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Last Updated: March 29, 2024

Claims for Patent: 7,741,077


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Summary for Patent: 7,741,077
Title:Method for the generation of genetically modified vertebrate precursor lymphocytes and use thereof for the production of heterologous binding proteins
Abstract: The present invention generally relates to the fields of genetic engineering and antibody production. In particular, it relates to the generation of genetically modified vertebrate precursor lymphocytes that have the potential to differentiate into more mature lymphoid lineage cells, and to the use thereof for the production of any heterologous antibody or binding protein.
Inventor(s): Grawunder; Ulf (Basel, CH), Melchers; Georg Friedrich (Grenzach-Wyhlen, DE)
Assignee: 4-Antibody AG (Basel, CH)
Application Number:10/499,631
Patent Claims:1. A method for the generation of vertebrate precursor B lymphocytes that can be used for the production of an antibody or an antigen-binding fragment(s) thereof, comprising: (a) genetically modifying isolated vertebrate precursor B lymphocytes, which (i) are derived from primary lymphoid organs, and (ii) have the potential to differentiate into mature B lymphoid lineage cells, by introducing at least one exogenous genetic element encoding at least one antibody or antigen-binding fragment(s) thereof; and (b) effecting differentiation of said genetically modified precursor B lymphocytes into mature lymphoid lineage cells either in vitro or in vivo, thereby generating B lymphocytes capable of producing an antibody or an antigen-binding fragment(s) thereof.

2. The method according to claim 1, further comprising the step of immortalizing the differentiated lymphocytes by: (a) fusing the differentiated lymphocytes to myeloma cells for the generation of hybridoma cells; (b) infecting the differentiated lymphocytes with transforming viruses; or (c) transfecting the differentiated lymphocytes with a vector construct ensuring expression of at least one transforming oncogene; thereby generating vertebrate precursor B lymphocytes capable of permanently producing said binding protein, or functional fragment(s) thereof.

3. The method according to claim 1, wherein the vertebrate precursor B lymphocytes are able to express at least one component of the lymphoid V(D)J recombination machinery and originate from jawed vertebrates comprising cartilaginous fish, bony fish, amphibians, reptilia, birds, and mammals including pigs, sheep, cattle, horses and rodents including mice, rats, rabbits and guinea pigs.

4. The method of claim 3, wherein said lymphocytes are murine precursor (pre) B lymphocytes.

5. The method according to claim 1, wherein said vertebrate precursor B lymphocytes are deprived of their potential to express endogenous antibodies or antigen-binding fragment(s) thereof, which is achieved by isolating/selecting vertebrate precursor B lymphocytes being deficient in expressing endogenous immunoglobulins or fragments thereof, and/or by introducing into said vertebrate precursor B lymphocytes at least one vector construct designed to functionally inactivate at least one allele of at least one genetic element, which is selected from the group consisting of: (a) the coding regions of the immunoglobulin heavy chain gene locus, including all or parts of the V, D, and J gene segments, and any of the coding regions for the constant region exons for .mu., .delta., .gamma., .epsilon. and .alpha. heavy chains, with or without their membrane spanning exons; (b) the coding regions of the immunoglobulin K and/or X light chain gene loci, including any of the V and J gene segment coding regions, as well as any of the constant region exons; (c) the cis-acting immunoglobulin heavy chain gene locus enhancer elements, including the heavy chain intron enhancer and the 3'.alpha. enhancer; (d) the cis-acting immunoglobulin light chain gene locus enhancer elements, including the .kappa. light chain intron enhancer (.kappa.iE), the 3'.kappa. enhancer, and the .lamda.2-4 and .lamda.3-1 enhancers; (e) the trans-acting recombination activating genes, RAG-1 and RAG-2, including their promoter and enhancer elements, as well as their coding regions; and (f) the trans-acting DNA repair genes essential for V(D)J recombination, including Ku70, Ku86, the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), DNA ligase IV, XRCC4 and Artemis, including their promoter and enhancer elements, as well as the coding regions of said genes.

6. The method according to claim 5, wherein said vector constructs include gene targeting vectors comprising regions of DNA sequence homology to said at least one genetic element enabling for homologous recombination.

7. The method according to claim 6, wherein said gene targeting vectors additionally comprise a pair of DNA recognition sequences for site-specific DNA recombination enzymes, flanking the positive selection marker, enabling deletion of said positive selection marker upon transfection and transient expression of nucleic acid sequences encoding at least one of the cognate recombinase enzymes.

8. The method according to claim 6, wherein said gene targeting plasmid vectors additionally comprise a negative selection marker enabling selection against transfectants in which said gene targeting vectors are randomly integrated into the genome by non-homologous recombination.

9. The method according to claim 6, wherein said regions of DNA sequence homology flank a positive selection marker enabling selection of positive transfectants.

10. The method according to claim 1, wherein said at least one exogenous genetic element encoding an antibody or functional fragment(s) thereof, is carried on a genetic construct selected from the group consisting of: (a) recombinant retroviral DNA constructs comprising promoter, enhancer and coding nucleic acid sequences operably linked to allow expression of at least one antibody, or antigen-binding fragment thereof, being either wild-type or having (a) designed mutation(s) in the primary amino acid sequence(s) or being artificial; (b) recombinant plasmid-based DNA constructs comprising promoter, enhancer and coding nucleic acid sequences operably linked to allow expression of at least one antibody, or antigen-binding fragment thereof, being either wild-type or having (a) designed mutation(s) in the primary amino acid sequence(s) or being artificial; (c) recombinant plasmid-based mini-immunoglobulin gene loci with unrearranged V, D and J gene segments operably linked to allow V(D)J recombination and subsequent expression of at least one heterologous antibody, or antigen-binding fragment thereof, being either wild-type or having (a) designed mutation(s) in the primary amino acid sequence(s); (d) bacterial, yeast or vertebrate artificial chromosomes comprising parts or all of immunoglobulin gene loci in germline configuration operably linked to allow V(D)J recombination and subsequent expression of at least one heterologous antibody or antigen-binding fragment thereof, being either wild-type or having (a) designed mutation(s) in the primary amino acid sequence(s); (e) bacterial, yeast or vertebrate artificial chromosomes comprising parts or all of at least one heterologous immunoglobulin gene locus in modified arrangement designed to allow V(D)J recombination and subsequent expression of at least one heterologous antibody, or antigen-binding fragment thereof, being either wild-type or having (a) designed mutation(s) in the primary amino acid sequence(s); and (f) trans-chromosome elements which are fragments of heterologous chromosomes harboring parts or all of immunoglobulin gene loci in germline configuration allowing V(D)J recombination and subsequent expression of at least one heterologous antibody, being wild-type with respect to the primary amino acid sequence(s).

11. The method according to claim 1, wherein the at least one exogenous genetic element encodes a native or modified human antibody, or (an) antigen-binding fragment(s) thereof.

12. The method according to claim 1 wherein the at least one exogenous genetic element encodes a heterologous antibody or heterologous antigen-binding fragment thereof.

13. The method according to claim 1, wherein the differentiation of vertebrate precursor B lymphocytes is effected in vitro by: (a) arresting proliferation of said vertebrate precursor B lymphocytes and inducing differentiation into mature lymphocyte lineage cells by cultivating in the absence of any precursor lymphocyte growth factor; and (b) inducing terminal lymphocyte differentiation by further cultivating said cells in the presence of at least one of the following components selected from: (i) soluble T cell related stimulating factors, comprising interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin-10, interleukin-13, TGF-.beta., and IFN-.gamma.; (ii) factors activating co-stimulatory receptors of B cells, comprising agonistic antibodies or active, recombinant ligands specific for CD40, B7-1 (CD80), B7-2 (CD86), complement receptors 1 (CD35) and 2 (CD21), LFA-1 (CD11a), LFA-3 (CD58), CD19, CD20, CD30, CD32, CD37, CD38, CD70, CD71, Ig.alpha. (CD79.alpha.), Ig.beta. (CD79.beta.), TAPA-1 (CD81), Fas (CD95), TNF-receptor1 (p55, CD120a), TNF-receptor2 (p75, CD120b), Ox-40 (CD134), and lymphotoxin-.beta. receptor; and (iii) B cell mitogenic factors, T cell independent antigens of type 1, and other polyclonal activators, including lipopolysaccharide (LPS), lipoproteins from gram negative bacteria, polyanions, poly-dIdC, pokeweed mitogen (PWM), and anti-immunoglobulin reagents; and combinations thereof.

14. The method according to claim 1, wherein said differentiation in vivo is effected upon transplantation of said genetically modified precursor B lymphocytes into a suitable vertebrate host.

15. The method according to claim 14, wherein said lymphocytes are co-transplanted into said host with naive or antigen primed T helper lymphocytes.

16. The method according to claim 14, wherein said differentiation in vivo is followed by immunization of said host with at least one desired immunogenic compound or composition.

17. The method according to claim 14, wherein said vertebrate host is a compatible host being deficient with respect to the generation of endogenous B cells, T cells, and/or NK (natural killer) cells, or a combination thereof.

18. The method according to claim 14, wherein the vertebrate host is selected from jawed vertebrates comprising cartilaginous fish, bony fish, amphibians, reptilia, birds, and mammals including pigs, sheep, cattle, horses and rodents including mice, rats, rabbits and guinea pigs.

19. The method according to claim 14, wherein said vertebrate host is mouse.

20. A method for the production of an antibody, antibodies or (an) antigen-binding fragment(s) thereof, comprising: (a) genetically modifying vertebrate precursor B lymphocytes, which (i) are isolated from primary lymphoid organs, and (ii) have the potential to differentiate into mature B lymphoid lineage cells, by introducing at least one exogenous genetic element encoding at least one antibody or antigen-binding fragment(s) thereof; or, alternatively, isolating genetically modified vertebrate precursor B lymphocytes from primary lymphoid organs, wherein said genetically modified vertebrate precursor B lymphocytes have the potential to differentiate into mature B lymphoid lineage cells, and carry at least one exogenous genetic element encoding at least one antibody or antigen-binding fragment thereof; (b) effecting differentiation of said genetically modified precursor B lymphocytes into mature lymphoid lineage cells either in vitro or in vivo, thereby generating genetically modified and differentiated vertebrate B lymphocytes capable of producing an antibody or (an) antigen-binding fragment(s) thereof; (c) effecting expression of the antibodies, antibody, or antigen-binding fragment(s) thereof.

21. The method according to claim 20, followed by the steps of: (a) isolating from said differentiated lymphocytes the at least one exogenous genetic element, and (b) placing said genetic element(s) in an expression system enabling production of said at least one binding protein, or functional fragment(s) thereof.

22. The method according to claim 20, wherein a monoclonal antibody is produced.

23. The method according to claim 20, wherein polyclonal antibodies are produced.

24. The method of according to claim 20 wherein said antibodies, antibody or antigen-binding fragment(s) are at least partially human.

25. The method according to claim 20, wherein the antibodies, antibody, or antigen-binding fragment(s) thereof to be produced are selected from the group consisting of: (a) antibodies being either membrane bound or secreted, and consisting of both heterologous heavy and light chain polypeptides in the stochiometric composition found in natural antibodies and consisting of any of the known heavy (.mu., .delta., .gamma., .alpha., .epsilon.) and/or light (.kappa. and .lamda.) chain isotypes; (b) antibodies with combinations of heavy and light chain polypeptides being completely human with respect to the primary amino acid sequence; (c) hybrid antibodies containing heterologous heavy or light chain polypeptides from different vertebrate species; (d) secreted Fab, scFv and F(ab')2 antibody fragments being either completely or partially heterologous; and (e) antigen-binding fragments of antibodies covalently coupled via linker peptides, resulting in bispecific or multispecific antibody fragments.

Details for Patent 7,741,077

Applicant Tradename Biologic Ingredient Dosage Form BLA Approval Date Patent No. Expiredate
Merck Sharp & Dohme Corp. INTRON A interferon alfa-2b For Injection 103132 06/04/1986 ⤷  Try a Trial 2039-03-29
Merck Sharp & Dohme Corp. INTRON A interferon alfa-2b For Injection 103132 ⤷  Try a Trial 2039-03-29
Merck Sharp & Dohme Corp. INTRON A interferon alfa-2b Injection 103132 ⤷  Try a Trial 2039-03-29
>Applicant >Tradename >Biologic Ingredient >Dosage Form >BLA >Approval Date >Patent No. >Expiredate

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