Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
First Claim
1. A method for obtaining secondary repertoire, somatically mutated human immunoglobulin V gene segments encoding immunoglobin V regions reactive with a pre-selected antigen the method comprising:
- antigenically stimulating a transgenic mouse with said pre-selected antigen said transgenic mouse having one or more transgenes containing DNA segments from unrearranged human immunoglobulin heavy and kappa light chain gene loci incorporated its germline DNA, said transgene or transgenes comprising a plurality of human V kappa gene segments, a plurality of human J kappa gene segments, a human kappa constant region gene segment, a plurality of human VH gene segments, a plurality of human DH gene segments, a plurality of human JH gene segments, a human mu switch segment, and a human mu constant region gene segment,wherein a first subset of B lymphocytes of said transgenic mouse produce IgM immunoglobulin molecules encoded by functional rearrangement of said immunoglobulin heavy and kappa light chain gene loci, said functionally rearranged immunoglobulin genes having variable region sequences including FR1, CDR1, FR2, CDR2, and FR3 portions from said human V kappa or VH gene segments of said unrearranged gene loci,and wherein a second subset of B lymphocytcs of said transgenic mouse produce non-IgM immunoglobulin molecules encoded by somatically mutated immunoglobulin heavy and kappa light chain gene loci, said somatically mutated immunoglobulin heavy and kappa light chin gene loci having variable region sequences including FR1, CDR1, FR2, CDR2, and FR3 portions of somatically mutated DNA sequences from said human V kappa or VH gene segments of said first subset; and
obtaining said somatically mutated human immunoglobulin V gene segments by (1) collecting somatically mutated V gene segments from said second subset of B lymphocytes or (2) immortalizing said second subset of B lymphocytes and then collecting somatically mutated V gene segments from said lymphocytes, wherein one or more of the somatically mutated V gene segments encode immunoglobulin V regions reactive with the preselected antigen.
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Abstract
The invention relates to transgenic non-human animals capable of producing heterologous antibodies and transgenic non-human animals having inactivated endogenous immunoglobulin genes. In one aspect of the invention, endogenous immunoglobulin genes are suppressed by antisense polynucleotides and/or by antiserum directed against endogenous immunoglobulins. Heterologous antibodies are encoded by immunoglobulin genes not normally found in the genome of that species of non-human animal. In one aspect of the invention, one or more transgenes containing sequences of unrearranged heterologous human immunoglobulin heavy chains are introduced into a non-human animal thereby forming a transgenic animal capable of functionally rearranging transgenic immunoglobulin sequences and producing a repertoire of antibodies of various isotypes encoded by human immunoglobulin genes. Such heterologous human antibodies are produced in B-cells which are thereafter immortalized, e.g., by fusing with an immortalizing cell line such as a myeloma or by manipulating such B-cells by other techniques to perpetuate a cell line capable of producing a monoclonal heterologous antibody. The invention also relates to heavy and light chain immunoglobulin transgenes for making such transgenic non-human animals as well as methods and vectors for disrupting endogenous immunoglobulin loci in the transgenic animal.
1673 Citations
21 Claims
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1. A method for obtaining secondary repertoire, somatically mutated human immunoglobulin V gene segments encoding immunoglobin V regions reactive with a pre-selected antigen the method comprising:
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antigenically stimulating a transgenic mouse with said pre-selected antigen said transgenic mouse having one or more transgenes containing DNA segments from unrearranged human immunoglobulin heavy and kappa light chain gene loci incorporated its germline DNA, said transgene or transgenes comprising a plurality of human V kappa gene segments, a plurality of human J kappa gene segments, a human kappa constant region gene segment, a plurality of human VH gene segments, a plurality of human DH gene segments, a plurality of human JH gene segments, a human mu switch segment, and a human mu constant region gene segment, wherein a first subset of B lymphocytes of said transgenic mouse produce IgM immunoglobulin molecules encoded by functional rearrangement of said immunoglobulin heavy and kappa light chain gene loci, said functionally rearranged immunoglobulin genes having variable region sequences including FR1, CDR1, FR2, CDR2, and FR3 portions from said human V kappa or VH gene segments of said unrearranged gene loci, and wherein a second subset of B lymphocytcs of said transgenic mouse produce non-IgM immunoglobulin molecules encoded by somatically mutated immunoglobulin heavy and kappa light chain gene loci, said somatically mutated immunoglobulin heavy and kappa light chin gene loci having variable region sequences including FR1, CDR1, FR2, CDR2, and FR3 portions of somatically mutated DNA sequences from said human V kappa or VH gene segments of said first subset; and obtaining said somatically mutated human immunoglobulin V gene segments by (1) collecting somatically mutated V gene segments from said second subset of B lymphocytes or (2) immortalizing said second subset of B lymphocytes and then collecting somatically mutated V gene segments from said lymphocytes, wherein one or more of the somatically mutated V gene segments encode immunoglobulin V regions reactive with the preselected antigen. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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15. A method for obtaining secondary repertoire V regions reactive with a preselected antigen, said V regions encoded by somatically mutated human immunoglobulin V gene segments, the method comprising:
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antigenically stimulating a transgenic mouse with said pre-selected antigen, said transgenic mouse having one or more transgenes containing DNA segments from unrearranged human immunoglobulin heavy and kappa light chain gene loci incorporated into its germline DNA, said transgene or transgenes comprising a plurality of human V kappa gene segments, a plurality of human J kappa gene segments, a human kappa constant region gene segment, a plurality of human VH gene segments, a plurality of human DH gene segments, a plurality of human JH gene segments, a human mu switch segment, and a human mu constant region gene segment, wherein a subset of B lymphocytes of said transgenic mouse produce IgM immunoglobulin molecules encoded by functional rearrangement of said immunoglobulin heavy and kappa light chain gene loci, said functionally rearranged immunoglobulin genes having variable region sequences including FR1, CDR1, FR2, CDR2, and FR3 portions from said human V kappa or VH gene segments of said unrearranged gene loci, and wherein a second subset of B lymphocytes of said transgenic mouse produce non-IgM immunoglobulin molecules encoded by somatically mutated immunoglobulin heavy and kappa light chain gene loci, said somatically mutated immunoglobulin heavy and kappa light chain gene loci having variable region sequences including FR1, CDR1, FR2, CDR2, and FR3 portions of somatically mutated DNA sequences from said human V kappa or VII gene segments; and obtaining said secondary repertoire V regions reactive with said preselected antigen, by (1) collecting said somatically mutated immunoglobulin V gene segments from said second subset of B lymphocytes, and expressing collected V region gene segments in a host cell, or (2) immortalizing cells of said second subset of B lymphocytes and collecting said secondary repertoire V regions. - View Dependent Claims (16, 17, 18, 19, 20, 21)
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Specification