Immunoglobulin compositions and methods
First Claim
1. A vector for reverting cell lines to a pro-B cell-like state or to a germline-like state, said vector comprising one or more immunoglobulin regions selected from the group consisting of V regions, D regions, and J regions, a 5′
- flanking region operably linked 5′
to a 5′
-most region and a 3′
flanking region operably linked 3′
to a 3′
-most region wherein said 5′ and
3′
flanking regions are capable of facilitating homologous recombination of said immunoglobulin regions into a cell having a V(D)J rearranged immunoglobulin gene.
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Accused Products
Abstract
Provided are methods for the preparation of immunoglobulins, including fully-human immunoglobulins, and immunoglobulins and compositions comprising immunoglobulins prepared by these methods. Also provided are libraries of immunoglobulins and immunoglobulin expressing cells as well as methods and vector constructs for preparation of these libraries. Immunoglobulins provided herein are useful inter alia as immunological and diagnostic agents and as therapeutic molecules in the treatment of diseases such as autoimmune diseases, heart disease, infections and cancers.
27 Citations
60 Claims
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1. A vector for reverting cell lines to a pro-B cell-like state or to a germline-like state, said vector comprising one or more immunoglobulin regions selected from the group consisting of V regions, D regions, and J regions, a 5′
- flanking region operably linked 5′
to a 5′
-most region and a 3′
flanking region operably linked 3′
to a 3′
-most region wherein said 5′ and
3′
flanking regions are capable of facilitating homologous recombination of said immunoglobulin regions into a cell having a V(D)J rearranged immunoglobulin gene. - View Dependent Claims (2, 4, 5, 6, 7, 8)
- flanking region operably linked 5′
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3. The vector of claim wherein said vector comprises two, three, four, five or more fused DJ regions.
- 9. A vector comprising a polynucleotide sequence encoding a recombination-promoting protein, or functional fragment, derivative, or variant thereof, selected from the group consisting of RAG-1 and RAG-2.
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14. A method for generating immunoglobulin heavy and/or light chains, said method comprising the steps of:
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(a) reverting a cell comprising a V(D)J rearranged immunoglobulin region by introducing into said cell a polynucleotide encoding V, D, and/or J regions of an immunoglobulin heavy and/or light chain, or fragment thereof, wherein said V, D, and/or J regions replace said V(D)J rearranged immunoglobulin region such that the introduced V, D, and/or J regions are in a pro-B cell-like or a germline-like state; and
(b) expressing in said reverted cell a polynucleotide sequence encoding a recombination-facilitating protein, or functional fragment, derivative or variant thereof, for a time and under conditions sufficient to induce rearrangement of the V, D, and/or J regions, wherein rearrangement of the V, D, and/or J regions facilitates expression of an immunoglobulin heavy and/or light chain. - View Dependent Claims (29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46)
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15. A method for generating immunoglobulin heavy chains, said method comprising the steps of:
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(a) reverting a cell comprising a V(D)J rearranged immunoglobulin region by introducing into said cell a polynucleotide encoding fused DJ regions of an immunoglobulin heavy chain, wherein said DJ regions replace said V(D)J rearranged immunoglobulin region such that the introduced fused DJ regions are in a pro-B cell-like state; and
(b) expressing in the reverted cell a polynucleotide sequence encoding a recombination-facilitating protein, or functional fragment thereof, for a time and under conditions sufficient to induce rearrangement of the germline V regions in the reverted cell with the introduced fused DJ regions, wherein rearrangement of the V and fused DJ regions facilitates expression of an immunoglobulin heavy chain. - View Dependent Claims (47, 48, 49, 50, 51, 52)
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16. A method for generating immunoglobulin light chains, said method comprising the steps of:
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(a) reverting a cell comprising a V(D)J rearranged immunoglobulin region by introducing into said cell a polynucleotide encoding J regions of an immunoglobulin light chain, wherein said J regions replace said V(D)J rearranged immunoglobulin region such that the introduced J regions are in a pro-B cell-like or a germline-like state; and
(b) expressing in the reverted cell a polynucleotide sequence encoding a recombination-facilitating protein, or functional fragment thereof, for a time and under conditions sufficient to induce rearrangement of the germline V regions in the reverted cell with the introduced J regions, wherein rearrangement of the V and J regions facilitates expression of an immunoglobulin light chain.
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17. A method for generating libraries of cells that produce an array of immunoglobulins wherein each immunoglobulin exhibits a particular antigen specificity, said method comprising the steps of:
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(a) providing a cell having a V(D)J rearranged immunoglobulin heavy and/or light chain region;
(b) introducing into the cell a polynucleotide encoding V, D, and/or J regions of an immunoglobulin heavy and/or light chain, or fragment thereof, wherein said V, D, and/or J regions replace said V(D)J rearranged immunoglobulin heavy and/or light chain region such that the introduced V, D, and/or J regions are in a pro-B cell-like or a germline-like state;
(c) culturing the cell under suitable conditions to generate a reverted cell population each member of which population comprises V, D, and/or J regions in a pro-B cell-like or a germline-like state;
(d) introducing into cells of the reverted cell population a polynucleotide sequence encoding a recombination-facilitating protein, or functional fragment, derivative or variant thereof; and
(e) culturing the resulting population of cells expressing the recombination-facilitating protein for a time and under conditions sufficient to induce rearrangement of the pro-B cell-like or germline-like V, D, and/or J regions, wherein rearrangement of the V, D, and/or J regions facilitates expression of an immunoglobulin heavy and/or light chain having a particular antigen specificity.
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18. A method for identifying in a cell an immunoglobulin having a desired antigen specificity, said method comprising the steps of:
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(a) reverting a cell comprising a V(D)J rearranged immunoglobulin region by introducing into said cell a polynucleotide encoding V, D, and/or J regions of an immunoglobulin heavy and/or light chain, or fragment thereof, wherein said V, D, and/or J regions replace said V(D)J rearranged immunoglobulin region such that the introduced V, D, and/or J regions are in a pro-B cell-like or a germline-like state;
(b) expressing in the reverted cell a polynucleotide sequence encoding a recombination-facilitating protein, or functional fragment, derivative, or variant thereof, for a time and under conditions sufficient to induce rearrangement of the pro-B cell-like or germline-like V, D, and/or J regions, wherein rearrangement of the V, D, and/or J regions facilitates expression of an immunoglobulin heavy and/or light chain; and
(c) screening the resulting V, D, and/or J region rearranged cells for an immunoglobulin having the desired antigen specificity. - View Dependent Claims (53, 54, 55, 56, 57, 58, 59, 60)
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19. A method for generating cell lines capable of producing immunoglobulins having a desired specificity, said method comprising the step of reverting a cell comprising a V(D)J rearranged immunoglobulin region by introducing into said cell a polynucleotide encoding V, D, a nd/or J regions of an immunoglobulin heavy and/or light chain gene, or fragment thereof, wherein said V, D, and/or J regions replace said V(D)J rearranged immunoglobulin region such that the introduced V, D, and/or J regions are in a pro-B cell-like or a germline-like state.
- 20. A method for generating cell lines capable of producing immunoglobulins having a desired specificity, said method comprising the step of reverting a cell comprising a V(D)J rearranged immunoglobulin region by introducing into said cell a polynucleotide encoding one or more fused DJ regions of an immunoglobulin heavy chain, wherein said fused DJ regions replace said V(D)J rearranged immunoglobulin region such that the introduced fused DJ regions are in a pro-B cell-like state.
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24. A method for producing immunoglobulins having a particular affinity or specificity for a target molecule, said method comprising the steps of:
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(a) generating a reverted lymphoma cell line capable of producing immunoglobulins;
(b) expressing a polynucleotide encoding a recombination-facilitating protein, or functional fragment, derivative, or variant thereof, in the reverted lymphoma cell line for a time and under conditions sufficient to induce a rearrangement of the genes encoding the immunoglobulins which facilitates the generation of a library of lymphoma cells which produce an array of immunoglobulins wherein each immunoglobulin exhibits a particular affinity or specificity; and
(b) screening for immunoglobulins having a desired affinity or specificity.
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25. A method for producing libraries of human monoclonal antibody-producing lymphoma cells, said method comprising the step of expressing in a reverted human lymphoma cell a polynucleotide encoding a recombination-facilitating protein for a time and under conditions sufficient to induce rearrangement of genes encoding human antibodies in the reverted lymphoma cells.
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26. A method for generating a library of lymphoma cell lines of human origin, each human lymphoma cell line capable of producing fully-human immunoglobulins of a particular specificity, said method comprising the steps of:
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(a) reverting a human lymphoma cell line comprising a V(D)J rearranged immunoglobulin region by introducing a polynucleotide encoding human V, D, and/or J regions of human immunoglobulin heavy and/or light chains into said human lymphoma cell line, wherein said V, D, and/or J regions replace said V(D)J rearranged immunoglobulin region such that the introduced V, D, and/or J regions are in a pro-B cell-like or germline-like state; and
(b) expressing in the reverted human lymphoma cell a polynucleotide sequence encoding a human recombination-facilitating protein, or a functional fragment, derivative or variant thereof, for a time and under conditions sufficient to facilitate rearrangement of immunoglobulin-encoding genes thus generating a library of human lymphoma cells each producing a fully-human immunoglobulin of a particular specificity.
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27. A method for generating a library of lymphoma cell lines of human origin, each human lymphoma cell line capable of producing fully-human immunoglobulin heavy chains of a particular specificity, said method comprising the steps of:
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(a) reverting a human lymphoma cell line comprising a V(D)J rearranged immunoglobulin region in by introducing a polynucleotide encoding fused DJ regions of a human immunoglobulin heavy chain into said cell line, wherein said DJ regions replace said V(D)J rearranged immunoglobulin region such that the introduced fused DJ regions are in a pro-B cell-like state;
(b) expressing in the reverted human cell a polynucleotide sequence encoding a human recombination-facilitating protein, or functional fragment, derivative or variant thereof, for a time and under conditions sufficient to induce rearrangement of the human lymphoma cell line germline V regions with the introduced fused DJ regions, wherein rearrangement of the V and fused DJ regions facilitates expression of a fully-human immunoglobulin heavy chain.
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28. A method for generating a library of lymphoma cell lines of human origin, each human lymphoma cell line capable of producing fully-human immunoglobulin light chains of a particular specificity, said method comprising the steps of:
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(a) reverting a human lymphoma cell comprising a V(D)J rearranged immunoglobulin region in by introducing a polynucleotide encoding J regions of a human immunoglobulin light chain into said human lymphoma cell, wherein said J regions replace said V(D)J rearranged immunoglobulin region such that the introduced J regions are in a pro-B cell-like or a germline-like state;
(b) expressing in the reverted human lymphoma cell line a polynucleotide sequence encoding a human recombination-facilitating protein, or functional fragment, derivative or variant thereof, for a time and under conditions sufficient to induce rearrangement of the germline V regions with the introduced J regions, wherein rearrangement of the V and J regions facilitates expression of a fully-human immunoglobulin light chain.
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Specification