Generation of xenogeneic antibodies
First Claim
1. A method for producing a xenogeneic immunoglobulin or analog thereof in a non-human animal host, said method comprising:
- immunizing said host with an immunogen under conditions to stimulate an immune response to said immunogen, whereby said host mounts an immune response to said immunogen and produces B-cells producing immunoglobulin specific for said immunogen, and isolating xenogeneic immunoglobulin produced bys aid host, wherein said host is characterized by
1) being substantially incapable of producing endogenous immunoglobulin heavy chain;
(2) being substantially incapable of producing endogenous immunoglobulin light chains; and
3) being capable of producing a xenogeneic immunoglobulin or analog thereof.
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Abstract
The subject invention provides non-human mammalian hosts characterized by inactivated endogenous Ig loci and functional human Ig loci for response to an immunogen to produce human antibodies or analogs thereof. The hosts are produced by multiple genetic modifications of embryonic cells in conjunction with breeding. Different strategies are employed for recombination of the human loci randomly or at analogous host loci. Chimeric and transgenic mammals, particularly mice, are provided, having stably integrated large, xenogeneic DNA segments. The segments are introduced by fusion with yeast spheroplasts comprising yeast artificial chromosomes (YACs) which include the xenogeneic DNA segments and a selective marker such as HPRT, and embryonic stem cells.
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Citations
78 Claims
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1. A method for producing a xenogeneic immunoglobulin or analog thereof in a non-human animal host, said method comprising:
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immunizing said host with an immunogen under conditions to stimulate an immune response to said immunogen, whereby said host mounts an immune response to said immunogen and produces B-cells producing immunoglobulin specific for said immunogen, and isolating xenogeneic immunoglobulin produced bys aid host, wherein said host is characterized by
1) being substantially incapable of producing endogenous immunoglobulin heavy chain;
(2) being substantially incapable of producing endogenous immunoglobulin light chains; and
3) being capable of producing a xenogeneic immunoglobulin or analog thereof. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 15)
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11. An immortalized non-human cell line genetically modified so as to lack the ability to produce immunoglobulin endogenous to the cell line and comprising xenogeneic immunoglobulin loci encoding at least one xenogeneic immunoglobulin heavy chain and a light chain;
wherein said xenogeneic immunoglobulin heavy and light chain loci are expressed. - View Dependent Claims (12, 13, 14)
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16. A genetically modified non-human animal comprising a modified genome selected from the group consisting of:
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a genome heterozygous or homozygous for a modification that results in the inability of at least one locus to produce endogenous immunoglobulin heavy or light chains;
a genome heterozygous or homozygous for a modification that results in the inability of at least one locus to produce endogenous immunoglobulin heavy and light chains;
a genome heterozygous for a modification that results in the inability of at least one locus to produce endogenous immunoglobulin heavy and light chains and hemizygous for the ability to produce xenogeneic immunoglobulin heavy chains;
a genome heterozygous for a modification that results in the inability of at least one locus to produce endogenous immunoglobulin heavy and light chains and hemizygyous for the ability to produce xenogeneic immunoglobulin light chains;
a genome homozygous for a modification that results in the inability to produce endogenous immunoglobulin heavy and light chains and homozygous for the ability to produce xenogeneic immunoglobulin heavy or light chains;
a genome homozygyous for a modification that results in the inability to produce endogenous immunoglobulin heavy and light chains and hemizygous for the ability to produce xenogeneic immunoglobulin heavy or light chains;
a genome homozygous or heterozygous for a modification that results in the inability of at least one locus to produce endogenous immunoglobulin heavy and light chains and hemizygous for the ability to produce xenogeneic immunoglobulin heavy and light chains;
a genome heterozygous for a modification that results in the inability of at least one locus to produce endogenous immunoglobulin heavy or light chain and hemizygous for a modification that results in the ability to produce xenogeneic immunoglobulin heavy and light chains;
a genome homozygous for a modification that results in the inability to produce endogenous immunoglobulin heavy or light chain and homozygous for a modification that results in the ability to produce xenogeneic immunoglobulin heavy and light chains; and
a genome homozygous for a modificatio that results in the inability to produce endogenous immunoglobulin heavy or light chain and hemizygous for a modification that results in the ability to produce xenogeneic immunoglobulin heavy and light chains. - View Dependent Claims (17, 18, 19, 20, 21, 22, 23, 24)
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- 25. A transgenic murine animal comprising a genome lacking the ability to produce endogenous immunoglobulin, said genome comprising a lesion in the J region of the heavy chain immunoglobulin loci, and a lesion in the constant and/or J regions of the light chain immunoglobulin loci.
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27. A method for producing a modified non-human animal, said animal having a xenogeneic DNA segment of at least 100 kb stably integrated into the genome of said animal, said method comprising:
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combining under fusing conditions yeast spheroplasts, said spheroplasts comprising a YAC having said xenogeneic DNA segment and a marker for selection, with embryonic stem cells of said animal, whereby said xenogeneic DNA segment becomes integrated into the genome of said embryonic stem cells;
selecting for embryonic stem cells carrying said xenogeneic DNA segment by means of the marker;
transferring said embryonic cells into a host blastocyst and implanting said blastocyst in a pseudopregnant animal recipient, and allowing said blastocyst to develope to term to produce a chimeric animal carrying said xenogeneic DNA segment; and
mating said chimeric animal with an animal of the same species to produce said modified animal carrying said xenogeneic DNA segment. - View Dependent Claims (28, 29, 30, 31, 32, 33, 34)
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- 35. A non-human animal heterozygous or homozygous for a xenogeneic genomic mammalian DNA segment of at least 100 kb, stably integrated in substantially intact form into the genome of said animal.
- 40. An embryonic stem cell comprising a genome having endogenous immunoglobulin heavy chain loci, and immunoglobulin light chain loci, said genome comprising a lesion in said endogenous immunoglobulin heavy chain and/or light loci, resulting in the incapacity of the immunoglobulin locus comprising said lesion to rearrange.
- 50. A murine embryonic stem cell comprising homozygotic alleles of immunoglobulin heavy chain loci, said loci comprising a lesion resulting in the incapacity of the immunoglobulin loci comprising said lesion to rearrange.
- 52. A murine embryonic stem cell comprising homozygotic alleles of immunoglobulin light chain loci, said loci comprising a lesion resulting in the incapacity of the immunoglobulin loci comprising said lesion to rearrange.
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54. A murine embryonic stem cell of a murine host said stem cell comprising a genome having immunoglobulin loci comprising J regions, said stem cell comprising a lesion in at least one of the J regions of the immunoglobulin locus resulting in the incapacity of said immunoglobulin locus to rearrange, said embryonic stem cell produced by the method comprising introducing homologous DNA into a murine stem cell in culture, wherein said homologous DNA comprises a region homologous with the J region of an immunoglobulin locus and a marker gene for insertion into said locus;
- and selecting for embryonic stem cells having undergone homologous recombination with said homologous DNA.
- View Dependent Claims (55, 56)
- 57. A murine embryonic stem cell comprising at least 100 kb of xenogeneic DNA.
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60. A method for modifying a genome of a recipient murine embryonic stem cell by homologous recombination with a large xenogeneic DNA genomic fragment previously manipulated in a yeast artificial chromosome (YAC), the improvement which comprises:
introducing at least one YAC into said murinie embryonic stem cell by spheroplast fusion, and selecting recipient cells comprising said genomic fraqment, wherein said YAC comprises a mammalian selectable or screenable gene, wherein said YAC is faithfully transmitted through the host germline, and said xenogeneic DNA fragment is transmitted in substantially intact form. - View Dependent Claims (61, 62, 63, 64, 65, 66, 67)
- 68. A murine animal heterozygous for a xenogeneic unrearranged mammalian DNA segment of at least 100 kb stably integrated into the genome of said murine animal.
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70. A human antibody molecule characterized by;
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comprising the protein sequences of the human immunoglobulin heavy and light chains;
specificity for an immunogen; and
having other than human glycosylation. - View Dependent Claims (71)
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72. A method for producing a genetically modified non-human animal, comprising interbreeding a first parent and a second parent, and recovering the progeny thereof, wherein the parents and progeny are selected from the group consisting of:
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first and second parents heterozygous for a genome modified to be incapable of producing endogenous immunoglobulin light chain, and progeny homozygous for said modified genome;
first and second parents heterozygous for a genome modified to be incapable of producing an endogenous immunoglobulin heavy chain, and progeny homozygous for said modified genome;
a first parent heterozygous for a genome modified to be incapable of producing an endogenous immunoglobulin light chain, a second parent heterozygous for a genome modified to be incapable of producing an endogenous immunoglobulin heavy chain and progeny heterozygous for said modified genome so as to be incapable of producing endogenous immunoglobulin heavy and light chains;
first and second parents heterozygous for a genome modified to be incapable of producing endogenous immunoglobuin light and heavy chains, and progeny homozygous for said modified genome;
a first parent hemizgyous for a genome modified to be capable of producing xenogeneic immunoglobulin heavy chain, a second parent heterozygous for a genome modified to be incapable of producing endogenous immunoglobulin light and heavy chains, and progeny heterozygous for said modified genome so as to be incapable of producing endogenous immunoglobulin light and heavy chains and hemizygous for a modified genome so as to be capable of producing xenogeneic immunoglobulin heavy chain;
first and second parents heterozygous for a genome modified to be incapable of producing endogenous immunoglobulin light and heavy chains and hemizygous for a genome modified to be capable of producing xenogeneic immunoglobulin heavy chain, and progeny
1) homozygous for said modified genome and
2) homozygous for said modification of being incapable of producing endogenous immunoglobulin light and heavy chains and also hemizygous for the modification of being capable of producing xenogeneic immunoglobulin heavy chain;
a first parent hemizygous for a genome modified to be capable of producing xenogeneic immunoglobulin light chain, a second parent heterozygous for a genome modified to be incapable of producing immunoglobulin heavy and light chains, and progeny heterozygous for said genome modified to be incapable of producing endogenous immunoglobulin light and heavy chains and hemizygous for said genome modified to be capable of producing xenogeneic immunoglobulin light chain;
first and second parents heterozygous for a genome modified to be incapable of producing endogenous immunoglobulin light and heavy chains and hemizygous for a genome modified to be capable of producing xenogeneic immunoglobulin light chain, and progeny
1) homozygous for said modified genome and
2) homozygous for said modification of being incapable of producing endogenous immunoglobulin light and heavy chains and also hemizygous for the modification of being capable of producing xenogeneic immunoglobulin light chain;
a first parent heterozygous for a genome modified to be incapable of producing endogenous immunoglobulin light and heavy chains and hemizygous for xenogeneic immunoglobulin heavy chain, a second parent heterozygous for a genome modified to be incapable of producing endogenous immunoglobulin light and heavy chains and hemizygous for the modification of being capable of producing xenogeneic immunogobulin light chain, and progeny homozygous and heterozygous for said genome modified to be incapable of producing endogenous immunoglobulin light and heavy chains and hemizygous for the modification of being capable of producing xenogeneic immunoglobulin light and heavy chains, first and second parents heterozygous for a genome modified to be incapable of producing endogenous immunoglobulin light and heavy chains and hemizgyous for a genome modified to be capable of producing xenogeneic immunoglobulin light and heavy chains, and progeny
1) homozygous for said modified genome, and
2) homozygous for a genome modified to be incapable of producing endogenous immunoglobulin heavy and light chains and hemizygyous for a genome modified to be capable of producing xenogeneic immunoglobulin light and heavy chains;
first and second parents homozygous for a genome modified to be incapable of producing endogenous immunoglobulin light and heavy chains and hemizygous for a genome modified to be capable of producing xenogeneic immunoglobulin light and heavy chains, and progeny homozygous for said modified genome;
a first parent heterozygous for a genome modified to be incapable of producing endogenous immunoglobulin heavy chain, a second parent hemizygyous for a genome modified to be capable of producing xenogeneic immunoglobulin light and heavy chains, and progeny heterozygous for a genome modified to be incapable of producing endogenous immunoglobulin heavy chain and hemizygyous for a genome modified to be capable of producing xenogeneic immunoglobulin light and heavy chains;
first and second parents heterozygous for a genome modified to be incapable of producing endogenous immunoglobulin heavy chain and hemizygous for a genome modified to be capable of producing xenogeneic immunglobulin light and heavy chains, and progeny
1) homozygous for said modified genome and
2) homozygous for a genome modified to be incapable of producing endogenous immunoglobulin heavy chain and hemizygous for a genome modified to be capable of producing xenogeneic immunoglobulin light and heavy chains;
a first parent heterozygous for a genome modified to be incapable of producing endogenous immunoglobulin light chain, a second parent hemizygyous for a genome modified to be capable of producing xenogeneic immunoglobulin light and heavy chains, and progeny heterozygous for a genome modified to be incapable of producing endogenous immunoglobulin light chain and hemizygyous for a genome modified to be capable of producing xenogeneic immunoglboulin light and heavy chain; and
first and second parents heterozygous for a genome modified to be incapable of producing endogenous immunoglobulin light chain and hemizgyous for a genome modified to be capable of producing xenogeneic immunoglobulin light and heavy chains, and progeny
1) homozygous for said modified genome, and
2) homozygous for a genome modified to be incapable of producing endogenous immunoglobulin light chain and hemizygous for a genome modified to be capable of producing xenogeneic immunoglobulin light and heavy chains. - View Dependent Claims (73, 74, 75, 76, 77, 78)
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Specification