Antibodies, variable domains & chains tailored for human use

Abstract

The invention relates to the provision of antibody therapeutics and prophylactics that are tailored specifically for human use. The present invention provides libraries, 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, heavy chains, polypeptides, nucleotide sequences, pharmaceutical compositions and uses are also provided by the invention.

1 . A method for producing a heavy chain, VH domain or an antibody specific to a target antigen, wherein the heavy chain, VH domain or antibody comprises an HCDR3 at least 20 amino acids in length, the method comprising
 providing a non-human vertebrate, optionally a mouse or a rat, that has a genome comprising an immunoglobulin heavy chain locus comprising unrearranged human gene segment JH6*02, one or more VH gene segments and one or more D gene segments upstream of a constant region; wherein the gene segments in the heavy chain locus are operably linked to the constant region thereof so that the vertebrate is capable of producing an antibody heavy chain produced by recombination of the human JH6*02 with a human D segment and a human VH segment,   wherein the JH6*02 gene segment comprises the following nucleotide sequence: ATTACTA CTACTACTAC GGTATGGACG TCTGGGGCCA AGGGACCACG GTCACCGTCT CCTCAG, and   wherein the vertebrate has been immunised with the target antigen and produces an antibody heavy chain specific for the target antigen wherein the variable domain of the heavy chain is the product of recombination between a human VH, D and JH6*02 and wherein the HCDR3 length is at least 20 amino acids, and   isolating from the non-human vertebrate the heavy chain, VH domain or an antibody specific to the target antigen or a cell producing the heavy chain, VH domain or antibody, wherein the heavy chain, VH domain or antibody comprises a HCDR3 that is derived from the recombination of human JH6*02 with a human VH gene segment and a human D gene segment, optionally wherein the constant region of the locus is a non-human vertebrate (e.g., mouse or rat) constant region, and the non-human constant region of the isolated heavy chain or antibody is replaced with a human constant region.   
     
     
         2 . The method of  claim 1  comprising isolating a heavy chain, VH domain or an antibody, wherein the HCDR3 length is at least 20 amino acids. 
     
     
         3 . The method of  claim 1 , comprising isolating a B-cell or hybridoma expressing a heavy chain VH domain that is identical to the VH domain of the heavy chain of  claim 2 . 
     
     
         4 . The method of  claim 1 , wherein the constant region is mouse or rat. 
     
     
         5 . The method of  claim 1 , comprising removing B lymphocytes from the vertebrate and selecting one or more B lymphocytes expressing antibodies that bind to the antigen, optionally immortalising said selected B lymphocytes or progeny thereof, optionally by producing hybridomas therefrom, and isolating an antibody expressed by the B lymphocytes. 
     
     
         6 . The method of  claim 5 , further comprising isolating from said B lymphocytes nucleic acid encoding said antibody. 
     
     
         7 . The method of any  claim 1 , wherein the constant region of the locus is a non-human vertebrate (e.g., mouse or rat) constant region, and the method further comprises replacing the non-human constant region of the isolated heavy chain or antibody with a human constant region. 
     
     
         8 . The method of  claim 6 , further comprising 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. 
     
     
         9 . The method of  claim 8 , further comprising inserting said nucleic acid into an expression vector and optionally a host cell. 
     
     
         10 . The method of  claim 9 , comprising expressing the heavy chain, VH domain or antibody from the host cell and providing an isolated heavy chain, VH domain or antibody. 
     
     
         11 . The method of  claim 1 , wherein the locus comprises one, more or all human D gene segments D3-9; D4-17; D3-10; D2-2; D5-24; D6-19; D3-22; D6-13; D5-12; D1-26; D1-20; D5-18; D3-16; D2-21; D1-14; D7-27; D1-1; D6-25; D2-14; and D4-23, optionally wherein the locus comprises one, more or all human D gene segments D3-9, D3-10, D6-19, D4-17, D6-13, D3-22, D2-2, D2-25 and D3-3. 
     
     
         13 . The method of  claim 1 , wherein the locus comprises a plurality of human D gene segments and the JH6*02 is in human germline configuration with respect to the 3′-most human D gene segment. 
     
     
         14 . The method of  claim 1 , wherein the locus comprises one, more or all of human gene segments selected from V3-21, V3-13, V3-7, V6-1, V1-8, V1-2, V7-4-1, V1-3, V1-18, V4-4, V3-9, V3-23, V3-11, V3-20, D3-9*01, D3-10*01, D6-19*01, D6-13*01, D1-26*01, IGHV1-8*01, IGHV4-61*01, IGHV6-1*01, IGHV4-4*02, IGHV1-3*01, IGHV3-66*03, IGHV3-7*01 and IGHV3-9*01. 
     
     
         15 . The method of  claim 1 , wherein the antibody heavy chain is a product of the recombination of JH6*02 with a human VH gene segment recited in  claim 14  and/or a D gene segment recited in  claim 11 . 
     
     
         16 . The method of  claim 1 , wherein all endogenous non-human vertebrate heavy chain variable region gene segments have been inactivated in the genome and/or wherein the genome is homozygous for said heavy chain locus. 
     
     
         17 . The method of  claim 1 , wherein the vertebrate is a mouse comprising functional heavy gene segments VH2-5, VH7-4-1, VH4-4, VHI-3, VHI-2, VH6-1, DI-1, D2-2, D3-9, D3-10, D4-11, D5-12, D6-13, DI-14, D2-15, D3-16, D4-17, D5-18, D6-19, DI-20, D2-21, D3-22, D4-23, D5-24, D6-25, DI-26, D7-27, JHI, JH2, JH3, JH4, JH5 and JH6, in 5′ to 3′ order,
 wherein the JH6 is the human JH6*02 variant, 
 wherein the insertion of human DNA is made between positions 114666435 and 114666436 on mouse chromosome 12; 
 wherein human functional heavy chain gene segments VH3-13, VH3-11, VH3-9, VH1-8, VH3-7 are inserted upstream of VH2-5; 
 wherein the mouse VH, D and JH gene segments are retained in the locus, immediately upstream of (5′ of) the inserted human heavy chain DNA; 
 and wherein the mouse VH, D and J segments are inverted to inactivate them, thereby producing mice in which only the human heavy chain variable region gene segments are active. 
 
     
     
         18 . The method of  claim 17  additionally comprising VH2-26, V H 1-24, VH 3-23, VH 3-21, VH3-20, VH1-18, and VH 3-15 inserted upstream (5′) of the 5′-most VH. 
     
     
         19 . The method of  claim 1 , wherein
 (i) the target antigen is an antigen of an infectious disease pathogen;   (ii) the target antigen is a receptor, and the antibody heavy chain specifically binds a receptor cleft; or   (iii) the target antigen is an enzyme, and the antibody heavy chain specifically binds the enzyme active site.   
     
     
         20 . A non-human vertebrate cell having a genome comprising:
 at least 3 human variable region gene segments of the same type, wherein at least two of the human gene segments are variants that are not identical to each other;   at least 2 different human variable region gene segments of the same type in cis at the same Ig locus;   at least 2 different human variable region gene segments of the same type in trans at the same Ig locus, or   first and second human Ig locus gene segments of the same type, wherein the first gene segment is a gene segment selected from any one of Tables 1 to 7 and 9 to 14 and the second gene segment is the corresponding reference sequence.   
     
     
         21 . A method of providing an enhanced human immunoglobulin variable region gene segment repertoire, the method comprising
 providing a population of non-human vertebrates comprising a repertoire of human variable region gene segments,   providing at least 2 different human variable region gene segments of the same type, wherein a first of said different gene segments is provided in the genome of a first vertebrate of the population, and a second of said different gene segments is provided in the genome of a second vertebrate of the population, wherein the genome of the first vertebrate does not comprise the second gene segment.   
     
     
         22 . A library of antibody-producing transgenic cells whose genomes collectively encode a repertoire of antibodies, wherein
 (a) a first transgenic cell expresses a first antibody having a chain encoded by a first immunoglobulin gene, the gene comprising a first variable domain nucleotide sequence produced following recombination of a first human unrearranged immunoglobulin gene segment;   (b) a second transgenic cell expresses a second antibody having a chain encoded by a second immunoglobulin gene, the second gene comprising a second variable domain nucleotide sequence produced following recombination of a second human unrearranged immunoglobulin gene segment, the first and second antibodies being non-identical;   (c) the first and second gene segments are different and derived from the genome sequences of first and second human individuals respectively, wherein the individuals are different; wherein the second human immunoglobulin gene segment is a polymorphic variant of the first human immunoglobulin gene segment and wherein the second gene segment is selected from the group consisting of a gene segment in any of Tables 1 to 7 and 9 to 14, and wherein   (d) the cells are non-human vertebrate cells.   
     
     
         23 . A non-human vertebrate cell comprising a genome that comprises
 (i) a transgenic heavy chain immunoglobulin locus, wherein the locus comprises a full human repertoire of functional VH, D and JH segments derived from the genome sequence of the same human individual, supplemented with one or more additional functional human VH, D and/or JH gene segment that is not found in the genome sequence of said human individual;   (ii) a transgenic kappa light chain immunoglobulin locus, wherein the locus comprises a full human repertoire of functional Vk and Jk segments derived from the genome sequence of the same human individual, supplemented with one or more additional functional human Vk and/or Jk gene segment that is not found in the genome sequence of said human individual; or   (iii) a transgenic lambda light chain immunoglobulin locus, wherein the locus comprises a full human repertoire of functional V lambda and J lambda segments derived from the genome sequence of the same human individual, supplemented with one or more additional functional human V lambda and/or J lambda gene segment that is not found in the genome sequence of said human individual.   
     
     
         24 . A transgenic immunoglobulin locus comprising a synthetic immunoglobulin gene haplotype, the haplotype comprising first and second human gene segments (each being a V, D or J), a switch region and a constant region, wherein
 a) the second gene segment is a polymorphic variant of the first gene segment; or   b) the first and second gene segments are derived respectively from genome sequence of individuals from different, first and second, ethnic populations according to the 1000 Genomes database and the second gene segment is not found in the first population according to the 1000 Genomes database; and
 wherein the constant region, and optionally the switch region, are non-human vertebrate constant and switch regions. 
   
     
     
         25 . A method of producing an antibody heavy chain, the method comprising providing an antigen-specific heavy chain variable domain; and
 combining the variable domain with a human heavy chain constant region to produce an antibody heavy chain comprising (in N- to C-terminal direction) the variable domain and the constant region;   wherein the human heavy chain constant region is an IGHAref, IGHAIa, IGHA2a, IGHA2b, IGHGIref, IGHG2ref, IGHG2a, IGHG3ref, IGHG3a, IGHG3b, IGHG4ref, IGHG4a, IGHDref, IGHEref, IGHMref, IGHMa or IGHMb constant region.   
     
     
         26 . A non-human cell (eg, a mouse cell or rat cell) comprising a genome that comprises a transgenic heavy chain immunoglobulin locus, wherein the locus comprises at least 42 (optionally at least 43, 44, 45, 46, 47, 48, 49, 50) functional human VH gene segments, one or more functional human D gene segments, one or more functional human JH gene segments operably connected upstream of a non-human vertebrate constant region (eg, a mouse constant region, eg, a Cmu and/or a C gamma). 
     
     
         27 . A non-human cell (eg, a mouse cell or rat cell) comprising a genome that comprises a transgenic kappa light chain immunoglobulin locus, wherein the locus comprises at least 39 functional human Vk gene segments and one or more functional human Jk gene segments operably connected upstream of a non-human vertebrate constant region (eg, a mouse constant region). 
     
     
         28 . A non-human cell (eg, a mouse cell or rat cell) comprising a genome that comprises a transgenic lambda light chain immunoglobulin locus, wherein the locus comprises at least 32 functional human V lambda gene segments and one or more functional human J lambda gene segments operably connected upstream of a non-human vertebrate constant region (eg, a mouse constant region).