Non-human transgenic animals expressing humanized antibodies and uses thereof

Abstract

The present invention provides a non-human mammal containing an endogenous λ light chain locus, an endogenous κ light chain locus and an endogenous heavy chain locus, each of which can be rearranged so that immunoglobulin heavy chain and light chain genes are formed and expressed in B cells after antigen challenge, but the loci have been mutated so that the ability to form functional immunoglobulin tetramers comprising rearranged heavy and light chains produced from the mutant loci is greatly reduced or eliminated.

Description

Field of the Invention

[0001] The present invention relates to improved methods and uses of the same for deriving and selecting a diverse library of functional affinity matured tetrameric immunoglobulins (comprising heavy and light chains) in response to antigenic challenge using transgenic non-human mammals.

[0002] Specifically, the present invention relates to a non-human mammal, preferably a mouse, that has been engineered so that its ability to produce mouse endogenous κ and / or λ light chain immunoglobulins is greatly reduced or its ability to compound with heavy chains is reduced, eliminated or blocked. The ability of the non-human mammal of the present invention to produce mouse functional endogenous heavy chains is also reduced. Therefore, its ability to form functional immunoglobulin tetramers comprising rearranged heavy and light chains produced by the mutant locus has been greatly reduced or eliminated. The present invention also discloses methods for producing such mammals and methods for using such mammals to produce human tetrameric antibodies and hybrid tetrameric antibodies using immunoglobulin heavy and light chain transgenes.

[0003] In the following description, all amino acid residue position numbers are given according to the numbering system devised by Kabat et al. ((1991), US Public Health Services publication No. 91-3242). Background of the Invention

[0005] Antibody

[0006] The structure of antibodies is well known in the art. Most natural antibodies are tetramers, comprising 2 heavy chains and 2 light chains. The heavy chains are connected to each other by disulfide bonds between the hinge domains roughly located in the middle of each heavy chain. The light chains associate with each heavy chain at the N-terminal side of the hinge domain. Each light chain is usually bound to its corresponding heavy chain by a disulfide bond close to the hinge domain.

[0007] When an antibody molecule is correctly folded, each chain folds into many distinct globular domains connected by relatively linear polypeptide sequences. L ) domain and constant (C κ or C λ ) domain. The heavy chain has a single variable domain V H , the first constant domain (C H 1), hinge domain and two or three additional constant domains. The constant domain of the heavy chain and the hinge domain together form what is commonly referred to as the antibody heavy chain constant region. H ) chain and light (V L The interaction of the heavy chain variable domains results in the formation of the antigen binding region (Fv). H 1 domain and the C of the light chain κ or Cλ The V domain facilitates the interaction between the heavy and light chains. In general, antigen binding requires both V H Need V again L , but studies have shown that heavy chain dimers and amino-terminal fragments remain active in the absence of light chains (Jaton et al. (1968) Biochemistry, 7, 4185-4195). Normally, the proportion of circulating lambda light chains is low, perhaps accounting for 2-5% of all light chains complexed into tetrameric immunoglobulins in plasma (Goldsby et al. (2003) Immunology, 5th edition, WH Freeman & Co NY).

[0008] In vitro manipulation of heavy chain immunoglobulin genes to construct new antibodies was first described in the 1980s. Most early antibody engineering studies used rearranged mouse immunoglobulin μ genes (IgM) generated against well-characterized antigens. The characteristics of this antibody are known to exist in V H The antigen binding specificity of the V domain was determined by the assembly and secretion of an unrelated light chain, as it was shown that antigen binding was retained (Neuberger and Williams (1986) Phil. Trans. R. Soc. Lond., A 317, 425-432). Using this system, it was shown that mouse antigen-specific V H The binding domain was obtained to contain mouse antigen-specific V H The resulting hybrid IgE retains antigen specificity and exhibits the expected effector activity of IgE (Neuberger et al. (1985) Nature, 314, 268-270). Other examples of heavy chain engineering include: encoding a mouse V that retains anti-phosphocholine activity H Hybrid mouse-human antibody genes of human / IgA or IgG antibody fusions (Morrison et al. (1984) PNAS, 81, 6851-6855); containing mouse V H and anti-cancer-associated antigen 17-1A antibody comprising a human IgG (γ3) constant region (Sun et al. (1987) PNAS, 84, 214-218); and an antibody comprising a human IgG (γ1) constant region and a mouse V H Anti-human T cell antibody (anti-CD7) with a domain (see Heinrich et al. (1989) J. Immunol., 143, 3589-97).

[0009] Normal human B cells contain a single immunoglobulin heavy chain locus on chromosome 14, from which the genes encoding the heavy chains are generated by rearrangement. In mice, the heavy chain locus is located on chromosome 12. The normal heavy chain locus contains multiple V gene segments, many D gene segments, and many J gene segments. Most VH The domains are encoded by V gene segments, but each V H The C-terminus of the domain is encoded by the D gene segment and the J gene segment. VDJ rearranges in B cells, followed by affinity maturation, to form a single V H The structural domain provides its antigen binding specificity.Sequence analysis of the normal H2L2 tetramer obtained from the heavy chain immunoglobulin containing a single V segment shows that the diversity of response antigen attack is mainly caused by the combination of VDJ rearrangement and somatic hypermutation (Xu and Davies (2000) Immunity, 13, 37-45). More than 50 human V gene segments are present in the human genome, of which only 39 have function.In normal diploid antibody-producing B cells, each cell produces antibody tetramer (H2L2) from a group of heavy chain and light chain antibody loci.Due to the process called allele exclusion, the use of another group of loci is non-productive (Singh et al. (2003) J. Exp. Med., 197, 743-750 and references therein).

[0010] Fully human antibodies (H2L2) can now be obtained from transgenic mice that respond to antigenic challenge. Such transgenic mice generally contain a single human heavy chain immunoglobulin locus and a separate human light chain immunoglobulin locus. The corresponding endogenous mouse heavy chain, kappa light chain, and optionally lambda light chain locus coding sequences are deleted or partially deleted. Thus, only human antibodies containing kappa light chains are produced in the presence of a low background of mouse / human antibodies containing human heavy chains and mouse lambda light chains (WO90 / 04036; WO93 / 12227; WO98 / 24893; US5877397, US5814318, and US6162963). Deleting segments of all endogenous mouse heavy and light chain immunoglobulin genes to completely exclude expression of endogenous heavy and light chain genes has been achieved, but technical difficulties remain, especially if the elimination of all lambda light chain coding sequences is considered necessary. Knockout of the murine lambda light chain coding sequence has been achieved by complete deletion of all functional V and J gene segments and the C1, C2 and C3 constant regions of the lambda locus, generating mice with a silenced lambda light chain locus (see EP 1399559).

[0011] A different approach is to restrict mouse B cell development and immunoglobulin secretion by disrupting the membrane exons of the genes encoding the mouse heavy chains. Thus, although the endogenous mouse heavy chain genes are functional because they are transcribed and undergo VDJ rearrangement in response to antigenic attack, further development is inhibited because IgM is never expressed on the cell surface of pre-B cells, resulting in a non-productive response to antigenic attack (Kitamura et al. (1991) Nature, 350, 423-426), even though both endogenous mouse κ and λ light chain genes remain structurally intact and functional (Tuaillon (2000) Molecular Immunology, 37, 221-231).

[0012] In the case where the endogenous mouse heavy chain and light chain loci remain functional, any other introduced immunoglobulin heavy chain transgenes are also regulated by allelic exclusion, so some B cells only functionally express mouse heavy chain and light chain loci, while other B cells only functionally express human heavy chain loci and mouse light chain loci (Nussenzweig et al. (1987) Science, 236, 816-819). In any single non-human transgenic animal, there is a highly diverse population of B cells expressing antibodies that are derived from all possible immunoglobulin loci in response to different antigenic attacks. Using established hybridoma technology, subsequent selection of antigen-specific antibodies using HAT selection (Davis et al. (1982) J. Immunol. Methods, 50, 161-171) cannot distinguish between hybridomas expressing one heavy chain immunoglobulin locus and hybridomas expressing another heavy chain immunoglobulin locus.

[0013] The regulatory elements present in the immunoglobulin heavy chain transgene contain the necessary tissue-specific enhancer elements to ensure B cell-specific expression in a copy number-dependent manner. The presence of the 5' intronic enhancer and the 3' LCR ensures that the transgene is active at all stages of B cell maturation (Guglielmi et al. (2003) Biochim. Biophys. Acta, 1642, 181-190). The addition of heavy and light chain-specific LCRs to the transgenic locus ensures that not only is expression B cell-specific, but also that expression is performed regardless of the site of integration into the genome (WO90 / 10077, Mills et al. (1997) J. Exp. Med., 186, 845-858 and Pettersson et al. (1997) Immunobiol., 198, 236-248)). Therefore, if the LCR is present, each transgene is functional regardless of its position in the genome. In the case of partial deletion of the LCR present in the transgene, the chromatin surrounding the transgene is only partially open to transcription at any point in time, resulting in position-effect mosaic expression and thus limited expression levels of the transgene across target tissues (Festenstein et al. (1996) Science, 23, 271(5252): 1123-5; Milot et al. (1996) Cell, 87(1), 105-14).

[0014] An alternative method for producing human immunoglobulins in a mouse background is to replace the murine immunoglobulin gene segments with human homologous gene segments. Thus, if only the mouse V, D, and J gene segments are replaced by human homologs, a population containing human V H and V L Functional mouse / human hybrid antibodies (WO94 / 04667) with structural domains and mouse constant (effector) regions. If all mouse gene segments are replaced by human homologs, complete human immunoglobulins (US6596541) will be produced after antigen attack. It is observed that an advantage of this method is that if only the coding region is exchanged, the resulting transgenic retains all mouse regulatory elements, thereby ensuring the maximum response to antigen attack. The method provides high-affinity human antibodies or mouse / human hybrid antibodies with high serum titers according to the final structure of the transgenic. However, in fact, it is a long and arduous task to replace all the individual V, D and J segments in the mouse genome by homologous recombination. Similarly, the construction of a heavy chain transgenic comprising all 39 functional human V, D and J segments and constant (effector) regions is technically very laborious.

[0015] Thus, there remains a need in the art for methods that do not rely on large segment deletions or multiple deletions of genomic DNA, which allow for (i) simplified generation of mice that have a greatly reduced ability to express endogenous heavy and light chain immunoglobulin genes in a B cell-specific manner in response to antigen challenge, or that express endogenous immunoglobulin heavy and / or light chain genes in B cells after antigen challenge, but encode immunoglobulin heavy and light chain proteins that lack the ability to assemble into functional immunoglobulin tetramers, resulting in a non-productive response to antigen challenge; and (ii) reproducible, simplified methods for constructing and B cell-specifically expressing multiple heavy chain transgenic loci that The loci, while collectively comprising all 39 human V gene segments, each comprise a preferred smaller group of V gene segments, each in combination with all D and J gene segments and some or all constant (effector) regions, whose functional expression is antigen-dependent and ultimately determined by allelic exclusion; and (iii) having the ability to: exclude hybridomas expressing residual endogenous mouse immunoglobulins and select for hybridomas that express and secrete assembled immunoglobulin tetramers comprising the complete repertoire of V segments present on the heavy chain transgenic locus, or to select for hybridomas that express a subset of V gene segments present on a heavy chain transgenic locus as opposed to another heavy chain transgenic locus. DETAILED DESCRIPTION OF THE INVENTION

[0017] According to a first aspect of the present invention, a non-human mammal is provided that contains an endogenous λ light chain locus, an endogenous κ light chain locus and an endogenous heavy chain locus, each locus being rearranged so that immunoglobulin heavy chain and light chain genes are formed and expressed in B cells after antigen attack, but the locus has been mutated so that the ability to form functional immunoglobulin tetramers comprising rearranged heavy chains and light chains produced from the mutated locus has been greatly reduced or eliminated.

[0018] In a non-human mammal, at least one of the endogenous λ light chain locus, the endogenous κ light chain locus, and the endogenous heavy chain locus may have been mutated by introducing a frameshift mutation, a polypeptide coding sequence, and / or one or more stop codons into one or each endogenous locus.

[0019] Preferably the mutation is an insertion of less than 50 nucleotides.

[0020] In non-human mammals, expression of at least one of the endogenous λ light chain locus, the endogenous κ light chain locus and the endogenous heavy chain locus may be substantially reduced by deleting part or all of the LCR at a locus or at each locus.

[0021] Preferably, the introduction is located at the endogenous kappa light chain locus.

[0022] Alternatively, the introduction is located at the endogenous lambda light chain locus.

[0023] In another alternative, the introduction is located at the endogenous kappa light chain locus and the endogenous lambda light chain locus.

[0024] Introduction can also be located at the endogenous heavy chain locus.

[0025] In a further alternative, the introduction may be at both the endogenous kappa light chain locus and the endogenous heavy chain locus.

[0026] In a yet further alternative, the introduction may be at both the endogenous lambda light chain locus and the endogenous heavy chain locus.

[0027] In a further alternative, the introduction may be located at the endogenous lambda light chain locus, the endogenous kappa light chain locus, and the endogenous heavy chain locus.

[0028] Preferably there is an introduction as defined above at the endogenous kappa light chain locus and a partial or complete LCR deletion as defined above at the endogenous lambda locus.

[0029] If desired, the endogenous heavy chain locus can be mutated so that heavy chain gene rearrangement, mRNA transcription, and protein synthesis occur, but B cell activation is blocked.

[0030] Preferably, the non-human mammal as defined above comprises a transgene comprising one or more heterologous kappa light chain gene loci and associated B cell specific regulatory elements.

[0031] The non-human mammal may also comprise a transgene comprising one or more heterologous lambda light chain gene loci and associated B cell-specific regulatory elements.

[0032] In a non-human mammal as defined above, the transgene comprising a heterologous light chain gene locus may comprise a dominant selectable marker gene.

[0033] The non-human mammal as defined above may further comprise a transgene comprising one or more heterologous heavy chain loci and associated B cell specific regulatory elements.

[0034] If desired, the non-human mammal may comprise two or more transgenes comprising two or more different heterologous heavy chain loci and associated B cell-specific regulatory elements.

[0035] In non-human mammals, the or each transgene comprising a heterologous heavy chain locus may comprise a dominant selectable marker gene.

[0036] In the non-human mammal, each heterologous heavy chain locus may comprise a CTCF binding site.

[0037] Preferably, the non-human mammal comprises a transgene comprising a heterologous kappa light chain locus and a transgene comprising one or more heterologous heavy chain loci.

[0038] Alternatively, the non-human mammal may comprise a transgene comprising a heterologous lambda light chain locus and a transgene comprising one or more heterologous heavy chain loci.

[0039] In a further alternative, the non-human mammal may comprise a transgene comprising a heterologous kappa light chain locus, a transgene comprising a lambda light chain locus, and a transgene comprising one or more heterologous heavy chain loci.

[0040] Preferably, each heterologous locus incorporates a cognate LCR.

[0041] Each heterologous locus is preferably a human locus.

[0042] However, each heterologous locus can be a hybrid locus comprising variable and constant regions derived from more than one species, such as a hybrid locus comprising human variable regions and rat or murine constant regions.

[0043] The non-human mammal may comprise a transgenic population comprising different populations of different heterologous heavy chain loci, wherein each transgenic population comprises a different dominant selectable marker gene.

[0044] Alternatively, the non-human mammal can comprise a transgene comprising a heterologous light chain locus and a transgene comprising a heterologous heavy chain locus, wherein the transgene comprising a heterologous light chain locus and the transgene comprising a heterologous heavy chain locus each comprise a different dominant selectable marker gene.

[0045] The non-human mammal is preferably a rodent, such as a mouse.

[0046] According to a second aspect, the present invention provides a method for producing an antigen-specific heterologous monoclonal antibody, the method comprising:

[0047] (a) immunizing a non-human transgenic mammal according to any one of the preceding claims with an antigen;

[0048] (b) preparing hybridomas or immortalized B cell lines that produce monoclonal antibodies, respectively, from B cells of the immunized transgenic mammal;

[0049] (c) optionally selecting at least one heterologous antibody-expressing hybridoma or immortalized B-cell line by using a dominant selectable marker gene present on a transgene comprising heterologous immunoglobulin light and heavy chain gene loci; and

[0050] (d) selecting at least one hybridoma or immortalized B cell line that produces an antibody that specifically binds to the antigen.

[0051] According to another aspect of the present invention, there is provided a method for obtaining mammalian (preferably human) antibodies from hybrid antibodies, the method comprising:

[0052] (a) performing the above method;

[0053] (b) selecting at least one V that specifically binds to the antigen and comprises a V vector of the selected species H and V L a hybridoma or immortalized B cell line that produces an antibody that binds to the domain;

[0054] (c) V H and V L The domains were cloned and sequenced;

[0055] (d) Make the selected H and V L recloning the sequence of the binding domain encoding sequence with a selected constant effector domain from the same species; and

[0056] (e) Co-expression of the recloned sequences encoding the heavy and light chain polypeptides of the desired species using expression vectors in a selected cell type.

[0057] According to another aspect of the present invention, a method for producing a non-human mammal as defined above is provided, the method comprising mutating the endogenous heavy chain locus, the endogenous κ light chain locus and optionally the endogenous λ light chain locus in a mammalian progenitor cell, and producing a mammal from the progenitor cell, wherein the mutation results in: in the mammal, each locus can be rearranged so that immunoglobulin heavy chain and light chain genes are formed and expressed in B cells after antigen attack, but the ability to form functional immunoglobulin tetramers comprising rearranged heavy chains and light chains produced by the mutant loci has been greatly reduced or eliminated.

[0058] Preferably the progenitor cells are non-human embryonic stem cells.

[0059] The non-human mammal is preferably a rodent, such as a mouse.

[0060] The present invention also provides the use of a preferably human antigen-specific heterologous functional immunoglobulin tetramer obtained using a non-human mammal or method as defined above as a medicament, diagnostic agent or reagent.

[0061] The inventors of the present invention unexpectedly overcame the limitations of the prior art by developing a simplified method for producing non-human mammals, particularly mice, in which the functional expression of endogenous kappa and / or lambda light chain genes is greatly reduced by causing constant region non-functionality by small insertion events, preferably in the kappa constant region and / or lambda constant region, resulting in frameshift or premature termination of mRNA translation, or by partial or complete deletion of the associated endogenous LCR. This is in stark contrast to alternative strategies that require functional silencing of endogenous immunoglobulin light chain genes by complete or partial deletion of some or all light chain gene coding sequences.

[0062] The strategy is equally applicable to the immunoglobulin heavy chain locus. Thus, for example, complete or partial deletion of the LCR will result in greatly reduced heavy chain gene expression. Preferably, the C H 1 domain, but alternatively in all immunoglobulin isotypes the constant region C H Introducing sequences that cause frameshift or premature termination of mRNA translation into region 1 will greatly reduce, eliminate or block the formation of immunoglobulin tetramers with light chains. Similarly, as previously described, if the expression of IgM on the cell surface of pre-B cells is blocked in vivo by premature termination of mRNA translation, further development is inhibited, resulting in a non-productive response to antigenic challenge (Kitamura et al. (1991) Nature, 350, 423-426). Introduction of a heavy chain immunoglobulin transgene rescues B cell expansion, and functional immunoglobulin tetramers containing transgenic-encoded heavy chains and endogenous mouse light chains circulate in plasma.

[0063] Thus, the ability of the endogenous light chain locus and / or heavy chain locus to produce heavy and light chains that interact and form functional immunoglobulin tetramers may be eliminated or greatly reduced upon introduction of a frameshift mutation, since frameshift mutations result in the synthesis of unrelated protein sequences and are generally accompanied by premature termination of protein synthesis due to the presence of out-of-frame stop codons.

[0064] This can be achieved by inserting exogenous DNA or a small amount of missing DNA in or upstream of the heavy chain or light chain polypeptide sequence responsible for forming a functional immunoglobulin tetramer. A preferred method is to insert a new sequence. The designed effective insertion event that causes frameshifting in the amino acid coding sequence and causes premature termination of translation of the coding mRNA can be limited to the introduction of a single nucleotide. Therefore, inserting one or more nucleotides (nucleocleotide) in the coding region can cause frameshifting. Alternatively, the in-frame insertion of a sequence encoding other peptides and containing a stop codon will also lead to the synthesis of truncated proteins (US5591669). Targeted insertion events can also include the introduction of selectable marker genes and other functional bodies, provided that all endogenous sequences are retained, and the resulting fusion protein destroys the formation of an immunoglobulin tetramer, or the presence of one or more in-frame stop codons causes premature termination of mRNA translation, resulting in the synthesis of truncated proteins that cannot form a functional immunoglobulin tetramer.

[0065] Preferably, the insertion event is located in the immunoglobulin kappa light chain constant region. Optionally, the insertion event is located in the immunoglobulin kappa and / or lambda light chain constant region. In fact, the insertion may contain any recombinase recognition site, such as a lox site. This alone can result in a frameshift. Adding additional nucleotides that ensure a frameshift or one or more stop codons can also increase the insertion sequence in disrupting the heavy and light chains through the heavy chain C HThe effect of dimerization of the interaction of the 1 domain with the light chain kappa and / or lambda constant region. The insertion may contain a single nucleotide, preferably less than 50 nucleotides. The insertion may only result in a frameshift, but is preferably designed so that one or more stop codons cause premature termination of mRNA translation. Insertion is performed by homologous recombination using the arms adjacent to the insertion site. Preferably, during the operation, a selection marker is added, followed by cutting, leaving only the recognition site and any other sequences inserted in situ in the genome, such as lox sites.

[0066] It is obvious to those skilled in the art that the synthesis of frameshifted and truncated proteins can also be achieved by deleting one or more nucleotides in the coding sequence and adding a minimal additional sequence comprising a stop codon. Preferably, the insertion or deletion event occurs in the constant region of the heavy and light chain genes, and not in the multiple V, D and J regions of the endogenous locus. The preferred choice is the kappa light chain constant region.

[0067] Thus, strategies that do not rely on large-scale gene deletions are not required to eliminate endogenous immunoglobulin gene rearrangements or mRNA transcripts. The same insertion strategy can be used to eliminate endogenous immunoglobulin gene rearrangements or mRNA transcripts. H The ability of endogenous heavy chain immunoglobulins to form functional tetrameric complexes with light chains is inhibited by a targeted insertion event in region 1 of the mitochondria. Preferably, a strategy similar to that described by Kitamura et al. (Kitamura et al. (1991) Nature, 350, 423-426) is used to block the expression of endogenous immunoglobulin heavy chain genes at the pre-B cell stage, so that endogenous heavy chains are not expressed on the surface of B cells and productive expression caused by B cell expansion is blocked, while the functional C complex of light chains with endogenous IgM is inhibited. H The association of 1 is inhibited by insertion events that result in the translation of kappa and / or lambda light chain mRNAs encoding light chain constant regions that are unable to functionally interact with immunoglobulin heavy chains, thereby preventing the formation of functional endogenous immunoglobulin tetramers.

[0068] If the functional assembly of endogenous immunoglobulin tetramers is impaired, then H Expansion of B cells with affinity-matured domains will be limited and dependent on the presence and expression of exogenous immunoglobulin heavy and light chain transgenes. The immunoglobulin transgenes will participate in the allelic exclusion process of the selected non-human mammalian host in a B cell-specific manner, resulting in productive response to antigenic challenge, expansion of B cells and circulating, transgene-encoded antigen-specific immunoglobulin tetramers.

[0069] The present invention also provides non-human mammals, wherein endogenous λ light chain gene expression is greatly reduced by deleting some or all λ light chain LCRs, and endogenous κ light chain gene expression is greatly reduced by deleting some or all κ light chain LCRs. In one embodiment of the present invention, only endogenous κ light chain expression is greatly reduced, or only λ light chain expression is greatly reduced, by deleting some or all relevant LCRs.

[0070] Endogenous light chain loci can remain functional because they can be rearranged and transcribed into functional mRNA, but the transcription level is greatly reduced by eliminating some or all endogenous LCR functionality (WO90 / 10077). LCR functionality can be removed by targeting genes at nuclease hypersensitive sites in mouse ES cells, or by cloning using nuclear transplantation (Soulter (1998) Nature, 394, 315-316) or iPS cells derived from other mammalian species (see Gottweiss. and Minger (2008) Nature Biotechnology, 26, 271-272) in the absence of ES cells. Alternatively, the destruction of heavy or light chains can be achieved by targeted mutagenesis, such as zinc finger nuclease technology and DNA repair (e.g., http: / / www.sigmaaldrich.com / life-science / functional-genomics-and-rnai / z inc-finger-nuclease-technology).

[0071] By deleting part or all of the κ light chain LCR, endogenous κ light chain gene expression can be greatly reduced, while the λ light chain gene can be functionally silenced following a deletion or insertion event.

[0072] By deleting part or all of the λ light chain LCR, endogenous λ light chain gene expression can be greatly reduced, while the κ light chain gene can be functionally silenced following a deletion or insertion event.

[0073] Following an LCR knockout or insertion event, the endogenous kappa light chain gene can be functionally silenced.

[0074] The present invention also provides a non-human mammal, wherein either or both of endogenous κ light chain gene expression and endogenous λ light chain gene expression are greatly reduced by deleting part or all of the κ light chain LCR and / or deleting part or all of the λ light chain LCR. By deleting part or all of the λ light chain LCR, the endogenous κ gene can be functionally silenced and the activity of the endogenous λ gene can be greatly reduced.

[0075] By deleting part or all of the κ light chain LCR, the expression of the κ light chain gene can be greatly reduced and the expression of the λ gene can be functionally silenced.

[0076] Only endogenous kappa light chain gene expression can be greatly reduced by knocking out the kappa chain LCR or functionally silenced by deletion or insertion events.

[0077] The above-mentioned non-human mammals in which endogenous κ and / or λ light chain gene expression is functionally silenced or endogenous κ and / or λ light chain gene expression is greatly reduced, and its endogenous heavy chain gene expression may also be reduced or functionally silenced. According to one embodiment, in the non-human mammals of the present invention, endogenous heavy chain gene expression is reduced after the deletion of some or all of the nuclease hypersensitive sites comprising LCR, or is functionally silent after the deletion or insertion event. It is preferred to block the expression of endogenous heavy chain genes at the pre-B cell stage so that the endogenous heavy chain is not expressed on the surface of the B cells, and a similar strategy as described by Kitamura et al. (Kitamura et al. (1991) Nature, 350, 423-426) is used to block the productive expression caused by B cell expansion.

[0078] The non-human mammals described above may further comprise one or more transgenes comprising heterologous heavy and light chain loci and associated B cell-specific regulatory elements (preferably comprising cognate LCRs).

[0079] In the context of the present invention, the term "heterologous" refers to a nucleotide sequence or locus described herein that is not endogenous to the mammal in which it is located.

[0080] Thus, the non-human mammal may comprise a transgene comprising a heterologous κ light chain locus and associated B cell-specific regulatory elements (preferably comprising an LCR) and / or a transgene comprising a heterologous λ light chain locus and associated B cell-specific regulatory elements (preferably comprising an LCR).

[0081] The presence of cognate LCRs is not required for B cell-specific expression. The inclusion of cognate LCRs within the locus ensures that high levels of transgene expression occur at each integration site, independent of random integration events that only some integration events occasionally occur within actively transcribed chromatin regions in B cells. The use of cognate LCRs significantly reduces the number of transgenic animals required for screening for antibody expression and allows insertion of more than one locus, ensuring that all inserted loci can be expressed at substantially normal levels in a B cell-specific manner. Thus, the use of LCR technology, combined with the unexpected observation that the allelic exclusion mechanism can distinguish endogenous immunoglobulin genes from multiple competing transgenes, opens a pathway for the assembly of transgenic non-human mammals containing one or more immunoglobulin heavy or light chain loci, each locus having a lower complexity of V genes than the endogenous genes and containing relatively tractable DNA fragments (<300 Kb) for in vitro assembly compared to endogenous loci (1-2 Mb). For example, 39 functional human immunoglobulin heavy chain V gene segments can be cloned into two or more immunoglobulin heavy chain loci. Each may contain different V gene segments, but have common D and J gene segments and constant (effector) regions. The addition of LCRs ensures that each is expressed in the same manner regardless of the integration site within the genome. Therefore, the addition of two or more small loci in this manner provides the same V gene complexity compared to a single more complex gene present on a single large gene fragment that is technically difficult to manipulate.

[0082] Each heterologous light chain locus can contain V L Gene segment, J gene segment and light chain constant region segment. L The J gene segment and the light chain constant region segment are derived from the same mammalian source, such as rodents, pigs, cattle, goats or sheep. Preferably, they are derived from humans.

[0083] Alternatively, the heterologous light chain locus can be a hybrid locus comprising a variable domain of mammalian origin (preferably human origin) and a constant (effector) region from a different mammal, such as, but not limited to, mouse, rat, hamster, rabbit, pig, goat, and cattle. If the host mammal is a mouse, then the constant region is preferably derived from a rodent, more preferably a mouse or rat. Such heterologous light chain loci contain V domains preferably from only one species. L and J segment and light chain constant region from another species.

[0084] If a hybrid kappa light chain transgene is desired, V LThe V, D and J gene segments of the heavy chain are preferably from the same species as the V, D and J gene segments of the heavy chain, and are preferably derived from humans. The kappa light chain constant region and the heavy chain constant region are also preferably derived from the same species, but the species is different from the species that provides the variable domains, and are preferably derived from rodents, preferably from rats or mice.

[0085] A characteristic of all light chain transgenes is that, following antigen challenge, the light chains rearrange in a B cell-specific manner and, following transcription and translation, the resulting light chains are capable of complexing with transgene-derived heavy chain immunoglobulins produced in the same B cell. Productive expression of immunoglobulin tetramers results in B cell expansion and accumulation of transgene-encoded antigen-specific tetravalent immunoglobulin complexes in serum in the absence of significant levels of endogenous immunoglobulin tetramers.

[0086] If endogenous lambda light chain expression is not functionally suppressed, low levels of host antibodies or hybrid antibodies containing endogenous lambda light chains can be detected. These can be discarded after screening the hybridoma supernatant.

[0087] In humans, there are 36 functional κV L Gene segment, 5 J L gene segment and a kappa light chain constant region ( http: / / imgt.cines.fr Preferably, the heterologous kappa light chain locus present in the transgenic non-human mammal of the invention comprises one or more human V L Gene segment, all people J L gene segments and a human kappa light chain constant region. Optionally, the human kappa light chain constant region may be replaced with an alternative mammalian kappa light chain constant region, preferably of rat or mouse origin.

[0088] The heterologous λ light chain locus present in the transgenic non-human mammal of the present invention preferably comprises mouse λ LCR and human λ light chain V1 and V2 gene segments, human λ J1, J2, J3 and J4 gene segments and human λ light chain C1, C2, C3 and C4 constant region segments (WO90 / 10077 and WO2003 / 000737). Optionally, the human λ light chain C1, C2, C3 and C4 constant region segments can be replaced with alternative λ light chain constant regions preferably of rat or mouse origin.

[0089] The heterologous heavy chain loci present in the transgenic non-human mammal of the invention preferably comprise a heavy chain immunoglobulin LCR (preferably of murine origin), one or more human V gene segments, one or more J gene segments, and one or more D gene segments. Preferably, there are 10 or more different human V gene segments and all human J and D gene segments.

[0090] The locus may also include one or more human constant (effector) regions, preferably μ and γ constant regions. Optional human constant effector regions may be replaced with effector regions from other non-human mammals. If the non-human mammal host is a mouse or rat, then preferably the constant (effector) region is derived from a rat or mouse. Compared with humans, the transmembrane domain of the mouse and rat B cell receptor complex (BCR) is 100% conservative. Therefore, after antigen attack, the mouse transgene of the antibody locus comprising rat constant (effector) region genes should play the same good role as the locus comprising mouse constant (effector) region genes, and may be superior to the locus comprising human constant (effector) region genes (De Franco et al. (1995) Ann. NY Acad. Sci., 766, 195-201). Transgenics may include heavy chains, κ and λ light chain LCRs preferably from mice or humans. LCRs that function in all mammalian species are known and can replace transgenic human or mouse LCRs (Li et al. (1999) Trends Genet., 10, 403-8).

[0091] If it is desired to produce fully human antibodies, the cloned human antigen-specific V derived from the hybrid antibody expressed by the hybridoma H and V L The binding domains can be fused to human constant heavy and light chain constant regions, thus resulting in a fully human tetrameric antibody of any species.

[0092] As a further refinement, each immunoglobulin kappa and / or lambda light chain gene locus may also comprise a dominant selectable marker gene.

[0093] The dominant selectable marker gene incorporated into the locus may have the same or different mechanisms of action. For the purposes of the present invention, any dominant selectable marker gene may be used provided that the expression of the gene provides a selective benefit for a hybridoma or a transformed B cell derived from a non-human transgenic mammal in the presence of a selective or toxic attack. Typically, the dominant selectable marker gene is prokaryotic in origin and is selected from genes that confer resistance to toxic drugs, such as puromycin (Vara et al. (1986) NAR, 14, 4617-4624), hygromycin (Santerre et al. (1984) Gene, 30, 147-156) and G418 (Colbere-Garapin et al. (1981) 150, 1-14), or includes genes that eliminate the need for certain nutrients so that their expression converts toxic substances into essential amino acids, such as converting indole into tryptophan or converting histidyl alcohol into histidine (Hartmann and Mulligan, (1988) PNAS, 85, 8047-8051).

[0094] A prerequisite for this aspect of the invention is that a dominant selectable marker is incorporated into the immunoglobulin light chain transgenic locus and co-expressed with the desired immunoglobulin light chain allele to ensure B cell-specific expression. Alternatively, homologous recombination combined with ES cells or nuclear transplantation methods can be used to insert drug resistance genes into endogenous or exogenous (transgenic) immunoglobulin loci (te Riele et al. (1992), PNAS, 89, 11, 5128-5132).

[0095] The non-human mammal may also comprise a transgene comprising a heterologous heavy chain locus and associated B cell-specific LCR and regulatory elements. There may be more than one different transgenic heavy chain locus, each comprising an LCR and regulatory elements.

[0096] Heavy chain loci, each comprising one or more V gene segments, one or more D gene segments, one or more J gene segments, and one or more constant (effector) regions, are introduced as transgenes, each locus comprising an associated LCR.

[0097] Each locus contains the necessary 5' and 3' regulatory elements to drive B cell-specific expression. In response to antigen challenge, each heavy or light chain locus is expressed in essentially the same manner as the endogenous locus, resulting in transgenic-encoded antigen-specific affinity-matured tetrameric immunoglobulins circulating in mouse serum.

[0098] Preferably, each heavy chain locus comprises one or more V gene segments, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 60 or more V gene segments, which can be derived from any vertebrate species, preferably a non-human mammal. Preferably, no more than 20 V gene segments are present at any single heavy chain locus.

[0099] In one embodiment, each locus may contain only one V gene segment. In an alternative embodiment of this embodiment, there are multiple V gene segments, each V gene segment is different from all other V gene segments. In this embodiment, the V gene segments on any locus may all be derived from organisms of the same species, for example, all V gene segments may be derived from humans. Alternatively, the V gene segments on any locus may be derived from organisms of different species, for example, some V gene segments are from humans, and others are from sheep, cattle, rabbits, camels (camelid) or even sharks. In a second alternative embodiment, each V gene segment is identical to all other V gene segments. Regardless of the number and nature of the V gene segments present, the remaining D and J gene segments on each locus may be identical or different from the segments on all other loci.

[0100] It is therefore envisaged that non-human mammals may contain multiple copies of the heavy chain locus. This has the advantage of optimizing the probability of productive rearrangement occurring in B cells, thus allowing optimal production of immunoglobulin heavy chains for antigen recognition.

[0101] In another embodiment, each locus comprises multiple V gene segments.

[0102] Preferably, the V gene segments are of human origin.

[0103] The term "V gene segment" encompasses any naturally occurring V gene segment derived from a vertebrate, including but not limited to sharks, rodents, camels and humans. The V gene segment must be able to recombine with a D gene segment, a J gene segment, and a gene segment encoding a heavy chain constant (effector) region to produce an immunoglobulin heavy chain antibody that is capable of complexing with a kappa or lambda immunoglobulin light chain when the rearranged nucleic acid is expressed in a B cell.

[0104] V gene segments also include within their scope any gene sequence encoding a natural or engineered homologue, derivative or protein fragment that is capable of recombining with a D gene segment, a J gene segment and a gene segment encoding a heavy chain constant region to produce an immunoglobulin heavy chain antibody capable of complexing with a kappa or lambda immunoglobulin light chain when the rearranged nucleic acid is expressed in a B cell. The V gene segment may be derived from, for example, a T cell receptor locus.

[0105] Preferably, the multiple heavy chain loci of the present invention comprise any number or combination of 39 functional human V gene segments and their engineered variants. They can be at any number of loci, such as 4 loci comprising 8 V gene segments plus 1 locus comprising 7 V gene segments; 7 loci comprising 4 V gene segments plus 1 locus comprising 3 V gene segments; or 39 loci each comprising 1 V gene segment.

[0106] The human V genes are divided into seven families VH 1-V H 7, and classify each gene into its own numbered family. The frequency of use of each gene depends on the different needs of a specific immune response. H Compared with genes from the 5 families, V H Therefore, in another preferred embodiment of the present invention, the V gene segment groups that have been proven to be useful for generating antibody responses against specific antigens are classified into separate loci, each containing a different dominant selection marker gene. The V gene segments can be classified by family, or they can be classified by their respective functions. For example, if V H If V genes from the V family 3 are shown to be useful for generating an immune response to a bacterial antigen, they can be used to generate loci that are particularly useful for generating heavy chain-only antibodies to a bacterial antigen. Alternatively, if it is shown that V genes from the V family 3 are useful for generating an immune response to a bacterial antigen, they can be used to generate loci that are particularly useful for generating heavy chain-only antibodies to a bacterial antigen. H 3 and V H If several individual genes of the 5 family are useful for generating an immune response against a bacterial antigen, they can be grouped together and used to generate a locus that is particularly useful for generating antibodies against a bacterial antigen.

[0107] In the context of the present invention, an "immunoglobulin heavy chain locus" refers to a minimal minilocus encoding a V gene comprising one or more V gene segments, one or more D gene segments and one or more J gene segments, and operably linked to one or more gene segments encoding heavy chain constant (effector) regions. H Domains. The major source of variability in the preferred antibody repertoire is the CDR3 region formed by the selection of the V, D, and J gene segments and by joining of the VD and DJ.

[0108] An advantage of the present invention is that the antibody repertoire and diversity obtained in rearranging V, D and J gene segments can be maximized by using multiple immunoglobulin heavy chain loci in the same transgenic non-human mammal by utilizing allelic exclusion. One of the loci is randomly selected to start recombination, and if the first recombination is unproductive, the next locus is followed, and so on, until a productive recombination is generated from one of the loci. This process of allelic exclusion ensures that virtually all V gene segments present at the combined loci can become part of the overall recombination process.

[0109] To increase the number of V H The probability that a gene segment is productively involved in VDJ rearrangement can disperse CTCF sites in V H between groups of gene segments.

[0110] Based on distance measurements made in B cells and in V H In the direction of the zone and throughout V HBased on the measurement of the V region, the immunoglobulin locus appears to have a three-dimensional folded structure in its normal conformation (Jhunjhunwala et al. (2008) Cell, 133, 265-279). This type of folded or looped structure explains the different V H This is why the regions can be used equally effectively even when arranged at completely different distances from the D, J, and constant regions of the immunoglobulin heavy chain locus.

[0111] It has also become increasingly clear recently that the folded structure formed by looping at many loci is mediated by a specific chromatin binding protein called CTCF. As confirmed by mutagenesis experiments, CTCF appears to be directly involved in the formation of chromatin loops (Splinter et al. (2006) Genes Dev., 20, 2349-2354). More recent studies have shown that cohesin, a protein complex that holds sister chromatids together, is present at CTCF binding sites (Wendt et al. (2008) Nature, 451, 796-801). Immunoglobulin V H The addition of many CTCF sites in the region (Kim et al. (2007) Cell, 128, 1231-1245; Denger, Wong, Jankevicius and Feeney (2009) J. Immunol., 182, 44-48) increased the V of the transgenic immunoglobulin heavy chain locus. H The probability that the region can fold correctly and allow efficient use of all the different V gene segments present at the locus.

[0112] Each transgene comprising a heterologous heavy chain locus may also comprise a dominant selectable marker. Preferably, the dominant selectable marker is different from the dominant selectable marker introduced into the kappa or lambda light chain locus.

[0113] For the purposes of the present invention, any dominant selectable marker gene may be used provided that expression of the gene provides a selective benefit to the hybridoma or transformed B cells derived from a non-human transgenic mammal in the presence of a selective or toxic challenge. Typically, the dominant selectable marker gene may be of prokaryotic origin and selected from genes that confer resistance to toxic drugs, such as puromycin (Vara et al. (1986) NAR, 14, 4617-4624), hygromycin (Santerre et al. (1984) Gene, 30, 147-156) and G418 (Colbere-Garapin et al. (1981) 150, 1-14), or include genes that eliminate the need for certain nutrients such that expression converts toxic substances into essential amino acids, such as indole into tryptophan or histidinol into histidine (see Hartmann and Mulligan (1988) PNAS, 85, 8047-8051).

[0114] It is essential for the present invention that the dominant selectable marker (if used) is located within the immunoglobulin heavy chain transgenic locus to ensure B cell-specific co-expression. Alternatively, homologous recombination combined with ES cells or nuclear transfer methods can be used to insert drug resistance genes into endogenous or exogenous (transgenic) immunoglobulin loci (e.g., te Riele, Robanus Maandag and Berns (1992), PNAS, 89, 11, 5128-5132).

[0115] The same dominant selectable marker gene can be incorporated into all heavy chain loci. Alternatively, different heavy chain loci or groups of heavy chain loci can comprise different dominant selectable marker genes.

[0116] Due to the co-expression of the functional dominant selection marker gene in the transgenic light chain locus, hybridomas or transformed B cells derived from transgenic mice expressing tetrameric antibodies of the present invention that do not contain cells expressing endogenous immunoglobulins can be selected, preferably transformed long-lived plasma cells (Slifka et al. (1998) Immunity, 8, 363-372). In addition, due to the presence and co-expression of different dominant selection markers in the heavy chain locus compared to the dominant selection markers incorporated into the light chain locus, hybridomas or transformed B cell lines expressing antibodies derived from a specific V segment group present on the transgenic heavy chain locus can also be selected. For example, the addition of a puromycin resistance gene to the κ light chain transgenic locus enables the selection of all cells expressing the κ light chain transgene. Alternatively, the addition of a G418 resistance gene to the heavy chain transgenic locus containing a preferred V gene segment enables the selection of all cells expressing the preferred V gene segment.

[0117] Specifically, the present invention provides a method for producing an antigen-specific heterologous monoclonal antibody, the method comprising:

[0118] (a) immunizing the above-mentioned non-human transgenic mammal with an antigen;

[0119] (b) preparing monoclonal antibody-producing hybridomas or immortalized B cell lines from B cells of the immunized transgenic mammal;

[0120] (c) selecting at least one hybridoma or immortalized B cell line expressing a heterologous antibody by using a dominant selectable marker gene present on a transgene comprising heterologous immunoglobulin light and heavy chain gene loci; and

[0121] (d) selecting at least one hybridoma or immortalized B cell line that produces an antibody that specifically binds to the antigen.

[0122] The present invention is described below, by way of example only, in detail with reference to the following drawings.

[0123] Attached pictures:

[0124] Figure 1A : 3' end of the mouse IgH locus.

[0125] This atlas was copied from the IMGT database ( http: / / imgt.cines.f ). The scale bar is in kilobases (kb). The green squares are functional V H Segment; red and yellow squares are non-functional V H Segment; orange square is J H Segment; blue squares are constant regions. The intronic IgH enhancer and LCR at the 3' end of the locus are not indicated.

[0126] Figure 1B : Strategies to disable IgH

[0127] The top row shows the mouse Cμ region with different exons including 2 exons encoding the membrane form of IgM. To the left are the J, D, and V H The top row shows the amino acid sequence of the normal M1 exon after recombinant DNA. The DNA sequence indicates the integration sequence. The stop codon is indicated in red, and the Spe I site is indicated in red and blue.

[0128] Figure 1C : Recombination that inactivates Cμ in ES cells

[0129] The figure shows two recombinant positive clones of ES cells obtained by PCR analysis covering the 3' side of the recombinant insert. The larger fragment corresponds to Figure 5 B The neo selection marker was inserted into the M1 exon at the position indicated.

[0130] Figure 1D : FACS analysis of Cμ knockout mice

[0131] After the M1 exon was interrupted by a stop codon and the neo gene, ES cells were introduced into blastocysts to obtain chimeras. These chimeras were grown to homozygosity and analyzed for the presence of B cells in the blood. The top two figures show FACS analysis of normal wild-type mice and mice with heterozygous interrupted M1 exon using B cell markers B220 and CD 19. The bottom two figures show two homozygous mice that do not show B220. + 、CD19 + Cells, i.e. non-functional B cells.

[0132] Figure 1E :Deletion of neo after breeding into recombinant mice

[0133] make Figure 5D mice were crossed with mice expressing the recombinase to delete the neo gene. The two lanes on the right show long PCR products of the neo gene that are higher than those of the parental animals, and the two lanes immediately to the left represent two heterozygous mice carrying the neo deletion and wild-type alleles. The four adjacent lanes on the left are wild-type mice, while the leftmost lane represents mice carrying a homozygous deletion of the neo gene and an inactivated M1 exon.

[0134] Figure 2 :Mouse Ig κ Map of gene loci

[0135] This atlas was copied from the IMGT database ( http: / / imgt.cines.f ). The scale is in kilobases (kb). The green square is V κ Segment; orange square is J κ segment; blue squares are constant regions; black circles are κ-enhancers and red circles are κ-LCR sequences.

[0136] Figure 3 :Mouse V κ Knockdown

[0137] LCR deletion in mice κ Protocol for functional inactivation of gene loci

[0138] Insertion of the lox neomycin resistance gene cassette into ES cells by homologous recombination replaces the 3' κ -LCR (bottom row). Treatment with cre recombinase (+cre) removes all sequences between the lox sites, leaving a single lox site at the kappa locus (top row).

[0139] Figure 4 :Insert mouse C κ Inactivate the kappa locus.

[0140] Loci (first row) and Figure 3 The last line indicates C κ Sequences at the 5' end of the exon (blue in the first row) with amino acid codes above the bases. The initial GG base pair is immediately adjacent to the splice acceptor site that codes for amino acid R after splicing. The middle row shows the insertion of a 34 base pair lox site insert (blue and red inverted repeats), which renders the codon usage of the constant region out of frame and generates a downstream stop codon (e.g., TGA in bold and underlined). The black circle is the κ-enhancer and the red circle is the κ-LCR sequence.

[0141] Figure 5 :Mouse C κ insert

[0142] Loci (first row) and Figure 3The last line indicates C κ 5' end sequence of the exon (blue in the first row) with the amino acid code indicated on the bases. The initial GG base pair is adjacent to the splice acceptor site that codes for amino acid R after splicing. The middle row shows the inserted 46 base pair insert containing lox sequences (blue and red inverted repeats) and 4 stop codons, which also makes the codon usage of the constant region out of frame and generates a downstream stop codon (e.g. TGA in bold and underlined). The black circle is the κ-enhancer and the red circle is the κ-LCR sequence.

[0143] Fig. 6A :Mouse C κ Constant region stop codon and frameshift insertion

[0144] Loci (first row) and Figure 3 The last line indicates C κ Partial exon sequence (black), with amino acid codes marked on the bases. The middle row represents C κ Partial sequence of the coding region. The upper row shows the insertion of a 44-base pair insert containing lox sequences (blue and red inverted repeats) and three stop codons, which also makes the codon usage of the constant region out of frame and generates a downstream stop codon (TAA). The black circle is the κ-enhancer, the red circle is the κ-LCR sequence, and the Hpa I site used for insertion is indicated in red.

[0145] Figure 6B : C κ Failed Reorganization

[0146] The gel shows Fig.13A In the ES cells shown, C κ Results of PCR amplification of the insertion sites of multiple recombinant clones. Clones 351 and 623 were positive and were injected into blastocysts to produce Ig κ Negative mice.

[0147] Figure 7 :Human Ig κ Gene locus.

[0148] This atlas was copied from the IMGT database ( http: / / imgt.cines.f ). The scale bar is in kilobases (kb). Green squares are functional VK segments; red and yellow squares are non-functional V κ Segment; orange square is J κ segment; blue squares are constant regions; black circles are κ-enhancers, and red circles are κ-LCR sequences.

[0149] Figure 8 : Hybrid human / rat Ig for transgenic effectκ Generation of gene loci

[0150] The 3' end of the locus was obtained from a mouse containing the mouse κ 3' enhancer (yellow). The mouse constant coding sequence was replaced with the rat constant coding sequence, including its 5' enhancer obtained from rat genomic DNA (red) by long-range PCR. κ The human segment of the sequence was obtained from the mouse (yellow) to maintain the proper spacing between the V and J regions. κ The segments were obtained from PACs covering this part of the human locus (green). The green squares are V κ The puromycin resistance gene present in the PAC vector is indicated in red, the black circle is the κ-enhancer, and the red circle is the κ-LCR sequence.

[0151] Fig. 9 :V H Map of the 4 heavy chain loci

[0152] This locus, which contains a neomycin selectable marker at the 5' end, was used as the starting material for the construction of a human / mouse hybrid locus. The locus was constructed as described in WO2008 / 035216. The scale is in kilobases. The locus contains 4 V H regions (1-46, 3-53, 3-23, 3-11), all human D segments, all human J segments and IgG constant regions and 3' human IgH LCR.

[0153] Fig.10 : 4 V H Generation of human / rat constant region IgH loci

[0154] Exists in V H The CeuI site of the 4 human locus (Figure 6) was used to generate a human / rat locus by adding the rat constant coding region and switch region amplified by PCR from rat genomic DNA. Similarly, the mouse LCR region was amplified from mouse genomic DNA as several fragments that were first cloned together to generate a complete mouse IgH LCR. H The 5' end of the 4 locus contains 4 V H Segments, all human D segments and all human J segments (Figure 6). All human sequences are shown in blue, all mouse sequences are shown in light green, rat sequences are shown in red, and the neomycin resistance gene is shown in purple.

[0155] Fig.11 : Deletion of the Igλ enhancer containing the λLCR

[0156] The lambda locus enhancer was knocked out by homologous recombination using a standard replacement vector using a hygromycin resistance gene flanked by sequences homologous to the region adjacent to the enhancer 2-4 and a blasticin S resistance gene flanked by segments homologous to the region adjacent to the enhancer 4-10. The replacement produced a lambda locus with a loss of enhancers and reduced expression. The hygromycin resistance gene is shown in red and the blasticin S resistance gene is shown in orange. Mouse V λ The A segment is shown in green, the J segment is shown in yellow, and the constant region is shown in blue. The spectrum is copied from the IGMT database.

[0157] Fig.12 : Human rat V for transgenic effect κ Locus

[0158] Gene loci and Figure 8 Same as, but has added other people V κ The resulting locus contains all frequently and moderately frequently used human V κ Green circles and green lines represent mouse κ-enhancer and intron; red squares and circles represent rat κ-constant region and enhancer; blue squares represent human V H segment; dark blue is puromycin selection marker.

[0159] Fig.13A : Generation of a human-rat hybrid locus

[0160] By changing V H The regions were connected together to form 17 consecutive human V H The mouse spacer was added to the locus containing all human D H and J H The 40 kb fragment of the human H With V H Keep an appropriate distance between the segments. Then add the V containing the SceI site H 6-1 segment. Then add the concatemer to V H 6-1. Finally, various rat constant regions and mouse LCR were added to the 3' side. The resulting locus contains all frequently and moderately frequently used human V H Section.

[0161] Fig. 13B : Starting construct for human / rat IgH locus

[0162] Gel indicates that V H6-1 The results of the first step of the construction of the human rat IgH locus. The lanes on the right represent the two cloned plasmids digested with NotI. The lanes (NotI x MluI) indicate that the plasmid in lane 1 has the correct orientation in the vector, while the plasmid in lane 2 has the wrong orientation. The plasmid in lane 1 is used in the next step of generating the locus.

[0163] Fig. 13C :V H concatemer

[0164] Gel showing 17 different V digested with XhoI / SalI H The labeled lane contains lambda phage DNA digested with BstEII.

[0165] Fig.14 : Transgenic human rat heavy chain immunoglobulin locus

[0166] Example of two heavy chain loci introduced into the same animal. The opposing selection is performed by allelic exclusion. H Alternatively, more heavy chain loci can be used to introduce other V segments. H Obviously, the same strategy can be used to increase the diversity of the kappa or lambda light chain loci.

[0167] Fig.15 : Human / rat lambda transgenic light chain locus

[0168] Figure shows an example of a human / rat locus. Its 3' end was obtained from a mouse (yellow) containing the mouse λ 3' LCR (yellow). The mouse constant coding sequence was replaced with a rat constant coding sequence (red) obtained from rat genomic DNA by long fragment PCR. λ 2-14 Downstream until people J κ The human segment of sequence was obtained from the mouse (yellow) to maintain the proper spacing between the V and J regions. The human J was obtained by long-range PCR of a human PAC covering this portion of the human locus (blue). λ Segments. Blue squares are V added individually or as a block group. λ The hygromycin resistance gene present on the PAC vector is shown in purple. Example

[0169] In the following examples, transgenic mice were generated that expressed hybrid human / rat heavy and light chain loci introduced as transgenes into fertilized eggs by microinjection (conventional transgenic process). The donor mice were modified to not express or very low express endogenous mouse heavy chain genes and mouse light chain genes. There are two light chain loci in mice, κ chain and λ chain, of which λ is only used for about 2% of mouse H2L2 antibodies. Therefore, the examples are in mice with IgH loci and only inactivated endogenous κ loci, or in mice with IgH and inactivated κ loci and deletion of regulatory sequences of the λ loci to further reduce the expression of the λ loci.

[0170] Methods for constructing heavy and light chain loci, generating and selecting transgenic mice after antigen challenge are essentially as described previously (Janssens et al. (2006) PNAS, 10, 103(41), 15130-5; WO2006 / 008548, WO2007 / 096779, GB0805281.3 and PCT application filed on June 11, 2008 claiming priority to GB0805281.3), retaining C at all heavy chain loci. H 1 domain. For obtaining a general method for expressing a functional heterologous immunoglobulin locus and / or a vertebrate (including mammals other than mice) with an engineered endogenous locus, see WO2006 / 047367. In the following examples, recombination is used in ES cells, and modified ES cells are used to produce mice with desired properties. However, the same method can be carried out in induced pluripotent stem cells (iPS cells), which are then used to produce mice (e.g., Boland, Hazen, Nazor, Rodriguez, Gifford, Martin, Kupriyanov and Baldwin (2009), 461, 7260, 91-4 and references therein). Alternatively, somatic cells or somatic stem cells are modified and then reprogrammed into iPS cells to produce modified mice. Similarly, modified hematopoietic stem cells can be transplanted into recipient mice lacking B cells to produce human antibodies or human hybrid antibodies.

[0171] Example 1

[0172] In this example, the IgH locus ( Figure 1A ) is inactivated, the difference being that a stop codon is introduced at the position of the amino acid preceding the position described by Kitamura et al. (Kitamura et al. (1991) Nature, 350, 423-426) μES (or iPS) cells are transfected with a construct that changes the second codon of the first membrane exon of the mouse IgM gene to a stop codon. This involves conventional steps including neo selection for transfection. An SpeI site is added to the recombination sequence to enable monitoring of successful recombination ( Figure 1B ES cells were then screened by Southern blotting to confirm successful recombinant clones. This yielded 10 correct recombinants (e.g. Figure 1C Of these, three were injected into mouse blastocysts to obtain chimeras, which were subsequently bred to obtain mice homozygous for the IgH mutation. FACS analysis of B cells from these mice (B220 vs. CD 19) showed that there were no B cells in the peripheral blood ( Figure 1D The mice were then crossed with mice expressing the recombinase to remove the neo gene ( Figure 1E ). Similarly, mouse Ig κ Locus ( Figure 2 ) inactivation or reduction ( Figure 2 , 3 , 4-6). The resulting κ-inactivated mice were crossed with heavy chain KO mice. Activity knockdown was achieved by replacing the 3'κ gene LCR with a neo resistance marker flanked by lox sites ( Figure 3 ). Optionally, the neo gene is removed by treatment with a recombinase.

[0173] Alternatively, the κ allele can be knocked out directly in IgH KO cells. κ inactivation can be achieved using several different strategies. The neo gene flanked by lox sites can be inserted into the C κ 5' end of the exon ( Figure 4 ), or by inserting a neo gene flanked by lox sites and other sequences encoding a stop codon ( Figure 5 and Fig. 6A ) to achieve the repression of κ gene activity. Recombinant ES cells treated with cre can make this sequence in an out-of-frame ( Figure 4 ), or contain a new stop codon ( Figure 5-6A ). Figure 6B express Fig. 6A The results of recombination in ES cells after transfection of the constructs shown above produced a C κ Two ES cell clones with the same lox allele (a total of 5 such clones were obtained). The cells were treated with the recombinase by standard transient transfection using an actin-driven recombinase plasmid to eliminate the region between the two lox sites. The cells were then used to generate mice by conventional methods, and the offspring were bred to obtain homozygous mice. Such mice contain B cells, in which the assembly of immunoglobulin tetramers containing kappa light chains is substantially destroyed or completely blocked.

[0174] Next, follow the previous example for people V H As described in the section, the most commonly used human Ig κ V κ Genes (evaluated using Ig database; http: / / imgt.cines.fr / ; See Figure 7 , 8 ) was amplified by standard PCR and subcloned between the XhoI / SalI sites. This allowed V κ The region is capable of multimerization and holds the multimers between the XhoI and SalI sites.

[0175] Similarly, the 3' end of the mouse κ locus, including the 3' κ enhancer, was linked to the rat constant (C κ ) region and rat 5' enhancer cloned together ( Figure 8 ). Next, clone into human J κ V κ and J κ The region between (17kb) ( Figure 2 ) to maintain V κ District and J κ Finally, human V κ Insert a PAC containing the puromycin selection marker ( Figure 8 ).

[0176] In the embodiment shown, the most commonly used V κ The segments (4-1, 3-11, 3-15, 3-20, and 1-39) are multimerized and linked to a region containing human J regions and mouse enhancers and rat C κ This produces a PAC vector consisting of the puro resistance marker gene, human V κ Segment and rat constant (C κ ) region composed of human-rat hybrid locus ( Figure 8 ).

[0177] Likewise, a hybrid human / rat IgH locus was constructed. Again, there are many possibilities with regard to starting material. In this example, the starting material was a 4-person V H District, all people D H and J H segment, two human constant regions and human LCR human PAC ( Fig. 9 and UK patent application number 0905023.8).

[0178] The PAC at the back has a unique CeuI meganuclease site between the J region and the constant region. To make it possible to easily construct a hybrid locus, the CeuI site is used to remove the human 3' end sequence, and these are replaced with rat constant regions and switch regions (Cμ, Cγ3, Cγ1 and Cγ2). These are amplified from rat genomic DNA by standard long fragment PCR. Finally, mouse heavy chain LCR is added. The regulatory sequence is amplified by mouse genomic DNA in 3 parts, and subcloned together to restore complete LCR, and added to the 3' side of the rat constant region ( Fig.10 ). Thus, the resulting hybrid IgH locus contains the neo selection marker, human V, D and J regions and rat constant regions with mouse regulatory sequences.

[0179] The hybrid locus insert was then isolated from the PAC as a large DNA fragment and injected into an IgH / Ig κ Mouse fertilized eggs from heterozygous or homozygous null mice produce IgH and Ig κ All of this was done by conventional methods (eg Janssens et al. (2006), supra).

[0180] Then, hybrid IgH and hybrid Ig κ Transgenic mice, obtained for endogenous mouse IgH and Ig κ Homozygous null for expression and for human / rat hybrid IgH and Ig κ These mice are then immunized by conventional methods to produce antigen-specific hybrid human / rat H2L2 antibodies. If monoclonal human / rat Ig is produced by hybridomas, dual selection with puromycin and neomycin ensures that only hybridomas containing both IgH and Ig κ Myeloma fusions of the gene locus.

[0181] It will be appreciated by those skilled in the art that various changes can be made to this method to produce hybrid transgenic mice, such as using different vectors, different selection markers, different recombination positions to inactivate mouse genes, or various changes can be made in the actual (conventional) cloning strategy of the hybrid locus. The same method can be used to produce any normal locus or hybrid locus using immunoglobulin DNA derived from any single mammalian species, or to obtain a hybrid locus using DNA from two or more species.

[0182] Example 2

[0183] This example is essentially the same as Example 1, except that the endogenous mouse Ig λ The expression frequency of the gene locus is further increased to obtain IgH / Ig κHigh frequency of H2L2 antibodies. This can be achieved by replacing the two Ig λ The regulatory area is realized ( Fig.11 ), in this case the hygromycin resistance gene and the TK-BSD gene.

[0184] The latter enables positive selection for blasticidin S resistance (Karreman, (1998) NAR, 26, (10), 2508-2510). This combination of markers, when replacing regulatory regions, enables positive selection in the recombination of two ES cells. The recombination is performed in the ES cells generated in Example 1, or alternatively, in normal ES cells in parallel and bred into mice as described in Example 1 above.

[0185] The resulting transgenic mice contain hybrid human rat IgH and Ig κ loci, for endogenous mouse IgH and Ig κ Negative for C κ ) and expressed Ig at very low levels λ After immunization and hybridoma production by conventional methods, the neo( Fig.10 ) and puro( Figure 8 ) selection, and hybridomas expressing only human rat hybrid H2L2 antibodies were selected for expressing transgenic hybrid loci.

[0186] Example 3

[0187] Example 3 is similar to the above example, but hybrid Ig κ The locus is modified by adding less frequently used V κ The section extends Fig.12 ; V κ 1-9, 1-33, 2-30, 2-28, 1-27, 1-5). Alternatively, additional mutated / modified V κ Segment or V λ The addition of other segments can be performed by using the same XhoI / SalI cloning strategy described above. Immunization of mice generated in this example allows for greater complexity in the response to immunization with antigen. κ section or use all of the above V L The combination of segments makes V L The number of zones further changes.

[0188] Example 4

[0189] Example 4 is similar to the method described in the above examples, but here by using 18 V H segments and five rat constant regions to generate a hybrid human / rat IgH locus ( Fig.13A ; People V H First, the central 70 kb DJ region of the human locus was extended at the 5' end with 8 kb from the mouse IgH intron to maintain V H Next, the first V segment with an artificial SceI meganuclease site of 10 kb was H The region (6-1) was cloned at the 5' end of the mouse intron sequence ( Fig.10 B) In a separate plasmid, all remaining V H The region was then inserted by XhoI / SalI V H Segments are cloned together ( Fig.10 C). You can also add more V H Segment or modified / mutated V H Segments are added to these loci. CTCF sites may also be included. In the example shown, three such sites were used. They are connected by V including the upstream CTCF site. H In addition, Fig.10 Compared with the loci in rat C α In this example, immunization allows for greater complexity in responses when immunized with antigen, particularly when combined with Example 3. H Multimer cloned into V H 6-1DJ plasmid, and then the rat constant region was added to complete the locus.

[0190] In all of these embodiments, the complexity of the response is further increased by adding the V segments as part of other heavy or light chain transgenic loci present in the same mouse. Since all loci undergo allelic exclusion (see WO2007 / 096779), only the preferred rearrangements are selected in vivo following antigen challenge, leading to B cell expansion and accumulation of antibodies in the serum. Fig.14 This shows an example of two heavy chain loci that can be introduced into the same animal, each of which can be exploited by allelic exclusion. Obviously, more V H Segment (including modified V H Segments), or even more loci can be introduced to increase the complexity of the transgenic immune repertoire. This method can also be applied to other species using V segments specific to these species.

[0191] Example 5

[0192] In this example, the human / rat Igλ locus was added by breeding with a mouse strain carrying the human / rat IgH and / or Ig κ The generation of the human / rat hybrid lambda locus is similar to that described in the previous examples for human / rat Ig κ The difference is caused by the paired Jλ and Cλ regions, so the two rat C λ The region was cloned into two individuals J λ district( Fig.15 ) by cloning the normal mouse sequence present at this position to maintain V λ and J λ The interval between segments (see Fig.11 ). In this embodiment, 2 people J λ and rat C λ Section with 4 people V λ Together, these comprise more than 80% of the human Igλ response. Regulatory sequences (LCR, Fig.15 ) was derived from mice to ensure optimal expression, and a selectable marker was added to the 5' end of the locus. As described above, the locus was isolated as a restriction fragment and injected into fertilized eggs to generate mice carrying the transgenic lambda locus.

[0193] In all of these embodiments, V from different species can be used. H 、V L Other single species antibodies or hybrid species antibodies can be produced by using V, D, J and constant regions. It is also obvious to those skilled in the art that once antigen-specific antibodies are identified, V, D, J and constant regions can be converted to V, D, J and constant regions by completely conventional methods. H DJ and V L The J region is cloned into an alternative constant region from the same species or from a different species.

Claims

1. A method for producing a transgenic mouse capable of producing a hybrid tetrameric antibody, the method comprising: mutating an endogenous heavy chain locus and an endogenous kappa light chain locus of a mouse progenitor cell, wherein the mutation comprises introducing a frameshift mutation, a polypeptide coding sequence, or one or more stop codons into each locus, and wherein the mutation in the endogenous heavy chain locus introduces a stop codon at the second codon of the M1 exon of the Cμ region of the endogenous heavy chain locus; Producing from the progenitor cells a mouse having a significantly reduced or abolished ability to form functional immunoglobulin tetramers comprising rearranged heavy and light chains generated from the mutant loci; and introducing the heterologous hybrid human / rat heavy and light chain loci as transgenes by microinjection into fertilized eggs provided by said mice having significantly reduced or abolished ability to form functional immunoglobulin tetramers comprising rearranged heavy and light chains generated from said mutant loci, wherein the heterologous heavy chain locus comprises a human variable region comprising one or more V gene segments, one or more J gene segments and one or more D gene segments, a rat constant region and a heavy chain immunoglobulin LCR of murine origin, and wherein the heterologous light chain locus comprises a transgenic locus comprising a heterologous κ light chain locus and associated B cell-specific regulatory elements and / or a transgenic locus comprising a λ light chain locus and associated B cell-specific regulatory elements, and wherein the heterologous light chain locus comprises a V L and J segments of human variable regions and rat constant regions.

2. A method for preparing an antigen-specific heterologous monoclonal antibody, the method comprising: (a) immunizing transgenic mice with an antigen; (b) preparing hybridomas or immortalized B cell lines each producing a monoclonal antibody from the B cells of the immunized transgenic mouse; and (c) selecting at least one hybridoma or immortalized B cell line that produces an antibody that specifically binds to the antigen, wherein the transgenic mouse comprises a mutated endogenous heavy chain locus and a mutated endogenous kappa light chain locus such that the ability to form functional immunoglobulin tetramers comprising rearranged heavy and light chains produced from the mutated loci is significantly reduced or eliminated, and wherein the mutation comprises the introduction of a frameshift mutation, a polypeptide coding sequence, or one or more stop codons into each locus, and wherein the mutation in the endogenous heavy chain locus introduces a stop codon into the second codon of the M1 exon of the Cμ region of the endogenous heavy chain locus, and wherein the transgenic mouse comprises a transgenic locus comprising a heterologous κ light chain locus and associated B cell-specific regulatory elements and / or a transgenic locus comprising a λ light chain locus and associated B cell-specific regulatory elements, and wherein the transgenic mouse comprises a transgenic locus comprising one or more heterologous heavy chain loci, wherein the heterologous light chain locus comprises a V L and J segments and rat constant regions, and wherein the heterologous heavy chain locus comprises a human variable region containing one or more V gene segments, one or more J gene segments and one or more D gene segments, a rat constant region, and a heavy chain immunoglobulin LCR of murine origin.

3. A method for obtaining antigen-specific human antibodies from hybrid antibodies, the method comprising: (a) immunizing transgenic mice with an antigen; (b) isolating B cells from the immunized transgenic mice and optionally preparing hybridomas or immortalized B cell lines each producing a monoclonal antibody; (c) selecting at least one isolated B cell, hybridoma or immortalized B cell line that produces an antibody that specifically binds to the antigen; (d) V of the antibody that specifically binds to the antigen H and V L The domains were cloned and sequenced; (e) Make V H and V L recloning selected sequences of binding domain encoding sequences with human constant effector domains; and (f) co-expressing the re-cloned sequences encoding human heavy and light chain polypeptides in cells using an expression vector, wherein the transgenic mouse comprises a mutated endogenous heavy chain locus and a mutated endogenous kappa light chain locus such that the ability to form functional immunoglobulin tetramers comprising rearranged heavy and light chains produced from the mutated loci is significantly reduced or eliminated, and wherein the mutation comprises the introduction of a frameshift mutation, a polypeptide coding sequence, or one or more stop codons into each locus, and wherein the mutation in the endogenous heavy chain locus introduces a stop codon into the second codon of the M1 exon of the Cμ region of the endogenous heavy chain locus, and wherein the transgenic mouse comprises a transgenic locus comprising a heterologous κ light chain locus and associated B cell-specific regulatory elements and / or a transgenic locus comprising a λ light chain locus and associated B cell-specific regulatory elements, and wherein the transgenic mouse comprises a transgenic locus comprising one or more heterologous heavy chain loci, wherein the heterologous light chain locus comprises a V L and J segments and rat constant regions, and wherein the heterologous heavy chain locus comprises a human variable region containing one or more V gene segments, one or more J gene segments and one or more D gene segments, a rat constant region, and a heavy chain immunoglobulin LCR of murine origin.

4. The method of claim 1, wherein more than one different transgenic heavy chain locus is introduced.

5. The method of claim 1 or 4, wherein the progenitor cells are embryonic stem cells.

6. The method of claim 2 or 3, wherein the one or more heterologous heavy chain loci each comprise 10 or more V gene segments.

7. The method of claim 6, wherein the one or more heterologous heavy chain loci each comprise 18 V gene segments.

8. The method of claim 7, wherein the one or more heterologous heavy chain loci each comprise 5 rat constant regions and a human V H Zones 6-1, 1-2, 4-4, 2-5, 3-07, 1-8, 4-39, 3-15, 1-18, 3-23, 3-30, 3-33, 3-48, 4-34, 3-49, 3-53, 4-59 and 1-69.

9. The method of any one of claims 1 to 3, wherein the heterologous κ light chain locus comprises a human J region, a mouse enhancer region, a rat CK region, and human Vκ segments 4-1, 3-11, 3-15, 3-20, and 1-39.

10. The method of claim 9, wherein the heterologous κ light chain locus comprises Vκ segments 4-1, 3-11, 3-15, 3-20, 1-39, 1-9, 1-33, 2-30, 2-28, 1-27, and 1-5.

11. The method of any one of claims 1 to 3, wherein the heterologous λ light chain locus comprises 4 human Vλ segments, 2 rat Cλ segments, and 2 human Jλ segments.

12. The method of any one of claims 1 to 3, wherein each of the heterologous kappa and lambda light chain gene loci comprises a dominant selectable marker gene.

13. The method of any one of claims 1 to 3, wherein each transgene comprising a heterologous heavy chain locus comprises a dominant selectable marker.

14. The method of claim 13, wherein the dominant selectable marker in the heterologous heavy chain locus is different from the dominant selectable marker introduced into the kappa or lambda light chain locus.

15. The method of any one of claims 1 to 3, wherein the stop codon introduced into the endogenous heavy chain locus is in the Cμ region at the second codon of the first membrane exon of the IgM gene.

16. The method of any one of claims 1 to 3, wherein the mutation in the endogenous kappa light chain locus is the introduction of a frameshift mutation.

17. The method of claim 16, wherein the frameshift mutation is in the kappa constant region of the endogenous kappa light chain locus.

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