Transgenic animals for producing specific isotypes of human antibodies via non-cognate switch regions

• US 7,049,426 B2
• Filed: 11/30/2001
• Issued: 05/23/2006
• Est. Priority Date: 06/10/1999
• Status: Expired due to Term

• Current Assignee: Abgenix Inc (Amgen Inc.)
• Original Assignee: Abgenix Inc (Amgen Inc.)
• Inventors: Green, Larry L., Davis, C. Geoffrey, Ivanov, Vladimir E.
• Primary Examiner(s): WEHBE, ANNE MARIE SABRINA
• Application Number:
  US09/999,321
• Publication Number:
  US 20030093820A1
• Time in Patent Office: 1,635 Days
• Field of Search: 536/23.1, 435/320.1, 435/23.53, 800/13, 800/18
• US Class Current: 536/23.53
• 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 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A01K 67/0276 Knock-out vertebrates
• CPC Class Codes: A01K 67/0278 Knock-in vertebrates, e.g. humanised vertebrates
• 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 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/241 Tumor Necrosis Factors
• CPC Class Codes: C07K 16/244 Interleukins [IL]
• CPC Class Codes: C07K 16/248 IL-6
• CPC Class Codes: C07K 16/2896 against molecules with a "CD"-designation, not provided for elsewhere
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 2510/00 : Genetically modified cells
• CPC Class Codes: C12N 2800/30 : Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• CPC Class Codes: C12N 2830/00 : Vector systems having a special element relevant for transcription
• CPC Class Codes: C12N 5/0606 : Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696

Abstract

The present invention provides fully human antibodies in a transgenic animal of a desired isotype in response to immunization with any virtually any desired antigen. The human immunoglobulin heavy chain transgene in the foregoing animals comprises a human constant region gene segment comprising exons encoding the desired heavy chain isotype, operably linked to switch segments from a constant region of a different heavy chain isotype, i.e., a non-cognate switch region. Said additional constant region segment comprises a switch region and human constant region coding segment, wherein the constant region coding segment is operably linked to a switch region that it is not normally associated with, i.e., a non-cognate switch region. In the transgenes of the invention, the non-cognate switch region may be a switch region from a different species than the constant region coding segment. The switch region and membrane exons of the invention may comprise a human gamma-2 constant region and the secreted constant region exons are from a human gamma-1 or a human gamma-4 constant region.

Claims

1. A transgene comprising a DNA fragment comprising a DNA sequence of a human D segment gene, a human J segment gene and a human constant region gene Cμ

, wherein said DNA fragment is operably linked to at least one human V segment gene, and wherein said DNA fragment further is operably linked to an additional constant region gene, said additional constant region gene comprising a human switch region, human CH1, C_hinge, CH2 and CH3 exons and human membrane exons, wherein said human switch region and said human membrane exons are from the same isotype and the human CH1, C_hinge, CH2 and CH3 exons are from a different isotype than said human switch region and said human membrane exons.

2. The transgene according to claim 1, wherein said human switch region and said human membrane exons are human gamma-2 sequences.

3. The transgene according to claim 2, wherein said human CH1, C_hinge, CH2 and CH3 exons encode a human constant region selected from the group consisting of:

a human gamma-1constant region, a human gamma-3 constant region, a human gamma-4 constant region, a human alpha-1 constant region, a human alpha-2 constant region and a human epsilon constant region.

4. The transgene according to claim 3, wherein the human CH1, C_hinge, CH2 and CH3 exons encode a human gamma-1 constant region.

5. The transgene according to claim 3, wherein the human CH1, C_hinge, CH2 and CH3 exons encode a human gamma-4 constant region.

6. The transgene according to any one of claims 1 and 15, wherein said DNA fragment is operably linked to a plurality of human VH genes.

7. The transgene according to any one of claims 1 and 15, wherein said DNA fragment is operably linked to at least 50% of the human germline VH genes.

8. The transgene according to any one of claims 1 and 15, wherein said DNA fragment is operably linked to at least 40 different human VH genes.

9. The transgene according to any one of claims 1 and 15, wherein said DNA fragment is operably linked to a sufficient number of different human VH genes so that the transgene is capable of encoding at least 1×

10⁵ different functional human immunoglobulin heavy chain sequences through V(D)J recombination, without taking into account junctional diversity or somatic mutation events.

10. The transgene according to any one of claims 1 and 15, wherein the number of human VH genes is sufficient to produce at least 50% of the B-cell population of a wild-type mouse in a transgenic mouse containing the transgene.

11. The transgene according to any one of claims 1 and 15, further comprising a mouse 3′

enhancer.

12. The transgene according to claim 11, wherein said mouse 3′

enhancer is an approximately 0.9 kb core fragment of a mouse germline 3′

enhancer.

13. The transgene according to claim 11, wherein said mouse 3′

enhancer is an approximately 4 kb fragment of a mouse germline 3′

enhancer.

14. The transgene according to claim 11, wherein said mouse 3′

enhancer is a locus control region.

16. The transgene according to any one of claims 1, and 15, wherein a loxP site is inserted 3′

of the switch region and 5′

of the CH1 exon.

15. A transgene comprising a DNA fragment comprising a DNA sequence of a human D segment gene, a human J segment gene and a human constant region gene Cμ

, wherein said DNA fragment is operably linked to at least one human V segment gene, and wherein said DNA fragment further is operably linked to an additional constant region gene, said additional constant region gene comprising;

(1) a human switch region;

(2) a region comprising human CH1, C_hinge, CH2 and CH3 exons; and

(3) human membrane exons, wherein said human switch region, said region comprising human CH1, C_hinge, CH2 and CH3 exons, and said human membrane exons are from three different isotypes.