Manipulation of Immunoglobulin Gene Diversity and Multi-Antibody Therapeutics

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, VH3-7*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 and the D is selected from the group D2-2*02, D3-9*01, D3-10*01 and D3-22*01, or D2-2*02, D3-9*01 and D3-10*01, or D3-9*01 and D3-10*01, or D1-26, D2-2, D3-9, D3-10, D3-22, D4-17, D6-13 and D6-19, or D1-26*01, D2-2*02, D3-9*01, D3-10*01, D3-22*01, D4-17*01, D6-13*01 and D6-19*01, or D2-2, D3-9, D3-10, D3-22, D4-17, D6-13 and D6-19, or D2-2*02, D3-9*01, D3-10*01, D3-22*01, D4-17*01, D6-13*01 and D6-19*01, or D1-26, D2-2, D3-10 and D6-19, or D2-2, 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 junctional mutations when compared to corresponding human germline V, D and J sequences.

A non-human vertebrate (optionally a mouse or a rat) or vertebrate cell whose genome comprises:

• (c) 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 upstream of a constant region;
• optionally wherein the heavy chain locus is according to (a) of claim 81;
• and
• (d) 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 VHJLCL or VHJλCL, wherein the CL is Cλ 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 rearranged VLDJHCL or V λDJHCL, wherein the CL is Cλ or CK);
• or
• (iii) one or more human VL gene segments selected from the group consisting of:
• a V λII gene family member, V λVII 4A, V λII 2.1, V λVII 4A, a V λ1 gene family member, a V λ3 gene family member, IGLV1S2, V λ3-cML70, IaIh2, IaIyI, Ia3h3, 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 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 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.

A non-human vertebrate or vertebrate cell whose genome comprises:

• (f) 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 upstream of a constant region;
• and
• (g)
• (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 antibody-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 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 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) 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 wherein said antibody binds to an antigen of an infectious disease pathogen, wherein optionally the variable regions of said antibody being 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;
• (h) Wherein the gene segments 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 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 derived from the light chain locus;
• (i) 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 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, VHII1-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;
• (j) 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 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 (VH3-23), VH3-21, LSG6.1, LSG12.1, DP77 (V3-21), 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.

A non-human vertebrate (optionally a mouse or a rat) or vertebrate cell whose genome comprises:

• (a)
• (i) An unrearranged immunoglobulin heavy chain locus comprising one or more human VL gene segments, one or more human D gene segments 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 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 produced by the recombination of a human J gene segment and optionally a human D gene segment with a human VL gene segment that is identical to (or mutant of) the 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 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);
• (b) 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
• (c) Wherein the gene segments 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 producing 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 VAII gene family member, VAVII 4A, VAII 2.1, VA.VII 4A, a VA1 gene family member, a VA3 gene family member, IGLV1S2, VA3-cML70, IaIh2, IaIyI, Ia3h3, Kv325, a VKI gene family member, LI-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;
• (e) Optionally when (a)(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 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 VA1 gene family member, a VA3 gene family member, IGLV1S2, VA3-cML70, IaIh2, IaIyI, Ia3h3, 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.

A 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 constructed with one or more of the following arrangements (5′ to 3′):—

• (a) [V (heavy, lambda or kappa)]-[two-turn RSS]-[one-turn RSS]-[D]-[JH], wherein said RSSs are in an opposite orientation;
• (b) [VH]-[D]-[two-turn RSS]-[one-turn RSS]-[J lambda], 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], wherein 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 orientation;
• (f) [V (heavy, lambda or kappa)]-[one-turn RSS]-[two-turn RSS]-[D]-[JH], wherein said RSSs are in an opposite orientation;
• (g) [VH]-[D]-[one-turn RSS]-[two-turn RSS]-[J lambda], wherein said RSSs 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 RSS]-[two-turn RSS]-[J lambda], wherein said RSSs are in an opposite orientation;
• (j) [V kappa]-[two-turn RSS]-[one-turn RSS]-[JH or J lambda], wherein said RSSs are in an opposite orientation.