments of the invention where specific gene segments are omitted are useful for avoiding dominance of certain human gene ts, such as those omitted from the genome In this way, the genome human gene segment repertoire is biased away from the dominance and this enables better use and sampling of the remaining human gene segment sequence space, thereby providing the chance of producing antibodies that may not be normally raised in a natural setting. n specific dies can be selected from vertebrates and cells with such genomes. In some examples, this may yield neutralising antibodies.

It is advantageous to include a plurality of different human VH gene segments, making up the human VH gene segment oire. This provides for good diversities of rearranged human variable regions from which to select leads. It is le, for example, to include an otherwise complete, functional repertoire of human VH gene segments. To this end, the human VH gene wo 2013/041846 PCT/G82012/052298 segment repertoire comprises, in one example, a plurality of human VH gene segments, eg, at least 7, 10, 15, 20, 15, 30, 35, 40 or 45 different human VH gene segments. This can be achieved, for example, using BACs harbouring stretches of unrearranged human variable region DNA comprising Vi-l gene segments - gous recombination and/or sRMCE being used to insert several stretches of such DNA from serial BACs into an endogenous heavy chain locus upstream of the constant region thereof in the genome of a non-human vertebrate ES cell (eg, mouse or rat ES cell), followed by development of one or more progeny vertebrates from such cells (and optional breeding to homozygosity of the heavy chain . In one ment, human DNA is inserted that es a first human VH (eg, VH1-69 and/or VH1-2) and flanking VH gene segments upstream and downstream of these. In a second ES cell genomic manipulation, the first VH is deleted from the genome, eg, using standard homolgous recombination techniques as is known in the art. In this way, one or more VH gene segments usually upstream and/or downstream of the deleted gene segment(s) in a ype human germline genome are retained so that they can be available to contribute to the subsequent rearranged human V region oire that is used for selection of leads. In another example, the l insertion of human DNA is made using stretches of DNA that already omit the first VH (eg, by deleting such hes using ineering of BACs in E coli, as is known in the art).

Similar techniques can be used (with appropriate BACs) for the omission of human D and/or] gene segments.

Thus, in an ment, VH gene segments that normally are upstream and/or downstream of the omitted human VH gene ts (or omitted D orJ as per other embodiments) in a wild-type germline human genome are included in the vertebrate or cell of the invention. For example, the human VH gene segment repertoire of the genome in the invention does not comprise VH1-69, but does comprise one, two, three or four human VH gene segments selected from VH2—10,VH3-72, VH3-73 and VH3-74. These are gene segments that are immediately upstream of VH 1-69 in a wild- type human germline heavy chain locus (see lMGT). For example, additionally or alternatively the human VH gene segment repertoire of the genome in the ion does not comprise VH1-69, but does comprise one, two, three, four or more (or all of) human VH gene segments selected from VH3-66, VH3-64, VH4-61, VH4—59, VH1-58, VH3-53, VH3-49, VH3-48, VH1-46 and VH1-45. These are gene segments that are immediately downstream of VH1-69 in a ype human germline heavy chain locus (see IMGT). Additionally or alternatively, the human VH gene segment repertoire of the genome in the invention does not comprise , but does comprise one, two, three four or wo 2013/041846 PCT/GBZOIZ/052298 more (or all of) human VH gene segments selected from VH2-5, 7-41, 4-4, 1—3, 1-2 and 6-1.

Additionally or alternatively, the human VH gene segment oire of the genome in the invention does not comprise VH1-69, but does comprise one, two, three, four, 5, 6, 7, 8, 9, 10 or more (or all of) human VH gene segments selected from VH2-S, 7-41, 4-4, 1—3, 1~2, 6-1, 3-7, 1-8, 3-9, 3-11 and 3- 13. Additionally or alternatively, the human VH gene segment repertoire of the genome in the invention does not comprise , but does comprise one, two, three, four, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more (or all of) human VH gene segments selected from VH2-5, 7-41, 4-4, 1- 3, 1—2, 6-1, 3-7, 1-8, 3-9, 3-11, 3-13, 3-15, 1-18, 3-20, 3-21, 3-23, 1-24 and 2-26. Additionally or atively, the human VH gene segment repertoire of the genome in the ion does not comprise VH1-69, but does comprise VH6-1 (which is commonly used in human immune responses, VH6-1 being the most proximal to the constant region in a wild-type human germline heavy chain locus) and/or VH3-23 (which is commonly used in human immune responses). In ment (eg, for generating VH, heavy chains or antibodies for ng and/or preventing an infectious disease, eg, HIV infection, in a human), VH1-2 is omitted in the genome or locus. In this case one, two, three or all human VH gene segments immediately 5’ and 3’ of VH1-2 in a wild-type germline human IgH locus (eg, see IMGT) are included in the genome, such as comprised by the same IgH locus upstream of human D and JH gene segments and a constant region. 24. The vertebrate or cell of clause 22 or 23, wherein the genome comprises a human JH gene segment repertoire that does not compriseJHG.

JH Bias . A non-human vertebrate (eg, a mouse or a rat) or a non-human vertebrate cell (eg, a mouse cell or a rat cell) whose genome comprises a human immunoglobulin JH gene segment repertoire that is biased to human JH6.

In an example, the repertoire is biased to human JH6*02 (IMGT nomenclature).

So, the inventors made a choice of human JH6*02 on the basis of (i) Containing YYG and YYGXDX motifs that is conserved across several vertebrate species; (ii) Provision of one less TAC codon than other human JH6 variant (an AID hotspot that risks stop codons) and a choice instead of a codon that preserves the YYG and YYGXDX motifs; (iii) Avoidance of a GGCA AID hotspot in the region of the HCDR3/FW4 junction; and wo 41846 PCT/G82012/052298 (iv) Common ence (and thus conservation and ability) in humans of the JH6*02 variant. 26. The vertebrate or cell of clause 25, wherein the genome comprises an unrearranged immunoglobulin heavy chain locus comprising a plurality of human JH6 gene segments; optionally wherein the genome is homozygous for said locus.

In an example, the plurality comprises or consists of a plurality of JH6*02 gene segments. 27. The vertebrate or cell of clause 26, wherein the heavy chain locus ses (in 5’ to 3’ order) human VH, D and JH gene segments and said JH6 gene segments are spaced from the D gene segmentls) by no more than two otherJH gene segments. 28. The vertebrate or cell of clause 25,26 or 27, wherein are no other JH gene segments in the locus between said human JH6 gene ts. 29. A non—human vertebrate (eg, a mouse or a rat) or a non-human vertebrate cell (eg, a mouse cell or a rat cell) whose genome comprises a human immunoglobulin JH gene segment repertoire that consists of one or more human JH6 gene segments.

In an example, all of the gene segments are JH6*02 gene segments.

. The vertebrate or cell of any one of clauses 25 to 29, wherein all of said gene segments are human germline gene segments. 31. The vertebrate or cell of any one of clauses 25 to 30, comprising different t JH6 gene segments.

In an example, the variants are all naturally—occurring (eg, appearing in the IMGT or 1000 Genome databases). In an other example, one or more variant is synthetic. wo 2013/041846 2012/052298 32. The vertebrate or cell of any one of clauses 25 to 31, wherein said gene segments are provided by homozygous immunoglobulin heavy chain loci.

In one embodiment, the biased JH gene segment(s) are selected from the IMGT database of variants or the 1000 Genomes se. 33. A monoclonal or polyclonal antibody composition or a population of dy-producing cells for ing such composition, wherein the composition or population is prepared by immunising at least one vertebrate according to any preceding clause with an antigen, wherein the antibody or antibodies have human heavy chain variable regions comprising non-human vertebrate AID- n somatic hypermutations, (eg, mouse or rat ttern mutations) when compared to corresponding human germline V, D and] sequences and/or non-human (eg, mouse or rat) terminal deoxynucleotidyl transferase (TdT)- pattern onal mutations when compared to corresponding human germline V, D and J sequences; wherein the ition comprises at least one antigen-specific antibody having a HCDR3 length of at least 20 amino acids (according to IMGT).

As will be readily apparent to the skilled person, A10 and TdT mutations can be determined using bioinformatics analysis to find the closest matching human germline gene segment(s) that correspond to a given variable domain sequence, aligning the sequences and determining the differences. AID has known hotspots for mutation (eg, see Annu. Rev. Biochem. 2007. 76:1~22; Javier M. Di Noia and Michael S. Neuberger, ular Mechanisms of Antibody Somatic Hypermutation" (in particular figure 1 and associated discussion on AID hotspots in mouse); and Curr Opin l. 1995 Apr;7(2):248-54, "Somatic utation”, Neuberger MS and Milstein C (in particular, discussion on hotspots in mouse), the disclosures of which are incorporated herein by reference). By carrying out the rd bioinformatics analysis, TdT mutations leg, to provide junctional muatations and diversity) can be determined, as will be familiar to the skilled person. ponding human germline V, D and J sequences can be according to the IMGT database or 1000 Genomes database, for example.

W0 2013/0418ch For example, the HCDR3 length is at least 20, 21, 22, 23, 24, 25, 26, 27,28, 29 or 30 amino acids.

For example, the HCDR3 length is from 20 to 23 or 24 to 30, eg, from 28 to 30 amino acids.

For example, the cells are B cells (eg, immortalised B cells) or hybridomas.

Optionally the antibodies of any aspect of the invention comprise human light chain variable regions. For example, the human light chain variable regions have non-human vertebrate AID- pattern c utations, (eg, mouse or rat AID-pattern mutations) when compared to corresponding human germline V, D and J sequences and/or non-human (eg, mouse or rat) terminal deoxynucleotidyl transferase (TdT)- pattern onal mutations when ed to corresponding human ne V, D and J sequences. 34 An isolated antibody that specifically binds an n, the antibody comprising human heavy chain variable regions and non-human constant regions, wherein the variable regions are derived from the ination in a non—human vertebrate of (i) a human VH gene segment selected from the group recited in clause 8 with (ii) a human D gene segment selected from the group recited in clause 1 and with a human JH gene segment (optionally 1H6),- wherein the antibody has a HCDR3 length of at least 20 amino acids ding to IMGT); and non—human vertebrate AID-pattern somatic hypermutations, (eg, mouse or rat AID-pattern mutations) when compared to corresponding human germline V, D and J sequences and/or non-human (eg, mouse or rat) terminal deoxynucleotidyl transferase (TdT)- pattern junctional ons when compared to corresponding human germline V, D and] sequences.

In examples, the VH is selected from the group VH1-2*02, VH1-3*01, VH1—8*01, VH1—18*01, VH2-S*10,VH3-7*01, VH3-9*01, VH3—11*01, VH3—13*01,VH3*21*03, VH3-23‘04, 02, VH6-1*01 and VH71*01 and/or the D is selected from the group DZ—Z‘OZ, 03-9*01, D3—10*Ol and D3-22*01, or D2-2*02, 03-9*01 and D3-10*01, or D3-9*Ol and D3-10*01, or 01-26, D2-2, 03-9, D3-10, 03-22, 04-17, 06-13 and D6-19, or Dl-26*01, D2-2‘02, DB-9*01, 01, 01, D4-17*01, D6-13*01 and D6-19*01, or 02-2, 03-9, D3-10, 03-22, 04-17, D6-13 and 06-19, or DZ-Z*02, DS-9*01, D3-10*01, DB-ZZ‘OI, D4-17*01, D6-13*01 and D6-19*01, or D1-26, 02-2, D3~10 and 06—19, or 02-2, 03-9 and 03—10.

. The antibody of clause 34, n the antibody is obtained or obtainable from a vertebrate according to any one of clauses 1 to 32.

In an embodiment, the antibody is obtained from said rate, or is a copy of such an antibody. 36. A method of isolating an antibody that binds a ermined antigen, the method comprising (a) providing a vertebrate (optionally a mouse or rat) according to any one of clauses 1 to 32; (b) immunising said vertebrate with said antigen; (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 lymphocytes or progeny thereof, optionally by producing hybridomas therefrom; and (e) isolating an antibody (eg, and lgG-type antibody) expressed by the B lymphocytes. 37. The method of clause 36, wherein in step (e) wherein the antibody has a HCDR3 length of at least 20 amino acids ding to IMGT).

The length can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids (according to IMGT), eg, from 20 to 23 amino acids (a produced in the examples). 38. The method of clause 36 or 37, comprising the step of isolating from said B lymphocytes nucleic acid encoding said dy that binds said n; optionally ging the heavy chain constant region nucleotide ce of the antibody with a nucleotide sequence encoding a human or humanised heavy chain constant region and optionally affinity ng the variable region of said antibody; and optionally inserting said nucleic acid into an expression vector and optionally a host. wo 2013/041846 PCT/GBZOIZ/052298 39. The method of clause 36, 37 or 38, further comprising making a copy, mutant or derivative (eg, humanised version) of the antibody ed by the method.

Humanisation can entail making the nt regions human. 40. The antibody composition, cell tion, antibody or method of any one of clauses 33 to 39, wherein the antigen is an antigen of an infectious disease pathogen; optionally wherein the pathogen is a virus or bacterium. 41. The antibody composition, cell population, antibody or method of clause 40, wherein pathogen is selected from the group consisting of Haemophilus nza, E coli, Neisseria meningitidis, a herpes family virus, cytomegalovirus (CMV), HIV and influenza virus. 42. The dy composition, cell population, dy or method of any one of clauses 33 to 41, wherein the n is a HIV gp120 antigen or a HIV gp41 n. 43. The antibody composition, cell population, antibody or method of any one of clauses 33 to 40, wherein the antigen comprises an active site or cleft, wherein the antibody having a HCDR3 length of at least 20 amino acids specifically binds to the active site or cleft of the antigen. 44. A pharmaceutical composition comprising an antibody or antibody composition according to any one of clauses 33 to 35 and 40 to 43, or an antibody produced by the method of any one of clauses 36 to 38, for treating and/or preventing an infectious disease in a human (eg, wherein the infectiOus disease is caused by a pathogen selected from the group consisting of Haemophilus influenza, E coli, Neisseria meningitidis, a herpes family virus, cytomegalovirus (CMV), HIV and influenza virus). 45. A repertoire of dy heavy chains (eg, provided by antibodies) comprising one or more heavy chains whose variable domain HCDR3 has a iength of at least 20 amino acids (according to IMGT) and derived from the recombination of a human VH, D and JH, wherein wo 2013/041846 PCT/G82012/052298 the VH is selected from the group VH1-2*02, VH1-3*Ol, VH1-8*01, VH1-18*01, VH2-5*10,VH3-7*01, VHS-9‘01, VH3-11*01, VH3— l3*01, VH3-21*03, VH3-23 *04, VH4-4’l‘02, VH6-1*Ol and VH71*01 and the D is selected from the group 02-2*02, D3-9*01, D3-10*01 and DS-ZZ‘Ol, or D2-2*02, D3-9*01 and D3-10*01, or 1 and 03—10*01, or D1-26, 02-2, 03-9, 03-10, 03-22, 04-17, D6-13 and 06-19, or 01-26*01, DZ-Z*02, 1, D3-10‘01, D3-22*01, D4-17*01, DG~13*01 and 06-19*01, or DZ-Z, D3-9, D3-10, D3-22, 04-17, 06-13 and 06-19, or 2, DS-9*01, 03-10*01, 03-22*Ol, D4-17‘01,D6-13*01 and 06-19*01, or D1-26, D2-2, 03-10 and 06—19, or DZ-Z, D3-9 and D3~10; and optionally the JH is JH6 (eg, JH6*02); Wherein (a) the heavy chain variable domain has been produced in vivo in a non-human vertebrate (eg, a mouse or a rat); and/or (b) the heavy chain variable domain comprises non-human vertebrate AID-pattern somatic hypermutations, (eg, mouse or rat AID-pattern mutations) when compared to corresponding human germline V, D and J sequences and/or non-human (eg, mouse or rat) terminal deoxynucleotidyl transferase (TdT)- pattern onal mutations when compared to corresponding human germline V, D and] sequences.

In an example, the heavy chain (or all heavy chains in the repertoire) comprise non—human vertebrate constant s (eg, mouse or rat constant regions). For example, the constant regions are gamma-type constant regions (eg, 1, gamma-2 or gamma-4 type).

In an example, the oire is a naive repertoire. This is shown in the examples section .

In an example, the repertoire is an immunised oire. This is shown in the examples n herein.

PCT/G82012/052298 In an example, the oire is an antigen-specific repertoire (eg, provided by a plurality of hybridomas). This is shown in the examples section herein.

The repertoire can be ed by B cells (eg, immortalised B cells).

The repertoire can be provided by hybridomas.

In an example, the vectors are harboured by host cells (eg, CHO or HEK293 cells or yeast cells).

The HCDR3 length can be 20, 21, 22, 23, 24, 25, 26, 27, 28,29 or 30 amino acids (according to IMGT), eg, from 20 to 23 amino acids (a produced in the examples).

In an example, in (a) the vertebrate is a vertebrate according to the invention. 46. A nucleic acid collection encoding the heavy chain repertoire of clause 45.

In an example, the nucleic acids are provided in respective s (eg, expression vectors, eg, E coli or CHO or HEK293 vectors). 47. A method of ing an antigen—specific heavy chain (eg, ed by an antibody), the method comprising exposing the oire of clause 45 to a predetermined antigen and selecting one or more heavy chains that specifically bind to the antigen, wherein one or more heavy chains is isolated that has a HCDR3 length of at least 20 amino acids.

Optionally, when the heavy chain has a non-human constant region, this is swapped for a human constant region, as is conventional in the art. Thus, the invention provides a human antibody heavy chain so produced (eg, provided in combination with a human light chain to produce a human antibody which is useful for human therapeutic and/or prophylactic use, eg, to treat and/or t an infectious disease in a human patient).

In an example of the vertebrate or cell of any aspect of the invention, the genome comprises an immunoglobulin light chain locus sing one or more human V gene segments and one or more wo 2013/041846 PCT/G32012/052298 human J gene segments upstream of a constant region (eg, a human or a mouse lambda or kappa constant region) For rearrangement and expression of heavy chains, the locus comprises control ts, such as an Ep and Sp between theJ gene segment(s) and the constant region as is known by the skilled person. in one example, a mouse Ep and Sp is included in the heavy chain locus between the JH repertoire and the constant region (ie, in 5' to 3' order the locus comprises the JH gene segment(s), Ep and Sp and constant region). In an example, the Eu and Sp are Ep and Sp of a mouse 129-derived genome (eg, a 129$v-derived genome, eg, 129$v/EV (such as 129S7Sv/Ev (such as from ABZ.1 or A822 cells obtainable from Baylor College of Medicine, Texas, USA) or 129$6$v/Ev))); in r example, the Ep and Sp are Eu and Sp of a mouse 6 —derived genome. In this respect, the locus can be constructed in the IgH locus of the genome of a cell selected from ABZ.1, A822, J7 and FH 14.

VGFl cells were established and described in Auerbach W, e JH, Fairchild-Huntress V, et 0/; Establishment and chimera analysis of 129/Sva- and C57BL/6-derived mouse embryonic stem cell lines. Biotechniques 2000; 29:1024—8, 30, 32, incorporated herein by reference. onally or alternatively, the nt region (or at least a Cu; or Cp and gamma constant s thereof) is a constant region (or Cp; or Cp and gamma constant regions thereof) is of a genome described in the paragraph immediately above.

A suitable source of human DNA sequences or gene segments will be readily apparent to the d person. For example, it is possible to collect 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 person. Alternatively, the skilled person is able to construct gene segment sequence by ing to one or more databases of human lg gene segment sequences disclosed herein.

In an example, the genome comprises all or some of the following human VH gene segments PCT/G82012/052298 lGHV6-l lGHV3-7 IGHVI—S IGHV3—9 IGHV3-11 IGHV3-13 IGHV1-18 IGHV3-30 lGHV4-39 lGHV4-59 Optionally also (i) and/or (ii) lGHVlQ lGHV2-5 and IGHV3-21 lGHVlQ IGHV2-5 lGHV3-21 PCT/GB2012I052298 IGHV1-24 For example, the genome comprises all or some of the following human VH gene segment variants |GHV6-1"‘01 |GHV3-7*01 |GHV1-8*01 |GHV3-9*01 11*01 |GHV3-l3*01 |GHV1-18*01 |GHV3—30*18 lGHV4-31*03 |GHV4-39*01 and |GHV4-59*01; Optionally also (iii) or (iv) (iil lGHVl-2*04 *1O and lGHV3-21*03 wo 2013/041846 PCT/GBZO]2/052298 liV) lGHVl-2*02 IGHV2-5*01 |GHV3-21*01 and lGHVl—24*01 For example, the genome comprises all or some of the following human JH gene segment variants IGHJ2*01 IGHJ3*02 IGHJ4*02 IGHJS*02 and For example, the genome comprises all or some of the following human D gene segments lGHDl—l IGHDZ-Z IGHDB—IO lGHDS-lZ lGH06-13 lGHDl-l4 wo 2013/041846 PCT/GBZOIz/052298 IGHDZ-lS lGHD3—16 IGHD4-17 IGHD6-19 IGHDZ-Zl IGHDS-24 IGHD1-26 and and optionaily also (v) or (vi) lGHDS-S (vi) IGHD3-3 IGHD4-4 IGHDS-S IGHD6—6 IGHDl-7 IGHD2-8 and IGHDZ-8 wo 2013/041846 It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments t 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, us equivalents to the specific procedures described herein. Such equivalents 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 ation or patent application was specifically and individually ted 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 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 definition that refers to only alternatives and "and/or." Throughout 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 As used in this ication 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 ining" (and any form of ning, such as "contains" and "contain") are inclusive or open—ended and do not exclude additional, unrecited ts or method steps The term “or combinations f" 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, AC8, 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, wo 2013/041846 PCT/GBZOIZ/052298 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 disclosure may be read in ation with any other part of the disclosure, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and d herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this ion have been described in terms of preferred ments, it will be apparent to those of skill in the art that variations 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 concept, spirit and scope of the invention. All such similar tutes and cations nt to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

The present invention is described in more detail in the following non limiting Examples (Examples 1- 3 being prophetic). Example 4 is a worked example.

EXAMPLES EXAMPLE 1 ”Recombineered BAC Vectors to add Polymorphic ons to the Mouse Genome” Figure 1 through 3 depict recombineering methods (see references above) that can be used to introduce polymorphic V-gene regions into genomic DNA. In one embodiment, a genomic fragment wo 2013/041846 PCT/GBZO]2/052298 from the human heavy chain region is inserted into a ial artificial chromosome (BAC) vector by standard techniques. Preferably, 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 including sequence searches of cially available ies or by hybridization to bacterial colonies ning 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 VH gene segments can be ed by polymorphic VH or VL gene segments. In this example, two steps are required. The first step es 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 bacteria can be selected for the absence of the rpsL gene by resistance to omycin. Short stretches of DNA homologous to sequences ng the endogenous VH gene exon are placed 5' and 3’ of the mp operon. In the ce of appropriate recombination factors per standard recombineering techniques (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 la) which are selected by resistance to ampicillin. 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 allele 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 , ically the rpsL portion.

In this example, the two step process as bed can be repeated for each of the endogenous VH gene segments or for as many endogenous gene segments that one wishes to replace with polymorphic V gene segments (Figure 1d). wo 2013/041846 PCT/GBZOIZ/052298 As is apparent, any rphic V gene segment can be ed in this manner and any endogenous V gene segment 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 s another method for creating a genomic fragment encoding polymorphic V gene segments. In this example, polymorphic V gene segments are inserted into a region of genomic DNA devoid of other genes, l 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 c fragment, recombineering techniques can be used to insert polymorphic V gene segments at intervals of, for e, 10-kb. In this example, a 150-kb genomic fragment might accommodate 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 gous to a specific site are used to flank the operon.

These are used by the recombineering system to insert the element specifically into the BAC genomic fragment and positive events are selected by resistance to ampicillin (Figure 2a). The second step replaces the operon in the c fragment with a polymorphic V gene segment by a similar 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 mp operon and therefore resistance to streptomycin (Figure 2c).

The two step technique for inserting polymorphic V gene segments into a specific site on the genomic fragment can be repeated multiple times resulting in a BAC genomic fragment with l polymorphic gene segments, including their promoter elements. It is nt that the examples shown in Figures 1 and 2 can be combined n the technique for insertion can be used to add extra rphic 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 bute to certain chromatin conformations, both important in wild-type gene expression.

PCT/GBZOIZ/052298 Figure 3 s 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 ed DNA fragments recombine with a homologous sequence using recombineering (Nat Rev Genet. 2001 Oct;2(10):769-79; Recombineering: a ul new tool for mouse functional genomics; Copeland NG, Jenkins NA, Court DL). The recombinases used in recombineering preferentially bind and use such short —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 imately 100 randomly picked (not selected) clones resulting from recombineering. A ve event in this example ing when one or more single nucleotide changes introduced into the single-stranded fragment get erred to the BAC insert containing V gene segments and surrounding c 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 tide changes, for example. An example of such nucleotide polymorphisms are depicted in Figure 5. Short single stranded regions that encompass the polymorphic nucleotide s can be chemically synthesized using rd techniques. The resulting single stranded DNA fragments are introduced into bacteria and via recombineering techniques approximately 1 in 100 BAC fragments will have incorporated the polymorphic nucleotides via homologous incorporation of the single stranded nt (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 VH1-69*01 gene segment into a VH1-69*02 gene segment (Figure 3b).

It is clear that this process 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 between existing V gene segments. As would be evident to one skilled in the art, a combination of these techniques can be used to create numerous variations of both rphic and endogenous human V gene segments.

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

ZOIZIOSZZ98 g Polymorphic V-regions to the Genome using SRMCE of Modified BACs” Modified BACs with polymorphic V gene segments created using the methods bed in Example 1 can be used to alter the genome of non—human mammals. These tions can result in an intact l6 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 d 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 ts into a genome is sequential recombinase mediated te exchange (SRMCE). The technique is described in W02011004192 (Genome Research Limited), which is incorporated here in its entirety by reference.

SRMCE provides for a locus modified 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 previous BAC insertion. In this example, the landing pad is inserted in the mouse IGH locus between the most 3'J gene segment and the Cu gene t and a previous BAC insertion via SRMCE techniques 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 ion of correct insertion of a second targeting BAC fragment. The icity of this insertion is provided by cre recombinase-mediated exchange between permissive lox sites. A iox site is permissive for recombination only with a compatible lox site. In this example, the loxP site will only recombine with onP and lox2272 will only recombine with iox2272. This provides directionality to the insertion 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. Resistance to puromycin results from the PCT/GBZOIZ/052298 juxtaposition of an active promoter element, PGK, with the puroTK coding region. Correct insertions are verified by standard techniques including PCR ofjunctions, PCR of internal elements, Southern ng, comparative genomic hybridization (CGH), sequencing and etc. In the example, correct on2272-lox2272 and loxP-loxP recombination also results in two intact sets of piggyBac ts 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 ”PBB'”. An appropriated oriented set of piggyBac elements are the substrate of piggyBac transposase which can catalyse recombination between the ts, resulting in deletion of intervening sequences as well as both elements. The DNA remaining after a piggyBoc transposition is left intact and is lacking any remnant of the piggyBac element. In the e, 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 t of the SRMCE method in this example is a IGH locus with several rphic 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 participate in the recombination events associated with B cell maturation yielding VDJ gene segments. These gene segments can then be transcribed and spliced to the mouse constant region. Translation of these transcripts will result in the production of an dy 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, transferring the injected embryo to a le recipient and breeding the chimeric offspring that result. The ed gene locus can be ated through breeding and made either heterozygous or homozygous depending on the genetic cross.

It is evident from the structure of the IGH locus ed in this example and by knowledge of the mechanisms involved in B cell receptor (BCR) and dy gene rearrangements that a large set of wo 2013/041846 PCT/G82012/052298 different ations 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 modified animals. In this example, several different human VH 1-69 polymorphs are orated to provide superhuman VH diversity. This particular VH gene t 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 expected to e 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 affinities for pathogen antigens. Examples of such ens include influenza virus, hepatitis C virus (HCV) and human immunodeficiency virus-1 (HIV-1) (see also table above).

EXAMPLE 3 "Align ment of 13 VH1-69 Alleles” Building a more diverse dy repertoire by incorporating additional V gene segment polymorphs requires availability of polymorphic alleles of V gene segments. One source of such alleles e sequence databases. In this example, 13 distinct alleles of the VH1-69 gene t are provided.

These allele sequences and comparisons are drawn from the ”IMmunoGeneTics" IMGT Information System (www.imgt.com) database. Figure 5 is a diagram of the alignment of alleles *02 h ‘13 with the *01 allele. The VH1-69*01 nucleotide and amino acid sequence is provided at the top of the figure. Where the ing alleles are identical to the *01 allele sequence a dash is inserted below the sequence. Nucleotide differences are noted alongside the appropriate allele and if the sequence change s in a protein coding change, the amino acid change is indicated above the triplet.

Figure 5 depicts between 1 and 4 amino acid changes for each allele in comparison to the *01 .

All of the amino acid changes occur in the part of the heavy chain protein encoding the complementarity ining regions (CDRs). These regions 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 binding characteristics for various antigens. In several reports, it has been observed that the VH1~69-enc0ded variable region of the heavy chain is often found in antibodies that bind influenza wo 2013/041846 PCT/082012/052298 virus, HCV and HIV-1 ns (see table above). Therefore incorporating the polymorphic V gene segments of this example into a transgenic animal model using the methods of Examples 1 and 2 would likely result in an antibody repertoire in said transgenic animal with more antibodies that bind to antigens associated with these and other pathogens. 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 8 cells from such an immunized mouse can be used to make a hybridoma library that can be screened for production of antibodies that bind the pathogen antigens. This library will be superior to libraries from ional transgenic mice for g such antibodies given the addition of polymorphic VH1-69 gene segments to the IGH locus in said transgenic 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, D,J segments can be from a plurality of human sources (optionally more than one human ethnic population).

Example 4: ”Transgenic Mice, B-cells, Hybridomas, Antibodies & Heavy Chains Based on Human JH6*OZ” A functional human gene segment repertoire (from VH2-26 to 1H6, see the IMGT database for the structure of the human lgH locus; lMGTre ertoire index. h ?section2l.ocusGenes&re ertoire:lOCus&s ecieszh roup=lGK ) was sectored by the inventors to produce two different transgenic heavy chain alleles (denoted 52F and 53F) and ponding mice. The transgenic alleles were expressed in the mice and the heavy chain repertoires were assessed at the RNA ript level. Deep sequence analysis was d out using ormatics 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 le region gene segments were inactivated by inversion (as per the method described in WOW“ "Vi/”07 this disclosure being incorporated herein by reference).

PCT/G32012/052298 cing of Human Donor DNA Samples: Identification of Conserved JH6*02 Variant 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 QIAamp DNA Mini Kit (Cat.No.51304, Qiagen).

PCR reactions were set up to y the JH6 region and PCR products were sequenced (PCR Oligos sequence: Fwd. 5’-AGGCCAGCAGAGGGTI'CCATG~3’ (SEQ ID NO: 444), Rev. 5’- GGCTCCCAGATCCTCAAGGCACB’ (SEQ ID NO: 445)).

Sequence is was carried out by comparing to the JH6 reference sequence from lMGT annotated database [[www.in1gt.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 donors.

The inventors also consulted the genomic sequences ly available for Jim Watson and Craig Venter at Ensembl human genome database [http://www.ensembl.org/]. These too ned 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 Identification of Suitable Human DNA Seguence BACs A series of human bacterial artificial chromosome (BAC) clones were identified from Ensemble (http:z(wwwensembiorgZindexhtml) or UCSC (http:[[genome.ucsc.edu[) human database searches based on gene name (IGH) or location (chromosome 14: 106026574—107346185). Seven human RPll BAC clones were selected, RP11—1065N8 BAC carrying human JH6*02. In total, the following BACs were identified as s of human lgH locus DNA: RP11-1065N8, 59319,RP11-141l7, RP-112H5, RP11-101624, RP11-12F16 and RP11-47P23.

With a r approach, ent BAC clones (eg, different RPll clone IDs or different sources from RP11) 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 lgH Loci Insertion of human heavy gene segments from a lst IGH BAC (RP11—1065N8) into the IGH locus of mouse A82.1 ES cells (Baylor College of Medicine) was performed to create a heavy chain allele denoted the $1 allele. The inserted human sequence corresponds to the sequence of human chromosome 14 from position 106494908 to position 106328951 and comprises functional heavy gene segments VH7—4v1,VH4-4, VH1-3, VH1-2, VHS-1, 01-1, 02—2, 03-9, 03-10, 04-11, 05-12, 06-13, 01-14, 02-15, 03-16, 04-17, 05-18, 06—19, 01-20, 02-21, 03-22, 04-23, 05-24, 06-25, 01-26, D7-27, 1H1, 1H2, 1H3, 1H4, JHS and JH6 (in 5’ to 3‘ order), n the JH6 was chosen to be the human JH6*02 variant. The insertion was made between positions 114666435 and 114666436 on mouse chromosome 12, which is upstream of the mouse Cu region. The mouse V”, D and JH gene segments were retained in the locus, immediately upstream of (5’ of) the inserted human heavy chain DNA.

A second , 52 was constructed in which more human onal VH gene segments were inserted upstream (5’) of the 5’-most VH inserted in the 51 allele by the sequential insertion of human DNA from a second BAC (BACZ). The inserted human sequence from BACZ corresponds to the sequence of human chromosome 14 from position 106601551 to position 106494909 and comprises functional heavy chain gene segments VH3—13,VH3-11,VH3-9, VH1-8, VH3-7. The mouse VH, 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 .

A third allele, 53 was constructed in which more human functional VH gene segments were inserted upstream (5’) of the S'vmost VH inserted in the $2 allele by the sequential insertion of human DNA from a third BAC (BACS). The inserted sequence corresponds to the sequence of human chromosome 14 from on 106759988 to on 106609301, and comprises functional heavy chain gene segments, VH2-26, VH1-24, VH3-23, VH3-21, VH3-20, , and VHS-15. The mouse V", D and JH gene ts were retained in the locus, immediately upstream of (5' of) the inserted wo 2013/041846 PCT/G82012/052298 human heavy chain DNA. In a subsequent step, these were inverted to vate them, thereby producing 53F mice in which only the human heavy chain variable region gene ts are active.

Mice bearing either the 52F or 53F 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 inactivating ce comprising neo" into the mouse JH- Cp. intron (to produce the ”HA" allele).

Specifically, the following alleles were included:- VH1-2*02, 01, VH1-8*01, VH1-18*01, VH2-5*10, 01, VH3—9*Ol, VHS-11*Ol, VH3- 13*01, VH3-21*03, VH3-23*04, VH4-4*02, 01 and VH71‘01 ‘01, D2-2*OZ, 1, 03-10*01, 03-22*01, D4-17*01, D6-13"01 and D6~19*01 Immunisation procedure Transgenic mice of the SZF or 53F genotype were primed with 20-40ug recombinant proteins obtained commercially or produced in house with Antigen 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 als with about half the amount of antigen in lete 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.

Hybridoma fusion procedure 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/Z Ag14 myeloma cells (HPA 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 96-well culture plates and left for about 10 days before screening. Media was changed 1-3 times during this period.

Screening Culture supernatants of the hybridoma wells above were screened using homogenious time resolved fluorescence assay (htrf) using Europium cryptate labelled anti-mouse IgG (Cisbio anti-mouse lg Europium Cryptate) and a biotin tagged target antigen with a commercially ble 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 ately counterscreened using an lgG- specific detection ELISA method. Positives identified by primary ELISA screen were immediately expanded to 24-well plates. Once cultures were ed to 24-well stage and reached conflueny, supernatants were ted using htrf or lgG-specific ELISA to confirm binding to target antigen.

Supernatant of such confirmed es were then also analysed by e plasmon resonance using a BioRad n XPR36 instrument. For this, dy expressed in the hybridoma cultures was captured on a biosensor GLM chip (BioRad 176-512) which had an anti—mouse lgG (GE Healthcare 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 ties were determined using the 1:1 model inherent to the BioRad ProteOn XPR36 analysis software.

Any clones with confirmed binding activity were used for preparing total RNA and ed by PCR to recover the heavy chain variable region sequences. Standard 5‘-RACE was carried out to analyse RNA transcripts from the transgenic heavy chain loci in the 52F and 53F mice. Additionally, deep sequence analysis 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 : first cDNA strand was synthesized using an oligo based on the Cmu region of the mouse IGH locus as a PCR template. PCR was performed wo 2013/041846 PCT/G32012/052298 using this oligo with an oligo dT-anchor primer. Then PCR t was cloned into pDrive vector (Qiagen) and then sequenced. 0 The second is from hybridomas generated through electro-fusion: total RNA was extracted from hybridoma lines of interest using standard Trlzol methods and frozen at -80 °C for long term storage. cDNA was generated from 100ng total RNA using standard Superscript Ill reverse transcriptase and a gene-specific reverse primer binding to all mouse lgG isotypes for heavy chain and a mouse kappa nt region primer for the light chain amplification. 2-3 ul of cDNA were then used as template in a PCR reaction using Pfu DNA rase and a panel of degenerate forward primers annealing to the leader sequence of the human immunoglobulin variable domain as well as one m0use pan-lgG reverse primer. PCR products were run out of a 1% e 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 lgG from sed 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 e complement of the e read, and aligning it against the forward read. The alignment scoring was 5 for a match, -4 for a mismatch, a gap open y of 10 and a gap extension y of 1. A consensus sequence was then produced by stepping through the alignment and comparing bases. When there was a eement the base with the highest quality value from sequencing was used.

The BLAST+ (Basic Local Alignment Search Tool) (Camacho C., Coulouris 6., Avagyan V., Ma N., Papadopoulos J., Bealer K., & Madden T.L. (2008) "BLAST+: architecture and applications." BMC Bioinformatics 102421 http://wwwncbi.nlm.nih.gov/pubmed/20003500) program ’blastn’ was then wo 2013/041846 PCT/G82012/052298 used to find the germline J 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 matched both a V and a J segment, the sequence between the two was then ed to a database of germline D segments in the mouse using ’blastn’ with a wordsize of 4 and the s 'blastn-short' and ’ungapped’. This was used to assign a D segment, if possible. The CDR3 was identified by searching for the conserved ”TATTACTGT" sequence in the V segment, and the ”CTGGGG" in theJ segment. If these motifs were not found, then up to 4 mismatches were allowed.

The IMGT definition of CDR3 was used, so the CDR3 length is calculated from after the ”TGT" in the V to before the ”T66” 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 ”TGA") were excluded.

The ty of the matching V, J and D ts as well as the CD R3 length from this assignment were then saved as a table for downstream analysis. The ratio of IGHJ6*02 used increased from the naive to sed mice, as well as being enriched in the sub-population of sequences with along HCDR3 (defined as consisting of 20 or more amino acids): All HCDR3>20 JH6*02% TotalCount JH6*02% ETotalCount %HCDR3>20 Naive 22.31% 1340 91.11% E 45 sed 37.50% 256 66.67% Hybridoma 36.13% 119 63.64% €11 This shows that the JH6*02 gene segment is ed for by immunisation, 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 specifically bound by long HCDR3 length antibodies. wo 2013/041846 PCT/632012/052298 Additionally, the is revealed that certain VH and D gene segments frequently yielded HCDR35 of long length (in all of naive, immunised and antigen-specific repertoires of heavy chains). See Table 2.

Tahlg 2A; Long HCDR3s from Na’ive Regertoirfi Average V CDR3Length Count IGHV1-2*02 21 3 IGHV1-18*01 21 5 |GHV3-7*01 22 3 lGHV6—1*01 21 5 IGHV3-9*01 20 2 IGHV2-5*10 20 1 4-l*01 21 3 |GHV1w3*01 21 5 |GHV4-4*02 20 3 IGHV3-13*01 22 1 IGHV3-23*04 20 1 IGHV1-8*01 21 10 IGHV3-21*03 23 3 Average D ngth Count IGHD2-2*02 20 1 |GHD3-9*Ol 21 13 IGHD3-10*01 21 26 lGHD6-13*01 20 1 IGHD4-17*01 22 2 lGHD6-19*01 23 1 IGHD3-22*01 20 1 CDR3Length (All Naive) Count 23 21 10 22 7 23 3 24 l 26 1 PCT/G820] 2/052298 T leZB: n HCDR3sfromlmmuni R rt ir Average V CDR3Length Count |GHV4-4*02 20 1 lGHV3-11*01 23 2 iGHV3-7*01 21 6 Average D CDR3Length Count |GHD2-2*02 22 2 |GHD3-10*01 22 5 19*01 20 1 lGHDl-26*01 20 1 CDR3Length (All Immunised) Count 24 l—‘NH r—s Table 2C: Long HCDR3s from n-Specific Repertoires Average V CD th Count |GHV4-4*02 20 2 IGHV1-3*01 21 lGHV3—11*01 21 lGHV3-7*01 22 |GHV1-8*01 22 |GHV3-20*d01 22 |GHV3-9*01 20 HHNHr—bw Average D CDR3Length Count IGHDZ-2*02 22 IGHD3-9*01 21 EGHD3I10*01 21 wo 2013/041846 PCT/G32012/052298 ngth (All Antigen-Specific) Count 4 22 2 21 4 24 1

Claims (5)

CLAIMS :

1. A method of producing an antibody with an HCDR3 sequence of at least 20 amino acids in length, wherein the antibody binds to an antigen of an infectious disease pathogen, the method comprising: 5 (a) providing a transgenic mouse or rat whose genome ses an immunoglobulin heavy chain locus comprising one or more human VH gene segments selected from the group consisting of: a VHIII gene family member (optionally, a VHIIIa or VHIIIb family member), a VHIV gene family member, VHIII 9.1 (VH3-15), VHIII VH26 3), VH3-21, LSG6.1, LSG12.1, DP77 (V3-21), VH H11, VH1GRR, ha3h2, VHI- 10 ha1c1, VHIII-VH2-1, VH4.18, ha4h3, Hv1051, 71-2, Hv1f10, VH4.11, 71-4, VH251, VH1-69 and a gene segment at least 80% identical; one or more human D gene segments and one or more human JH gene segments upstream of a nt region; n the heavy chain locus ses VH and/or D gene segments capable of yielding HCDR3s of 20 or more amino acids in length, comprising VH gene segments IGHV1- 15 2*02, IGHV1-18*01, IGHV3-7*01, IGHV6-1*01, IGHV3-9*01, IGHV2-5*10, IGHV71*01, IGHV1-3*01, IGHV4-4*02, IGHV3-13*01, IGHV3-23*04, IGHV1-8*01, IGHV3-21*03 and/or IGHV3-11*01, and/or comprising D segments IGHD2-2*02, IGHD3-9*01, IGHD3-10*01, IGHD6-13*01, IGHD4-17*01, IGHD6-19*01, IGHD3-22*01 and/or IGHD1-26*01, and an immunoglobulin light chain locus comprising one or more human V gene 20 segments and one or more human J gene ts upstream of a constant region, n the gene segments in the heavy chain locus are operably linked to the constant region thereof, and the gene segments in the light chain locus are ly linked to the nt region thereof, so that upon immunisation the mouse or rat is capable of producing an antibody comprising heavy chains produced by recombination of the heavy 25 chain locus and light chains produced by recombination of the light chain locus; (b) immunising said mouse or rat with said antigen; (c) removing B lymphocytes from the mouse or rat and selecting one or more B lymphocytes expressing antibodies that bind to the antigen; (d) optionally immortalising said ed B lymphocytes or progeny thereof, ally by 30 producing hybridomas therefrom; and (e) isolating an dy expressed by the B lymphocytes.

2. A method according to claim 1, comprising the step of isolating from said B lymphocytes a nucleic acid encoding said dy that binds said antigen; optionally exchanging the heavy chain 35 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 cell. 0_1 (GHMatters) P96496.NZ 18-May-16 3. A method according to claim 1 or claim 2, further comprising making a mutant or derivative of the antibody produced by the method of claim 1 or claim 2. 5 4. A method of producing a polyclonal antibody mixture, the method sing carrying out a method according to any one of claims 1 to 3 by separately immunising first and second mice or rats with antigen and combining the anti-antigen antibodies isolated from each mouse or rat, or mutants or derivatives of said antibodies, to produce a polyclonal antibody mixture; optionally wherein the following (i), (ii) or (iii) apply separately or the following (i) and (ii) or (i) and (iii) apply in combination: 10 (i) the mice or rats are immunised with the same n or different antigens, ally wherein the different antigens are expressed by the same pathogenic organism or a family member thereof; (ii) prior to sation the light chain loci of the mice or rats n the identical VH gene repertoire, optionally a single VH gene, and optionally the identical J oire; optionally the light chain loci of the mice or rats are identical prior to immunisation; 15 (iii) prior to immunisation the light chain loci of the mice or rats contain the identical rearranged VJ or VDJ repertoire, optionally a single VJ or VDJ; optionally the light chain loci of the mice or rats are identical prior to immunisation. 5. A method of producing a polyclonal antibody mixture, the method comprising carrying out a 20 method according to any one of claims 1 to 4 by immunising one or a plurality of mice or rats with first and second antigens and combining the anti-antigen antibodies isolated from each mouse or rat, or mutants or derivatives of said antibodies, to produce a polyclonal antibody mixture; optionally wherein the following (i), (ii) or (iii) apply separately or the following (i) and (ii) or (i) and (iii) apply in combination: 25 (i) the antigens are expressed by the same pathogenic organism, or a family member thereof; (ii) prior to immunisation the light chain loci of the mice or rats contain the identical VH gene repertoire, optionally a single VH gene, and ally the identical J repertoire; optionally the light chain loci of the mice or rats are identical prior to immunisation; (iii) prior to immunisation the light chain loci of the mice or rats contain the cal nged 30 VJ or VDJ repertoire, optionally a single VJ or VDJ; optionally the light chain loci of the mice or rats are identical prior to immunisation. 6. A method of producing host cells capable of sing a onal antibody mixture, the method comprising, in a method according to claim 2: 35 (a) sing one or a plurality of mice or rats with first and second antigens, optionally wherein the different antigens are sed by the same pathogenic organism or a family member f; (b) isolating nucleic acid encoding first and second anti-antigen antibodies from B lymphocytes from said mice or rats; 7759020_1 (GHMatters) P96496.NZ 18-May-16 (c) determining the nucleotide sequences of the heavy and light chain variable regions of the first antibody; (d) determining the tide sequence of the heavy variable region and ally the light chain le region of the second antibody; 5 (e) inserting the heavy chain variable region coding sequence of each antibody into a heavy chain expression vector; ally wherein the constant region coding sequence of each heavy chain is ged for a nucleotide sequence that encodes a human or humanised constant region; (f) inserting the light chain variable region coding sequence of the first antibody into a light chain expression ; optionally wherein the constant region coding sequence of the light chain of the 10 first antibody is exchanged for a nucleotide sequence that encodes a human or humanised constant region; (g) optionally inserting the light chain variable region coding sequence of the second antibody into a light chain expression vector; optionally wherein the constant region coding sequence of the light chain of the second antibody is exchanged for a nucleotide sequence that encodes a human or 15 humanised constant region; and (h) introducing each expression vector into a host cell and ressing antibody chains in a mixture of said host cells to produce antibodies, each antibody comprising one or both of said heavy chain variable regions and a light chain; optionally wherein the expression vectors are introduced er into the same host cell, optionally a CHO cell or HEK293 cell, so that the cell is capable of 20 expressing antibody light chains and heavy chains, such that the cell or a plurality of the host cells express antibodies, each comprising one or both of said heavy chain variable regions and a light chain; (i) optionally: prior to immunisation the light chain loci of the mice or rats n the identical VH gene repertoire, 25 ally a single VH gene segment, and optionally the identical J repertoire, optionally a single J gene segment; optionally the light chain loci of the mice or rats are identical prior to immunisation; or prior to immunisation the light chain loci of the mice or rats contain the identical rearranged VJ or VDJ repertoire, optionally a single VJ or VDJ; optionally the light chain loci of the mice or rats are identical prior to immunisation. 7. A method of ing a monoclonal or polyclonal antibody mixture, the method comprising carrying out a method according to claim 6 and expressing a monoclonal antibody or polyclonal e of said antibodies; optionally followed by isolating an antibody comprising the heavy chain le region of the first and/or second antibodies. 8. A method according to any one of claims 1 to 7, wherein each mouse or rat used for immunisation is provided by: (a) isolating from a human blood or tissue, optionally B cytes, PBMCs, bone marrow, spleen, tonsil or lymph node, sample a B lymphocyte that expresses an antibody that binds the 7759020_1 (GHMatters) P96496.NZ 18-May-16 antigen, ally wherein said serum or tissue was from a human individual suffering, susceptible to, or recovered from, a disease or condition caused or mediated by an organism harbouring or secreting said antigen; or from a human individual harbouring said organism; (b) determining which human germline VH gene segment was recombined in the human to 5 produce the tide sequence of said B lymphocyte that encodes the heavy chain variable region of the antibody; (c) constructing a transgenic mouse or rat wherein said human germline VH gene segment is provided in a light chain locus thereof according to claim 1; and (d) providing said transgenic mouse or rat for immunisation in a method according to any one of 10 claims 1 to 7. 9. Use of a transgenic mouse or rat for producing an antibody with an HCDR3 sequence of at least 20 amino acids in length, wherein the antibody binds to an antigen of an infectious disease pathogen, wherein the mouse or rat genome comprises human VH, D and J gene ts ed 15 in a heavy chain locus upstream of the endogenous mouse or rat constant region, wherein the VH gene segments comprise IGHV1-2*02, IGHV1-18*01, IGHV3-7*01, IGHV6-1*01, IGHV3-9*01, IGHV2-5*10, IGHV71*01, 3*01, IGHV4-4*02, 13*01, IGHV3-23*04, IGHV1-8*01, IGHV3-21*03 and/or IGHV3-11*01, and wherein the D segments comprise IGHD2-2*02, IGHD3-9*01, IGHD3-10*01, IGHD6-13*01, IGHD4- 20 17*01, IGHD6-19*01, IGHD3-22*01 and/or 26*01. 10. An isolated antibody that specifically binds an antigen of an infectious disease pathogen, the dy sing human heavy chain variable s and non-human constant regions, n the variable s are derived from the recombination in a man vertebrate of 25 (i) a human VH gene segment selected from IGHV1-2*02, IGHV1-18*01, IGHV3-7*01, IGHV6-1*01, IGHV3-9*01, IGHV2-5*10, IGHV71*01, IGHV1-3*01, IGHV4-4*02, IGHV3-13*01, IGHV3-23*04, IGHV1-8*01, IGHV3-21*03 and/or IGHV3-11*01 with (ii) a human D gene segment selected from IGHD2-2*02, IGHD3-9*01, IGHD3-10*01, IGHD6-13*01, IGHD4-17*01, IGHD6-19*01, IGHD3-22*01 and/or IGHD1-26*01 and with 30 (iii) a human JH gene segment, optionally JH6; wherein the antibody has an HCDR3 sequence of at least 20 amino acids in length ; and non-human vertebrate AID-pattern somatic hypermutations, optionally mouse or rat AID-pattern mutations, when compared to corresponding human ne V, D and J sequences and/or non-human, optionally mouse or rat, terminal deoxynucleotidyl transferase (TdT)- pattern onal mutations when 35 compared to corresponding human germline V, D and J sequences. 11. Use of a onal or polyclonal antibody produced by the method of any one of claims 1 to 5 or 7 or an antibody according to claim 10 in the manufacture of a medicament for treating and/or preventing an infectious disease. 7759020_1 (GHMatters) .NZ 18-May-16 12. A method according to any one of claims 1 to 8, wherein the antigen is an antigen expressed by a bacterial or viral infectious disease pathogen. 5 13. Use according to claim 9 or claim 11, wherein the antigen is an n expressed by a bacterial or viral infectious disease pathogen. 14. An antibody according to claim 10, wherein the antigen is an antigen expressed by a bacterial or viral infectious disease pathogen. 15. A method according to claim 12, wherein the antigen is an antigen of HIV. 16. Use according to claim 13, wherein the antigen is an antigen of HIV. 15 17. An antibody according to claim 14, wherein the n is an antigen of HIV. 18. A method ing to claim 12, wherein the n is an antigen of malaria. 19. 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