WO2009013620A2 - Recombinaison homologue - Google Patents

Recombinaison homologue Download PDF

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Publication number
WO2009013620A2
WO2009013620A2 PCT/IB2008/002572 IB2008002572W WO2009013620A2 WO 2009013620 A2 WO2009013620 A2 WO 2009013620A2 IB 2008002572 W IB2008002572 W IB 2008002572W WO 2009013620 A2 WO2009013620 A2 WO 2009013620A2
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Prior art keywords
heavy chain
human
antigen
gene segments
modified
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PCT/IB2008/002572
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WO2009013620A3 (fr
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Frank Grosveld
Rick Janssens
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Erasmus University Medical Center Rotterdam
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Priority claimed from GB0711244A external-priority patent/GB0711244D0/en
Priority claimed from GB0805281A external-priority patent/GB0805281D0/en
Application filed by Erasmus University Medical Center Rotterdam filed Critical Erasmus University Medical Center Rotterdam
Publication of WO2009013620A2 publication Critical patent/WO2009013620A2/fr
Publication of WO2009013620A3 publication Critical patent/WO2009013620A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Humanized animals, e.g. knockin
    • 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; CARE OF BIRDS, FISHES, INSECTS; 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; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; 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; CARE OF BIRDS, FISHES, INSECTS; 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; CARE OF BIRDS, FISHES, INSECTS; 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; CARE OF BIRDS, FISHES, INSECTS; 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype

Definitions

  • the present invention relates to methods for the production of heavy chain-only antibodies in vivo in non-human mammals from natural and modified heavy chain immunoglobulin genes lacking CHl functionality in response to antigen challenge.
  • human heavy chain-only antibodies and soluble human VH binding domains generated using the methods of the present invention are also described.
  • the invention also provides non-human mammals suited to the expression of diverse vertebrate heavy chain only gene loci lacking CHl functionality and hybrid heavy chain-only loci comprising mixtures of V, D and J gene segments and combinations of constant effector regions lacking CHl functionality sourced from diverse vertebrate species suited to the production of heavy chain-only antibodies and soluble VH domains.
  • antibodies The structure of antibodies is well known in the art. Most natural antibodies are tetrameric, comprising two heavy chains and two light chains. The heavy chains are joined to each other via disulphide bonds between hinge domains located approximately half way along each heavy chain. A light chain is associated with each heavy chain on the N-terminal side of the hinge domain. Each light chain is normally bound to its respective heavy chain by a disulphide bond close to the hinge domain.
  • each chain fold When an antibody molecule is correctly folded, each chain folds into a number of distinct globular domains joined by more linear polypeptide sequences.
  • V L variable
  • C L constant
  • Heavy chains have a single variable domain V H , a first constant domain (C H I ), a hinge domain and two or three further constant domains.
  • the heavy chain constant domains and the hinge domain together form what is generally known as the constant region of an antibody heavy chain. Interaction of the heavy (V H ) and light (V L ) chain variable domains results in the formation of an antigen binding region (Fv).
  • variable domains of both heavy (V H ) and light (V L ) chains some short polypeptide segments show exceptional variability. These segments are termed hypervariable regions or complementarity determining regions (CDRs). The intervening segments are called framework regions (FRs). In each of the V H and V L domains, there are three CDRs (CDRl -CDR3).
  • Antibody classes differ in their physiological function. For example, IgG plays a dominant role in a mature immune response. IgM is involved in complement fixing and agglutination. IgA is the major class of Ig in secretions - tears, saliva, colostrum, mucus - and thus plays a role in local immunity. The effector functions of natural antibodies are provided by the heavy chain constant region.
  • IgA In mammals, there are five types of antibody: IgA, IgD, IgE, IgG and IgM, with 4 IgG and 2 IgA subtypes present in humans.
  • IgA can be found in areas containing mucus (e.g. in the gut, in the respiratory tract or in the urinogenital tract) and prevents the colonization of mucosal areas by pathogens.
  • IgD functions mainly as an antigen receptor on B cells.
  • IgE binds to allergens and triggers histamine release from mast cells (the underlying mechanism of allergy) and also provides protection against helminths (worms).
  • IgG in its four isotypes provides the majority of antibody-based immunity against invading pathogens.
  • IgM is expressed on the surface of B cells and also in a secreted form with very high affinity for eliminating pathogens in the early stages of B cell mediated immunity (i.e. before there is sufficient IgG to eliminate the pathogens).
  • Normal B cells contain a heavy chain locus from which the gene encoding a heavy chain is produced by rearrangement.
  • a normal heavy chain locus comprises a plurality of V gene segments, a number of D gene segments and a number of J gene segments. Most of a V H domain is encoded by a V gene segment, but the C terminal end of each V H domain is encoded by a D gene segment and a J gene segment.
  • VDJ rearrangement in B-cells, followed by affinity maturation, provides a rearranged gene encoding each V H domain. Sequence analysis of H 2 L 2 tetramers demonstrates that diversity results from a combination of VDJ rearrangement and somatic hypermutation and that diversity in the CDR3 region is sufficient for most antibody specificities [see ref 2].
  • heavy chain-only antibody devoid of light chain
  • man Heavy Chain Disease
  • murine model systems Analysis of heavy chain disease at the molecular level showed that mutations and deletions at the level of the genome could result in inappropriate expression of the heavy chain C H I domain, giving rise to the expression of heavy chain-only antibody lacking the ability to bind light chain [3,4].
  • camelids as a result of natural gene mutations, produce functional IgG2 and IgG3 heavy chain-only dimers which are unable to bind light chain due to the absence of the C H I domain, which mediates binding to the light chain [5].
  • a characterising feature of the camelid heavy chain-only antibody is a particular subset of camelid V H domains, which provides improved solubility relative to human and normal camelid V H domains.
  • the particular subset of camelid V H domains are usually referred to as V HH domains.
  • camelid V 11H domains found in heavy chain-only antibodies are also characterised by a modified CDR3.
  • This CDR3 is, on average, longer than those found in non-camelid antibodies and is a feature considered to be a major influence on overall antigen affinity and specificity, which compensates for the absence of a V L domain in the camelid heavy chain-only antibody [7, 8].
  • the heavy chain locus in the camelid germline comprises gene segments encoding some or all of the possible heavy chain constant regions.
  • a rearranged gene transcript encoding a V HH DJ binding domain is spliced onto the 5' end of a transcribed gene segment encoding a hinge domain, to provide a rearranged gene encoding a heavy chain which lacks a C H I domain and is therefore unable to associate with a light chain.
  • Camelid V HH domains contain a number of characteristic amino acids at positions 37, 44, 45 and 47 [see ref 9]. These conserved amino acids are thought to be important for conferring solubility on heavy chain-only antibodies [9]. Only certain camelid V H domains are V HH domains with improved solubility characteristics. In contrast human V H domains derived from display libraries lack these characteristic amino acid changes at the V H /V L interface and consequently are less soluble or "sticky" relative to camelid V HH domains [10]. Unfortunately, the results of efforts to engineer or camelise human V H domains in vitro remains unpredictable since the introduction of camelising mutations in the human V H domain at the V H /V L interface alone is not sufficient to improve solubility in a predictable manner. It would appear that the introduction of features to enhance solubility may have to be compensated for by as yet undefined mutations elsewhere in the V H domain to maintain solubility and structural stability [see review 9].
  • Heavy chain-only monoclonal antibodies can be recovered from B-cells of camelid spleen by standard cloning technology or from B-cell mRNA by phage or other display technology [10]. Heavy chain-only antibodies derived from camelids are of high affinity. Sequence analysis of mRNA encoding heavy chain-only antibody demonstrates that diversity results primarily from a combination of VDJ rearrangement and somatic hypermutation [H].
  • camelid VHH and human soluble V H domains An important and common feature of natural camelid VHH and human soluble V H domains is that each domain binds as a monomer with no dependency on dimerisation with a V L domain. These camelid VHH and soluble V H binding domains appear particularly suited to the production of blocking agents and tissue penetration agents and eliminate the need to derive scFv in vitro when constructing antibody-based binding complexes (see WO/9923221 and PCT/GB2005/002892).
  • Antibody-based products are usually derived from natural tetrameric antibodies.
  • routes of derivation e.g. from transgenic mice
  • routes of manufacture e.g. from transgenic mice
  • product-specific substances of matter e.g. from transgenic mice
  • scFv molecules comprise only the variable domains of the heavy (V H ) and light (V L ) chains linked by a peptide linker to form a single molecule and are usually obtained by screening display libraries (e.g. phage display or emulsion display). Alternatively, they are engineered from natural antibodies by cloning the nucleic acid regions encoding the V H and V L domains into a transcription unit. scFv molecules obviously have a much smaller molecular weight and lack the constant region effector functions of natural antibodies. scFv molecules are often optimized in vitro.
  • Antibody-based products will represent a high proportion of new medicines launched in the 21st century. Monoclonal antibody therapy is already accepted as a preferred route for the treatment for rheumatoid arthritis and Crohn's disease and there is impressive progress in the treatment of cancer. Antibody-based products are also in development for the treatment of cardiovascular and infectious diseases. Most marketed antibody- based products recognise and bind a single, well-defined epitope on the target ligand (e.g. TNF ⁇ ).
  • TNF ⁇ target ligand
  • Manufacture of antibody-based products for therapy remains dependent on mammalian cell culture.
  • the assembly of a tetrameric antibody and subsequent post-translational glycosylation processes preclude the use of bacterial systems, although yeast engineered to produce mammalian glycosylation patterns shows promise as an alternative to mammalian cell-based production systems.
  • Production costs and capital costs for manufacture of antibody-based products by mammalian cell culture are high and threaten to limit the potential of antibody-based therapies in the absence of acceptable alternatives.
  • a variety of transgenic organisms are capable of expressing fully functional antibodies. These include plants, insects, chickens, goats and cattle.
  • Functional antibody fragments can be manufactured in E. coli but the product generally has low solubility and serum stability unless pegylated during the manufacturing process.
  • V 11H domains have been selected from randomised camelid V HH domains in display libraries derived from heavy chain-only antibody produced naturally from antigen challenge of camelids. These high affinity V HH domains have been incorporated into antibody-based products. These V HH domains, display a number of differences from classical V H domains derived from H2L2 antibodies, in particular a number of mutations that ensure improved solubility of the heavy chains in the absence of light chains. Most prominent amongst these changes is the presence of charged amino acids at positions 44, 45 and 47. It is supposed that these changes compensate for the absence of V L through the replacement of hydrophobic residues by more hydrophilic amino acids, thereby maintaining solubility in the absence of the V H /V L interaction [for review see ref 9 and other references cited therein].
  • Ward et al. [10] demonstrated unambiguously that cloned murine V H domains, when expressed as soluble protein monomers in an E. coli expression system, retain the ability to bind antigen with high affinity.
  • Ward et al. [10] describe the isolation and characterisation of V H domains derived from natural murine and human H2L2 antibodies and set out the potential commercial advantages of this approach when compared with classic monoclonal antibody production (see last paragraph). They also recognise that V H domains isolated from heavy chains which normally associate with a light chain lack the solubility of the natural tetrameric antibodies. Hence Ward et al. [10] used the term "sticky" to describe these molecules and proposed that this "stickiness" can be addressed through the design of V H domains with improved solubility properties.
  • V H solubility has subsequently been addressed using combinations of randomized and site-directed approaches using phage display.
  • Davies and Riechmann [12] and others have incorporated some of the features of V HH domains from camelid heavy chain-only antibodies in combination with phage display the goal to improve solubility whilst maintaining binding specificity.
  • V H binding domains have been derived from display libraries, intrinsic affinities for antigen remain in the low micromolar to nanomolar range, in spite of the application of affinity improvement strategies involving, for example, affinity hot spot randomisation [13].
  • affinity improvement strategies involving, for example, affinity hot spot randomisation [13].
  • the engineering of mammalian V H domains to improve solubility remains unpredictable.
  • the introduction of "camelising" mutations is insufficient to provide predictable outcomes and further mutations are required if enhanced solubility is to be obtained in the absence of aggregation [9].
  • Human V H derived from H2L2 antibodies or camelid V HH domains produced in vivo unlike V H produced from naive libraries using array technologies, have the advantage of improved characteristics in the CDR3 region of the normal antibody binding site as a result of somatic mutations introduced as a result of affinity maturation, in addition to diversity provided by D and J gene segment recombination.
  • Camelid V HH whilst showing benefits in solubility relative to human V H , is antigenic in man and must be generated by immunisation of camelids or by phage display technology using camelid sequences.
  • soluble heavy chain-only antibodies were derived from an antibody heavy chain locus in a germline (i.e. non-rearranged) configuration that contained two llama V HH (class 3) gene segments coupled to all of the human D and J gene segments and gene segments encoding human constant regions.
  • the gene segments encoding each of the constant regions had a deletion of the C H I domain to prevent the binding of light chain.
  • the locus contained the heavy chain immunoglobulin LCR at the 3' end and other intragenic enhancer elements to ensure a high level of expression in cells of the B lineage [17].
  • transgenic non-human mammals which, when challenged with antigen, results in the B-cell specific production of heavy chain-only antibody from a heavy chain-only immunoglobulin locus lacking CHl functionality comprising natural or engineered V gene segments (see PCT IB2007/003647) which, when recombined with D and J segments in transgenic non- human mammals in response to antigen challenge, generate functional, soluble, antigen- specific, heavy chain-only antibodies, and a source of soluble VH domains.
  • V, D J and heavy chain constant effector regions are of human origin.
  • B-cell activation reflects a normal wild type response. THE INVENTION
  • the present inventors have surprisingly overcome the limitations of the prior art and shown that fully-human, soluble V H domains can be derived by the incorporation of natural or engineered human V segments into a heavy chain locus wherein the host V, D and J gene segments are replaced by V, D and J segments of human origin, and the host immunoglobulin heavy chain effector constant regions replaced by immunoglobulin heavy chain constant effector regions (devoid of CHl), preferably of human origin.
  • a similar strategy can be used to generate soluble VH domains representative of any vertebrate species, or hybrid soluble VH domains comprising mixtures of V, D and J gene segments and constant effector regions derived from disparate vertebrate species.
  • Mouse heavy chain constant effector genes may be replaced by homologous recombination with mouse heavy chain genes lacking CHl, resulting in the generation of murine heavy chain only antibodies devoid of light chains.
  • CHl regions present in the endogenous murine immunoglobulin heavy chain gene locus can be removed individually by homologous recombination with the same outcome.
  • the mouse heavy chain constant effector gene regions may be replaced with any alternative heavy chain constant effector region lacking CHl functionality. All approaches will generate a heavy chain-only antibody. All three approaches will permit the isolation and characterisation of murine soluble VH domains by B-cells following antigen challenge.
  • the selection of functional V segments occurs in the non-human mammalian host as a result of VDJ rearrangement followed by antigen binding and affinity maturation of the selected heavy chain-only antibody V H domain in a B-cell dependent manner.
  • the elimination of CHl functionality ensures that light chain cannot bind, that normal host production of H2L2 antibody is disrupted and that antibody production in response to antigen challenge is limited to heavy chain-only antibody encoded by the modified endogenous heavy chain locus following allelic exclusion.
  • the presence or otherwise of functional host immunoglobulin light chains is immaterial to the invention, since the absence of a functional CHl domain prevents the assembly of H2L2 immunoglobulin tetramers.
  • the present invention provides a method for producing a heavy chain-only antibody in a transgenic non-human mammal comprising challenging with an antigen a transgenic non-human mammal having a endogenous heavy chain locus modified by homologous recombination which: lacks gene segments encoding a CHl domain or has been engineered to prevent expression of a functional CHl domain; and when expressed in response to antigen challenge, produces an affinity-matured heavy chain-only antibody devoid of CHl, having a soluble VH domain encoded by a VH gene segment which includes a preferred V gene segment incorporated as a result of VDJ rearrangement into said VH gene.
  • transgenic non-human mammal is intended to include any non-human mammal whose chromosomal structure has been modified such that it differs from the endogenous chromosomal structure which is naturally occurring.
  • one or more of the endogenous V gene segments are replaced using homologous recombination by heterologous natural, modified or engineered V gene segments or modified or engineered homologous V gene segments.
  • all the endogenous V gene segments are removed and replaced by one or more heterologous natural, modified or engineered V gene segments or modified or engineered homologous V gene segments.
  • homologous recombination is intended to include the reciprocal exchange of any chromosomal region between the endogenous chromosome and an exogenous nucleic acid source.
  • the term is intended to encompass the reciprocal exchange of any nucleic acid segment, including the reciprocal exchange of a short segment as well as the exchange of an entire gene locus.
  • one or more of the endogenous D gene segments is replaced using homologous recombination by heterologous natural, modified or engineered D gene segments or modified or engineered homologous D gene segments.
  • one or more of the endogenous J gene segments is replaced using homologous recombination by heterologous natural, modified or engineered J gene segments or modified or engineered homologous J gene segments.
  • one or more of the endogenous constant region gene segments is replaced using homologous recombination by heterologous natural, modified or engineered constant region gene segments or modified or engineered homologous constant region gene segments, all said constant region gene segments lacking CHl functionality.
  • the constant region may not be replaced but may have part or all of its CHl region modified or removed by homologous recombination so as to prevent subsequent association of the expressed heavy chain with immunoglobulin light chains.
  • V segments, D segments, J segments and/or constant region gene segments are of human origin, but V, D and J segments and constant regions can also be from other vertebrate species.
  • the transgenic non-human mammal may be produced by the targeted replacement of host heavy chain gene segments, preferably by human gene segments.
  • Targeted gene replacement occurs in a cell derived from the non-human mammal of choice, preferably an embryonic stem cell, a somatic cell or an iPS cell (see Aoi T, Yae K, Nakagawa M, Ichisaka T, Okita K, Takahashi K, Chiba T, Yamanaka S. Generation of Pluripotent Stem Cells from Adult Mouse Liver and Stomach Cells. Science. 2008 Feb 14; [Epub ahead of print] and references therein).
  • the non-human mammal is then derived from the modified cell in which the endogenous heavy chain locus in the non-human mammal now comprises natural or engineered V gene segments, D and J gene segments, preferably of human origin, replacing the equivalent non-human mammal host gene segments.
  • V gene segments, D and J gene segments preferably of human origin
  • each is engineered such that CHl functionality is eliminated.
  • each introduced immunoglobulin heavy chain constant effector region is separately engineered such that CHl functionality is eliminated.
  • the approach can be applied using V, D and J gene segments and/or constant regions lacking CHl functionality from potential any other vertebrate species, including mixtures of said gene segments and constant regions.
  • the present invention provides the method as described above, wherein said transgenic non-human mammal is produced by: modifying in vitro by homologous recombination an endogenous heavy chain immunoglobulin locus in an embryonic stem cell, a somatic cell or an iPS cell derived from said transgenic non human mammal; selecting cells comprising functionally modified endogenous heavy chain immunoglobulin sequences; deriving from said cells or nuclei from said cells transgenic non-human mammals comprising functionally-modified endogenous heavy chain immunoglobulin sequences; generating genetically modified animals from such modified cells by cloning (e.g. see Campbell KH, Mc Whir J, Ritchie WA, Wilmut I.
  • cloning e.g. see Campbell KH, Mc Whir J, Ritchie WA, Wilmut I.
  • the invention provides a method for producing a transgenic non-human mammal comprising a modified endogenous heavy chain locus which minimally lacks gene segments encoding a CHl domain or has been engineered to prevent expression of a functional CHl domain, and optionally comprises V, D and J gene and constant region genes (lacking CHl functionality) naturally absent from said non-human mammal and when expressed in response to antigen challenge, produces a heavy chain-only antibody devoid of CHl, having a soluble VH domain encoded by a VH gene segment which includes a preferred V gene segment incorporated and modified as a result of VDJ rearrangement and affinity maturation into said VH gene comprising: modifying in vitro by homologous recombination an endogenous heavy chain immunoglobulin locus in an embryonic stem cell or somatic cell derived from said non- human mammal; selecting cells comprising functionally modified endogenous heavy chain immunoglobulin sequences; deriving from said cells or nuclei from said cells transgenic non-
  • the transgenic non-human mammals as used by the invention are rodents and most preferably they are mice.
  • the invention also provides a method for the production of antigen-specific affinity- matured heavy chain-only antibody, and soluble VH binding domains following antigen challenge by: generating B-cell hybridomas; selecting cells expressing antigen-specific affinity matured heavy chain-only antibody; and isolating and characterising antigen-specific affinity matured heavy chain-only antibody secreted by hybridoma cells.
  • the invention provides a method for the production of antigen-specific affinity matured soluble VH binding domains from antigen-specific, affinity-matured heavy chain-only antibodies by challenging said transgenic non-human mammal with an antigen and
  • the invention provides use of a transgenic non-human mammal according to the invention for the production of affinity-matured human heavy chain-only antibodies, and soluble human VH domains.
  • the invention also provides the use of a transgenic non-human mammal according to the invention for the production of affinity- matured heavy chain-only antibodies, and soluble VH domains, of non-human vertebrate species or hybrid complexes where the V, D and J segments and the constant region are derived from two or more different species.
  • the non-human mammal comprises two modified immunoglobulin heavy chain loci lacking CHl functionality at least one of which is as defined above.
  • the V gene segments are natural or engineered V gene segments.
  • the locus includes multiple natural or engineered D gene segments.
  • the locus includes multiple natural or engineered J gene segments.
  • the locus comprises one or more gene segments encoding a constant region devoid of CHl functionality.
  • the locus contains multiple immunoglobulin heavy chain constant region gene segments each lacking CHl functionality.
  • each gene segment encoding a constant region is human.
  • each V, D and J gene segment is of human origin
  • each V gene segment is a natural V gene segment, or is a modified V gene carrying favourable mutations which may enhance solubility and stability either introduced as a result of affinity maturation in vivo, or based on structure/function analysis in silico.
  • a V gene segment is a gene segment derivable from vertebrates which maybe incorporated into a soluble VH domain as a result of VDJ rearrangement in a B-cell dependent manner. These include, but are not limited to, V gene segments derived from immunoglobulin heavy and light chain gene loci and T-cell receptor gene loci.
  • VH domain in the context of the present invention refers to a heavy chain antigen binding domain derived from a natural H2L2 antibody, or a heavy chain antigen binding domain derived by screening naive VDJ arrays for antigen binding domains followed by affinity maturation and/or engineering in vitro. Such VH domains are characteristically "sticky' and prone to aggregation in solution.
  • a "soluble VH domain" in the context of the present invention refers to an expression product of a V gene segment when recombined with a D gene segment and a J gene segment present in a heavy chain-only immunoglobulin locus lacking CHl functionality which undergoes affinity maturation in response to antigen challenge.
  • the soluble V H domain derivable following antigen challenge as used herein remains in solution and is active in a physiological medium and at physiological temperature in mammals without the need for any other factor to maintain solubility.
  • the solubility and stability of the soluble V H domain may be improved or further improved as a result of affinity maturation and somatic mutation following VDJ recombination.
  • the solubleV ⁇ domain is able to bind antigen as a monomer and, when expressed with an effector constant region, may be produced in mono-specific, bi-specific, multi-specific, bi-valent or multivalent forms, dependent on the choice and engineering of the effector molecules used (e.g. IgG, IgA IgM etc.) or alternative mechanisms of dimerisation and multimerisation (see PCT/GB2005/002892 and PCT/GB2007/000258).
  • the properties of the soluble V H domain expressed from the locus may be altered or improved by selection of V, D and/or J gene segments which encode sequences with the required characteristics prior to incorporation into the desired locus.
  • the incorporation of natural V gene segments into the locus requires that, following VDJ rearrangement, affinity maturation and natural selection provide soluble heavy chain-only antibodies from activated B-cells in a non-human mammal of choice. B cells failing to produce soluble antibodies will not survive, whilst those which do will undergo further natural selection in vivo through affinity maturation and the incorporation of favourable mutations in the V H gene segment but also the D and J gene segments following VDJ rearrangement. The resulting antibodies will be both soluble and show high antigen specificity.
  • affinity maturation may further improve stability and solubility characteristics in addition to providing for antigen specificity.
  • Preferred D and J segments, whether natural or engineered, may also be incorporated into the locus.
  • the method of the present invention represents an improvement over the prior art in that there is no requirement to generate non-human host mammals deficient in the production of competing H2L2 immunoglobulin tetramers.
  • Host immunoglobulin light chain expression is irrelevant due the absence of CHl functionality from the modified host heavy chain immunoglobulin locus.
  • heavy chain-only antibodies are produced in the absence of competition from natural H2L2 immunoglobulins.
  • a V H heavy chain-only antibody will only be produced if the V H domain, translated from natural or engineered, recombined V, D and J gene segments, is soluble.
  • the method also allows for the use of host heavy chain gene regulatory elements, thus for example ensuring that positional effects seen when introduced transgenes are expressed in the absence of locus control regions are eliminated.
  • Soluble V H domains can be analysed by sequencing the V H domains found in B cells producing soluble, high affinity antibodies. This may allow the identification of further somatic mutations in the V gene segment, but also D and J gene segments which impart increased solubility and stability. Once identified, these mutations can be incorporated into new V, D and J gene segments. These can again be incorporated into a heavy chain locus, which can then be expressed in further transgenic non-human mammals generated by homologous recombination or as transgenes and the process of selecting soluble V H domains repeated.
  • the invention provides further scope for the engineering of the heavy chain constant effector gene regions, this may include but is not limited to the incorporation or substitution of individual amino acids or amino acid sequences for example to introduce disulphide groups or eliminate T-cell epitopes; the incorporation of additional binding domains, alternative dimerisation domains, additional effector functions(e.g. enzymes, toxins, marker protiens), recognition sites for the post-translational modification of the encoded protein (e.g. glycosylation, phosphorylation, pegylation, proteolytic modification, radionucleotide attachment). Any modification may occur with the proviso that B-cell maturation and affinity maturation of the resulting soluble VH domain occurs following antigen challenge and that CHl functionality is absent.
  • additional binding domains e.g. enzymes, toxins, marker protiens
  • additional effector functions e.g. enzymes, toxins, marker protiens
  • recognition sites for the post-translational modification of the encoded protein e.g. glycosylation,
  • the transgenic non-human mammal is preferably a rodent such as a guinea pig, rat or mouse. Mice are especially preferred.
  • the mammal is of the genus Leporidae, preferably a rabbit.
  • Alternative mammals such as pigs, goats, sheep, cows or other animals may also be employed.
  • the mammal is a mouse.
  • transgenic non-human animals are generated using murine embryonic stem (ES) cell technology and, in the absence of ES cells, by cloning (either by nuclear transfer (see e.g. Solter D. Dolly is a clone— and no longer alone. Nature. 1998;394:315- 6) or iPS cells (see e.g. Gott Stamm H, Minger S. iPS cells and the politics of promise. Nat Biotechnol. 2008;26:271-2).
  • ES murine embryonic stem
  • a functional heavy chain-only antibody locus comprising entirely host sequences can be generated by the functional inactivation of the CHl domain from target heavy chain immunoglobulin effector constant regions.
  • Hybrid antibodies may be devised through the incorporation of V, D, J and constant effector region gene segments from disparate non-human mammals or vertebrates.
  • telomeres For example, using homologous recombination, one could insert lox or fit recombination sites at each end of the locus using standard recombination technology in ES cells (e.g. see www.ncrr.nih.gov/newspub/KOMP_Lloyd_l-18-2007.ppt and EP0658197). After treating the recombined ES cells with ere (acting on lox sites) or flp (acting on frt sites) recombinase, respectively, the entire murine locus would be removed.
  • the homologous recombination of very large loci is not efficient and hence the engineering may be done in a number of smaller steps, each time replacing parts of the locus with a new human (or other non-human vertebrate) part.
  • BACs or PACs instead of BACs or PACs, YACs and recombination in yeast could be used to introduce the lox sites into the human locus.
  • the recombination could be done at different positions of the murine locus, for example the human locus may not contain the human LCR when the 3' lox site is introduced to the 5' side of the murine LCR. As a result, expression of the human locus would be driven by the murine LCR.
  • the recombination could be carried out in a non-human mammalian somatic cell rather than ES cells.
  • the nucleus of the recombined cell could be used to generate mice using nuclear transfer cloning using standard procedures known to the person skilled in the art (http://www.liebertonline.eom/toc/clo/9/l).
  • the host somatic cells could be of any mammalian origin and be used to delete its immunoglobulin locus and use the nuclei for a nuclear transfer-mediated cloning of that mammal.
  • the non- human mammalian host is a rodent.
  • the locus to be recombined and replace the endogenous locus could be from a mammal other than human to produce antibodies of that particular species.
  • a number of variations are possible in the above scheme using homologous recombination for locus recombineering, particularly in host species lacking ES cell technology.
  • transgenic mice comprising heavy and light chain loci in a mouse background where the V, D and J regions of the murine heavy chain and the V and J regions of the murine light chain loci have been engineered such that these regions comprise the equivalent human gene segments or sequences is known (see EP 1399575 and www.Regeneron.com). Whilst a painstaking approach, the identical strategy may be used to generate a functional heavy chain locus in transgenic mammals.
  • the host heavy chain loci are selectively engineered so that natural or engineered V gene segments of the species of choice replace host heavy chain V segments.
  • Host D and J segments are similarly replaced and the host heavy chain constant regions are either replaced by constant regions of choice (devoid of C H I functionality) or the C H I domains are deleted from the host heavy chain loci.
  • the inserted sequences of choice are natural human V gene segments, optionally engineered (see PCT/IB2007/003647) to optimize the biophysical characteristics of the soluble V H domains derived subsequently in response to antigen challenge.
  • the host is a rodent, preferably a rat or mouse, allowing the application of standard laboratory molecular and cellular techniques for characterization of the resultant antibodies.
  • the host may potentially be any mammal for example sheep, pig, cow, goat, rabbit, horse, cat, dog or rodent.
  • the methods of generating heavy chain-only antibodies as described above may be of particular use in the generation of human heavy chain antibodies and soluble VH domains for human therapeutic use, as often the administration of antibodies to a species of vertebrate which is of different origin from the source of the antibodies results in the onset of an immune response against those administered antibodies.
  • species specific or hybrid heavy chain-only antibodies and soluble VH domains may be generated for diagnostic, agricultural and veterinary applications, and for diverse industrial applications such as catalysts and cleansing agents, in food and cosmetic applications.
  • the antibodies produced by the method of the invention have the advantage over those of the prior art in that they are of substantially a single or known class.
  • a further aspect of the invention provides a transgenic non-human mammal comprising one or more heterologous V H heavy chain loci as defined above.
  • the transgenic non-human mammal may be engineered to have a reduced capacity to produce antibodies that include light chains.
  • Such a non-human mammal is particularly suited to the introduction of additional heavy chain only gene loci by transgenesis. Where the complexity of heavy chain-only loci is increased, allelic exclusion determines which of multiple heavy chain-only loci are expressed in a B-cell specific manner following antigen challenge (see PCT / IB2007/001491).
  • Antibody-producing cells may be derived from transgenic non-human mammals as defined herein and used, for example, in the preparation of hybridomas for the production of heavy chain-only antibodies as herein defined.
  • nucleic acid sequences may be isolated from these transgenic non-human mammals and used to produce heavy chain-only chain antibodies and soluble VH domains. These maybe used as building blocks to construct soluble VH domain binding complexes using recombinant DNA techniques which are familiar to those skilled in the art (see for example WO/9923221, WO2004/058821, PCT/GB2005/002892 and references therein).
  • antigen-specific heavy chain-only antibodies may be generated by immunisation of a transgenic non-human mammal as defined herein.
  • the invention also provides a method for the production of heavy chain- only antibodies.
  • This may be a direct response to an environmental antigen (e.g. pathogen or allergen) or as a result of immunisation with a target antigen.
  • Antibodies and fragments thereof may be may be isolated, characterised and manufactured using well-established methods known to those skilled in the art. These antibodies are of particularly use in the methods described in PCT/GB2005/002892.
  • FIG. 1 Replacing the murine VH region.
  • the left hand side of the figure shows the complete murine IgH locus.
  • On the top line an homologous recombination insertion construct containing a selectable marker (green box) and lox 51 1 sites (blue boxes).
  • On bottom it shows a similar insertion construct but containing an extra loxP site.
  • Homologous recombination and treatment by transient ere recombinase expression leads to the removal of all sequences between the most extreme lox 51 1 sites yielding the locus in the top right hand corner).
  • This locus is subsequently cassette exchanged with a human VH construct containing human V, D and J regions flanked by Iox511 and a loxP site. It also includes a eukaryotic cell selectable marker to provide for easy selection of hybridomas after fusion (green box). The resulting chimaeric locus with humanVH, D, J and murine constant regions is shown at the bottom. Drawings are not to (relative) scale.
  • FIG. 1 Deletion of the murine CHl regions.
  • the locus generated in Figure 1 is further modified by homologous recombination deleting CHl regions using frt sites (purple boxes) and a selection marker gene different from that already present in the locus (green boxes). That selection marker is removed by (transient) flp recombinase expression (step boxed 2) resulting in the locus shown in the middle of the figure. This is further modified by the same principle (steps 3, 4, etc). Drawings are not to (relative) scale.
  • FIG. 3 Deletion of the murine CHl domains from the constant regions.
  • the start of the strategy is similar to that shown in Figure 2 in that an frt site is introduced somewhere (here just 3' of the IgGl constant region) in the locus.
  • This is subsequently caseete exchanged via loxP/frt and cre/flp treatment (step boxed 4) with a construct that contains the homologous exchange sites surrounding the same part of the murine heavy chain locus but from which the CHl regions have been removed by recombination in bacteria (see e.g. janssens et al 2006). Cassette exchange will lead to the locus show at the bottom. Drawings are not to (relative) scale
  • FIG. 4 Replacement of the murine constant regions with human constant regions.
  • the strategy is similar to that shown in the figures above, but this time inserting a selectable marker by homologous recombination into the 3'region of the murine locus containing a selectable marker (different from the one already present) with a loxP and frt site (Step boxedl). This will lead to the locus shown in line 2.
  • Treatment with ere recombinase (step boxed 2) will lead to the locus show in line three where all of the sequences between the loxP sites is removed.
  • This locus is further modified by cassette exchange or regular homologous recombination using the loxP and frt sites with a construct that contains human CHl deleted constant regions flanked by loxP and frt.
  • Regular homologous (red lines) or Cre/flp recombinase expression (black lines) leads to a cassette exchange and a completely human locus shown at the bottom. Drawings are not to (relative) scale.
  • FIG. 5 Replacement of the murine constant regions with human constant regions.
  • the strategy is similar to that shown in the figures above, but this time inserting a selectable marker by homologous recombination into the 3 'region of the murine locus containing a selectable marker (different from the one already present) with only an fit site (Step boxedl).
  • This can be immediately cassette exchanged with a human constant region containing the same loxP and frt sites in a subsequent step (boxed T).
  • the resulting locus is a completely human heavy chain only antibody coding locus. Drawings are not to (relative) scale.
  • FIG. 6 Generation of poly-proteins, multivalent heavy chain only antibodies or variants thereof by replacing the CH2 and CH3 domains with other dimerising domains.
  • the top line shows two heavy chain only antibodies with specificities VHl and VH2 respectively. From these the soluble VHl and VH2 domains can be derived by routine cloning methods. The soluble VHl and VH2 domains are combined into poly-proteins as shown in the two examples at the left of the bottom line structures using (preferably non- immunogenic) hinge sequences.
  • VH2 is made into multivalent heavy chain only antibodies by adding the VH2 via a hinge sequence to the carboxy terminus of the VHl heavy chain only antibody (third from left, bottom line) resulting in a tetravalent bi-specific heavy chain only antibody.
  • Additional soluble VH domains could be added in addition to either the N or C terminus of the antibody (not shown).
  • CH2 and CH3 domains are replaced by other dimerisation domains (e.g. the jun leucine zipper).
  • the murine V,D and J gene segments are spread over several megabases of the murine genome and the most effective manner to remove these from the genome is to use cre/lox (or flp/frt) recombination technology. In this example only the ere system is used.
  • First a region to the most 5' murine V,D,J region is isolated by PCR form a murine YAC containing the 5' end of the immunoglobulin locus. This can either be done by standard cloning technology or standard long range PCR technology.
  • a selectable marker e.g.
  • flank-neo-flank sequences is preferably provided with a counterselectable marker (such as the thymidine kinase gene, TK).
  • TK thymidine kinase gene
  • Clones with correctly recombined loci are subsequently identified by standard means (PCR and/or Southern blots). The procedure is repeated for a region flanking the 3' side of the most 3' J region using another selectable marker (e.g. puromycin) and with the same lox sequence followed by a different sequence lox sites (e.g lox 51 1 at the 5' end versus lox 51 1 plus lox P at the 3 'end, Blue boxes and brown box Figure 1 boxed 2; and see Lauth et al., Characterization of Cre-mediated cassette exchange after plasmid microinjection in fertilized mouse oocytes, Genesis 27, 153-8 and references therein). This will result in an ES cell that contains lox sites before the V region and past the J region.
  • another selectable marker e.g. puromycin
  • the cassette that is recombined into the murine locus is any combination of new V regions, D regions and J regions. In addition it would preferably contain a selectable marker that enables an easier selection of hybridomas after immunisation.
  • the final locus shown as the end result in Figure 1 can now be used for a number of purposes and two of these are described here, firstly the deletion of CHl regions from the remaining murine constant regions in this example ( Figure 2) resulting in a hybrid human/m urine IgH locus that would result in the production of hybrid heavy chain only antibodies due to the absence of CHl.
  • the locus is used to generate a locus that also contains novel (preferably human) constant regions lacking CHl .
  • the next stage of this example is the deletion of the CHl region from the murine constant region(s) ( Figure 2).
  • the first step in this procedure ( Figure 2, boxed 1) is the generation of a construct that contains a selectable marker different from the marker left in the locus in the modified VH locus in Figure 1.
  • This selectable marker is cloned between fit sites (or lox sites other than loxP or Iox511) and the region flanking the CHl region of the murine IgM constant region by standard procedure (e.g. Janssens et al., 2006).
  • the CHl deleted region is subsequently introduced into the murine locus from Figure 1 ( Figure 2 , top line) by homologous recombination (boxed 1 step completed).
  • CHl regions need to be replaced it may be advantageous to replace several constant regions lacking CHl in one recombination (or cassette exchange).
  • the CHl regions could first be deleted from a PAC or BAC containing several constant regions in their normal configuration by standard homologous recombination in bacteria (e.g. see Janssens et al 2006 and references therein).
  • Several modified constant region could than be introduced to replace the normal mouse constant regions in a fashion similar to that described above or by a cassette exchange over large distances, e.g.
  • the murine CHl region is replaced by novel human CHl deleted constant domains.
  • Figure 4 shows this is achieved through the introduction of a selection marker and a loxP and frt site in the locus shown in Figure 1 by homologous recombination by standard procedures (step 1, boxed 1 in Figure 4). This results in the locus that contains two loxP sites ( Figure 2 line 2 and using a selectable marker different from the one already present at the 5' end of the locus). Treat ment with ere leads to the locus lacking a constant region ( Figure 4, line 3). In the next step a new set of constant regions (lacking CHl) is introduced to generate a completely human Ig locus that would generate completely human heavy chain only antibodies.
  • the constant regions could be from other species and the procedure could be carried out in steps. It is also obvious from the previous example that the procedure could be carried out by leaving out the loxP site in step 1 ( Figure 5) and introduce only an frt site at the 3 'end of the locus. This is cassette exchanged in a second step (using cre/flp cassette exchange step boxed 2, Figure 5) with a cassette containing human constant regions lacking CHl domains. The result would as in Figure 4 result in a locus expressing completely human heavy chain only antibodies. This latter exchange is favourable over the procedure in Figure 4 as it requires less steps but has the disadvantage that there is not intermediate locus completely deficient of a constant region that would also be useful for applications requiring a Ig deficient mouse background.
  • heavy chain only antibodies or soluble human VH binding domains have been generated by immunizing animals that carry any of the transgenic loci described in the examples above (or modifications thereof), these can be used to generate a number of other binding proteins, such as poly-proteins, multivalent heavy chain only antibodies or variants thereof by replacing the CH2 and CH3 domains with other dimerising domains ( Figure 6).
  • poly-proteins soluble VH domains could be joined together using non immunogenic linker sequences, for example as shown in Figure 6 (see also WO/9923221 for camelid VHH polyproteins), one soluble VH domain with specificity 1 linked to a soluble VH domain with specificity 2 linked to another soluble VH domain of specificity 1.
  • VHI could be specific for a particular antigen whereas VH2 recognizes a stable blood protein, such that the trimeric soluble VH polyprotein would have a longer half life.
  • soluble VH domains with a second specificity or the same specificity could be added to the heavy chain only antibody using non immunogenic linkers to generate tetra-valent multispecific or tetra-valent monospecific heavy chain only antibodies (see PCT/GB2005/002892). Again many variations are possible including adding more than one soluble VH domain.
  • other dimerisation domains could be used. Many such other dimerisation sequences are known and many variations can be generated (see PCT/GB2007/000258).

Abstract

L'invention concerne des procédés permettant de produire des anticorps uniquement à chaîne lourde in vivo chez des mammifères non humains à partir de gènes d'immunoglobuline à chaîne lourde manquant de fonctionnalité CHI en réponse à un test de provocation antigénique. L'invention concerne également des anticorps à chaînes lourde humains uniquement et des domaines de liaison VH humains solubles générés par des procédés selon l'invention, en particulier. L'invention concerne également des mammifères non humains appropriés pour l'expression de divers locus de gène à chaîne lourde uniquement de vertébrés manquant de fonctionnalité CHI, et de mélanges comprenant des locus à chaîne lourde uniquement hybrides de segments de gènes V, D et J et des combinaisons de régions effectrices constantes manquant de fonctionnalité CHI émanant de diverses espèces de vertébrés appropriées pour la production d'anticorps à chaînes lourdes uniquement et de domaines VH solubles.
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