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  • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
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  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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  • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
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  • A01K2217/00—Genetically modified animals
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  • A01K2217/00—Genetically modified animals
  • A01K2217/07—Animals genetically altered by homologous recombination
  • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
• • A—HUMAN NECESSITIES
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  • A01K2217/15—Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
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  • A01K2217/00—Genetically modified animals
  • A01K2217/20—Animal model comprising regulated expression system
  • A01K2217/206—Animal model comprising tissue-specific expression system, e.g. tissue specific expression of transgene, of Cre recombinase
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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  • A01K2227/00—Animals characterised by species
  • A01K2227/10—Mammal
  • A01K2227/105—Murine
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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  • A01K2267/01—Animal expressing industrially exogenous proteins
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  • C07K16/00—Immunoglobulins [IG], 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
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
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  • C07K2317/35—Valency
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  • C07—ORGANIC CHEMISTRY
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
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  • 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
  • C12N2015/8518—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic expressing industrially exogenous proteins, e.g. for pharmaceutical use, human insulin, blood factors, immunoglobulins, pseudoparticles
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  • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
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  • C12N2800/00—Nucleic acids vectors
  • C12N2800/90—Vectors containing a transposable element

Definitions

• a method of producing in a non-human vertebrate comprising (in N- to C- terminal direction) an antibody variable domain, an optional linker and an epitope binding moiety, wherein the variable domain and the epitope binding moiety specifically bind respective first and second epitopes), the method comprising
• E and E' form a second binding site that specifically binds a second epitope when E' is an antibody variable domain (eg, E is a VH and E' is a VL; or E is a VL and E' is a VH).
• E' is an antibody variable domain
• a non-human vertebrate eg, a mouse or a rat
• a non-human vertebrate cell eg, a mouse cell or a rat cell or a hybridoma, the cell genome comprising (in 5' to 3' direction) a rearranged variable region and a non-mu constant region; wherein the non-mu constant region comprises (in 5' to 3' direction) a nucleotide sequence encoding an optional linker and the rearranged variable region which encodes a first variable domain, for expressing polypeptide chains comprising (in N- to C- terminal direction) an epitope binding site comprising second and first antibody variable domains derived respectively from a rearranged second variable domain nucleotide sequence and from the first variable domain nucleotide sequence, wherein the second nucleotide sequence has been rearranged in the cell.
• the non-mu constant region comprises (in 5' to 3' direction) a nucleotide sequence encoding an optional linker and the rearranged variable region which encodes a first variable domain, for expressing polypeptide chains comprising
• the in vivo antibody production machinery of the non-human vertebrate or cell of the invention can be dedicated to H2 antibody production without any significant 4-chain antibody (H2L2) production hampering the extent of H2 antibody response following immunisation and ultimately the desirable levels of H2 that are obtainable.
• H2L2 4-chain antibody
• This advantage enables this isotype-switched, antigen/epitope-specific in vivo response to develop from the desirable recombined VDJ repertoire and B-cell compartments arising from the retention of endogenous IgM stage as further discussed below.
• configurations and aspects of the invention retain the ability to harness the endogenous antibody and B-cell development control mechanisms of the non-human vertebrate or cell. This includes the ability to harness endogenous terminal deoxynucleotidyl transferase (TdT) activity and junctional mutation as well as proper B-cell development (including at early stages of development when IgM is expressed) to yield good size B-cell compartments and good size and quality repertoires of recombined VDJ diversity from which to subsequently select and mature isotype-switched heavy chains produced from the antibody heavy or light chain locus of the invention.
• TdT terminal deoxynucleotidyl transferase
• RNA produced from the transgenic heavy chain locus 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). These can optionally comprise a constant region.
• 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.
• 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 or antibody will be prepared by at least one purification step.
• 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 lOpM or less.
• the SPR is carried out at a physiological salt level, eg, 150mM NaCI.
• an antibody heavy chain locus comprising (in 5' to 3' direction) a rearranged or unrearranged variable region, a first switch (eg, an S-mu, eg a human or endogenous S-mu), a mu constant region (eg, comprising a CHI gene segment), a second switch (eg, an S-gamma, eg a human or endogenous S-gamma), and a non-mu (eg, gamma) constant region; wherein the heavy chain locus of each cell is capable of undergoing IgM to the non-mu (eg, IgG) isotype switching; and optionally the non-mu constant region lacks a CHI gene segment and/or a hinge nucleotide sequence; and
• the non-mu constant region comprises (in 5' to 3' direction) a nucleotide sequence encoding an optional linker and a predetermined epitope binding moiety, wherein following isotype switching the vertebrate expresses multivalent polypeptide chains comprising (in N- to C- terminal direction) an antibody variable domain derived from said variable region of the heavy chain locus, an optional linker and an epitope binding moiety, wherein the variable domain and the epitope binding moiety specifically bind respective first and second epitopes.
• linker An example of a linker is (PAS)n(GS)m. In another embodiment, a linker is (GGGGS)n(GS)m. In another embodiment a linker is (TVAAPS)n(GS)m. In another embodiment a linker is
• a method of producing multivalent (eg, bivalent, eg, bispecific) polypeptide chains comprising (in N- to C- terminal direction) an antibody variable domain, an optional linker and an epitope binding moiety, wherein the variable domain and the epitope binding moiety specifically bind respective first and second epitopes (eg, wherein the epitopes are different or the same), the method comprising
• dimer of polypeptides according to the invention wherein the dimer lacks antibody light chains.
• the dimer may provide a multivalent (eg, multispecific) heavy chain-only (H2) antibody that is produced, matured and selected in vivo in this format.
• V is an antibody variable domain (eg, a VL) that specifically binds a first epitope
• a non-human vertebrate eg, a mouse or a rat
• the vertebrate comprises lymphocytic cells, each cell genome comprising
• the or each epitope binding moiety or domain is selected from the group consisting of an antibody variable domain (eg, a VL or a VH, an antibody single variable domain (domain antibody or dAb), a camelid VHH antibody single variable domain, a shark immunoglobulin single variable domain (NA V), a NanobodyTM or a camelised VH single variable domain); a T-cell receptor binding domain; an immunoglobulin superfamily domain; an agnathan variable lymphocyte receptor (J Immunol; 2010 Aug l;185(3):1367-74; "Alternative adaptive immunity in jawless vertebrates;
• NA V are another type of immunoglobulin single variable domain which were identified in cartilaginous fish including the nurse shark. These domains are also known as Novel Antigen Receptor variable region (commonly abbreviated to V(NAR) or NARV). For further details see Mol. Immunol. 44, 656-665 (2006) and US20050043519A.
• epitope binding moieties and domains include proteins which have been used as a scaffold to engineer different target antigen binding properties include human ⁇ -crystallin and human ubiquitin (affilins), kunitz type domains of human protease inhibitors, PDZ- domains of the Ras-binding protein AF-6, scorpion toxins (charybdotoxin), C-type lectin domain (tetranectins) are reviewed in Chapter 7 - Non-Antibody Scaffolds from Handbook of Therapeutic Antibodies (2007, edited by Stefan Dubel) and Protein Science 15:14-27 (2006). Epitope binding moieties and domains of the present invention could be derived from any of these alternative protein domains.
• (iii) means for turning off light chain expression, wherein functional light chains are not expressed after isotype switching to said non-mu-type antibodies.
• the means for turning off light chain expression comprises a nucleotide sequence (eg, a light chain variable region, J region and/or a CL region of the light chain locus) that is essential for functional light chain expression from said light chain locus, wherein the nucleotide sequence is functional in immature B-cells expressing IgM, but the nucleotide sequence is absent or substantially non-functional in mature B-cells expressing said non-mu-type antibodies.
• a nucleotide sequence eg, a light chain variable region, J region and/or a CL region of the light chain locus
• kappa chain expression is turned off using this means.
• lambda chain expression is turned off using this means.
• kappa and lambda chain expression is turned off using this means. Where a mouse system is used, lambda chain expression is normally low (around 5%), so it may not be necessary to turn off lambda chain expression as long as kappa chain expression is turned off when isotype-switched chains are being expressed.
• the non-mu constant region comprises (in 5' to 3' direction) a nucleotide sequence encoding an optional linker and a predetermined epitope binding moiety (or a plurality of epitope binding domains, eg, two or three with optional linkers; eg, in place of a CHI domain), wherein following isotype switching the vertebrate expresses multivalent (eg, bivalent, eg, bispecific) polypeptide chains comprising (in N- to C- terminal direction) an antibody variable domain derived from said variable region of the heavy chain locus, an optional linker and an epitope binding moiety, wherein the variable domain and the epitope binding moiety specifically bind respective first and second epitopes (eg, wherein the epitopes are different or the same).
• multivalent eg, bivalent, eg, bispecific
• polypeptide chains comprising (in N- to C- terminal direction) an antibody variable domain derived from said variable region of the heavy chain locus, an optional link
• the invention provides improved methods for the in vivo production of isotype-switched non-mu H2 antibodies and heavy chains as follows.
• a light chain variable region and a constant region eg, a CK or CA
• IgM antibodies eg, in immature B-cells
• 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)
• serial recombinase mediated cassette exchange - sRMCE - eg, see the Kymab Limited PCTs disclosed herein
• the skilled person can build up transgenic loci 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 mother.
• germline transmission and any necessary subsequent breeding and crossing the skilled person can arrive at progeny vertebrates and cells bearing a gene locus of the invention.
• 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 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 E coli).
• 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
• the invention relates to use of a non-human vertebrate of the present invention in the analysis of the likely effects of drugs and vaccines in the context of an antibody 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 vertebrate of the invention and monitoring one or more of: the immune response, the safety profile; the effect on disease.
• the lambda chain repertoire comprises at least 20, 25, 30, 35 or 40 different human VA gene segments.
• derivative antibodies/chains/polypeptides are antibodies/chains/ polypeptides that have one or more mutations compared to the isolated antibody or chain or polypeptide (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 a antibody or chain or polypeptide following conjugation to a toxic payload or reporter or label or other active moiety.
• mice according to the invention can for example express heavy chains from splenic B-cells in a mouse that produces a normal proportion or percentage of mature splenic B-cells, eg as determined by FACS.
• splenic B-cells produced by the mouse of the invention are mature B-cells.
• Splenic B-cells are B220 + and express B220 at relatively high levels as the skilled person will know.
• Mature splenic B-cells express B220 and IgD, both at relatively high levels as will be known by the skilled person.
• IgM expression is relatively low in mature splenic B- cells, again as is known in the art. For example, see J Exp Med. 1999 Jul 5;190(l):75-89; "B cell development in the spleen takes place in discrete steps and is determined by the quality of B cell receptor-derived signals"; Loder F et al.
• the mouse produces a normal ratio of Tl, T2 and mature splenic B-cells, eg, as determined by FACS.
• the mouse of the invention produces about 40-70% mature splenic B-cells, 15-35% splenic Tl cells; and 5-10% splenic T2 cells (percentage with reference to the total splenic B220-positive (high) population).
• normal is meant comparable to a
• normal is meant comparable to bone marrow B-cell production in a mouse (eg, a naive mouse) expressing only mouse antibody chains, eg, a mouse whose genome comprises only wild-type functional Ig heavy and light chain loci, eg, a wild-type mouse.
• At least 90% of the heavy chains are heavy chains comprising human variable regions.
• at least 90, 95, 96, 97, 98, 99 or 99.5% or 100% of the heavy chains comprise human variable regions, ie, variable regions derived from the recombination of human VH with human D and JH gene segments.
• VK1-5, VK1-6, VK1-8 and VK1-9 (i) VK5-2 and VK4-1); or
• a non-human vertebrate eg, a mouse or a rat
• whose genome comprises an Ig locus comprising (in 5' to 3' orientation)
• an unrearranged human variable region eg, a heavy chain variable region
• a rearranged sequence of a second variable domain eg, a VL domain
• sequence (i) is capable of undergoing rearrangement so that the vertebrate produces antibody chains each having an epitope binding site that comprises first and second variable domains derived from sequences (i) and (iii), wherein there is no further antibody domain between the first and second variable domains.
• an antibody heavy chain locus comprising (in 5' to 3' direction) a rearranged or unrearranged variable region (eg, a heavy chain variable region), a first switch, a mu constant region, a second switch and a non-mu (eg, gamma) constant region; wherein the heavy chain locus of each cell is capable of undergoing IgM to the non-mu (eg, IgG) isotype switching; and
• the vertebrate expresses polypeptide chains comprising (in N- to C- terminal direction) (a) an antibody variable domain derived from said variable region of the heavy chain locus, an optional linker, an epitope binding moiety and a non-mu (eg, gamma) constant domain (eg, gamma Fc) or (b) an antibody variable domain derived from said variable region of the heavy chain locus, a non-mu (eg, gamma) constant domain (eg, gamma Fc), an optional linker and an epitope binding moiety, wherein the variable domain and the epitope binding moiety specifically bind the respective first and second epitopes.
• polypeptide chains comprising (in N- to C- terminal direction) (a) an antibody variable domain derived from said variable region of the heavy chain locus, an optional linker, an epitope binding moiety and a non-mu (eg, gamma) constant domain (eg, gamma Fc) or (b
• a non-human vertebrate eg, a mouse or a rat
• the vertebrate comprises lymphocytic cells whose genomes comprise an antibody heavy or light chain locus comprising (in 5' to 3' direction) a rearranged variable region and a non-mu constant region;
• step (d) providing the nucleotide sequence of the epitope binding moiety in the non-mu constant region of heavy chain loci in the genome of the vertebrate, wherein the polypeptide chains selected in step(b) bind the first and second epitopes.
• each somatically mutated variable region in step (a) is provided by a non-mu-type (eg, IgG-type) heavy chain that lacks a CHI domain.
• a multispecific polypeptide chain obtained by the method of any one of clauses 19 to 23 or a derivative thereof that specifically binds the first and second epitopes.
• a bivalent (eg, bispecific) antibody comprising a dimer of a heavy chain, wherein each heavy chain is a polypeptide according to any one of clauses 25, 27, 28 and 30 to 33 and has in N- to C- terminal direction) the structure
• V- N-E-L-CH1 wherein N is L together with an optional antibody hinge region; M is an optional antibody hinge region; optionally wherein the constant domains are human constant domains.
• a target antigen eg, human EpCAM or human CD19
• said target antigen is an antigen of a target cell other than said human immune effector cell.
• a non-human vertebrate eg, a mouse or a rat
• the vertebrate comprises lymphocytic cells, each cell genome comprising an antibody heavy chain locus comprising (in 5' to 3' direction) a rearranged or unrearranged variable region and a mu constant region comprising a nucleotide sequence encoding an optional linker and a predetermined epitope binding moiety
• the vertebrate expresses multivalent polypeptide chains comprising (in N- to C- terminal direction) an antibody variable domain derived from said variable region of the heavy chain locus, an optional linker and an epitope binding moiety, wherein the variable domain and the epitope binding moiety specifically bind respective first and second epitopes.
• the common light chain comprises, for example, a human VK1-39JK or VK3-20JK (eg, VK1-39JK5 or VK3-20JK1) or VpreB (such as human VpreB).
• VK1-39JK or VK3-20JK eg, VK1-39JK5 or VK3-20JK1
• VpreB such as human VpreB
• FIG. 3 is a schematic representation of an improved route to in vivo production of heavy chain- only antibodies in non-human vertebrates according to the invention.
• the figure shows the architecture of heavy chain loci in a transgenic vertebrate.
• Each heavy chain locus comprises a variable region comprising human VH, D and JH gene segments upstream of an endogenous (eg, mouse or rat) S-mu switch and an endogenous C-mu region comprising constant region gene segments (CHI, CH2, CH3, H (hinge) and M (membrane regions)).
• an endogenous eg, mouse or rat
• C-mu region comprising constant region gene segments
• S-gamma switch eg, an endogenous switch
• a gamma-1 constant region eg, an endogenous gamma-1 constant region
• figure 6 shows a schematic method for producing bi-specific heavy chains and antibodies as well as heavy chain loci in non-human vertebrate (eg, mouse or rat) genomes for producing these in vivo.
• non-human vertebrate eg, mouse or rat
• binding moiety(ies) of BM are functional without need for light chain pairing.
• the BM nucleotide sequence can be positioned immediately 5' of the CH2 gene segment in the non-mu constant region (and thus immediately N-terminal of the CH2 or Fc in the product heavy chain and antibodies), or it can replace CH2 or CH3, or it can be 3' of CH3 (ie, at the C-terminus of the Fc in a resulting product).
• the BM can encode optional linker-dAb (where the dAb is of known antigen binding specificity); the resulting 4-chain product is a mAb with a dAb at the C-terminus of each heavy chain.
• DNA fragments including a synthetic common light chain genomic sequence (a promoter, a leader exon, a intron and an exon encoding the common light chain), a mouse genomic intronic ⁇ region, and a lox2272/66 and lox2272/71-floxed mouse CK region are ligated together to generate the insertion sequence (see sequence below).
• the insertion sequence is further subcloned into a vector, pBR322 with loxP-PB3'LTR and PB3' LTR-promoter-less PuroATK gene-lox5171 to form an insertion vector.
• AKTTP AKTTPRLGG; or EEGEFSEA
• each of L2 and L3 is separately selected from VEGGSGGSGGSGGSGGVD; or (GGGGS); or (GGGGS) 2 ; or (GGGGS) 3 ; or (GGS) 3 ; or AKTTP; or AKTTPRLGG; or EEGEFSEA.

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• 2012
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