Chimeric chains for receptor-associated signal transduction pathways

• US 5,359,046 A
• Filed: 12/09/1992
• Issued: 10/25/1994
• Est. Priority Date: 12/14/1990
• Status: Expired due to Fees

• Current Assignee: Cell Genesys Incorporated (ANI Pharmaceuticals, Inc.), Regents of the University of California (University of California)
• Original Assignee: Cell Genesys Incorporated (ANI Pharmaceuticals, Inc.), Regents of the University of California (University of California)
• Inventors: Zsebo, Krisztina, Capon, Daniel J., Weiss, Arthur, Roberts, Margo R., Irving, Brian A.
• Primary Examiner(s): ZISKA, SUZANNE E
• Assistant Examiner(s): ZISKA, SUZANNE E
• Application Number:
  US07/988,194
• Time in Patent Office: 685 Days
• Field of Search: 435/6, 435/69.1, 435/69.5, 435/69.7, 435/240.2, 435/240.1, 435/320.2, 435/235, 435/7, 435/70.21, 435/70.2, 536/23.1-23.53, 530/350, 530/351, 530/387, 530/388, 424/85.1-85.8, 424/93, 514/2, 514/8, 514/12
• US Class Current: 536/23.4
• CPC Class Codes: C07K 14/7051 T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 14/70514 CD4
• CPC Class Codes: C07K 14/70535 Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/32 fusions with soluble part of a cell surface receptor, "decoy receptors"

Abstract

Chimeric proteins and DNA sequence encoding chimeric proteins are provided, where the chimeric proteins are characterized by an extracellular domain capable of binding to a ligand in a non-MHC restricted manner, a transmembrane domain and a cytoplasmic domain capable of activating a signaling pathway. The extracellular domain and cytoplasmic domain are not naturally found together. Binding of ligand to the extracellular domain results in transduction of a signal and activation of a signaling pathway in the cell, whereby the cell may be induced to carry out various functions relating to the signalling pathway. A wide variety of extracellular domains may be employed as receptors, where such domains may be naturally occurring or synthetic. The chimeric DNA sequences may be used to modify lymphocytes as well as hematopoietic stem cells as precursors to a number of important cell types.

Claims

1. Chimeric DNA sequence encoding a membrane bond protein, said chimeric DNA comprising in reading frame:

a DNA sequence encoding a signal sequence which directs the membrane bound protein to the surface membrane;

a DNA sequence encoding a non-MHC restricted extracellular binding domain of a surface membrane protein selected from the group consisting of CD4, CD8, immunoglobulin and single-chain antibody that binds specifically to at least one ligand, wherein said ligand is a protein on the surface of a cell or a viral protein;

a transmbembrane domain from a protein selected from the group consisting of CD4, CD8, immunoglobulin, single-chain antibody, the CD3 zeta chain, the CD3 gamma chain, the CD3 delta chain and the CD3 epsilon chain; and

a cytoplasmic signal-transducing domain of a protein that activates an intracellular messenger system selected from the group consisting of the CD3 zeta chain, the CD3 gamma chain, the CD3 delta chain and the CD3 epsilon chain,

wherein said extracellular domain and cytoplasmic domain are not naturally joined together and said cytoplasmic domain is not naturally joined to an extracellular ligand-binding domain, and when said chimeric DNA is expressed as a membrane bound protein in a selected host cell under conditions suitable for expression, said membrane bond protein initiates signalling in said host cell.

2. DNA according to claim 1 wherein said extracellular domain is the heavy chain of an immunoglobulin, by itself or in conjunction with a light chain, or truncated portioins of said heavy chain and said light chain containing ligand binding activity.

3. DNA according to claim 1 wherein said extracellular domain in CD8.

4. DNA according to claim 1 wherein said extracellular domain is CD4.

5. DNA according to claim 4 wherein said extracellular domain is a single-chain antibody, or portion thereof containing ligand binding activity.

6. A DNA sequence according to claim 5, wherein said single-chain antibody recognizes an antigen selected from the group consisting of viral antigens and tumor cell associated antigens.

7. A DNA sequence according to claim 6 wherein said single-chain antibody is specific for the HIV env glycoprotein.

8. A DNA sequence according to claim 3, 4 or 7 where said cytoplasmic domain is zeta.

9. A DNA sequence according to claim 1, wherein said transmembrane domain is naturally joined to said extracellular domain.

10. A DNA sequence according to claim 1, wherein said transmembrane domain is naturally joined to said cytoplasmic domain.

11. An expression cassette comprising a transcriptional initiation region, a DNA sequence according to claim 1 under the transcriptional control of said transcriptional initiation region, and a transcriptional termination region.

12. An expression cassette according to claim 9, wherein said transcriptional initiation region is functional in a mammalian host.

13. A retroviral RNA or DNA construct comprising an expression cassette according to claim 12.

14. A cell comprising a DNA sequence according to claim 1.

15. A cell according to claim 14, wherein said cytoplasmic domain is the CD3 zeta chain.

16. A cell according to claim 15, wherein said extracellular domain is the heavy chain of an immunogloboulin itself or in conjunction with a light chain, or truncated portions thereof containing ligan finding activity

17. A cell according to claim 15 wherein said extracellular domain is CD8.

18. A cell according to claim 15, wherein said extracellular domain is CD4.

19. A cell according to claim 14, wherein said cell is a mammalian cell.

20. A cell according to claim 19, wherein said mammalian cell is a human cell.

21. A cell according to claim 14 wherein said cell is a hematopoietic stem cell.