EP2831245A1 - Vertébré non-humain transgénique pour l'expression d'anticorps entièrement humains à commutation isotypique - Google Patents

Vertébré non-humain transgénique pour l'expression d'anticorps entièrement humains à commutation isotypique

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Publication number
EP2831245A1
EP2831245A1 EP13711120.9A EP13711120A EP2831245A1 EP 2831245 A1 EP2831245 A1 EP 2831245A1 EP 13711120 A EP13711120 A EP 13711120A EP 2831245 A1 EP2831245 A1 EP 2831245A1
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EP
European Patent Office
Prior art keywords
human
vertebrate
cell
constant region
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP13711120.9A
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German (de)
English (en)
Inventor
Jasper Clube
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Kymab Ltd
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Kymab Ltd
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Filing date
Publication date
Priority claimed from US13/433,084 external-priority patent/US9445581B2/en
Priority claimed from US13/434,361 external-priority patent/US9253965B2/en
Priority claimed from GB1207814.3A external-priority patent/GB2501753A/en
Priority claimed from GB1208708.6A external-priority patent/GB2502127A/en
Priority claimed from GBGB1216795.3A external-priority patent/GB201216795D0/en
Application filed by Kymab Ltd filed Critical Kymab Ltd
Publication of EP2831245A1 publication Critical patent/EP2831245A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/461—Igs containing Ig-regions, -domains or -residues form different species
    • C07K16/462—Igs containing a variable region (Fv) from one specie and a constant region (Fc) from another
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/15—Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/01—Animal expressing industrially exogenous proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/20—Pseudochromosomes, minichrosomosomes
    • C12N2800/204—Pseudochromosomes, minichrosomosomes of bacterial origin, e.g. BAC
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT

Definitions

  • the present invention relates to the humanisation of antibodies and heavy chains in a way that produces useful human heavy chain repertoires by harnessing endogenous control of junctional mutation and affinity maturation in the context of desired constant regions in vivo in non-human vertebrate systems.
  • the invention provides non-human vertebrates, cells, populations and methods useful for humanising chimaeric antibodies and heavy chains in vivo.
  • Using the present invention it is possible straightforwardly and rapidly to obtain antigen-specific antibodies and heavy chains that are fully human (ie, comprising human variable and constant regions) and have undergone junctional mutation and affinity maturation in vivo by harnessing a mouse or other non- human vertebrate system.
  • antibodies and heavy chains are humanised - and selected - totally in vivo, and as such the present invention harnesses in vivo filtering for expressibility (and provides possibly for aspects of affinity and biophysical characteristics to be factored into in vivo selection) in the context of the desired human variable and constant region pairings. This is avoids problems of down-grading antibody characteristics when humanising the constant region of chimaeric antibodies and heavy chains in vitro.
  • the present invention allows the skilled person to select antibodies and heavy chains directly in the format that they will be used for subsequent human therapeutic and prophylactic use.
  • the state of the art provides methods for producing antibodies in vitro (eg, using phage, ribosome or yeast display) or in vivo (eg, using non-human vertebrates (eg, mice and rats) and cells comprising transgenic immunoglobulin loci).
  • in vivo systems eg, XenomouseTM
  • human transgenic heavy chain loci which comprise human variable regions (human VH, D and JH gene segments) upstream of human constant regions (eg, human mu upstream of human gamma constant gene segments).
  • human variable regions human VH, D and JH gene segments
  • constant regions eg, human mu upstream of human gamma constant gene segments
  • transgenic animals eg, the VelocimouseTM
  • VelocimouseTM chimaeric heavy chain loci in which a human variable region is upstream of endogenous (eg, mouse or rat) constant regions (ie, mouse mu constant region upstream of gamma constant region, in germline configuration).
  • endogenous eg, mouse or rat
  • constant regions ie, mouse mu constant region upstream of gamma constant region, in germline configuration
  • transgenic vertebrates and use of these to generate antibodies and nucleic acids thereof following antigen immunisation are known in the art, eg, see US7501552 (Medarex); US5939598 & US6130364 (Abgenix); WO02066630, WO2011163311 & WO2011163314 (Regeneron); WO2011004192 & WO2011158009 (Kymab Limited); WO2009076464,
  • US2008/0196112A1 discloses transgenic animals comprising a single, predetermined human rearranged VDJ from a lead antibody, together with one or more human constant region genes in a locus. There are no repertoires of unrearranged V, D and J and recombination and junctional diversity to produce a repertoire of VH domains and H chains and antibodies is not addressed.
  • antigen-binding affinity e.g, in cell lines such as CHO or HEK293 cells
  • half-life and/or biophysical characteristics eg, melting temperature, solution state, resistance to aggregation etc
  • biophysical characteristics e.g, melting temperature, solution state, resistance to aggregation etc
  • the present invention provides a solution by providing for the recombination, humanisation and selection of antibodies entirely in vivo by harnessing the control mechanisms of non-human vertebrate systems.
  • the invention provides non-human vertebrates, cells, populations and methods useful for humanising chimaeric antibodies to produce fully human antibodies that have undergone variable region gene segment recombination, junctional mutation, somatic hypermutation and isotype switching in vivo and that are selected by an in vivo system.
  • Using the present invention it is possible straightforwardly and rapidly to obtain antigen-specific antibodies that are fully human (ie, comprising human variable and constant regions) and have undergone recombination, affinity maturation in vivo.
  • Such antibodies are humanised - and selected - totally in vivo, and as such the present invention harnesses in vivo filtering for expressibility (and affinity and biophysical characteristics factors) in the context of the desired human variable and constant region pairings. This is avoids problems of down-grading antibody characteristics when humanising the constant region of chimaeric antibodies in vitro.
  • the present invention does this in a way that harnesses endogenous control of B-cell development and antibody production and selection.
  • the invention achieves this by ensuring that these functions are carried out in situ in a non-human vertebrate system in an endogenous IgH locus where heavy chain development and selection passes through an IgM stage that is matched with the non-human vertebrate system.
  • the invention adapts an endogenous IgH locus by targeted insertion of unrearranged human variable region gene segments therein in functional relationship upstream of an endogenous mu constant region (ie, the endogenous Cmu or an inserted exogenous Cmu of the same species of non-human vertebrate).
  • endogenous control of recombination and IgM heavy chain development and selection can be harnessed to provide a useful pool of properly developed B-cells expressing chimaeric human V/endogenous Cmu heavy chains.
  • Provision of such a good repertoire of cell-surface expressed IgM heavy chains is advantageous since it provides the non-human system with a useful and diverse repertoire from which to select and clonally mature B-cells for subsequent isotype switching to a human non-mu constant region.
  • a useful repertoire of human non-mu (eg, gamma) heavy chains is provided that have undergone rigorous selection by the endogenous non-human system in vivo for antigen binding and expressibility.
  • the skilled person is thus able to select and isolate one or more desired heavy chains (or antibodies comprising a heavy chain) that are specific to a target antigen, knowing that these have been selected in a totally human format for compatibility with in vivo systems.
  • one or more biophysical characteristics eg, melting temperature, low aggregatability, expressibility etc as will be known by the skilled person
  • a non-human vertebrate eg, a rodent, a mouse, a rat or a rabbit
  • a non-human vertebrate cell eg, a rodent cell, a mouse cell, a rat cell or rabbit cell
  • whose genome comprises a gene locus for expression of antibody heavy chains, the locus comprising
  • an endogenous mu constant region for expression of IgM antibody heavy chains comprising endogenous mu heavy chain constant domains and human variable domains;
  • variable region is provided as a targeted insertion of the human variable region gene segments upstream of the endogenous mu constant region in an endogenous IgH locus such that the variable region gene segments are able to recombine for expression and selection in the context of an endogenous mu constant region.
  • the mu constant region is not endogenous, but is of a different non-human vertebrate species (ie, different to the species of vertebrate or cell).
  • the vertebrate or cell is a mouse and the mu constant region is a non-mouse rodent (eg, rat) mu constant region.
  • the vertebrate or cell is a rat and the mu constant region is a non-rat rodent (eg, mouse) mu constant region.
  • the vertebrate or cell is of a first non-human vertebrate species and the mu constant region is of a second (ie, different) non-human vertebrate species.
  • the mu constant region is of the same non-human species as the vertebrate or cell, but is of a different strain (eg, different mouse or rat strain where the vertebrate is a mouse or rat, or the cell is a mouse or rat cell).
  • a different strain eg, different mouse or rat strain where the vertebrate is a mouse or rat, or the cell is a mouse or rat cell.
  • the invention also provides embodiments wherein the IgH locus is provide not by targeted insertion, but by insertion of a transgene and random integration into the genome or integration in a location that is not the endogenous wild-type IgH locus.
  • an IgH locus comprising human variable region and human non-mu constant region can be provided in a yeast artificial chromosome (YAC), PAC or other vector known in the art for accommodating large inserts.
  • YAC yeast artificial chromosome
  • PAC yeast artificial chromosome
  • the vector can be introduced into an ES cell (eg, mouse or rat ES cell) and the IgH locus can integrate into the genome of the cell.
  • the humanised non-mu constant region is totally human (ie, comprises only human constant domain genes).
  • a non-human vertebrate eg, a rodent, a mouse, a rat or a rabbit
  • a non-human vertebrate cell eg, a rodent cell, a mouse cell, a rat cell or rabbit cell
  • whose genome comprises a gene locus for expression of antibody heavy chains, the locus comprising
  • an endogenous mu constant region for expression of IgM antibody heavy chains comprising endogenous mu heavy chain constant domains and human variable domains;
  • variable region gene segments are able to recombine for expression and selection in the context of an endogenous mu constant region.
  • the invention also provides:-
  • a non-human vertebrate eg, a mouse or a rat
  • a non-human vertebrate cell eg, a mouse cell or a rat cell
  • whose genome comprises a gene locus for expression of antibody heavy chains, the locus comprising (a) a human variable region comprising human variable region gene segments for expression of human variable domains;
  • an endogenous mu constant region for expression of IgM antibody heavy chains comprising endogenous mu heavy chain constant domains and human variable domains;
  • the invention provides a non-human vertebrate (eg, a mouse or a rat) or a non-human vertebrate cell whose genome comprises an antibody heavy chain locus comprising (in 5' to 3' direction) a variable region, a first switch , an endogenous mu constant region, a second switch and a human non-mu (eg, gamma) constant region; wherein the heavy chain locus of each cell is capable of undergoing switching from IgM to the non-mu (eg, IgG) isotype.
  • a non-human vertebrate eg, a mouse or a rat
  • a non-human vertebrate cell whose genome comprises an antibody heavy chain locus comprising (in 5' to 3' direction) a variable region, a first switch , an endogenous mu constant region, a second switch and a human non-mu (eg, gamma) constant region; wherein the heavy chain locus of each cell is capable of undergoing switching from IgM to
  • the invention retains the ability to harness the endogenous antibody and B-cell development control mechanisms of the non-human vertebrate or cell.
  • TdT terminal deoxynucleotidyl transferase
  • this entails switching to a human constant region in resulting antibodies, which is beneficial for producing drugs for human therapeutic use.
  • a vertebrate of the invention can be immunised with a predetermined therapeutic target antigen (eg, a human, viral or bacterial antigen) and antibodies (eg, human IgG) or heavy chains (eg, human gamma-type heavy chains) can be isolated that (i) have undergone junctional and somatic mutation, and thus affinity maturation in vivo in response to antigen challenge, (ii) are fully human, ie, have human variable and constant domains, and (iii) have been selected by a totally in vivo system for expression and ability to bind antigen.
  • a predetermined therapeutic target antigen eg, a human, viral or bacterial antigen
  • antibodies eg, human IgG
  • heavy chains eg, human gamma-type heavy chains
  • the invention employs in vivo systems to direct the skilled person to viable human antibodies and heavy chains that can be expressed well in vivo and which are specific for a predetermined antigen.
  • the method is simple and avoids multi-step combinations of in vivo and in vitro production and engineering as per the prior art methods.
  • the selected antibodies have had their antigen binding affinities tuned by nature - rather than by man.
  • vertebrates and cells of the invention provide good sources for fully human antigen-specific antibodies and heavy chains with therapeutically-amenable affinities without the need to perform laborious in vitro affinity maturation (or risk downgrading desirable characteristics due to in vitro manipulation).
  • the present invention enables the skilled person to design vertebrates and cells to produce fully human antibodies of a specific, desired isotype in a predictable way.
  • the skilled person is able to design the heavy chain loci in a vertebrate of the invention so that all gamma constant region gene segments are human ones.
  • the skilled person will know that all IgG-type antibodies or heavy chains selected from the vertebrate will be fully human in their H chains and will have been subjected to endogenous antibody development (eg, affinity maturation) and in vivo expression and selection. Going a step further, the skilled person could design a vertebrate in which just the IgGl isotype gene segments are human.
  • IgGl isotype antibodies will be fully human and matured, expressed and selected in vivo. This is useful for tailoring antibody sub-types, such as IgGl or lgG4, to particular human therapeutic indications, as explained further below.
  • the gene locus of the invention comprises only human constant regions, with the exception of the endogenous mu constant region. This is useful for producing isotype- switched heavy chains in which the constant regions are predictably always human.
  • the gene locus of the invention comprises only human constant region gene segments, with the exception of the constant region gene segments of the endogenous mu constant region.
  • the skilled person will reliably know at the outset that all switched heavy chains and antibodies will bear human heavy chain constant domains.
  • the gene locus of the invention comprises gamma constant region gene segments and all of the gamma constant region gene segments are human. This is useful for producing isotype-switched gamma heavy chains in which the constant regions are predictably always human.
  • the gene locus of the invention comprises gamma constant region gene segments and all of the delta constant region gene segments are human. This is useful for producing isotype-switched delta heavy chains in which the constant regions are predictably always human.
  • the gene locus of the invention comprises epsilon constant region gene segments and all of the delta constant region gene segments are human. This is useful for producing isotype-switched epsilon heavy chains in which the constant regions are predictably always human.
  • the gene locus of the invention comprises alpha constant region gene segments and all of the delta constant region gene segments are human. This is useful for producing isotype- switched alpha heavy chains in which the constant regions are predictably always human.
  • the invention also enables the skilled person to tailor other characteristics of antibody constant regions to particular therapeutic or prophylactic uses.
  • the variants may encode mutant human Fc regions that are either activated (eg, for cell-killing applications such as oncology or treatment of inflammatory conditions) or inactivated (eg, when activation due to cross-linking of cell-surface antigens by antibody is undesirable, eg, as seen when the target antigen is CD40 or CD40 ligand).
  • the application recognises the desirability of exploring repertoires of variant human constant domains of antibodies when paired and selected with human variable domains in vivo. Such variants are seen in different populations of humans (eg, encoded by different variants of gamma constant region gene segments as seen in different ethnic human populations). Similarly, this concept can be applied to synthetic variants of human constant domains and corresponding human constant region gene segments (eg, variants that have been mutated in different ways to inactivate Fc, or to activate Fc). To this end, the invention provides:-
  • a population of vertebrates of the invention wherein the population comprises (i) a first vertebrate wherein the antibody heavy chain locus comprises one or more first human non-mu gene segment(s) (eg, a human CHI, CH2 or CH3; or an Fc);
  • first human non-mu gene segment(s) eg, a human CHI, CH2 or CH3; or an Fc
  • the antibody heavy chain locus comprises one or more second human non-mu gene segment(s) (eg, a human CHI, CH2 or CH3; or encoding an Fc); and wherein the first and second gene segments are the same type of constant region gene segment(s) (eg, both CHI gene segments or both encoding an Fc) and the second gene segment(s) is a variant of the first gene segment(s).
  • second human non-mu gene segment(s) eg, a human CHI, CH2 or CH3; or encoding an Fc
  • the first and second gene segments are the same type of constant region gene segment(s) (eg, both CHI gene segments or both encoding an Fc) and the second gene segment(s) is a variant of the first gene segment(s).
  • the immunoglobulin loci of the vertebrates of the population differ only in the repertoire of said human constant regions or constant region gene segments.
  • the invention also provides a method of isolating a human antibody, the method comprising immunising a vertebrate or population of vertebrates according to any preceding claim with an antigen and isolating an antibody from said vertebrate or a vertebrate of the population, wherein the isolated antibody specifically binds to the antigen and comprises a fully human heavy chain of said non-mu isotype.
  • the invention also provides:-
  • a method of obtaining a humanised and affinity matured antigen-specific antibody heavy chain comprising humanising the heavy chain in vivo in a non-human vertebrate (eg, a mouse or a rat) comprising functional RAG and activation induced cytidine deaminase (AID) by immunising the vertebrate with the antigen and obtaining recombination of VH,D and JH gene segments in vivo, somatic hypermutation and isotype switching in a B-cell of the vertebrate from an endogenous mu isotype to a human non-mu isotype, wherein a repertoire of affinity matured antigen-specific non- mu antibody heavy chains are produced and expressed by the vertebrate, the non-mu constant domains of the heavy chains being human constant domains, the method further comprising isolating one or more of said humanised heavy chains.
  • a non-human vertebrate eg, a mouse or a rat
  • AID functional RAG and activation
  • non-human vertebrate ES cell eg, an ES cell disclosed herein
  • the skilled person will be able to provide for the provision of functional RAG and AID. These will be matched with the endogenous mu for good harnessing of the endogenous control mechanisms.
  • a method of obtaining a humanised and affinity matured antigen-specific antibody heavy chain comprising humanising the heavy chain in vivo in a non-human vertebrate (eg, a mouse or a rat) comprising a functional activation induced cytidine deaminase (AID) by immunising the vertebrate with the antigen and obtaining somatic hypermutation and isotype switching in a B-cell of the vertebrate from an endogenous mu isotype to a human non-mu isotype, wherein an affinity matured antigen-specific antibody heavy chain is produced and expressed by the vertebrate, the non-mu constant domains of the heavy chain being human constant domains, the method further comprising isolating said humanised heavy chain.
  • a non-human vertebrate eg, a mouse or a rat
  • AID functional activation induced cytidine deaminase
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the isolated antibody heavy chain obtained in the method of the invention or a copy or derivative thereof; optionally wherein the heavy chain, copy or derivative is provided by an antibody that specifically binds the antigen.
  • the invention also provides a method of treating or preventing a medical condition or disease in a human associated or caused by said antigen, the method comprising administering to the human the antigen-specific antibody or heavy chain obtained in the method described above.
  • the invention also provides the antigen-specific antibody or heavy chain obtained in the method of the invention for use in the treatment or prophylaxis of a medical condition or disease in a human associated or caused by said antigen.
  • the invention also provides the use of the antigen-specific antibody or heavy chain obtained in the method of the invention in the manufacture of a medicament for use in the treatment or prophylaxis of a medical condition or disease in a human associated or caused by said antigen.
  • FIGURES
  • Figure 1 is a schematic showing the arrangement of the constant regions in wild-type human and mouse genomes (Dev Comp Immunol. 2006; 30(l-2):119-35; Schroeder HW Jr);
  • Figure 2A-2D is a schematic showing the arrangement of gamma, delta, alpha, mu and epsilon isotype human constant regions as found in genomic heavy chain loci (see I MGT database;
  • Figure 3A-3D is a schematic showing the arrangement of gamma, delta, alpha, mu and epsilon isotype mouse constant regions as found in genomic heavy chain loci (see IMGT database;
  • the present invention relates to humanisation of antibodies in vivo.
  • the invention provides non-human vertebrates, cells, populations and methods useful for humanising chimaeric antibodies in vivo.
  • antigen-specific antibodies that are fully human (ie, comprising human variable and constant regions) and have undergone affinity maturation in vivo.
  • antigen-specific antibodies that are fully human (ie, comprising human variable and constant regions) and have undergone affinity maturation in vivo.
  • such antibodies are humanised - and selected - totally in vivo, and as such the present invention harnesses in vivo filtering for expressibility (and factors in affinity and biophysical characteristics) in the context of the desired human variable and constant region pairings. This is avoids problems of down-grading antibody characteristics when humanising the constant region of chimaeric antibodies in vitro.
  • 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 gene locus for expression of antibody heavy chains, the locus comprising (a) a human variable region comprising human variable region gene segments for expression of human variable domains;
  • an endogenous mu constant region for expression of IgM antibody heavy chains comprising endogenous mu heavy chain constant domains and human variable domains;
  • a humanised non-mu constant region downstream of the mu constant region for expression of non-mu antibody heavy chains comprising human non-mu constant domains and human variable domains.
  • the locus is, in the preferred embodiment, a heavy chain locus of the genome, ie, it is in the germline location of a heavy chain locus of said type of vertebrate or cell.
  • the humanised locus of the invention can be constructed by inserting human constant region DNA into the endogenous (ie, non-human vertebrate) heavy chain locus downstream of the endogenous mu constant region.
  • the locus of the invention is synthetically constructed elsewhere in the genome (ie, not in the endogenous heavy chain locus location) and may be on a different chromosome to that chromosome harbouring the heavy chain locus in a wild-type vertebrate or cell.
  • the locus of the invention is constructed at the Rosa26 locus.
  • the gene locus of the invention comprises all of the elements required for expression of heavy chains and isotype switching in the vertebrate or cell, such a human variable region comprising a plurality of of VH gene segments, a plurality of D gene segments and a plurality of JH gene segments upstream of (ie, 5' of) a S-mu switch (eg, the endogenous S-mu), an endogenous mu constant region comprising endogenous mu constant gene segments (eg, CHI, CH2 and CH3), a second switch (eg, an endogenous switch; eg, a gamma switch) and a (ie, one or more) human non-mu constant region.
  • a human variable region comprising a plurality of of VH gene segments, a plurality of D gene segments and a plurality of JH gene segments upstream of (ie, 5' of) a S-mu switch (eg, the endogenous S-mu), an endogenous mu constant region comprising endogenous
  • the human non-mu constant region is a gamma-1 constant region comprising from CH2 to (and including) the poly-A 3' of M2 as shown in figure 3.
  • the non-mu constant region is a gamma-1 constant region comprising from CHI to (and including) the poly-A 3' of M2 as shown in figure 3.
  • the non-mu constant region is a gamma-1 constant region comprising from the hinge region (H) to (and including) the poly-A 3' of M2 as shown in figure 3.
  • the human constant region lacks a CHI gene segment but includes one or more other human constant gene segments (eg, CH2 and CH3); optionally wherein the constant region is a gamma constant region.
  • a human gamma (eg, gamma-1, gamma-2, gamma-3 or gamma-4) constant region
  • a human gamma (eg, gamma-1, gamma-2, gamma-3 or gamma-4) constant region
  • a human gamma (eg, gamma-1, gamma-2, gamma-3 or gamma-4) constant region
  • a human gamma (eg, gamma-1, gamma-2, gamma-3 or gamma-4) constant region
  • a human gamma (eg, gamma-1, gamma-2, gamma-3 or gamma-4) constant region excluding a CHI gene segment;
  • a human gamma (eg, gamma-1, gamma-2, gamma-3 or gamma-4) constant region
  • CH I (and optionally also a CH2 and CH3) gene segment
  • a human gamma (eg, gamma-1, gamma-2, gamma-3 or gamma-4) constant region
  • a human gamma (eg, gamma-1, gamma-2, gamma-3 or gamma-4) constant region
  • a human gamma (eg, gamma-1, gamma-2, gamma-3 or gamma-4) constant region
  • a human delta constant region comprising or consisting of the sequence from CH I to (and including) CH3, CH-S, M l or M2;
  • a human delta constant region comprising or consisting of the sequence from hinge H I or H2 to (and including) CH3, CH-S, M l or M2;
  • a human delta constant region comprising or consisting of the sequence from CH2 to (and including) CH3, CH-S, M l or M2;
  • a human delta constant region comprising or consisting of the sequence from CH3 to (and including) CH-S, M l or M2;
  • a human delta constant region comprising a CHI (and optionally also a CH2 and CH3) gene segment;
  • a human delta constant region comprising a CH2 (and optionally also a CH3) gene segment;
  • a human delta constant region comprising a CH3 gene segment
  • a human delta constant region comprising CH2-CH3 gene segments and excluding a CHI gene segment
  • a human alpha constant region comprising or consisting of the sequence from CH I to (and including) CH3, M or the poly-A im mediately 3' of M or CH3;
  • a human alpha constant region comprising or consisting of the sequence from hinge (H) to (and including) CH3, M or the poly-A im mediately 3' of M or CH3;
  • a human alpha constant region comprising or consisting of the sequence from CH2 to (and including) CH3, M or the poly-A im mediately 3' of M or CH3;
  • a human alpha constant region comprising or consisting of the sequence from CH3 to (and including) M or the poly-A im mediately 3' of M or CH3; 23. A human alpha constant region excluding a CHI gene segment;
  • a human alpha constant region comprising a CHI (and optionally also a CH2 and CH3) gene segment;
  • a human alpha constant region comprising a CH2 (and optionally also a CH3) gene segment;
  • a human alpha constant region comprising a CH3 gene segment
  • a human alpha constant region comprising CH2-CH3 gene segments and excluding a CHI gene segment
  • the alpha constant region is, in one example, a human alpha-1 constant region. In another example the constant region is a human alpha-2 constant region.
  • a human epsilon constant region comprising or consisting of the sequence from CHI to (and including) CH3, CH4, M or the poly-A immediately 3' of M or CH4;
  • a human epsilon constant region comprising or consisting of the sequence from hinge (H) to (and including) CH3, CH4, M or the poly-A immediately 3' of M or CH4;
  • a human epsilon constant region comprising or consisting of the sequence from CH2 to (and including) CH3, CH4, M or the poly-A immediately 3' of M or CH4;
  • a human epsilon constant region comprising or consisting of the sequence from CH3 to (and including) CH4, M or the poly-A immediately 3' of M or CH4;
  • a human epsilon constant region comprising or consisting of the sequence from CH4 to (and including) M or the poly-A immediately 3' of M or CH4;
  • a human epsilon constant region comprising a CHI (and optionally also a CH2, CH3 and CH4) gene segment;
  • a human epsilon constant region comprising a CH2 (and optionally also a CH3 and CH4) gene segment; 36. A human epsilon constant region comprising a CH3 (and optionally also a CH4) gene segment;
  • a human epsilon constant region comprising a CH4 gene segment
  • a human epsilon constant region comprising CH2-CH3-CH4 gene segments and excluding a CHI gene segment.
  • a nucleotide sequence comprising or consisting of the sequence from human gamma-3 to (and including) human gamma-4 constant regions;
  • a nucleotide sequence comprising or consisting of the sequence from human gamma-3 to (and including) human gamma-2 constant regions;
  • a nucleotide sequence comprising or consisting of the sequence from human gamma-3 to (and including) human gamma-1 constant regions;
  • a nucleotide sequence comprising or consisting of the sequence from human gamma-1 to (and including) human gamma-4 constant regions;
  • a nucleotide sequence comprising or consisting of the sequence from human gamma-1 to (and including) human gamma-2 constant regions;
  • a nucleotide sequence comprising or consisting of the sequence from human gamma-2 to (and including) human gamma-4 constant regions;
  • a nucleotide sequence comprising or consisting of the sequence from human delta to (and including) human alpha-2 constant regions;
  • a nucleotide sequence comprising or consisting of the sequence from human delta to (and including) human epsilon regions;
  • a nucleotide sequence comprising or consisting of the sequence from human delta to (and including) human gamma-4 constant regions;
  • a nucleotide sequence comprising or consisting of the sequence from human delta to (and including) human gamma-1 constant regions;
  • a nucleotide sequence comprising or consisting of the sequence from human delta to (and including) human gamma-3 constant regions.
  • Stretches of human DNA can be inserted in one piece or in serial insertions into the genome of a non-human vertebrate or cell (eg, ES cell) genome.
  • a plurality of bacterial artificial chromosomes can be constructed using standard recombineering techniques which between them comprise the entire sequence of human constant region DNA to be inserted, each BAC containing a portion of that stretch of DNA.
  • serial insertions from serial BACs eg, using standard homologous recombination (eg, see the Regeneron and Ablexis PCTs disclosed herein) or serial recombinase mediated cassette exchange - sRMCE - (eg, see the Kymab Limited PCTs disclosed herein) the skilled person can build up the one or more human constant regions downstream of the endogenous mu constant region in the genome.
  • serial BACs eg, using standard homologous recombination (eg, see the Regeneron and Ablexis PCTs disclosed herein) or serial recombinase mediated cassette exchange - sRMCE - (eg, see the Kymab Limited PCTs disclosed herein)
  • the skilled person can build up the one or more human constant regions downstream of the endogenous mu constant region in the genome.
  • the ES cell can be used, as is standard, to implant in a donor blastocyst, which is then implanted into a foster
  • the skilled person can arrive at progeny vertebrates and cells bearing the gene locus of the invention in which one or a plurality of human non-mu constant regions are downstream of an endogenous mu constant region.
  • the gene locus may be present in a homozygous state (eg, a gene locus of the invention at each heavy chain allele) or heterozygous state (eg, with the second allele being an inactivated endogenous heavy chain so that only heavy chain expression from the first allele - bearing the gene locus of the invention - provides expression of heavy chains).
  • different gene loci of the invention are provided at the first and second alleles of the heavy chain locus in the genome, so that different combinations of human isotype switched heavy chains can be expressed by the combination of loci.
  • the genome also comprises transgenic light chain loci that express human light chains (or in the alternative at least human light chain variable regions) and endogenous light chain expression is substantially inactive; also endogenous heavy chain expression is substantially inactive.
  • This embodiment advantageously provides a genome for predictable expression of only fully human antibodies (or at least fully human heavy chains and fully human light chain variable regions in the alternative) and no endogenous antibody chains, from which pool of antibodies the skilled person can select (eg, after immunisation with target antigen) non-mu type antibodies (eg, gamma type) that have undergone mutation and maturation and selection by the in vivo machinery of the vertebrate or cell.
  • human IgG sub-types IgGl, lgG2, gG3 and lgG4 exhibit differential capacity to recruit immune functions, such as antibody-dependent cellular cytotoxicity (ADCC, e.g., IgGl and lgG3), antibody-dependent cellular phagocytosis (ADCP, e.g., IgGl, lgG2, lgG3 and lgG4), and complement dependent cytotoxicity (CDC, e.g., IgGl, lgG3).
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • CDC complement dependent cytotoxicity
  • Sub-type-specific engagement of such immune functions is based on selectivity for Fc receptors on distinct immune cells and the ability to bind Clq and activate the assembly of a membrane attack complex (MAC).
  • MAC membrane attack complex
  • relative affinity for FcY receptors is high for IgGl and lgG3, however, there is minimal affinity for lgG2 (restricted to the FcYRIIa 131H polymorphism), and lgG4 only has measurable affinity for FcYRI.
  • the key contact residues for receptor binding have been mapped to the amino acid residues spanning the lower hinge and CH2 region.
  • standard protein engineering techniques some success in enhancing or reducing the affinity of an antibody preparation for Fc receptors and the Clq component of complement has been achieved.
  • lgG2 is least capable of binding the family of Fc receptors.
  • lgG2 as the starting point, efforts have been made to find a mutant with diminished effector functions but which retains FcRn binding, prolonged stability, and low immunogenicity.
  • Improved mutants of this nature may provide improved antibody therapeutics with retained safety.
  • Human IgGl therapeutic antibodies that bind to cell surface targets are able to engage effector cells that may mediate cell lysis of the target cell by antibody-dependent cellular cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). These mechanisms occur through interaction of the CH2 region of the antibody Fc domain to FcyR receptors on immune effector cells or with Clq, the first component of the complement cascade.
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement dependent cytotoxicity
  • Table 1 shows the activities of different human gamma sub-types. The skilled person may choose accordingly to promote or dampen-down activity depending upon the disease setting in humans of interest. For example, use of an insertion comprising human gamma-1 constant region is desirable when one wishes to isolated totally human heavy chains and antibodies that have relatively high complement activation activity by the classical pathway and FcYRl recognition in human patients.
  • Table 1 Summary of function correlated with human gamma sub-
  • lgG2 constant regions are well suited to producing antibodies and heavy chains according to the invention for binding to cytokines or soluble targets in humans, since lgG2 is essentially FcYRIJI I silent, FcYRIIa-active and has little Complement activity.
  • IgGl constant regions have wide utility for human therapeutics, since IgGl antibodies and heavy chains are FcYRI,ll,lll- active and have complement activity. This can be enhanced by using a human gamma-1 constant region that has been activated by engineering as is known in the art.
  • the human non-mu constant region comprises CH2 or CH2 and CH3 gene segments encode for an activated Fc region. This is usseful for, eg, treating or preventing inflammation or a inflammatory disease or condition. This embodiment is also useful where cell killing in the human patient is desirable, eg, for oncology applications.
  • the human non-mu constant region comprises CH2, or CH2 and CH3, gene segments encode for an inactivated Fc region.
  • This is usseful for, eg, applications where triggering following cross-linking is undesirable. This may be useful, for example where cross-linking of cell-surface antigens is to be avoided after administration of an antibody produced according to the invention (eg, where the target antigen against which the antibody or heavy chain is raised is a T-cell costimulatory antigen such as CD40 or CD40 ligand).
  • constant region and gene segments to include in the human constant region(s) may also take into account the desirability for good binding to human FcRn in the human patient, so that the heavy chain or antibody comprising this is efficiently recycled and a desirable drug half-life is achieved.
  • gamma-type human constant regions are desirable, since IgG antibodies and heavy chains bind efficiently to human FcRn in vivo.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcRs Fc receptors
  • cytotoxic cells e.g., Natural Killer (NK) cells, neutrophils, and macrophages
  • Ligand specific high-affinity IgG antibodies directed to the surface of target cells stimulate the cytotoxic cells and are required for such killing. Lysis of the target cell is extracellular, requires direct cell-to-cell contact, and does not involve complement.
  • ADCC ability of any particular antibody to mediate lysis of the target cell by ADCC can be assayed.
  • an antibody of interest is added to target cells displaying the target ligand in combination with immune effector cells, which may be activated by the antigen antibody complexes resulting in cytolysis of the target cell. Cytolysis is generally detected by the release of label (e.g., radioactive substrates, fluorescent dyes or natural intracellular proteins) from the lysed cells.
  • label e.g., radioactive substrates, fluorescent dyes or natural intracellular proteins
  • useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • ADCC activity of the antibody of interest may be assessed in vivo, e.g., in an animal model, such as that disclosed in Clynes et al, 1998, PNAS USA 95:652, the contents of which are incorporated by reference in its entirety.
  • “Complement-directed cytotoxicity” or CDC refers to the form of cytotoxicity in which the complement cascade is activated by the complement component Clq binding to antibody Fc.
  • Fc refers to the protein comprising (in N- to C-terminal direction) an immunoglobulin CH2 and CH3 domain.
  • the CH2 and CH3 domains can form at least a part of the constant region of an antibody or heavy chain.
  • Constant region as used herein when referring to the gene locus of the invention, is a reference to a stretch of DNA sequence comprising gene segments (constant region gene segments) that combine in vivo with recombined VDJ sequence in the locus.
  • the VDJ yield (optionally following hypermutation), together with the constant region gene segments RNA transcripts in the vertebrate or cell from which can be produced antibody heavy chains (eg, following splicing of RNA), each heavy chains comprising an variable domain and one or more constant domains.
  • antibody heavy chains eg, following splicing of RNA
  • Endogenous indicates that the constant region etc is a type of constant region etc that is normally found in the vertebrate or cell (as opposed to an exogenous constant region whose sequence is not normally found in such a vertebrate or cell, eg a human sequence).
  • the endogenous constant region can be those encoded by the wild-type genome of the non-human vertebrate or cell. So, in an example wherein the vertebrate is a mouse, the endogenous constant region would be a mouse constant region. Going further, the endogenous regions are, in an example, strain-matched to the vertebrate or cell.
  • the vertebrate or cell is a mouse 129 ES cell, the endogenous constant region would be mouse 129 constant region.
  • the vertebrate or cell is a JM8 strain mouse or mouse cell, the endogenous constant region would be mouse JM8 constant region.
  • the vertebrate or cell is a Black 6 mouse or mouse cell, the endogenous constant region would be mouse Black 6 constant region.
  • the gene locus in the vertebrate or cell of the invention comprises (in 5' to 3' direction) one or more membrane-encoding exons (M) and a polyA downstream of the non-human human constant region gene segments, wherein the vertebrate is capable of expressing membrane- bound heavy chain or antibody of said non-mu isotype. Additionally or alternatively, the locus comprises a polyA immediately downstream of the last human constant region gene segment. The locus may further comprise a splice site between the latter polyA and the one or more membrane- encoding exons, wherein the vertebrate or cell is capable of expressing both membrane-bound and secreted forms of the non-mu heavy chain or antibody.
  • the locus comprises a switch 5' of the mu constant region (eg, the endogenous S-mu is retained) and a second switch 5' of the (or each) human non-mu constant region.
  • the second switch can be the human switch usually present with the human constant region (eg, when the human constant region is a gamma-1 constant region, the human gamma-1 switch is retained).
  • the switch can be the corresponding endogenous non-human vertebrate switch, in this example an endogenous S- gamma (eg, S-gamma-1), eg, where a nucleotide sequence corresponding to human DNA from CHI to the M2 or polyA 3' of M2 of a human gamma constant region is inserted downstream of an endogenous S-gamma in the vertebrate or cell genome.
  • an endogenous S- gamma eg, S-gamma-1
  • a nucleotide sequence corresponding to human DNA from CHI to the M2 or polyA 3' of M2 of a human gamma constant region is inserted downstream of an endogenous S-gamma in the vertebrate or cell genome.
  • Switch sequences are known in the art, for example, see Nikaido et al, Nature 292: 845-848 (1981) and also WO2011004192, US7501552, US6673986, US6130364, WO2009/076464 and US6586251, eg, SEQ ID NOs: 9-24 disclosed in US7501552.
  • Commercial human and mouse BAC libraries eg, the RPC-11 and Caltech A, B, C and D libraries
  • suitable immunoglobulin locus sequences eg, sequences of switches or constant regions.
  • human DNA samples can be obtained de novo from consenting donors (eg, using cheek swabs) and DNA sequences obtained from the samples.
  • the vertebrate or cell of the invention comprises an antibody heavy chain locus that comprises (in 5' to 3' direction) a rearranged or unrearranged variable region, a first switch (eg, an S-mu switch, eg an endogenous S-mu), an endogenous mu constant region, a second switch (an S-gamma switch; eg, endogenous S-gamma) and a human constant region of said non-mu isotype; wherein the heavy chain locus is capable of undergoing switching from IgM to the non-mu isotype.
  • a first switch eg, an S-mu switch, eg an endogenous S-mu
  • an endogenous mu constant region e.g, an endogenous mu constant region
  • a second switch an S-gamma switch
  • a human constant region of said non-mu isotype e.g, endogenous S-gamma
  • this can be provided by inserting one or more human non-mu constant region sequences downstream (3') of an endogenous mu region in the genome of a non-human vertebrate or cell (eg, ES cell).
  • a non-human vertebrate or cell eg, ES cell.
  • the human sequences are provided within the location of the endogenous heavy chain locus and expression from the human sequence can be effectively controlled using endogenous antibody and B-cell mechanisms.
  • heavy chain variable regions are produced and selected in context of non-human (eg, mouse) mu constant regions.
  • non-human eg, mouse
  • the invention subsequently enables switching to a human non-mu C region, which enables one to produce human constant domains in product heavy chains which have been affinity matured by somatic hypermutation effected by endogenous activation induced cytidine deaminase (AID).
  • variable regions that still work well are selected in vivo by the mouse and amplified following immunisation with a predetermined antigen. This retains the advantage of harnessing endogenous antibody and B-cell production and control, together with affinity maturation and in vivo amplification of selected B-cells expressing desirable antibodies/heavy chains in a way that harnesses the mouse (or other non- vertebrate) system to select for good, fully-human antibodies and heavy chains.
  • the vertebrate or cell of the invention does not express endogenous antibody heavy chains of said non-mu isotype.
  • this is effected using standard methods to delete or inactivate endogenous heavy chain variable and constant regions.
  • heavy chains (and antibodies comprising these) of the predetermined human non-mu isotype eg, IgG antibodies
  • the vertebrate or cell will always comprise human constant regions.
  • the human non-mu constant region is a gamma constant region (eg, gamma-1, -2, 3 or -4).
  • the constant region can be a delta, alpha (eg, alpha-1 or -2) or epsilon constant region.
  • the gamma constant region comprises CH2 and CH3 gene segments for encoding an antibody Fc region.
  • the constant region also comprises a CHI gene segment of the human constant region. This is useful for producing heavy chains that are to form part of 4-chain antibodies (classic H2L2 antibodies).
  • the constant region lacks a CHI gene segment. This is useful for producing heavy chains that are to form part of heavy-chain only antibodies (H2 antibodies) which are devoid of light chains.
  • the genome comprises a light chain locus comprising human VL and JL gene segments upstream of a constant region for expression of light chains comprising human variable regions; optionally wherein the constant region is a human light chain constant region.
  • the genome comprises a light chain locus comprising human VK and JK gene segments upstream of an endogenous or human kappa constant region in the endogenous kappa locus for expression of kappa light chains comprising human variable regions.
  • the vertebrate is a mouse
  • the lambda locus is inactive and/or comprises human VX and JX upstream of an endogenous or human lambda constant region.
  • endogenous kappa chain expression is inactive.
  • the vertebrate or cell does not substantially express endogenous light chains.
  • Inactivation of endogenous antibody, antibody chain or gene segment usage is, for example, substantially complete inactivation or prevention (substantially 100%, ie, essentially none (eg, less than 10, 5, 4, 3, 2, 1 or 0.5%) of the endogenous antibody chain etc (eg, no endogenous heavy chains etc) is expressed). This can be determined, for example, at the antibody chain (protein) level by assessing the antibody repertoire produced by the non-human vertebrate or cell or at the nucleotide level by assessing mRNA transcripts of antibody gene loci, eg, using RACE .
  • inactivation is more than 50% (ie, 50% or less of the antibodies or transcripts are of an endogenous antibody chain), 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%.
  • endogenous heavy chain expression is substantially inactivated such that no more than 85%, 90%, 95%, 96%, 97%, 98% or 99% of the heavy chain repertoire of the vertebrate is provided by endogenous heavy chains.
  • endogenous heavy chain expression is substantially inactivated such that substantially none of the heavy chain repertoire of the vertebrate is provided by endogenous heavy chains.
  • endogenous kappa chain expression is substantially inactivated such that no more than 85%, 90%, 95%, 96%, 97%, 98% or 99% of the kappa chain repertoire of the vertebrate is provided by endogenous kappa chains.
  • endogenous kappa chain expression is substantially inactivated such that substantially none of the kappa chain repertoire of the vertebrate is provided by endogenous kappa chains.
  • endogenous lambda chain expression is substantially inactivated such that no more than 85%, 90%, 95%, 96%, 97%, 98% or 99% of the lambda chain repertoire of the vertebrate is provided by endogenous lambda chains.
  • endogenous lambda chain expression is substantially inactivated such that substantially none of the lambda chain repertoire of the vertebrate is provided by endogenous lambda chains.
  • the human gamma constant region does not comprise a CHI gene segment.
  • light chain expression is inactive when the non-mu heavy chains are expressed. This is useful for producing heavy-chain only antibodies of the non-mu isotype.
  • the locus comprises a replacement of endogenous non-mu constant region gene segments (eg, CH2 and CH3 gene segments) with corresponding human constant region gene segments (eg, CH2 and CH3 gene segments) of the same non-mu isotype; optionally wherein an endogenous switch of said non-mu isotype is retained.
  • endogenous non-mu constant region gene segments eg, CH2 and CH3 gene segments
  • human constant region gene segments eg, CH2 and CH3 gene segments
  • the locus comprises an insertion of a human gamma constant region nucleotide sequence comprising sequence from the human gamma S- gamma CHI, hinge or CH2 gene segment to (and including) the human gamma CH3 gene segment, wherein the insertion replaces a corresponding sequence of an endogenous gamma constant region in the genome of the vertebrate or cell.
  • This is advantageous for maintaining the human CH2 and CH3 and inter-gene segment sequences in relative genomic or germline configuration for proper expression of human non-mu Fc in the product heavy chain or antibodies.
  • Proper Fc function is desirable for Fc receptor interactions and recycling in human patients receiving the chain or antibody or a derivative drug thereof, as discussed above.
  • Replacement here means functional or physical replacement, wherein the endogenous constant region of said non-mu isotype is nonfunctional or absent in the final genome and the human constant region is functional and present.
  • the human constant region is in human germline configuration.
  • the term "germline configuration" refers to a germline genomic configuration.
  • human immunoglobulin gene segments present in the gene locus of the invention are in a germline configuration when the relative order of the human gene segments is the same as the order of corresponding gene segments in a human germline genome.
  • the gene locus of the invention comprises hypothetical human immunoglobulin constant region gene segments A, B and C, these would be provided in this order (5' to 3' in the locus) when the corresponding gene segments of a human germline genome comprises the arrangement 5'-A-B-C-3'; and optionally the inter-gene segments too are in germline configuration (ie, the nucleotide sequence from A to C corresponds to a stretch of contiguous nucleotide sequence from A to C in a human germline genome).
  • available databases and sources of human germline sequence are discussed below.
  • the human Ig locus elements are in germline configuration when the relative order of the elements is the same as the order of corresponding elements in a human germline genome and human sequences between the elements are included, these corresponding to such sequences between corresponding elements in the human germline genome.
  • the transgenic locus comprises human elements in the arrangement 5'-A-Sl-B-S2-C-S3-3', wherein A, B and C are human immunoglobulin gene segments and S1-S3 are human inter-gene segment sequences, wherein the corresponding arrangement 5'-A-Sl-B-S2-C-S3-3' is present in a human germline genome.
  • this can be achieved by providing in a transgenic immunoglobulin locus of the invention a DNA insert corresponding to the DNA sequence from A to C in a human germline genome (or the insert comprising the DNA sequence from A to C).
  • the arrangements in human germline genomes and immunoglobulin loci are known in the art (eg, see the IMGT, Kabat and other antibody resources).
  • endogenous inter-gene segment sequences have been retained so that the heavy chain locus comprises said human non-mu constant region gene segments flanked by endogenous constant region inter-gene segment sequences. This is useful for preserving any endogenous, non-human regulatory elements between gene segments - for control using endogenous mechanisms for class switching and antibody/chain production in the vertebrate or cell.
  • the gene locus comprises a replacement of endogenous gamma constant region gene segments (eg, gamma CH2 and CH3 gene segments) with human gamma constant region gene segments (eg, gamma CH2 and CH3 gene segments), but endogenous sequences between gamma constant regions have been retained. This is useful for preserving any endogenous, non-human regulatory elements between gene segments - for control using endogenous mechanisms for class switching and antibody/chain production in the vertebrate or cell.
  • endogenous gamma constant region gene segments eg, gamma CH2 and CH3 gene segments
  • human gamma constant region gene segments eg, gamma CH2 and CH3 gene segments
  • the invention also provides a general combinatorial or repertoire approach that enables the skilled person to design an antibody/heavy chain production and selection scheme that combines human mutated variable regions with a plurality of different human constant domains (and/or synthetic mutants thereof).
  • This scheme straight-forwardly harnesses the in vivo non-human vertebrate system to perform combinations, affinity maturation, and selection by expression and amplification of target antigen-specific heavy chains that are productive and in a fully human configuration.
  • the skilled person is provided with a way of obtaining a repertoire of matured, target antigen- specific human heavy chain variable domains paired with a repertoire of human constant regions.
  • the skilled person will know that all members of the repertoire represent productive combinations of human variable and constant heavy chain regions, in that they can be expressed to detectable and selectable levels in an in vivo system.
  • the repertoire eg, a repertoire provided in a population of non-human vertebrates of the invention; or a repertoire of B-cells isolated from such vertebrates; or a repertoire of antibodies or heavy chains isolated from such vertebrates or cel ls; or a repertoire of nucleotide sequences encoding such antibodies or heavy chains
  • the skilled person is able to select one or more antibodies or heavy chains (or corresponding nucleotides sequences) that bind the target antigen. Selection is performed on the basis of a desired antibody/chain characteristic, such as antigen binding affinity and/or a biophysical characteristic.
  • said gene locus is an antibody heavy chain locus that comprises one or more first human non-mu gene segment(s) (eg, a human CHI, CH2 or CH3; or an Fc) or a first human non-mu constant region;
  • first human non-mu gene segment(s) eg, a human CHI, CH2 or CH3; or an Fc
  • said gene locus is an antibody heavy chain locus that comprises one or more second human non-mu gene segment(s) (eg, a human CH I, CH2 or CH3; or encoding an Fc) or a second human non-mu constant region; and wherein the first and second gene segments are the same type of constant region gene segment(s) (eg, both CHI gene segments or both encoding an Fc) and the second gene segment(s) is a variant of (ie, is a mutant of) the first gene segment(s).
  • second human non-mu gene segment(s) eg, a human CH I, CH2 or CH3; or encoding an Fc
  • the first and second gene segments are the same type of constant region gene segment(s) (eg, both CHI gene segments or both encoding an Fc) and the second gene segment(s) is a variant of (ie, is a mutant of) the first gene segment(s).
  • this aspect of the invention is useful, for example, because it enables the skilled person to use the vertebrates to select for desirable human V regions (and those that express and perform well, have good biophysical characteristics etc) in vivo in the context of a repertoire of naturally-occurring human constant region polypmorphic variants - thus one can include the most common human variants (eg, as present in the 1000 Genomes database; www.100Q g enomes.org) to arrive at human antibodies and heavy chains that have wide application in human medicine, or can be selected (according to the C region variant distribution in the human ethnic and other populations) to be tailored to specific human populations. Also variants may have differing expression and/or biophysical characteristics - thus allowing one to have a pool of human antibodies and heavy chains from which to select good drug candidates for human use.
  • the most common human variants eg, as present in the 1000 Genomes database; www.100Q g enomes.org
  • variants may have differing expression and/or biophysical characteristics - thus allowing one to have
  • the second gene segment(s) is derived from the genome sequence of different human individuals, for example sequences appearing in the 1000 Genomes database
  • the variant can, in one example, have no more than 15, 10, 9, 8, 7, 6, 5,4, 3, 2 or 1 mutations compared to the corresponding first gene segment(s) or region.
  • the second gene segment(s) or region is a synthetic mutant of the first gene segment(s) or region.
  • the second gene segment(s) or region has no more than 15, 10, 9, 8, 7, 6, 5,4, 3, 2 or 1 mutations compared to the corresponding first gene segment(s) or region.
  • the skilled person can test out mutations in vivo in combination with a repertoire of human heavy chain variable regions. Combinations that are not productive will not be expressed and/or proliferate (as regulated by the vertebrates' own in vivo machinery) and productive combinations are then provided in one of said repertoires for selection of one or more desirable antibodies or heavy chains bearing human constant regions.
  • mutant variants of a desired type of constant region eg, to produce a collection of constant region encoding variant gamma, eg, gamma-1, constant regions.
  • the mutants in one example, are made with the aim of finding Fc inactivating variants that work well with the selected human variable regions.
  • the mutants are made with the aim of finding Fc activating variants that work well with the selected human variable regions. Inactivation or activation of Fc can be tested (as is known in the art) after one or more antibodies or heavy chains have been selected from the population or repertoire.
  • one or both of the first and second gene segment(s) or regions encodes an inactivated human gamma Fc.
  • one or both of the first and second gene segment(s) or regions encodes an activated human gamma Fc.
  • the population of the invention provides an overall heavy chain repertoire comprising the heavy chain sequence repertoires of the first and second vertebrates.
  • the vertebrates in the population can be immunised with the same antigen in a method of selecting and isolating one or more heavy chains (eg, provided as part of antibodies) that specifically bind to the antigen.
  • VH gene segments of a repertoire can, in one embodiment, be recombined VH, ie, provided as part of a variable region sequence derived from the recombination of human VH with D and JH (eg, where the VH, D and JH are human).
  • the population comprises a third said non-human vertebrate, wherein the human constant region gene segment repertoire is different from those of the first and second vertebrates, whereby the third vertebrate can produce a heavy chain sequence repertoire that is different from the heavy chain sequence repertoire produced by the first and second vertebrates.
  • the population provides an overall heavy chain repertoire comprising the heavy chain sequence repertoires of the first, second and third vertebrates.
  • the vertebrates in the population can be immunised with the same antigen in a method of selecting and isolating one or more heavy chains (eg, provided as part of antibodies) that specifically bind to the antigen.
  • VH gene segment repertoire provided by said population comprises a substantially complete repertoire of functional human VH gene segments; optionally providing at least 6 different human JH gene segments, 27 different human D segments and at least 40 different human VH gene segments.
  • the VH gene segment repertoire provided by said population comprises at least 20, 25, 30, 35 or 40 different human VH gene segments.
  • the method of the invention comprises the step of immunising the vertebrates of the population with the same antigen (eg, a human antigen).
  • the vertebrates are a population and are used as such.
  • immunisation of two, more or all of said vertebrates is separated by no more than 12, 9, 6, 5, 4, 3, 2 or 1 months or 3, 2 or 1 week or 6, 5, 4, 3, 2, or 1 day.
  • the vertebrates are a population and are used as such.
  • the method of the invention comprises the step of selecting one or more heavy chains or antibodies from each of said immunised vertebrates on the basis of a common desired antibody or heavy chain characteristic (eg, binding affinity for said antigen), wherein the selected antibodies comprise human non-mu heavy chains, or the selected heavy chains are human.
  • a common desired antibody or heavy chain characteristic eg, binding affinity for said antigen
  • the vertebrates share the same genetic background, with the exception of the heavy chain loci thereof (and optionally one or more of the light chain loci thereof).
  • vertebrates are derived from transgenic non-human vertebrate ancestor embryonic stem cells that have been genetically modified to include human immunoglobulin locus DNA, the ancestor stem cells being identical or related (eg, clonally related); optionally wherein the genome of the ancestor stem cells comprise a common sequence junction that is a junction between a non-human vertebrate sequence and a human sequence (eg, the ancestor genomes comprise a common transgenic immunoglobulin locus or a common human/non-human vertebrate (eg, human/mouse or human/rat) DNA junction).
  • the genomes comprise a common junction within or at the boundary of one or more of their immunoglobulin chain loci (eg, heavy chain loci and/or light chain loci).
  • the vertebrates of the population are mice whose genomes comprise a common human-mouse DNA junction within their heavy chain loci and/or one or more light chain loci. This is indicative that the mice form a population.
  • the vertebrates can all share the same genetic background with the exception of one or more human gene segment repertoires in their genomes.
  • the invention provides an animal house or a laboratory containing a population according to the invention.
  • vertebrates of the population can be housed in the same cage or in the same collection of cages in the same animal house, building or laboratory.
  • the cages or vertebrates themselves may be labelled so that they are part of the same population or experiment. They may be owned by the same owner, eg, the same company, or in the control of a single person or company. They may be allocated for use in the same research programme or series of related research experiments aimed at discovering one or more antibodies or antibody chains against a common antigen or related antigens.
  • the vertebrates provide a population and are used as such.
  • vertebrates are discussed in the context of the same research programme or immunisation schedule or experiment or set of experiments in a laboratory notebook or a set of laboratory notebooks that relate to the same research programme or immunisation schedule or experiment or set of experiments.
  • a programme, schedule or experiment(s) may relate to immunisation of the vertebrates of a population with the same antigen.
  • An aspect provides a method of isolating an antibody, heavy chain or nucleotide sequence encoding said antibody, the method comprising
  • an immunogenic amount of the antigen is delivered in the method of the invention.
  • the invention also relates to a method for detecting a target antigen comprising detecting an antibody or heavy chain produced as above or a derivative thereof with a secondary detection agent which recognises a portion of that antibody/chain.
  • Isolation of the antibody in step (b) can be carried out using conventional antibody selection techniques, eg, panning for antibodies against antigen that has been immobilised on a solid support, optionally with iterative rounds at increasing stringency, as will be readily apparent to the skilled person.
  • step (b) the amino acid sequence of the heavy chain variable region is mutated to improve affinity for binding to said antigen. Mutation can be generated by conventional techniques as will be readily apparent to the skilled person, eg, by error-prone PCR. Affinity can be determined by conventional techniques as will be readily apparent to the skilled person, eg, by surface plasmon resonance, eg, using BiacoreTM.
  • step (b) the amino acid sequence of the heavy chain variable region is mutated to further improve one or more biophysical characteristics of the antibody, eg, one or more of melting temperature, solution state (monomer or dimer), stability and expression (eg, in CHO or f coli).
  • An aspect provides an antibody or heavy chain produced by the method of the invention, optionally for use in human medicine, eg, for treating and/or preventing a medical condition or disease in a human patient.
  • the invention provides a method of isolating a human antibody, the method comprising immunising a vertebrate or population of vertebrates according to any preceding claim with an antigen (eg, human antigen or viral or bacterial antigen) and isolating an antibody from said vertebrate or a vertebrate of the population, wherein the isolated antibody specifically binds to the antigen and comprises a fully human heavy chain of said non-mu isotype.
  • an antigen eg, human antigen or viral or bacterial antigen
  • composition comprising the isolated antibody or a copy or derivative thereof.
  • the method provides a method of obtaining a humanised and affinity matured antigen-specific antibody heavy chain, the method comprising humanising the heavy chain in vivo in a non-human vertebrate (eg, a mouse or a rat) comprising a functional activation induced cytidine deaminase (AI D) by immunising the vertebrate with the antigen and obtaining somatic hypermutation and isotype switching in a B-cell of the vertebrate from an endogenous mu isotype to a human non-mu isotype, wherein an affinity matured antigen-specific antibody heavy chain is produced and expressed by the vertebrate, the non-mu constant domains of the heavy chain being human constant domains, the method further comprising isolating said humanised heavy chain.
  • a non-human vertebrate eg, a mouse or a rat
  • AI D functional activation induced cytidine deaminase
  • variable domain of the heavy chain is a human variable domain.
  • the vertebrate is according to any aspect of the invention as described above.
  • the method provides a pharmaceutical composition comprising the isolated antibody heavy chain obtained in the method or a copy or derivative thereof; optionally wherein the heavy chain, copy or derivative is provided by an antibody that specifically binds the antigen.
  • the method comprises the step of isolating a B-cell from said immunised vertebrate (or an immunised vertebrate of said population), wherein the B-cell expresses said isolated antibody or heavy chain; and optionally immortalising the B-cell.
  • the method comprises the step of isolating a nucleotide sequence from said immunised vertebrate or B-cell, wherein the nucleotide sequence encodes said isolated antibody or a heavy chain thereof.
  • the method provides a vector (optionally in a host cell) comprising the nucleotide sequence of the invention or a copy thereof, or a derivative thereof; optionally comprising up to 15, 10, 9,8,7,6,5,4,3, or 1 mutations.
  • the derivative specifically binds target antigen.
  • the method provides a method of treating or preventing a medical condition or disease in a human associated or caused by said antigen, the method comprising administering to the human the antigen-specific antibody or heavy chain obtained in the method.
  • the method provides the antigen-specific antibody or heavy chain obtained in the method for use in the treatment or prophylaxis of a medical condition or disease in a human associated or caused by said antigen.
  • the method provides the use of the antigen-specific antibody or heavy chain obtained in the method in the manufacture of a medicament for use in the treatment or prophylaxis of a medical condition or disease in a human associated or caused by said antigen.
  • a human gene segment eg, a V or J gene segment
  • an immunoglobulin chain comprising sequence encoded by the constant region of the locus.
  • antibody includes monoclonal antibodies (including full length antibodies which have an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, as well as antibody fragments (e.g., Fab, F(ab')2).
  • antibody also includes H2 antibodies that comprise a dimer of a heavy chain (5'- VH-(optional Hinge)-CH2-CH3-3') and are devoid of a light chain (akin to naturally-occurring H2 antibodies; see, eg, Nature. 1993 Jun 3;363(6428):446-8; Naturally occurring antibodies devoid of light chains;
  • RNA produced from the transgenic heavy chain locus of the invention encodes for heavy chains that re devoid of a CHI gene segment and comprise no functional antibody light chain.
  • RNA produced from the transgenic heavy chain locus encodes for VH single variable domains (dAbs; domain antibodies) with one or more human constant domains.
  • Antibodies, according to the invention comprise one or more human constant domains of non-mu isotype.
  • the heavy chain sequence repertoire encodes for heavy chains that re devoid of a CH I gene segment and comprise no functional antibody light chain.
  • the heavy chain sequence repertoire encodes a repertoire of VH single variable domains (dAbs; domain antibodies) with one or more human constant domains.
  • a repertoire comprises a plurality of different members (thus, for example, a heavy chain repertoire comprises a plurality of different heavy chain sequences, such as sequences differing in their variable regions and/or human non-mu constant regions).
  • a repertoire comprises or consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 10 3 , at least 10 4 , at least 10 5 , at least 10 s , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , at least 10 12 , at least 10 13 , or at least 10 14 members.
  • a repertoire of antibody heavy chains or antibodies comprises or consists of at least 100, at least 10 3 , at least 10 4 , at least 10 5 , at least 10 s , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 , at least 10 , at least 10 , at least 10 , or at least 10 antibody chain sequences or antibodies respectively.
  • a repertoire comprises or consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 10 3 , at least 10 4 , at least 10 5 , at least 10 s , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , at least 10 12 , at least 10 13 , or at least 10 14 different members.
  • a repertoire of gene segments comprises or consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, at least 15, at least 20, at least 30, at least 40, at least 50 gene segments.
  • all of the gene segments are different from each other; or all of the gene segments of the same type (eg, CH2) are different from each other.
  • a population comprises a plurality of different members.
  • a population of non-human vertebrates eg, mice or rats
  • a population of non-human vertebrates comprises a plurality of vertebrates wherein at least two or more of the vertebrates comprise non-identical genomes.
  • the genomes differ in their respective repertoire of human constant region gene segments.
  • a population of non-human vertebrates comprises or consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least lO 3 non- human vertebrates.
  • the genomes of vertebrates are different.
  • each vertebrate is a non-human mammal.
  • the vertebrate is a mouse, rat, rabbit, Camelid (eg, a llama, alpaca or camel), chicken, lamprey or shark.
  • all vertebrates are of the same vertebrate species, eg, all mice or all rats.
  • the human gene regions or segments may be derived from the same individual or different individuals, or be synthetic (synthetically mutated human gene segments) or represent human consensus sequences.
  • a non-human vertebrate of any configuration of the invention is able to generate a diversity of at least 1 X 10 s different functional non-mu immunoglobulin sequence combinations.
  • Human variable regions are suitably inserted upstream of a non-human vertebrate mu constant region, the latter together with downstream human constant region(s) comprising all of the DNA required to encode the full constant region or a sufficient portion of the constant region to allow the formation of an effective non-mu antibody/heavy chain capable of specifically recognising a target antigen.
  • the endogenous mu non-human vertebrate constant region herein is optionally the endogenous host wild-type constant region located at the wild type locus, as appropriate for the heavy chain.
  • the human constant region DNA is suitably inserted on mouse chromosome 12, immediately downstream of the mouse heavy chain mu constant region, where the vertebrate is a mouse.
  • the insertion of human variable region DNA is targeted to the region between the J4 exon and the endogenous ⁇ locus in the mouse genome IgH locus, and in one aspect is inserted between coordinates
  • 114,667,090 and 114,665,190 suitably at coordinate 114,667,091, after 114,667,090.
  • NCBI m37 for the mouse C57BL/6J strain, e.g. April 2007 ENSEM BL Release 55.37h, e.g. NCBI37 July 2007 (NCBI build 37) (e.g. UCSC version mm9 see www.genome.ucsc.edu and http://genome.ucsc.edu/FAQ/FAQreleases.html) unless otherwise specified.
  • Human nucleotides coordinates are those corresponding to GRCh37 (e.g. UCSC version hg 19, http://genome.ucsc.edu/FAQ/FAQreleases.html), Feb 2009 ENSEMBL Release 55.
  • Rat nucleotides are those corresponding to RGSC 3.4 Dec 2004 ENSEMBL release 55.34w, or Baylor College of Medicine HGSC v3.4 Nov 2004(e.g., UCSC rn4, see www.genome.ucsc.edu and http://genome.ucsc.edu/FAQ/FAQreleases.html) unless otherwise specified.
  • the host non-human vertebrate mu constant region for forming the chimaeric IgM antibody/heavy chain may be at a different (non endogenous) chromosomal locus.
  • the inserted human DNA ie, the human variable V,D,J and C gene segments, may then be inserted into the non-human genome at a site which is distinct from that of the naturally occurring heavy locus. Endogenous heavy chain expression is inactivated.
  • Reference to location of a human variable region, constant region or a gene segment upstream or downstream of an endogenous mu constant region means that there is a suitable relative location of the antibody nucleotide elements to enable formation of mu and then non-mu heavy chains in vivo in the vertebrate.
  • the inserted human DNA and endogenous constant region and any other endogenous sequences are in operable connection with one another for antibody or antibody chain production.
  • the Kabat Database (G. Johnson and T. T.Wu, 2002; World Wide Web (www) kabatdatabase.com). Created by E. A. Kabat and T. T. Wu in 1966, the Kabat database publishes aligned sequences of antibodies, T-cell receptors, major histocompatibility complex (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 subgroup classification, and the generation of variability plots. See also Kabat, E. A.,Wu, T. T., Perry, H., Gottesman, K., and Foeller, C.
  • MHC major histocompatibility complex
  • IMGT the International ImMunoGeneTics Information System ® ; M.-P. Lefranc, 2002; World Wide Web (www) imgt.cines.fr).
  • IMGT is an integrated information system that specializes in antibodies, T cell receptors, and MHC molecules of all vertebrate species.
  • IMGT includes three sequence databases (IMGT/LIGM-DB, IMGT/MHC-DB,
  • IMGT/PRIMERDB one genome database
  • IMGT/GENE-DB one genome database
  • V-BASE (I. M. Tomlinson, 2002; World Wide Web (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 Althaus and Werner Muller) that allows the assignment of rearranged antibody V genes to their closest germline gene segments.
  • Antibodies Structure and Sequence (A. C. R. Martin, 2002; World Wide Web (www)
  • AAAAA A Ho's Amazing Atlas of Antibody Anatomy; A. Honegger, 2001; World Wide Web (www) unizh.cn/ ⁇ antibody.
  • This resource includes tools for structural analysis, modeling, and engineering. It adopts a unifying scheme for comprehensive structural alignment of antibody and T-cell-receptor sequences, and includes Excel macros for antibody analysis and graphical representation.
  • WAM Web Antibody Modeling; N. Whitelegg and A. R. Rees, 2001; World Wide Web (www) antibody.bath.ac.uk). Hosted by the Centre for Protein Analysis and Design at the University of Bath, United Kingdom. Based on the AbM package (formerly marketed by Oxford Molecular) to construct 3D models of antibody Fv sequences using a combination of established theoretical methods, this site also includes the latest antibody structural information.
  • Therapeutic Antibody Human Homology Project which aims to correlate clinical efficacy and antiimmunoglobulin responses with variable region sequences of therapeutic antibodies.
  • the Antibody Resource Page (The Antibody Resource Page, 2000; World Wide Web (www) antibodyresource.com). This site describes itself as the "complete guide to antibody research and suppliers.” Links to amino acid sequencing tools, nucleotide antibody sequencing tools, and hybridoma/cell-culture databases are provided.
  • This resource provides an overview on antibody humanization technology.
  • the most useful feature is a searchable database (by sequence and text) of more than 40 published humanized antibodies including information on design issues, framework choice, framework back-mutations, and binding affinity of the humanized constructs.
  • Samples from which B-cells can be obtained include but are not limited to blood, serum, spleen, splenic tissue, bone marrow, lymph, lymph node, thymus, and appendix.
  • Antibodies and immunoglobulin chains can be obtained from each of the previous-mentioned samples and also from the following non-limiting list of B-cells, ascites fluid, hybridomas, and cell cultures.
  • a human variable region of a heavy chain can be derived from recombination of human VH, D and JH gene segments and this reflects the in vivo recombination of these gene segments in, for example, a transgenic heavy chain locus according to the invention with any accompanying mutation (eg, junctional mutation).
  • a constant region or gene segment sequence is derived from a human individual if, for example it is an identical nucleotide copy of that region or segment as found in the genome of that individual (or found in a ethnic or geographical population of humans of which the individual is a member).
  • Populations and genomic variant immunoglobulin gene sequences can be found, eg, in the 1000 Genomes database or by sampling DNA from humans and sequencing.
  • the genome of a or each vertebrate or cell has been modified to prevent or reduce the expression of fully-endogenous antibody or heavy chains.
  • suitable techniques for doing this can be found in WO2011004192, US7501552, US6673986, US6130364, WO2009/076464, EP1399559 and US6586251, the disclosures of which are incorporated herein by reference.
  • all or part of the non-human vertebrate VDJ region is inactivated by inversion in the endogenous heavy chain immunoglobulin locus of the mammal, optionally with the inverted region being moved upstream or downstream of the endogenous Ig locus.
  • 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 Ig locus.
  • all or part of the non-human vertebrate VJ region is inactivated by inversion in the endogenous lambda chain immunoglobulin locus of the mammal, optionally with the inverted region being moved upstream or downstream of the endogenous Ig locus.
  • the endogenous heavy chain locus is inactivated in this way as is one or both of the endogenous kappa and lambda loci.
  • the or each vertebrate has been generated in a genetic background which prevents the production of mature host B and T lymphocytes, optionally a RAG-l-deficient and/or RAG-2 deficient background. See US5859301 for techniques of generating RAG-1 deficient animals.
  • immunoglobulin (Ig) is used interchangeably with "antibody” herein.
  • An "isolated” antibody or heavy chain is one that has been identified, separated and/or recovered from a component of its production environment (e.g., naturally or recombinantly).
  • the isolated polypeptide is free of association with all other components from its production environment, eg, so that the antibody has been isolated to an FDA-approvable or approved standard.
  • Contaminant components of its production environment such as that resulting from recombinant transfected cells, are materials that would typically interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
  • the polypeptide will be purified: (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, an isolated polypeptide, chain or antibody will be prepared by at least one purification step.
  • antibody fragment comprises a portion of an intact antibody, preferably the antigen binding and/or the variable region of the intact antibody.
  • antibody fragments include dAb, Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules and multispecific antibodies formed from antibody fragments.
  • an antibody that "specifically binds to" or is “specific for" a particular polypeptide, antigen, or epitope is one that binds to that particular polypeptide, antigen, or epitope without substantially binding to other polypeptides, antigens or epitopes.
  • binding to the antigen or epitope is specific when the antibody binds with a K D of 100 ⁇ or less, 10 ⁇ or less, 1 ⁇ or less, 100 nM or less, eg, 10 nM or less, 1 nM or less, 500 pM or less, 100 pM or less, or ⁇ or less.
  • the binding affinity (K D ) can be determined using standard procedures as will be known by the skilled person, eg, binding in ELISA and/or affinity determination using surface plasmon resonance (eg, BiacoreTM or KinExATM solution phase affinity measurement which can detect down to fM affinities (Sapidyne Instruments, Idaho)).
  • surface plasmon resonance eg, BiacoreTM or KinExATM solution phase affinity measurement which can detect down to fM affinities (Sapidyne Instruments, Idaho)
  • “Pharmaceutically acceptable” refers to approved or approvable by a regulatory agency of the USA Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.
  • a “pharmaceutically acceptable carrier, excipient, or adjuvant” refers to an carrier, excipient, or adjuvant that can be administered to a subject, together with an agent, e.g., any antibody or antibody chain described herein, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent.
  • the genome comprises an antibody light chain locus which comprises all or part of the human IgX locus including at least one human JX region and at least one human CX region, optionally Q6 and/or Q7.
  • the light chain locus comprises a plurality of human JX regions , optionally two or more of ⁇ ⁇ 1, ⁇ ⁇ 2, ⁇ ⁇ 6 and ⁇ ⁇ 7, optionally all of ⁇ ⁇ 1, ⁇ ⁇ 2, ⁇ ⁇ 6 and ⁇ ⁇ 7.
  • the human lambda immunoglobulin locus comprises a unique gene architecture composed of serial J-C clusters.
  • the invention in optional aspects employs one or more such human J-C clusters.
  • the light chain locus comprises at least one human J 3 ⁇ 4 -Q cluster, optionally at least Jx7-Q7.
  • transgenes are facilitated by being able to use all or part of the human lambda locus such that the transgene comprises one or more J-C clusters in germline configuration, advantageously also including intervening sequences between clusters and/or between adjacent J and C regions in the human locus. This preserves any regulatory elements within the intervening sequences which may be involved in VJ and/or JC recombination and which may be recognised by endogenous AID (activation-induced deaminase).
  • AID activation-induced deaminase
  • nucleotide sequence encoding the antibody or heavy chain of the invention, optionally wherein the nucleotide sequence is part of a vector.
  • Suitable vectors will be readily apparent to the skilled person, eg, a conventional antibody expression vector comprising the nucleotide sequence together in operable linkage with one or more expression control elements.
  • An aspect provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or heavy chain of the invention and a diluent, excipient or carrier, optionally wherein the composition is contained in an IV container (eg, and IV bag) or a container connected to an IV syringe.
  • An aspect provides the use of the antibody or heavy chain of the invention in the manufacture of a medicament for the treatment and/or prophylaxis of a disease or condition in a human patient.
  • the invention relates to humanised antibodies and antibody heavy chains produced according to the present invention, and use of said antibodies in human medicine.
  • the invention also relates to a pharmaceutical composition comprising such an antibody or heavy chain and a pharmaceutically acceptable carrier or other excipient.
  • the invention relates to use of a population of non-human vertebrates of the present invention in the analysis of the likely effects of drugs and vaccines in the context of a human antibody heavy chain repertoire.
  • the invention also relates to a method for identification or validation of a drug or vaccine, the method comprising delivering the vaccine or drug to a population of vertebrates of the invention and monitoring one or more of: the immune response, the safety profile; the effect on disease.
  • the invention also relates to a kit comprising an antibody, heavy chain or antibody derivative as disclosed herein and either instructions for use of such antibody, derivative or chain or a suitable laboratory reagent, such as a buffer, antibody detection reagent.
  • a suitable laboratory reagent such as a buffer, antibody detection reagent.
  • the invention also relates to a method for making an antibody, or part thereof, the method comprising providing:
  • each vertebrate is a non-human vertebrate, mouse or rat, whose genome comprises
  • endogenous antibody heavy and light chain expression has been inactivated; and optionally wherein said genome is homozygous for said heavy and light chain loci.
  • the kappa chain transgenic loci comprises a substantially complete human functional VK and JK repertoire; and the lambda chain transgene comprises a substantially complete human functional VX and JX repertoire.
  • the gene locus of the invention is a heavy chain locus which comprises a S-mu switch (eg, endogenous S-mu) 5' of the endogenous mu constant region and a human gamma constant region3' of the endogenous mu constant region, with a S-gamma switch (eg, a human or endogenous S-gamma) between the Cmu and Cgamma regions.
  • a S-mu switch eg, endogenous S-mu
  • the C-mu region and switches are mouse 129 C-mu region and switches; or the C-mu region and switches are mouse Black 6 C-mu region and switches.
  • S-mu is a rat Smu and the C-mu region is mouse and optionally there is a mouse S- gamma 3' of the C-mu region.
  • each vertebrate is a mouse whose genetic background is selected from mouse strains C57BL/6, M129 such as 129/SV, BALB/c, and any hybrid of C57BL/6, M129 such as 129/SV, or BALB/c.
  • each of these vertebrates have the same genetic background but two or more of the vertebrates of the population differ in their human gene segment repertoires (eg, in their human C region gene segment repertoires).
  • each gene locus of the invention are human JH gene segments; optionally wherein each heavy chain locus comprises a substantially complete functional repertoire of human JH gene segments.
  • each gene locus of the invention comprises at least 2, 3, 4, 5 or 6 different human JH gene segments.
  • each gene locus of the invention are human D gene segments; optionally wherein each heavy chain locus comprises a substantially complete functional repertoire of human D gene segments.
  • each gene locus of the invention comprises at least 5, 10, 15, 20, 25, 26 or 27 different human D gene segments.
  • the heavy chain loci of said vertebrates comprise identical human D and JH gene segment repertoires, but differ in their VH gene repertoires.
  • each heavy chain locus comprises at least two human JH gene segments selected from the group consisting of Jl, J2, J3, J4, J5 and J6; optionally all of the gene segments of the group.
  • each vertebrate comprises human VH gene segments selected from the group consisting of V6-1, Vl-2, Vl-3, V4-4, V7-41, V2-5, V3-7, Vl-8, V3-9, V3-11, V3-15, Vl-18, V3-20, V3- 21, V3-23, Vl-24, V2-26, V4-28, V3-30, V4-31, V3-33, V4-34, V4-39, V3-43, Vl-45, Vl-46, V3-48, V3- 49, V5-51, V3-53, Vl-58, V4-59, V4-61, V3-64, V3-66, Vl-69, V2-70, V3-72, V3-73 and V3-74;
  • VH gene repertoire comprises a substantially complete human functional VH gene repertoire.
  • endogenous antibody heavy chain expression has been inactivated in the vertebrate or cell of the invention.
  • endogenous heavy chains ie, heavy chains whose variable regions are derived from recombination of non-human vertebrate V, D and J gene segments.
  • VL gene segments of a repertoire can, in one embodiment, be recombined VL, ie, provided as part of a variable region sequence derived from the recombination of human VL with JL (eg, where the VI and JL are human).
  • each transgenic light chain locus are human JL gene segments; optionally wherein each light chain locus comprises a substantially complete functional repertoire of human JK or JX gene segments (eg, each transgenic locus comprises human VX gene segments and JX gene segments, optionally a substantially complete functional repertoire of human JX gene segments; or each transgenic locus comprises human VK gene segments and JK gene segments, optionally a substantially complete functional repertoire of human JK gene segments).
  • the kappa chain repertoire provided by said population comprises a substantially complete repertoire of functional human VK gene segments; optionally providing at least 5 different human JK gene segments and at least at least 40 different human VK gene segments.
  • the kappa chain repertoire provided by said population comprises at least 20, 25, 30, 35 or 40 different human VK gene segments.
  • the lambda chain repertoire provided by said population comprises a substantially complete repertoire of functional human VX gene segments; optionally providing at least 5 different human JX gene segments and at least 40 different human VX gene segments.
  • the lambda chain repertoire provided by said population comprises at least 20, 25, 30, 35 or 40 different human VX gene segments.
  • each light chain locus of the vertebrate or cell comprises at least 2, 3, 4, 5 or 6 different human JK or JX gene segments.
  • the light chain loci of said vertebrate or cell comprise identical human J L gene segment repertoires, but differ in their VL gene repertoires.
  • endogenous antibody kappa and/or lambda l ight chain expression has been inactivated in the vertebrate or cell .
  • said transgenic light chain loci of the vertebrate or cell are kappa light chain loci (at the endogenous kappa loci, ie, corresponding to the position of a kappa locus in a wild-type non- human vertebrate genome).
  • a transgenic kappa locus can comprise human VK gene segments and JK gene segments upstream of a constant region (eg, a CH, CX or CK gene segment; optionally which is an endogenous or human gene segment).
  • a transgenic kapppa locus can comprise human VX gene segments and JX gene segments upstream of a constant region (eg, a CH, CX or CK gene segment; optionally which is an endogenous or human gene segment).
  • said transgenic light chain loci of the vertebrate or cell are lam bda light chain loci (at the endogenous lambda loci, ie, corresponding to the position of a lambda locus in a wild-type non-human vertebrate genome).
  • a transgenic lambda locus can comprise human VK gene segments and JK gene segments upstream of a constant region (eg, a CH, CX or CK gene segment; optionally which is an endogenous or human gene segment).
  • a transgenic lambda locus can comprise human VX gene segments and JX gene segments upstream of a constant region (eg, a CH, CX or CK gene segment; optionally which is an endogenous or human gene segment).
  • examples of a desirable antibody/chain characteristic are affinity for binding a predetermined antigen or epitope (eg, as determined by surface plasmon resonance), completion with a known antibody for binding to a predetermined antigen or epitope, epitopic specificity of the antibody (eg, as determined by X-ray crystallography, competition with a known antibody for antigen binding wherein the known antibody specifically binds to the antigen (eg, as determined by surface plasmon resonance, eg, BiacoreTM), performance in ELISA or another immunoassay, a desirable biophysical characteristic (eg, melting temperature, pi, solution state, degree of aggregation, storage profile etc).
  • affinity is determined by surface plasmon resonance.
  • Methods of immunisation for use in the invention are well known to the skilled person and may involve a classic prime-boost regime, RIMMS or any other protocol.
  • An adjuvant may be administered with the antigen, as is known in the art.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody or heavy chain selected as described above or a derivative thereof that binds said antigen, together with a pharmaceutically acceptable diluent, carrier or excipient.
  • derivative antibodies/chains are antibodies/chains that have one or more mutations compared to the isolated antibody or chain (eg, to improve antigen- binding affinity and/or to enhance or inactivate Fc function) Such mutants specifically bind the antigen.
  • Mutation or adaptation to produce a derivative includes, eg, mutation to produce Fc enhancement or inactivation.
  • a derivative can be an antibody following conjugation to a toxic payload or reporter or label or other active moiety.
  • the vertebrate is (or vertebrates of the populatio are) naive (ie, not immunised with a predetermined antigen, as the term is understood in the art; for example, such a vertebrate that has been kept in a relatively sterile environment as provided by an animal house used for R&D).
  • the vertebrates have been immunised with a predetermined antigen, as the term is understood in the art; for example, such a vertebrate that has been kept in a relatively sterile environment as provided by an animal house used for R&D).
  • the vertebrates have been immunised with a predetermined antigen
  • the predetermined antigen eg, an antigen bearing a human epitope.
  • the population comprises naive and immunised vertebrates.
  • the vertebrates have been immunised with the same antigen (eg, a human antigen).
  • the same antigen eg, a human antigen
  • the vertebrates are naive.
  • the vertebrates have a common collection of light chain loci.
  • the kappa chain loci alleles are identical in the vertebrates and/or the lambda chain loci alleles are identical in the vertebrates. This simplifies construction of vertebrate variants for producing the population and also simplifies breeding.
  • the method comprises comprising selecting one or more antibody heavy chains (eg, as part of an antibodies) from said population or repertoire according to a desired characteristic (eg, affinity for biding an antigen).
  • a desired characteristic eg, affinity for biding an antigen
  • the cell is a B-cell, hybridoma, ES cell or iPS cell.
  • ES cells and iPS cells can be used to develop corresponding non-human vertebrates (vertebrates of the invention).
  • the vertebrate species is selected from human, mouse, rat, rabbit, guinea pig, chicken, a fish, a bird, a reptile, a Camelid, bovine, chimpanzee, a non-human primate and a primate.
  • the vertebrate is a mouse or rat.
  • a non-human vertebrate eg, a mouse or a rat
  • a non-human vertebrate cell eg, a mouse cell or a rat cell
  • whose genome comprises a gene locus for expression of antibody heavy chains, the locus comprising
  • an endogenous mu constant region for expression of IgM antibody heavy chains comprising endogenous mu heavy chain constant domains and human variable domains;
  • variable region is provided as a targeted insertion of the human variable region gene segments upstream of the endogenous mu constant region in an endogenous IgH locus such that the variable region gene segments are able to recombine for expression and selection in the context of an endogenous mu constant region.
  • the vertebrate or cell of aspect 1 comprising an antibody heavy chain locus that comprises (in 5' to 3' direction) an unrearranged human variable region, a first switch, an endogenous mu constant region, a second switch and a human constant region of said non-mu isotype; wherein the heavy chain locus is capable of undergoing switching from IgM to the non-mu isotype; optionally wherein the first switch is a rodent (eg, mouse or rat) Smu or the endogenous Smu of the IgH locus.
  • the human variable region comprises a plurality of unrearranged human VH gene segments; optionally at least 5 different human VH gene segments or a substantially complete human functional VH gene repertoire.
  • variable region comprises human VH gene segments selected from the group consisting of V6-1, Vl-2, Vl-3, V4-4, V7-41, V2-5, V3-7, Vl-8, V3-9, V3-11, V3-15, VI- 18, V3-20, V3-21, V3-23, Vl-24, V2-26, V4-28, V3-30, V4-31, V3-33, V4-34, V4-39, V3-43, VI- 45, Vl-46, V3-48, V3-49, V5-51, V3-53, Vl-58, V4-59, V4-61, V3-64, V3-66, Vl-69, V2-70, V3- 72, V3-73 and V3-74;wherein the VH gene repertoire comprises a substantially complete human functional VH gene repertoire.
  • the human variable region comprises at least 10 human D gene segments selected from the group consisting of Dl-1, D2-2, D3-3, D4-4, D5-5, D6-6, Dl-7, D2-8, D3-9, D3-10, D4-11, D5-12, D6-13, Dl-14, D2-15, D3-16, D4-17, D5-18, D6-19, Dl-20, D2-21, D3-22, D4-23, D5-24, Dl-26, D6-25 and D7-27; optional ly all of the gene segments of the group.
  • the vertebrate or cell of any preceding aspect wherein the human variable region comprises a plurality of unrearranged human J H gene segments; at least 2 different human J H gene segments or a substantially complete functional repertoire of human D gene segments.
  • the vertebrate or cell of any preceding aspect wherein the non-mu constant region is a human gamma constant region.
  • the vertebrate or cell of aspect 8 wherein the gamma constant region comprises CH 2 and CH3 gene segments for encoding an antibody Fc region.
  • the vertebrate or cell of aspect 9 wherein the CH2 and CH3 gene segments encode for an inactivated Fc region.
  • the locus comprises an insertion of a human gamma constant region nucleotide sequence comprising sequence from the human gamma CH2 gene segment to the human gamma CH3 gene segment, wherein the insertion replaces a corresponding sequence of an endogenous gamma constant region in the genome of the vertebrate or cell.
  • endogenous inter-gene segment sequences have been retained so that the heavy chain locus comprises said human non-mu constant region gene segments flanked by endogenous constant region inter-gene segment sequences.
  • the locus comprises a replacement of endogenous gamma constant region gene segments (eg, gamma CH2 and CH3 gene segments) with human gamma constant region gene segments (eg, gamma CH2 and CH3 gene segments), but endogenous sequences between gamma constant regions have been retained.
  • endogenous gamma constant region gene segments eg, gamma CH2 and CH3 gene segments
  • human gamma constant region gene segments eg, gamma CH2 and CH3 gene segments
  • a first vertebrate wherein the locus is an antibody heavy chain locus that comprises one or more first human non-mu C gene segment(s) (eg, a human CHI, CH2 or CH3; or an Fc) or a first human constant region;
  • a second vertebrate wherein the locus is an antibody heavy chain locus that comprises one or more second human non-mu C gene segment(s) (eg, a human CHI, CH2 or CH3; or encoding an Fc) or a second constant region; and wherein the first and second gene segments or regions are the same type of C gene segment(s) (eg, both CHI gene segments or both encoding an Fc) or regions and the second gene segment(s) is a variant of the first gene segment(s).
  • the population of aspect 22, wherein the first and second constant regions are gamma constant regions of the same sub-type (eg, both gamma-2 or gamma-3).
  • the population of aspect 22, wherein one or both of the first and second gene segment(s) encodes an inactivated human gamma Fc.
  • the population of aspect 22, wherein one or both of the first and second gene segment(s) encodes an activated human gamma Fc.
  • a method of isolating an antibody or human heavy chain comprising immunising a vertebrate or population of vertebrates according to any preceding aspect with an antigen and isolating a non-mu antibody or heavy chain from said vertebrate or a vertebrate of the population, wherein the isolated antibody or heavy chain specifically binds to the antigen and comprises (or is) a fully human heavy chain of said non-mu isotype.
  • a pharmaceutical composition comprising the isolated antibody or heavy chain recited in aspect 27 or a copy or derivative thereof.
  • a method of obtaining a humanised and affinity matured antigen-specific antibody heavy chain comprising humanising the heavy chain in vivo in a non-human vertebrate (eg, a mouse or a rat) comprising functional RAG and activation induced cytidine deaminase (AI D) by immunising the vertebrate with the antigen and obtaining recombination of VH,D and JH gene segments in vivo, somatic hypermutation and isotype switching in a B-cell of the vertebrate from an endogenous mu isotype to a human non-mu isotype, wherein a repertoire of affinity matured antigen-specific non-mu antibody heavy chains are produced and expressed by the vertebrate, the non-mu constant domains of the heavy chains being human constant domains, the method further comprising isolating one or more of said humanised heavy chains.
  • a non-human vertebrate eg, a mouse or a rat
  • AI D activation induced
  • variable domain of each isolated heavy chain is a human variable domain.
  • the method of aspect 29 or 30, wherein the vertebrate is according to any one of aspects 1 to 21.
  • a pharmaceutical composition comprising an isolated antibody heavy chain obtained in the method of aspect 29, 30 or 31 or a copy or derivative thereof; optionally wherein the heavy chain, copy or derivative is provided by an antibody that specifically binds the antigen.
  • the method of any one of aspects 27, 29, 30 or 31 comprising the step of isolating a B-cell from said immunised vertebrate (or an immunised vertebrate of said population), wherein the B-cell expresses a said isolated antibody or heavy chain; and optionally immortalising the B-cell.
  • any one of aspects 27, 29, 30, 31 or 32 comprising isolating a nucleotide sequence from said immunised vertebrate or B-cell, wherein the nucleotide sequence encodes a said isolated antibody or a heavy chain thereof.
  • a vector (optionally in a host cell) comprising the nucleotide sequence recited in aspect 34 or a copy thereof.
  • a method of treating or preventing a medical condition or disease in a human associated or caused by said antigen comprising administering to the human the antigen- specific antibody or heavy chain obtained in the method of any one of aspects 21, 23, 24, 25, 27 and 28.
  • the antigen-specific antibody or heavy chain obtained in the method of any one of aspects 27, 29, 30 or 31 for use in the treatment or prophylaxis of a medical condition or disease in a human associated or caused by said antigen is useful in the treatment or prophylaxis of a medical condition or disease in a human associated or caused by said antigen.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), "including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps
  • A, B, C, or combinations thereof refers to all permutations and combinations of the listed items preceding the term.
  • A, B, C, or combinations thereof is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.
  • expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, M B, BBC, AAABCCCC, CBBAAA, CABABB, and so forth.
  • BB BB
  • AAA AAA
  • M B BBC
  • AAABCCCCCC CBBAAA
  • CABABB CABABB
  • compositions, populations, vertebrates, antibodies, repertoires and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that 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 substitutes and modifications apparent 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.
  • WO2011004192 & WO2011158009 can be used with cre-lox-mediated, transposon-mediated (eg, using piggyBac) or homologous recombination-mediated deletion of endogenous non-mu constant region sequences, in order to replace endogenous non-mu with human constant region gene segments.
  • Modified BACs with human C gene segments for example created using the methods described in Example 1, can be used to alter the genome of non-human mammals (eg, mice or rats). These alterations can result in an intact Ig heavy chain locus in which normal im munoglobulin gene segment recombination results in VDJ combinations which includes human VH, D and J H gene segments.
  • An example of how to insert human V, D and J gene segments into a non-human vertebrate genome is disclosed in the Regeneron and Kymab PCT applications cited above. These methods are also useful for inserting human C region sequences.
  • Standard recombineering methods can be used to introduce human C gene segments and regions into BACs for use in inserting non-human vertebrate genomic DNA into the genome of a non-human vertebrate embryonic stem cell (ES cell), eg, using standard homologous recombination or RMCE.
  • ES cell non-human vertebrate embryonic stem cell
  • RMCE homologous recombination or RMCE.
  • a genomic fragment containing a human constant region nucleotide sequence (encoding one or more human constant domains) of a human genomic sequence is inserted into a bacterial artificial chromosome (BAC) vector by standard techniques.
  • BAC bacterial artificial chromosome
  • such a BAC which can range in size from 20-kb to 200-kb or more, can be isolated from commercially available libraries of BACs (eg, Caltec A, B, C or D human BAC library or RPCI-11 human BAC library, Invitrogen) by standard techniques including sequence searches or by hybridization to bacterial colonies containing BACs to identify those with a BAC of interest.
  • libraries of BACs eg, Caltec A, B, C or D human BAC library or RPCI-11 human BAC library, Invitrogen
  • a BAC is chosen that contains a human gam ma-1 constant region nucleotide sequence comprising from the CH I gene segment to the polyA immediately 5' of M2 of a human genomic sequence.
  • the endogenous gamma-1 constant region gene segments can be replaced by the corresponding human gam ma-1 constant region gene segments.
  • two steps are required. The first step replaces the C domain coding exon of an endogenous C 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 streptomycin. Short stretches of DNA homologous to sequences flanking the endogenous C gene exon are placed 5' and 3' of the rpsL-Amp operon. In the presence of appropriate recombination factors per standard recombineering techniques (eg, homologous recombination) recombination between the operon fragment and the BAC will result in replacement of the endogenous C gene exon with the operon which are selected by resistance to ampicill in.
  • the second step uses the same homologous sequences in order to replace the inserted operon with a corresponding human C gene segment. Successful integrations of the human gene segment are selected in bacteria that become resistant to streptomycin due to the loss of the operon, specifically the rpsL portion.
  • the two step process as described can be repeated for each of the endogenous C gene segments until the desired endogenous non-mu constant region has been humanised by replacement of endogenous constant region gene segments with human counterparts.
  • the method can be used similarly to replace an entire endogenous non-mu constant region (including gene segments and inter-gene segment sequences) with the corresponding human non-mu constant region sequence; or just the endogenous C gene segments of the non-mu constant region can be placed (leaving endogenous inter-gene segment sequences in place between the human C gene segments).
  • genetically-manipulated stem cells can be inserted into blastocysts and implanted into foster mothers in order to give rise to live-born progeny animals bearing human C gene segments in heavy chain loci according to the invention.
  • Standard breeding can yield progeny that are homozygous for the heavy chain locus of the invention and in which endogenous heavy chain expression (ie, chains comprising endogenous variable regions) has been inactivated using conventional techniques.
  • sRMCE sequential recombinase mediated cassette exchange
  • the insertion is between the mouse C-mu and mouse C-gamma regions (eg, between mouse Cmu and Cdelta or between mouse Cdelta and mouse gamma-1).
  • a human gamma-1 constant region is built up in the genome of the ES cell (and optionally the mouse gamma-1 constant region or all mouse non-mu constant regions are deleted or replaced similarly with the corresponding human constant region sequences).
  • serial BACs are used with sRMCE to insert the entire human sequence from the human delta or gamma-3 constant region to (and including) the human gamma A2 constant region.
  • mice constant region from delta or gamma-1 to (and including) alpha is deleted (or just the mouse gamma constant regions are deleted).
  • BAC insertion via SRMCE techniques are also used for the addition of human VH, DH and J gene segments immediately downstream of the 3'-most mouse JH gene segment. Endogenous mouse VDJ is inactivated or deleted.
  • An alternative embodiment is to replace the endogenous mouse C gamma-1 with a 'landing pad' via homologous recombination.
  • a construct containing human C gamma-1 exons 1 to 4 fused to mouse C gamma-1 transmembrane and cytoplasmic exons, replaces the endogenous C gamma-1 segments.
  • the construct is assembled by PCR from human and mouse BACS exploiting a conserved SPGK amino acid motif at the 3' end of exon 4 in both human and mouse C gamma-1, which aids construction using PCR.
  • the rationale for using a chimeric C gamma-1 construct is that the secretory form of IgGl is fully human whilst the membrane-bound form of IgGl maintains normal mouse signalling.
  • a further embodiment would be to use the same landing pad and a similar human-mouse C gamma- 1 construct described previously, but in this example the construct lacks human C gamma-1 exon 1 and so ablates heavy and light chain pairing, thus a heavy-chain only IgGl is expressed.
  • ES cell clones with correct insertions are selected from a pool of clones without insertions or with non-productive insertions by resistance to puromycin as described in WO2011004192. Resistance to puromycin results from the juxtaposition of an active promoter element, PGK, with the puroTK coding region. Correct insertions are verified by standard techniques including PCR of junctions, PCR of internal elements, Southern blotting, comparative genomic hybridization (CGH), sequencing and etc. In the example, as described in WO2011004192, correct lox2272-lox2272 and loxP-loxP recombination also results in two intact sets of piggyBac elements that did not exist prior to insertion.
  • An intact piggyBac element is comprised of a set of inverted repeats which are depicted in the figure by "PB5"' and "PB3"'.
  • An appropriated oriented set of piggyBac elements are the substrate of piggyBac transposase which can catalyse recombination between the elements, resulting in deletion of intervening sequences as well as both elements.
  • the DNA remaining after a piggyBac transposition is left intact and is lacking any remnant of the piggyBac element.
  • 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.
  • each vector comprises a stretch of genomic human constant region DNA flanked by homology arms.
  • the homology arms would have the sequence of the DNA in the recipient ES cell genome that flanks the insertion site (for example, flanking a mouse constant region sequence to be replaced by insertion of the human DNA).
  • serial homologous recombination steps optionally using BACS with overlapping homology arms as described in WO2009076464
  • the human constant region(s) can be built up in a process that replaces mouse constant region DNA.
  • insertion of human DNA can be in a separate step from deletion of mouse constant region DNA (eg, using site-specific recombination such as cre-lox or transposon-mediated deletion such as using piggyBac).
  • the final product is an ES cell whose genome comprises a transgenic IgH locus with a plurality of human VH, D and JH gene segments upstream of a mouse S-mu switch, mouse mu constant region, a gamma switch (optionally the mouse S-gamma is retained, or the human S-gamma can be inserted together with the other human gamma constant region sequences) and a human gamma constant region (eg, at least a human gamma-1 constant region).
  • Expression of mouse variable regions is inactivated (eg, by inversion of the mouse V, D, J as described in WO2011004192 or by deletion of all or part (eg, the J gene segments) of the mouse IgH variable region gene segments.
  • mice are developed from the transgenic ES cells (the mice being preferably homozygous for the transgenic heavy chain locus).
  • 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 suitable recipient and breeding the chimeric offspring that result.
  • the modified gene locus can be propagated through breeding and made either heterozygous or homozygous depending on the genetic cross.
  • the human variable region gene segments are positioned such that they can participate in the recombination events associated with B cell maturation yielding VDJ gene segments and mouse C- mu constant domains. Isotype switching to a human constant region (eg, C gamma-1) will yield transcripts that encode fully human, mature heavy chains comprising human variable regions (with somatic hypermutation caused by mouse AID) and human constant domains.
  • a human constant region eg, C gamma-1
  • Productive transcripts will be expressed by the progeny mice (ie, the mouse's in vivo antibody-producing machinery is harnessed to select for productive transcripts that can be expressed) and the skilled person will know at the outset that all gamma-1 type chains will be human (where the heavy chain loci have been humanised to include human gamma-1 constant region and exclude mouse gamma-1 constant region).
  • Anti-available antibodies can be used to select and isolated specific human IgG isotypes (eg, rabbit anti-human IgGl, Catalogue Number: AP20578PU-N from Acris Antibodies GmbH; mouse anti-human lgG2, Catalogue Number: AM08152AP-N from Acris Antibodies GmbH; mouse anti-human lgG3, Catalogue Number: MCA516P or sheep-anti human Ig3, Catalogue Number: AP05355HR-N from AbD Serotec; mouse anti-human lgG4, Catalog NumberMCA2098P from AbD Serotec).
  • human IgG isotypes eg, rabbit anti-human IgGl, Catalogue Number: AP20578PU-N from Acris Antibodies GmbH; mouse anti-human lgG2, Catalogue Number: AM08152AP-N from Acris Antibodies GmbH; mouse anti-human lgG3, Catalogue Number: MCA516P or sheep-anti human Ig3, Catalog
  • progeny mice are immunised with a human target antigen or a viral or bacterial antigen and IgGl antibodies are selected for antigen binding isolated. These will have fully human heavy chains and be specific for the target antigen.
  • the progeny mice are engineered (using standard techniques, eg, as disclosed in the Kymab Limited or Regeneron PCT applications disclosed above) to include humanised light chain loci (eg, kappa and/or lambda light chain loci in which the mouse constant and variable regions have been deleted or inactivated and there has been inserted human light chain variable region VL and JL gene segments and optionally also human constant regions)
  • the isolated IgGl antibodies will have fully human variable region and, if the light chain loci bear only human constant region genes, the IgGl antibodies will be fully human, well expressed and selected by an in vivo system.
  • mice will specifically bind target antigen when the mice have been immunised and IgGl have been selected against said target.
  • light chain expression is inactivated in progeny mice, eg, by deletion of the mouse kappa (and optionally also lambda) variable region or part thereof (eg, JL gene segments) or by deletion of kappa (and optionally also lambda) constant regions in the light chain loci.
  • human gamma constant regions are inserted (eg, for all IgG subtypes, or at least for IgGl), in which the human CHI gene segments (and optionally hinge regions) are omitted.
  • CHI can be retained (eg, see WO2011072204, the disclosure of which is incorporated herein by reference).
  • heavy-chain only antibodies H2 antibodies
  • H2 antibodies heavy-chain only antibodies
  • These heavy-chain only antibodies are fully human (human variable and constant domains, eg human gamma-1 constant domains when the heavy chain loci in the mice have been humanised by insertion of human gamma-1 constant regions and inactivation or deletion of mouse gamma-1 constant regions).
  • heavy chain-only antibodies can be selected that are specific for the antigen of interest, fully human, well expressed and selected by an in vivo system.

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Abstract

La présente invention concerne l'humanisation d'anticorps in vivo. L'invention concerne des vertébrés non-humains, des cellules, des populations et des procédés utiles pour l'humanisation d'anticorps chimériques in vivo. La présente invention permet d'obtenir directement et rapidement des anticorps spécifiques d'antigènes qui sont complètement humains (c'est-à-dire, comprenant des régions variables et constantes humaines) et qui ont subi une recombinaison, une diversification jonctionnelle, une maturation d'affinité et une commutation isotypique in vivo dans un système vertébré non-humain. En outre, de tels anticorps sont humanisés (par exemple, totalement humains) - et sélectionnés - totalement in vivo, et, à ce titre, la présente invention exploite un filtrage in vivo pour leur aptitude à être exprimés, leur affinité et leurs caractéristiques biophysiques dans le contexte des appariements de régions variables et constantes humaines souhaitées. Ceci permet d'éviter des problèmes de sous-classement des caractéristiques d'anticorps lors de l'humanisation de la région constante d'anticorps chimériques in vitro.
EP13711120.9A 2012-03-28 2013-03-18 Vertébré non-humain transgénique pour l'expression d'anticorps entièrement humains à commutation isotypique Withdrawn EP2831245A1 (fr)

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US13/433,084 US9445581B2 (en) 2012-03-28 2012-03-28 Animal models and therapeutic molecules
US13/434,361 US9253965B2 (en) 2012-03-28 2012-03-29 Animal models and therapeutic molecules
GB1207814.3A GB2501753A (en) 2012-05-04 2012-05-04 Human antibodies
GB1208708.6A GB2502127A (en) 2012-05-17 2012-05-17 Multivalent antibodies and in vivo methods for their production
GBGB1216795.3A GB201216795D0 (en) 2012-09-20 2012-09-20 Human antibodies,heavy chains & repertoires selected in non-human in vivo systems
PCT/GB2013/050683 WO2013144567A1 (fr) 2012-03-28 2013-03-18 Vertébré non-humain transgénique pour l'expression d'anticorps entièrement humains à commutation isotypique

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