JP2007054076A - Transgenic non-human animal capable of producing heterogenous antibodies - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a transgenic non-human animal capable of producing heterogenous antibodies. <P>SOLUTION: This is a mouse including a human heavy chain immunoglobulin variable region not rearranged where the variable region includes a plurality of human V<SB>H</SB>gene segments, a plurality of human D gene segments and a plurality of human J<SB>H</SB>gene segments and the B cells of the mouse express the chimeric antibody including the human immune globulin heavy chain variable region and the chimeric antibody including the mouse immune globulin heavy chain constant region. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention provides the following:

FIG. 1 shows complementarity determining regions CDR1, CDR2 and CDR3 and framework regions FR1, FR2, FR3 and FR4 in unrearranged genomic DNA and in mRNA expressed from a rearranged immunoglobulin heavy chain gene. Represents. FIG. 2 represents the human λ chain locus. FIG. 3 represents the human kappa chain locus. FIG. 4 represents the human heavy chain locus. FIG. 5 represents a transgene construct containing a rearranged IgM gene linked to a 25 kb fragment containing human γ3 and γ1 constant regions followed by a 700 bp fragment containing the rat chain 3 'enhancer sequence. FIG. 6 is a restriction map of the human kappa chain locus representing fragments that can be used to form light chain transgenes by in vivo homologous recombination. FIG. 7 shows the preparation of pGP1. FIG. 8 shows the structure of the polylinker contained in pGP1. FIG. 9 shows the fragments used to make the human heavy chain transgenes of the present invention. FIG. 10 shows the production of pHIG1 and pCON1. FIG. 11 shows the human Cγ1 fragment inserted into pRE3 (rat enhancer 3 ′) to form pREG2. FIG. 12 shows the production of pHIG3 'and pCON. FIG. 13 shows a fragment containing the human D region segment used to produce the transgene of the present invention. FIG. 14 shows the production of pHIG2 (D segment-containing plasmid). FIG. 15 shows a fragment encompassing the human Jκ and human Cκ gene segments used to make the transgene of the present invention. FIG. 16 shows the structure of pEμ. FIG. 17 shows the production of pKapH. FIG. 18 shows the creation of a positive-negative selection vector for functional disruption of the mouse endogenous immunoglobulin heavy chain locus. FIG. 19 shows the creation of a positive-negative selection vector for functional disruption of the mouse endogenous immunoglobulin heavy chain locus. FIG. 20 shows the creation of a positive-negative selection vector for functional disruption of the mouse endogenous immunoglobulin heavy chain locus. FIG. 21 shows the creation of a positive-negative selection vector for functional disruption of the mouse endogenous immunoglobulin heavy chain locus. FIG. 22 shows the creation of a positive-negative selection vector for functional disruption of the mouse endogenous immunoglobulin heavy chain locus. FIG. 23 shows the creation of a positive-negative selection vector for functional disruption of the mouse endogenous immunoglobulin light chain locus. FIG. 24 shows the creation of a positive-negative selection vector for functional disruption of the mouse endogenous immunoglobulin heavy chain locus. FIG. 25 shows the structure of the kappa chain targeting vector. FIG. 26 shows the structure of a mouse heavy chain targeting vector. FIG. 27 shows the structure of a mouse heavy chain targeting vector. FIG. 28 shows a map of the vector pGPe. FIG. 29 shows the structure of vector pJM2. FIG. 30 shows the structure of vector pCOR1. FIG. 31 shows the transgene constructs of pIGM1, pHC1 and pHC2. FIG. 32 shows the structure of pγe2. FIG. 33 shows the structure of pVGEl. FIG. 34 shows the assay results of human Ig expression in pHCl transgenic mice. FIG. 35 shows the structure of pJCKl. FIG. 36 shows the production of a synthetic heavy chain variable region. FIG. 37 is a schematic representation of the heavy chain small locus constructs pICM1, pHC1 and pHC2. FIG. 38 is a schematic representation of the heavy chain small locus component pIGG1 and the kappa light chain small locus components pKC1, pKVe1 and pKC2. FIG. 39 represents a strategy for rearranging functionally rearranged light chain genes. FIG. 40 represents the serum ELISA results. FIG. 41 represents the results of an ELISA assay of sera from 8 transgenic mice. FIG. 42 is a schematic representation of plasmid pBCE1. FIG. 43 shows the immune response of the transgenic mice of the present invention against KLH-DNP by measuring IgG and IgM levels specific for KLH-DNP (37A), KLH (37B) and BSA-DNP (37C). To express. FIG. 44 shows ELISA data demonstrating the presence of an antibody that binds to human carcinoembryonic antigen (CEA) and comprises human μ chain; each panel was obtained from mice on the indicated date after immunization Shown are reciprocal serial dilutions from pooled serum samples. FIG. 45 shows ELISA data demonstrating the presence of an antibody that binds to human carcinoembryonic antigen (CEA) and comprises a human gamma chain; each panel was obtained from mice at the indicated date after immunization Shown are reciprocal dilutions from pooled serum samples. FIG. 46 shows the aligned variable region sequences of 23 randomly selected cDNAs generated from mRNA obtained from lymphoid tissue of HC1 transgenic mice immunized with human carcinoembryonic antigen (CEA). Shown relative to germline transgene sequence (top row). In each row, nucleotide changes to the germline sequence are indicated above the changes in the deduced amino acid sequence (if any). Regions corresponding to heavy chain CDR1, CDR2 and CDR3 are shown. Nucleotides that are not encoded by germ cells are shown in capital letters. The deduced amino acid sequence is given below the nucleotide sequence using conventional single letter notation. FIG. 47 shows an aligned variable region sequence of 23 randomly selected cDNAs generated from mRNA obtained from lymphoid tissue of HC1 transgenic mice immunized with human carcinoembryonic antigen (CEA). Shown relative to germline transgene sequence (top row). In each row, nucleotide changes to the germline sequence are displayed above the changes in the deduced amino acid sequence (if any). Regions corresponding to heavy chain CDR1, CDR2 and CDR3 are shown. Nucleotides that are not encoded by germ cells are shown in capital letters. The deduced amino acid sequence is given below the nucleotide sequence using conventional single letter notation. FIG. 48 shows the aligned variable region sequences of 23 randomly selected cDNAs generated from mRNA obtained from lymphoid tissues of HC1 transgenic mice immunized with human carcinoembryonic antigen (CEA). , Shown relative to germline transgene sequence (top row). In each row, nucleotide changes to the germline sequence are displayed above the changes in the deduced amino acid sequence (if any). Regions corresponding to heavy chain CDR1, CDR2 and CDR3 are shown. Nucleotides that are not encoded by germ cells are shown in capital letters. The deduced amino acid sequence is given below the nucleotide sequence using conventional single letter notation. FIG. 49 shows an aligned variable region sequence of 23 randomly selected cDNAs generated from mRNA obtained from lymphoid tissue of HCl transgenic mice immunized with human carcinoembryonic antigen (CEA). Shown relative to germline transgene sequence (top row). In each row, nucleotide changes to the germline sequence are displayed above the changes in the deduced amino acid sequence (if any). Regions corresponding to heavy chain CDR1, CDR2 and CDR3 are shown. Nucleotides that are not encoded by germ cells are shown in capital letters. The deduced amino acid sequence is given below the nucleotide sequence using conventional single letter notation. FIG. 50 shows an aligned variable region sequence of 23 randomly selected cDNAs generated from mRNA obtained from lymphoid tissue of HC1 transgenic mice immunized with human carcinoembryonic antigen (CEA). Shown relative to germline transgene sequence (top row). Over each row, nucleotide changes to the germline sequence are displayed above the changes in the deduced amino acid sequence (if any). Regions corresponding to heavy chain CDR1, CDR2 and CDR3 are shown. Nucleotides that are not encoded by germ cells are shown in capital letters. The deduced amino acid sequence is given below the nucleotide sequence using conventional single letter notation. FIG. 51 shows an aligned variable region sequence of 23 randomly selected cDNAs generated from mRNA obtained from lymphoid tissue of HC1 transgenic mice immunized with human carcinoembryonic antigen (CEA). Shown relative to germline transgene sequence (top row). In each row, nucleotide changes to the germline sequence are displayed above the changes in the deduced amino acid sequence (if any). Regions corresponding to heavy chain CDR1, CDR2 and CDR3 are shown. Nucleotides that are not encoded by germ cells are shown in capital letters. The deduced amino acid sequence is given below the nucleotide sequence using conventional single letter notation. FIG. 52 shows the data of FIG. 40 in histogram format; putative amino acid residue positions are shown as ordinates (left is amino terminal direction, right is carboxy terminal direction) and the frequency of sequence variation is horizontal Shown as coordinates. FIG. 53 shows the nucleotide sequence of a human DNA fragment named vk65.5 containing the Vκ gene segment; the Vκ coding region deduced amino acid sequence is also shown; the splicing sequence and the recombination signal sequence (heptamer / nonamer) are framed. Shown in FIG. 54 shows the nucleotide sequence of a human DNA fragment named vk65.8 containing the Vκ gene segment; the Vκ coding region deduced amino acid sequence is also shown; the splicing sequence and the recombination signal sequence (heptamer / nonamer) are in frame Shown in FIG. 55 shows the nucleotide sequence of a human DNA fragment named vk65.15 containing the Vκ gene segment; the Vκ coding region deduced amino acid sequence is also shown; the splicing sequence and the recombination signal sequence (heptamer / nonamer) are framed. Shown in FIG. 56 shows the formation of a light chain small locus by phase 1 recombination between two co-injected multiple fragments. The ELISA results for monoclonal antibodies reactive with CEA and non-CEA antigens showing a specific life of antigen binding are shown. 10 shows the DNA sequence of 10 CDNA amplified by PCR to amplify a transcript with human VDJ and mouse constant region sequences. Shows ELISA results for various dilutions of sera from mice supporting both human heavy chain minilocus and human kappa minilocus transgenes; mice immunized with human CD4 And the data shown represent antibodies reactive with human CD4 and having human κ, human μ, or human γ epitopes, respectively. Shown is the relative distribution of lymphocyte staining for human μ or mouse μ as determined by FACS for the three mouse genotypes. Shown is the opposite distribution of lymphocyte staining for human or mouse kappa as determined by FACS for the three Mawas genotypes. Shows the relative distribution of lymphocyte staining for mouse λ as determined by FACS for the three mouse genotypes. Shown is the relative distribution of lymphocyte staining for mouse λ or human κ, as determined by FACS for the four mouse ectotypes. The amounts of human μ, human γ, human κ, mouse μ, mouse γ, mouse κ, and mouse λ chain in the non-immunized serum are shown. Shown is a scatter plot showing the amount of human μ, human γ, human κ, mouse μ, mouse γ, mouse κ, and mouse λ chains in the sera of unimmunized 0011 mice of various genotypes. 1 shows anti-human CD4 titer in 0011 mice. Shows antibody titers containing human mu, human gamma, or human kappa chains in anti-CD4 antibodies in sera collected 3 or 7 weeks after immunization following immunization of 0011 mice with human CD4 . Human heavy chain minilocus transgenes PHC1 and PHC2, and light chain minilocus transgenes pKCl, pKCle and light chain minilocus transgenes produced by homologous recombination of PKC2 and Co4 at the indicated sites. FIG. Storbeta1. (1989) shows a linkage map of the murine lambda light chain locus taken from above. The dotted box represents a pseudogene. 4 schematically represents inactivation of the mouse λ locus by homologous gene targeting. A map of the BALB / c mouse heavy chain locus taken from "Immunoglobulin Gene", Honjo, T, AIt, FW, and Rabbits TH (eds) Academic Press, NY (1989) p129 is shown. The structural gene is shown in a closed box in the upper line. The second and third lines indicate the restriction sites with the displayed symbols. The nucleotide sequence of the mouse heavy chain locus α constant region gene is shown. It shows the construction of a frameshift vector (plasmid B) for introducing a two bp frameshift into the murine heavy chain locus J ₄ gene. Figure 3 shows isotype-specific responses of transgenic animals undergoing hyperimmunity. The relative levels of reactive human μ and γ1 are shown by a colorimetric ELISA assay (y uniaxial). We have injected 3 male 7- to 10-week-old male HC1 line 57 transgenic animals (# 1991, # 2356, # 2357) in a homozygous JHD background by intraperitoneal injection of CEA in Freund's adjuvant. ). The figure depicts the binding of 250-fold dilutions of pooled serum (collected prior to each injection) to CEA-coated microtiter wells. FIG. 5 shows the expression of the transgene-encoded γ1 isotype mediated by class switch recombination. The genomic structure of the integrated transgene in two different human γ1-expressing hybridomas is consistent with recombination of the μ and γ1 switch regions. FIG. 75 shows a Southern plot of PacI / SfiI digested DNA isolated from three transgene expressing hybridomas. From left to right: clone 92-09A-5Hl-5, human γ1 / μ; clone 92-90A-4G2-2, human γ1 / μ; clone 92-09A-4F7-A5-2, human γ1, μ. All three hybridomas are derived from 7 month old mice (mouse # 1991) hemizygous for HC1-57 integration and homozygous for JHD disruption. The plot is hybridized with a probe derived from a 2.3 kb BglII / SfiI DNA fragment spanning the 3 'half of the human γ1 switch region. Although no switch products can be found in the μ-expressing hybridomas, the two γ1-expressing hybridomas 92-09A-5H1-5 and 92-09A-4G2-2 are 5.1 kb and 5.3 kb PacI / SriI, respectively. Contains the switch product that results in the fragment. FIG. 76 is a diagram of two possible deletion mechanisms that can produce a class switch from μ to γ1. The human μ gene is flanked by 400 bp direct repeats (σμ and Σμ) that can recombine to delete μ. Class switching by this mechanism always produces a 6.4 kb PacI / SfiI fragment, whereas class switching by recombination of the μ and γ1 switch regions shows a variation in size between individual switch events, Generate 4 kb and 7 kb PacI / SfiI fragments. The two γ1-expressing hybridomas studied in FIG. 75 appear to have undergone recombination between the μ and γ1 switch regions. Figure 2 shows a chimeric human / mouse immunoglobulin heavy chain produced by a transswitch. Transswitch product cDNA clones were a mixture of spleen and lymph node RNA isolated from hyperimmunized HCl transgenic-JHD mice (# 2357; see legend to FIG. 74 for description of animals and immunization regimes) Was generated by reverse transcription and PCR amplification. Partial nucleotide sequences of 10 randomly picked clones are shown. Lowercase letters represent the encoded germline, and uppercase letters indicate nucleotides that cannot be assigned to known germline sequences. These may be somatic mutations, N nucleotides or truncated D segments. The double-sided type represents a mouse gamma array. It shows that the rearranged VH251 transgene undergoes somatic mutation in the hyperimmunized one. 78. Partial nucleotide sequence of IgG heavy chain variable region cDNA clone from CH1 strain 26 mouse showing primary response to antigen in FIG. 78 and secondary response in FIG. 79. Germline sequences are shown at the top; nucleotide changes from germline are represented for each clone. One cycle indicates identity with the germline sequence and capital letters indicate that no germline source is determined. The arrangement is organized according to the use of the J segment. The germline sequence for each of the J segments is shown. Lower case letters in the CDR3 sequence represent identity to known D segments contained within the HC1 transgene. The assigned D segments are listed at the end of each array. Unassigned sequences can be derived from N region additions or somatic mutations. Or, in some cases, they are simply too short to distinguish random N nucleotides from known D segments. Figure 78 Primary Response: 13 randomly picked VH251 single γl cDNA clones. Four weeks old female HCl strain 26-JHD mice (# 2599) were injected once with KLH and complete Freund's adjuvant. Spleen cell RNA was isolated after 5 days. The overall frequency of somatic mutations within the V segment is 0.06% (2/3, 198 bp). Secondary response of FIG. 79: 13 randomly picked VH251-γl cDNA clones. Two-month-old female HCl strain 26-JHD mice (# 3204) were injected with HEL and Freund's adjuvant three times over a month (the primary injection was complete adjuvant, and boosters were given at 1 and 3 weeks). Spleen and lymph node RNA were isolated after 4 months. The overall frequency of humoral mutations within the V segment is 1.6% (52/3, 198 bp). Extensive somatic mutations have been shown to be restricted to the γ1 sequence: somatic mutations and class switches occur within the same population of B cells. Partial nucleotide sequence of VH251 cDNA clone isolated from spleen and lymph node cells of HC1 line 57 transgenic-JHD mice (# 2357) immunized to CEA (see FIG. 68 for immunization plan). FIG. 80: Randomly picked VH251-μ cDNA clone of IgM: 23. Nucleotide sequence of a 156 bp segment containing CDRs 1 and 2 surrounding residues. The overall level of somatic mutation is 0.1% (5 / 3,744 bp). FIG. 81: Randomly picked VH251-γ1 cDNA clone of IgG: 23. Nucleotide sequence of a segment containing CDRs 1-3 and surrounding residues. The overall frequency of somatic mutations within the V segment is 1.1% (65/5, 658 bp). For comparison with the μ sequence in FIG. 80; the mutation frequency for the first 156 nucleotides is 1.1% (41/3, 588 bp). See legends in FIGS. 80 and 81 for explanation of symbols. VH51P1 and VH56P1 show extensive somatic mutations in mice that have not been immunized. Partial nucleotide sequence of an IgG heavy chain variable region cDNA clone from a female HC2 line 2550 transgenic JHD mouse (# 5250) 9 weeks after immunization. The overall frequency of somatic mutations in the 19VH56pl segment is 2.2% (101/4, 674 bp). The overall frequency of somatic mutations within a single VH51p1 segment is 2.0% (5/246 bp). See legend to Figures 78 and 79 for explanation of symbols. Double transgenic mice with the endogenous Ig locus analyzed contain human IgMκ positive B cells. FACS of cells isolated from the spleens of 4 mice with different genotypes. Left column: control mice (# 9944, females 6 weeks old, JH +/−, JCκ +/−; heterozygous wild type mouse heavy chain and κ single light chain loci, non-transgenic). Column 2: Human heavy chain transgenic (# 9877, female JH − / −, JCκ − / −, HC2 strain 2550 ± at 6 weeks of age; homozygous for split mouse heavy chain and κ single light chain loci Junction, semi-junction for HC2 transgene). Column 3: Human κ single light chain transgenic (# 9878, female JH − / −, JCκ − / −, KCo4 strain 4437+ at 6 weeks of age; for the heavy and κ single light chain loci of the disrupted mice Homozygous, semi-joint for KCo4 transgene). Right column: double transgenic (# 9879, 6 weeks old female, JH − / − mJCκ − / −, HC2 line 2550+, KCo4 line 4437+; About the heavy chain and κk single light chain locus of the divided mice Homozygous, semi-jointed for HC2 and KCo4 transgenes) Top: Splenocytes stained for expression of mouse λ light chain (x axis) and human κ light chain (y axis). Second stage: splenocytes stained for expression of human μ heavy chain (x axis) and human kappa light chain (y axis), third stage: mouse μ heavy chain (x axis) and mouse kappa light chain ( Spleen cells stained for expression on the y-axis). Bottom: Histogram of splenocytes stained for expression of mouse B220 antigen (logarithmic fluorescence: x axis: cell number: y axis). For each of the two color illustrations, the relative number of cells in each of the displayed inlaids is given as a percentage of the e-parameter gate based on propidium iodide staining and light scattering. The fractions 1 to 1 of B220 + cells in each of the samples displayed in the lower row are given as the white fraction of the lymphocyte light scattering gate. Secreted immunoglobulin levels in the sera of double transgenic mice. Mouse T and λ and human μ, γ and κ from 18 individual HC2 / KCo42 double transgenic mice homozygous for endogenous heavy chain and κ single light chain locus disruptions. Mice: (+) HC2 strain 2550 (up to 5 HC2 copies per integration), KCo4 strain 4436 (1-2 KCo4 copies per integration); (◯) HC2 strain 2550, KCo4 strain 4437 (single integration) (×) HC2 line 2550, KCo4 line 4583 (up to 5 KCo4 copies per integration); (□) HC2 line 2572 (30-50 HC2 copies per integration, KCo4 line 4437; (Δ) HC2 line 5467 (20-30 HC2 copies per integration, KCo4 line 4437). 1 shows a human antibody response to a human antigen. FIG. 85: Primary response to recombinant human soluble CD4. Human IgM and human kappa light chain levels have been reported for sera before bleeding (O) and after immunization (●) from four double transgenic mice. Figure 86: A switch to human IgG occurs in vivo. Using peroxodase conjugated polyclonal anti-human IgG used in the presence of 1.5 μ / ml extra IgE, κ and 1% normal mouse serum to inhibit non-specific cross-reactivity, Human IgG (circle) was examined. Human kappa light chains (squares) were detected using a polyclonal anti-human kappa reagent conjugated with peroxidase in the presence of 1% normal mouse serum. Representative results from one mouse (# 9344; HC2 line 2550, KCo4 line 4436) are shown. Each point represents the average of duplicate wells minus background absorption. Figure 2 shows FACS analysis of human PBL using hybridoma supernatants that discriminate human CD4 + lymphocytes from human CD8 lymphocytes. Figure 2 shows human α-CD41gManfIgG in the sera of transgenic mice. Shown are competitive binding experiments comparing the α-human CD4 hybridoma monoclonal, 2Cll-8 of transgenic mice with RPA-TA and Leu-3A monoclonals. Production data for Ig expression of cultured 2Cll-8 hybridomas is shown. 1 schematically shows the structure of a homologous recombination targeting transgene for deleting genes such as heavy chain constant region genes. The transgenic mice of the invention are immunized with a human antigen selected from Tables B and C below. The transgenic mouse produces a human antibody that binds to the human antigen. Hybridomas that secrete monoclonal antibodies against the selected human antigens are produced using B cells from the immunized transgenic mice.

Claims (30)

A mouse comprising an unrearranged human heavy chain immunoglobulin variable region comprising:
The variable region includes a plurality of human _VH gene segments, a plurality of human D gene segments, and a plurality of human _JH gene segments;
The mouse B cell expresses a chimeric antibody comprising a human immunoglobulin heavy chain variable region and a mouse immunoglobulin heavy chain constant region. 2. The mouse of claim 1 further comprising an unrearranged human light chain immunoglobulin variable region and a human light chain constant region. The mouse of claim 2, wherein the light chain variable region comprises a plurality of _VL gene segments and a plurality of _JL gene segments. The mouse of claim 2, wherein the chimeric antibody comprises a human light chain. The mouse of claim 4, wherein the human light chain comprises a kappa constant region. The mouse of claim 4, wherein the human light chain comprises a λ constant region. 7. The mouse of any one of claims 1-6, wherein the heavy chain immunoglobulin variable region is from 2 to about 10 _VH gene segments, at least 10 D gene segments, and at least 6 _JHs. A mouse containing a gene segment. The mouse according to any one of claims 1 to 6, wherein the mouse immunoglobulin heavy chain constant region includes a γ constant region. 7. The mouse according to any one of claims 1 to 6, wherein the human heavy chain immunoglobulin variable region further comprises a cis-acting transcriptional regulatory region comprising at least one promoter. A rearranged human heavy chain immunoglobulin variable region comprising a plurality of V _H gene segments, a plurality of D gene segments and a plurality of J _H gene segments, and a relocation comprising a plurality of _VL gene segments and a plurality of J _L gene segments. A mouse comprising an arranged human light chain immune variable region comprising:
A mouse wherein the B lymphocytes of the mouse express a chimeric antibody comprising a human immunoglobulin altered region and a mouse immunoglobulin constant region by rearranging at least one non-rearranged variable region. The mouse according to claim 10, wherein the chimeric antibody comprises a human heavy chain variable region and a mouse heavy chain constant region. 11. The mouse of claim 10, wherein the chimeric antibody comprises a human light chain variable region and a mouse light chain constant region. 11. The mouse according to claim 10, wherein the chimeric antibody comprises a human heavy chain variable region and a mouse heavy chain constant region, and a human light chain variable region and a mouse light chain constant region. The mouse according to any one of claims 10 to 13, which has been immunized with a predetermined antigen. A hybridoma comprising a second cell fused with the mouse B lymphocyte of claim 14 and capable of immortalizing the B lymphocyte, wherein the chimera specifically binds to the predetermined antigen is produced. Hybridoma. A method for obtaining a chimeric antibody that binds to a predetermined antigen, comprising:
(A) immunizing the mouse according to any one of claims 1, 2, and 10; and (b) obtaining a chimeric antibody that binds to the predetermined antigen from the mouse. The method according to claim 16, comprising:
Isolating a nucleic acid molecule encoding a human heavy chain variable region of said chimeric antibody, and operably linking said nucleic acid molecule to a human constant region. The method according to claim 16, comprising:
Isolating a nucleic acid molecule encoding a human light chain variable region of the chimeric antibody, and operably linking the nucleic acid molecule to a human constant region. The method according to claim 16, comprising:
Isolating a nucleic acid molecule encoding a human heavy chain variable region of the chimeric antibody, and operably linking the nucleic acid molecule to a nucleic acid molecule encoding a human constant region. The method according to claim 16, comprising:
Isolating a nucleic acid molecule encoding a human light chain variable region of the chimeric antibody, and operably linking the nucleic acid molecule to a nucleic acid molecule encoding a human constant region. The method according to claim 16, comprising:
Isolating a nucleic acid molecule encoding a human heavy chain variable region and a human light chain variable region of the chimeric antibody, and linking the nucleic acid molecule to a nucleic acid molecule encoding a human constant region. Method. 24. The method of any one of claims 17-21, comprising producing a human antibody by expressing in a host cell a nucleic acid molecule operably linked to a nucleic acid encoding a human constitutive region. how to. 23. The method of claim 22, further comprising isolating the expressed human antibody. A method for preparing a human antibody comprising:
Culturing a host cell, the host comprising:
(I) a human immune blobulin heavy chain recombination construct comprising a nucleic acid molecule encoding a human heavy chain variable region derived from mouse B lymphocytes operably linked to a human heavy chain constant region (ii) a human light chain constant region A human immune blobulin light chain recombination construct comprising a nucleic acid molecule encoding a human light chain variable region derived from mouse B lymphocytes operably linked to
The mouse B lymphocyte has a human heavy chain immunoglobulin heavy chain variable region comprising a plurality of V _H gene fragments, a plurality of human D gene fragments, and a plurality of human J _H gene fragments, and not rearranged; Derived from a mouse having a human heavy chain immunoglobulin light chain variable region comprising a plurality of _VL gene fragments, a plurality of human D gene fragments, and a plurality of human J _L gene fragments;
The culturing step is performed under conditions that allow the human heavy chain and the human light chain to express and form antibodies.
Method. 25. The method of claim 24, wherein the mouse B lymphocyte expresses a full length human heavy chain. 25. The method of claim 24, wherein the mouse B lymphocyte expresses a full length human light chain. 25. The method of claim 24, wherein the B lymphocyte expresses a chimeric heavy chain comprising a human variable region and a mouse homeostasis region. 25. The method of claim 24, wherein the B lymphocyte expresses a chimeric light chain comprising a human variable region and a mouse constant region. 25. The method of claim 24, comprising isolating the expressed human antibody.
Method. Functionally disrupted endogenous heavy chain homozygous allele pair, functionally disrupted endogenous kappa
A transgenic mouse comprising a pair of light chain homozygous alleles, at least one copy of a human immunoglobulin heavy chain transgene, and at least one copy of a human immunoglobulin kappa light chain transgene, said transgenic mouse Transgenic mice in which about 95% of the splenic B cells in human express human cell surface IgMκ and the transgenic mice make a human antibody response following immunization with an antigen.

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