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US 11,564,380 B2Granted
Animal models and therapeutic molecules
AssigneeKymab Limited
InventorsAllan Bradley, E-Chiang Lee, Qi Liang, Wei Wang, Glenn Friedrich
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Abstract
The invention discloses methods for the generation of chimaeric human-non-human antibodies and chimaeric antibody chains, antibodies and antibody chains so produced, and derivatives thereof including fully humanised antibodies; compositions comprising said antibodies, antibody chains and derivatives, as well as cells, non-human mammals and vectors, suitable for use in said methods.
Claims34
A method of providing an antigen-specific polypeptide comprising a human IgH variable region, an antigen-specific antibody comprising said polypeptide, a cell producing said polypeptide, a nucleic acid encoding said polypeptide or said human IgH variable region and/or a biological sample comprising said polypeptide, the method comprising:
- (a) delivering an antigen to a transgenic mouse and expressing said polypeptide, and
- (b) isolating from said transgenic mouse of step (a) said antigen-specific polypeptide, said antigen-specific antibody comprising said polypeptide, said cell producing said polypeptide, said nucleic acid encoding said polypeptide or said human IgH variable region and/or said biological sample comprising said polypeptide,
- wherein said transgenic mouse of step (a) comprises a germline comprising a homozygous chimeric immunoglobulin heavy chain (IgH) locus comprising unrearranged human IgH variable region gene segments, at an endogenous IgH locus upstream of an enhancer and a constant (C) region comprising an endogenous CH gene segment,
- wherein said unrearranged human variable region gene segments in said chimeric IgH locus are operably linked to said enhancer and said C region;
- wherein said chimeric IgH locus comprises in 5′ to 3′ transcriptional orientation:
- (i) said unrearranged human immunoglobulin heavy chain variable region gene segments comprising human IgH V gene segments, human D gene segments and human JH gene segments comprising a human 3′ JH gene segment,
- (ii) a chimeric JC intron comprising human intronic JC DNA and mouse JC intronic DNA upstream of said enhancer, wherein said human JC intronic DNA is DNA that is naturally downstream of a human JH6 gene segment in a wild type human IgH locus, wherein said mouse JC intronic DNA is DNA that is naturally upstream of mouse Eμ in a wild type human IgH locus, and wherein said mouse JC intronic DNA is truncated relative to wild type mouse JC intronic DNA between a mouse JH4 gene segment and mouse Eμ, wherein the 3′ end of said human 3′JH gene segment is less than 2 kb upstream of the 5′ end of said truncated mouse JC intronic DNA;
- and
- (iii) said operably linked enhancer and said C region;
- wherein said chimeric IgH locus is functional to undergo human IgH variable gene segment rearrangement, and
- wherein the germline of said transgenic mouse comprises endogenous mouse IgH variable region DNA, wherein the mouse variable region DNA is upstream of said truncated mouse JC intronic DNA, wherein said mouse is non-functional to produce mouse Ig heavy chain comprising a mouse heavy chain variable region and a mouse heavy chain constant region;
- wherein said transgenic mouse of step (a) is functional to form rearranged human VH, D and JH gene segments and to express chimeric immunoglobulin heavy chain polypeptide comprising a human VH region and a mouse C region, and
- wherein said transgenic mouse of step (a) comprises rearranged human VH, D and JH gene segments, and expresses said antigen-specific polypeptide and
- wherein said transgenic mouse is capable of breeding with another said transgenic mouse to produce a subsequent generation mouse,
- said subsequent generation mouse comprising in its germline a homozygous IgH locus comprising unrearranged human IgH gene segments at an endogenous IgH locus upstream of an enhancer and a mouse Ig constant region (C) gene segment, wherein the germline of said subsequent generation mouse comprises mouse IgH variable region DNA upstream of mouse JC intronic DNA.
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A method of providing an antigen-specific polypeptide comprising a human IgH variable region, an antigen-specific antibody comprising said polypeptide, a cell producing said polypeptide, a nucleic acid encoding said polypeptide or said human IgH variable region and/or a biological sample comprising said polypeptide, the method comprising:
- a) providing a transgenic mouse to which an antigen has been delivered and which expresses said polypeptide, and
- b) isolating from said transgenic mouse of step (a) said antigen-specific polypeptide, said antigen-specific antibody comprising said polypeptide, said cell producing said polypeptide, said nucleic acid encoding said polypeptide or said human IgH variable region and/or said biological sample comprising said polypeptide,
- wherein said transgenic mouse comprises a germline comprising a homozygous chimeric immunoglobulin heavy chain (IgH) locus comprising unrearranged human IgH variable region gene segments at an endogenous IgH locus upstream of an enhancer and a constant (C) region comprising an endogenous IgH C gene segment,
- wherein said unrearranged human variable region gene segments in said chimeric IgH locus are operably linked to said enhancer and said C region;
- wherein said chimeric IgH locus comprises in 5′ to 3′ transcriptional orientation:
- (i) said unrearranged human immunoglobulin heavy chain variable region gene segments comprising human IgH V gene segments, human D gene segments and human JH gene segments comprising a human 3′ JH gene segment,
- (ii) a chimeric JC intron comprising human JC intronic DNA and mouse JC intronic DNA upstream of said enhancer, wherein said human JC intronic DNA is DNA that is naturally downstream of a human JH6 gene segment in a wild type human IgH locus, wherein said mouse JC intronic DNA is DNA that is naturally upstream of mouse Eμ in a wild type mouse IgH locus, and wherein said mouse JC intronic DNA is truncated relative to wild type mouse JC intronic DNA between a mouse JH4 gene segment and mouse Eμ, wherein the 3′ end of said human 3′JH gene segment is less than 1 kb upstream of the 5′ end of said truncated mouse JC intronic DNA;
- and
- (iii) said operably linked enhancer and C region;
- wherein said chimeric IgH locus is functional to undergo human IgH variable gene segment rearrangement, and wherein a transgenic mouse that has not been contacted with said antigen is functional to form rearranged human VH, D and JH gene segments and to express chimeric immunoglobulin heavy chain polypeptide comprising a human VH region and a mouse C region, and
- wherein the germline of said transgenic mouse comprises endogenous mouse IgH variable region DNA upstream of said truncated mouse JC intronic DNA, wherein said mouse is non-functional to produce mouse Ig heavy chain comprising a mouse heavy chain variable region and a mouse heavy chain constant region;
- wherein after delivery of said antigen to said transgenic mouse, said transgenic mouse comprises rearranged human VH, D and JH gene segments, and expresses said antigen-specific polypeptide, and
- wherein said transgenic mouse is capable of breeding with another said transgenic mouse to produce said subsequent generation mice,
- said subsequent generation mouse comprising in its germline a homozygous IgH locus comprising unrearranged human IgH gene segments at an endogenous IgH locus upstream of an enhancer and a mouse Ig constant region (C) gene segment, wherein the germline of said subsequent generation mouse comprises mouse IgH variable region DNA upstream of mouse JC intronic DNA.
11 more elements
A method of providing an antigen-specific polypeptide comprising a human IgH variable region, an antigen-specific antibody comprising said polypeptide, a cell producing said polypeptide, a nucleic acid encoding said polypeptide or said human IgH variable region and/or a biological sample comprising said polypeptide, the method comprising:
- isolating from a transgenic mouse that has been delivered with an antigen and which expresses said polypeptide;
- said antigen-specific polypeptide, said antigen-specific antibody comprising said polypeptide, said cell producing said polypeptide, said nucleic acid encoding said polypeptide or said human IgH variable region and/or said biological sample comprising said polypeptide,
- wherein said transgenic mouse comprises a germline with a chimeric immunoglobulin heavy chain (IgH) locus comprising unrearranged human IgH variable region gene segments at an endogenous IgH locus upstream of an enhancer and a constant (C) region comprising an endogenous IgH C gene segment,
- wherein said unrearranged human variable region gene segments in said chimeric IgH locus are operably linked to said enhancer and said C region;
- wherein said chimeric IgH locus comprises in 5′ to 3′ transcriptional orientation:
- (i) said unrearranged human immunoglobulin heavy chain variable region gene segments comprising human IgH V gene segments, human D gene segments and human JH gene segments comprising a human 3′ JH gene segment,
- (ii) a chimeric JC intron comprising human JC intronic DNA and mouse JC intronic DNA upstream of said enhancer, wherein said human JC intronic DNA is DNA that is naturally downstream of a human JH6 gene segment in a wild type human IgH locus, wherein said mouse JC intronic DNA is DNA that is naturally upstream of mouse Eμ in a wild type mouse IgH locus, and wherein said mouse JC intronic DNA is truncated relative to wild type mouse JC intronic DNA between a mouse JH4 gene segment and mouse Eμ, wherein the 3′ end of said human 3′JH gene segment is less than 2 kb upstream of the 5′ end of said mouse truncated JC intronic DNA;
- and
- (iii) said operably linked enhancer and C region;
- wherein said chimeric IgH locus is functional to undergo human IgH variable gene segment rearrangement, and
- wherein the germline of said transgenic mouse comprises endogenous mouse IgH variable region DNA upstream of said truncated mouse JC intronic DNA, wherein said mouse is non-functional to produce mouse Ig heavy chain comprising a mouse heavy chain variable region and a mouse heavy chain constant region;
- wherein said transgenic mouse is functional to form rearranged human VH, D and JH gene segments and to express chimeric immunoglobulin heavy chain polypeptide comprising a human VH region and a mouse C region, and wherein said transgenic mouse after delivery of said antigen comprises rearranged human VH, D and JH gene segments, and expresses said antigen-specific polypeptide, and
- wherein said transgenic mouse is capable of breeding with another said transgenic mouse to produce said subsequent generation mice,
- said subsequent generation mouse comprising in its germline a homozygous IgH locus comprising unrearranged human IgH gene segments at an endogenous IgH locus upstream of an enhancer and a mouse Ig constant region (C) gene segment, wherein the germline of said subsequent generation mouse comprises mouse IgH variable region DNA upstream of mouse JC intronic DNA.
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Dependencies are inferred from the claim text.