Transgenic mammals having human Ig loci including plural Vh and Vk regions and antibodies produced therefrom

• US 20030070185A1
• Filed: 02/19/2002
• Published: 04/10/2003
• Est. Priority Date: 12/03/1996
• Status: Active Grant

• Current Assignee: Aya Jakobovits, Larry Green, Michael J. Mendez, Raju Kucherlapati, Susan Klapholz
• Original Assignee: Aya Jakobovits, Larry Green, Michael J. Mendez, Raju Kucherlapati, Susan Klapholz
• Inventors: Jakobovits, Aya, Green, Larry, Kucherlapati, Raju, Mendez, Michael J., Klapholz, Susan
• Granted Patent: US 7,064,244 B2
• Time in Patent Office: Days
• US Class Current: 800/14
• CPC Class Codes: A01K 2207/15 Humanized animals
• CPC Class Codes: A01K 2217/00 Genetically modified animals
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2217/072 maintaining or altering function, i.e. knock in
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• CPC Class Codes: A01K 67/0278 Knock-in vertebrates, e.g. humanised vertebrates
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: C07K 14/415 from plants
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/005 constructed by phage libraries
• CPC Class Codes: C07K 16/241 Tumor Necrosis Factors
• CPC Class Codes: C07K 16/244 Interleukins [IL]
• CPC Class Codes: C07K 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/24 : containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/73 : Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• CPC Class Codes: C07K 2317/92 : Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C12N 15/8509 : for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 2800/206 : of yeast origin, e.g. YAC, 2u

Abstract

The present invention relates to transgenic non-human animals that are engineered to contain human immunoglobulin gene loci. In particular, animals in accordance with the invention possess human Ig loci that include plural variable (V_H and Vκ) gene regions. Advantageously, the inclusion of plural variable region genes enhances the specificity and diversity of human antibodies produced by the animal. Further, the inclusion of such regions enhances and reconstitutes B-cell development to the animals, such that the animals possess abundant mature B-cells secreting extremely high affinity antibodies.

Claims

1. A transgenic non-human mammal having a genome, the genome comprising modifications, the modifications comprising:

an inactivated endogenous immunoglobulin (Ig) locus, such that the mammal would not display normal B-cell development;

an inserted human heavy chain Ig locus in substantially germline configuration, the human heavy chain Ig locus comprising a human mu constant region and regulatory and switch sequences thereto, a plurality of human J_H genes, a plurality of human D_H genes, and a plurality of human V_H genes; and

an inserted human kappa light chain Ig locus in substantially germline configuration, the human kappa light chain Ig locus comprising a human kappa constant region, a plurality of Jκ

genes, and a plurality of Vκ

genes,

wherein the number of V_H and Vκ

genes inserted are selected to substantially restore normal B-cell development in the mammal.

2. The mammal of claim 1, wherein the heavy chain Ig locus comprises a second constant region selected from the group consisting of human gamma-1, human gamma-2, human gamma-3, human gamma-4, alpha, epsilon, and delta.

3. The mammal of claim 1, wherein the number of V_H genes is greater than about 20.

4. The mammal of claim 1, wherein the number of Vκ

genes is greater than about 15.

5. The mammal of claim 1, wherein the number of D_H genes is greater than about 25, the number of J_H genes is greater than about 4, the number of V_H genes is greater than about 20, the number of Jκ

genes is greater than about 4, and the number of Vκ

genes is greater than about 15.

6. The mammal of claim 1, wherein the number of D_H genes, the number of J_H genes, the number of V_H genes, the number of Jκ

genes, and the number of Vκ

genes are selected such that the Ig loci are capable of encoding greater than about 1×

10⁵ different functional antibody sequence combinations.

7. The mammal of claim 1, wherein in a population of mammals B-cell function is reconstituted on average to greater than about 50% as compared to wild type.

16. A method for the production of human antibodies, comprising:

inoculating a mammal of any one of claims 1-10 with an antigen;

collecting and immortalizing lymphocytic cells to obtain immortal cell lines secreting human antibodies that specifically bind to the antigen with an affinity of greater than 10⁹ M<sup>−

1</sup>; and

isolating the antibodies from the immortal cell lines.

8. In a transgenic non-human mammal having a genome that comprises modifications, the modifications rendering the mammal capable of producing human immunoglobulin molecules but substantially incapable of producing functional endogenous antibody molecules, the improvement comprising:

insertion into the genome of the mammal of sufficient human V_H, D_H, J_H, Vκ

, and Jκ

genes such that the mammal is capable encoding greater than about 1×

10⁶ different functional human immunoglobulin sequence combinations, without accounting for junctional diversity or somatic mutation events.

9. In a transgenic non-human mammal having a genome that comprises modifications, the modifications rendering the mammal capable of producing human immunoglobulin molecules but substantially incapable of producing functional endogenous antibody molecules, which modifications, with respect to the mammal'"'"'s incapacity to produce functional endogenous antibody molecules would not allow the mammal to display normal B-cell development, the improvement comprising:

insertion into the genome of the mammal of sufficient human V_H, D_H, J_H, Vκ

, and Jκ

genes such that the mammal is capable of encoding greater than about 1×

10⁶ different functional human immunoglobulin sequence combinations and sufficient V_H and Vκ

genes to substantially restore normal B-cell development in the mammal.

10. In the mammal of claim 9, wherein in a population of mammals B-cell function is reconstituted on average to greater than about 50% as compared to wild type.

11. A transgenic non-human mammal having a genome, the genome comprising modifications, the modifications comprising:

an inactivated endogenous heavy chain immunoglobulin (Ig) locus;

an inactivated endogenous kappa light chain Ig locus;

an inserted human heavy chain Ig locus, the human heavy chain Ig locus comprising a nucleotide sequence substantially corresponding to the nucleotide sequence of yH2; and

an inserted human kappa light chain Ig locus, the human kappa light chain Ig locus comprising a nucleotide sequence substantially corresponding to the nucleotide sequence of yK2.

17. The method of claim 11, wherein the antigen is IL-8.

18. The method of claim 11, wherein the antigen is EGFR.

19. The method of claim 11, wherein the antigen is TNF-α

.

20. An antibody produced by the method of claim 11.

25. Transgenic mice and transgenic offspring therefrom produced through use of the improvement of claim 19.

12. A transgenic non-human mammal having a genome, the genome comprising modifications, the modifications comprising:

an inactivated endogenous heavy chain immunoglobulin (Ig) locus;

an inserted human heavy chain Ig locus, the human heavy chain Ig locus comprising a nucleotide sequence substantially corresponding to the nucleotide sequence of yH2; and

an inserted human kappa light chain Ig locus, the human kappa light chain Ig locus comprising a nucleotide sequence substantially corresponding to the nucleotide sequence of yK2.

21. An anti-IL-8 antibody produced by the method of claim 12.

13. A transgenic non-human mammal having a genome, the genome comprising modifications, the modifications comprising:

an inactivated endogenous heavy chain immunoglobulin (Ig) locus;

an inactivated endogenous kappa light chain Ig locus;

an inserted human heavy chain Ig locus, the human heavy chain Ig locus comprising a nucleotide sequence substantially corresponding to the nucleotide sequence of yH2 without the presence of a human gamma-2 constant region; and

an inserted human kappa light chain Ig locus, the human kappa light chain Ig locus comprising a nucleotide sequence substantially corresponding to the nucleotide sequence of yK2.

22. An anti-EGFR antibody produced by the method of claim 13.

14. A transgenic non-human mammal having a genome, the genome comprising modifications, the modifications comprising:

an inactivated endogenous heavy chain immunoglobulin (Ig) locus;

an inactivated endogenous kappa light chain Ig locus;

an inserted human heavy chain Ig locus, the human heavy chain Ig locus comprising a nucleotide sequence substantially corresponding to the nucleotide sequence of yH2 without the presence of a human gamma-2 constant region; and

an inserted human kappa light chain Ig locus, the human kappa light chain Ig locus comprising a nucleotide sequence substantially corresponding to the nucleotide sequence of yK2.

23. An anti-TNF-α

antibody produced by the method of claim 14.

15. A transgenic non-human mammal having a genome, the genome comprising modifications, the modifications comprising:

an inactivated endogenous heavy chain immunoglobulin (Ig) locus;

an inserted human heavy chain Ig locus, the human heavy chain Ig locus comprising a nucleotide sequence substantially corresponding to the nucleotide sequence of yH2 without the presence of a human gamma-2 constant region; and

an inserted human kappa light chain Ig locus, the human kappa light chain Ig locus comprising a nucleotide sequence substantially corresponding to the nucleotide sequence of yK2.

24. In a method for the production of transgenic mice, the transgenic mice having a genome, the genome comprising modifications, the modifications comprising insertion of a plurality of human variable regions, the improvement comprising:

insertion of the human variable regions from a yeast artificial chromosome.

26. In a transgenic mammal, the transgenic mammal comprising a genome, the genome comprising modifications, the modifications comprising an inserted human heavy chain immunoglobulin transgene, the improvement comprising:

the transgene comprising selected sets of human variable region genes that enable human-like junctional diversity and human-like complementarity determining region 3 (CDR3) lengths.

27. In the improvement of claim 26, wherein the human-like junctional diversity comprises average N-addition lengths of 7.7 bases.

28. In the improvement of claim 26, wherein the human-like CDR3 lengths comprise between about 2 through about 25 residues with an average of about 14.