CD20-specific redirected T cells and their use in cellular immunotherapy of CD20+ malignancies

• US 6,410,319 B1
• Filed: 10/20/1999
• Issued: 06/25/2002
• Est. Priority Date: 10/20/1998
• Status: Expired due to Term

• Current Assignee: City of Hope
• Original Assignee: City of Hope
• Inventors: Raubitschek, Andrew, Jensen, Michael C., Wu, Anna
• Primary Examiner(s): Ketter, James
• Assistant Examiner(s): LI, QIAN JANICE
• Application Number:
  US09/421,295
• Time in Patent Office: 979 Days
• Field of Search: 435/372, 435/347.2, 435/375, 435/69.1, 435/320.1, 435/455, 435/325, 435/326, 435/328, 435/332, 435/343, 435/343.1, 435/344, 536/23.4
• US Class Current: 435/343.1
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 39/4611 T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
• CPC Class Codes: A61K 39/4632 T-cell receptors [TCR]; antibody T-cell receptor constructs
• CPC Class Codes: A61K 39/464424 CD20
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 14/7051 T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 16/2887 against CD20
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 2502/30 tumour cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0636 T lymphocytes

Abstract

Genetically engineered, CD20-specific redirected T cells expressing a cell surface protein-having an extracellular domain comprising a receptor which is specific for CD20, an intracellular signaling domain, and a transmembrane domain. Use of such cells for cellular immunotherapy of CD20⁺ malignancies and for abrogating any untoward B cell function. In one embodiment, the cell surface protein is a single chain FvFc:ζ receptor where Fv designates the V_H and V_L chains of a single chain monoclonal antibody to CD20 linked by peptide, Fc represents a hinge-CH₂-CH₃ region of a human IgG₁, and ζ represents the intracellular signaling domain of the zeta chain of human CD3. A method of making a redirected T cell expressing a chimeric T cell receptor by electroporation using naked DNA encoding the receptor.

Claims

1. A DNA construct encoding a CD20-specific chimeric T cell receptor wherein the receptor consists of an intracellular signaling domain selected from the group consisting of the human CD3 zeta chain, Fcγ

RIII and Fcε

RI, a transmembrane domain and an extracellular domain, the extracellular domain comprising a CD20-specific receptor.

2. A plasmid expression vector containing a DNA construct of claim 1 in proper orientation for expression.

3. The DNA construct of claim 1 wherein the CD20-specific receptor comprises the Fv region of a single chain monoclonal antibody to CD20.

4. The DNA construct of claim 3 wherein the intracellular signaling domain is the intracellular signaling domain of the zeta chain of human CD3.

5. The DNA construct of claim 4 wherein the CD20-specific chimeric receptor comprises scFvFc:

ζ

, wherein scFv designates the V_H and V_L chains of a single chain monoclonal antibody to CD20, Fc represents at least part of a constant region of an IgG₁, and ζ

represents the intracellular signaling domain of the zeta chain of human CD3.

6. The DNA construct of claim 5 wherein the extracellular domain scFvFc and the intracellular signaling domain ζ

are linked by the transmembrane domain of human CD4.

7. The DNA construct of claim 6 wherein the chimeric receptor is amino acids 21-633 of Seq. ID No. 2.

8. A plasmid expression vector containing the DNA construct of any of claims 3-7.

9. Genetically engineered CD20-specific T cells which express and bear on the cell surface membrane a CD20-specific chimeric receptor consisting of an intracellular signaling domain selected from the group consisting of the human CD3 zeta chain, Fcγ

RIII and Fcε

RI, a transmembrane domain and an extracellular domain, the extracellular domain comprising a CD20-specific receptor.

10. CD20-specific T cells of claim 9 which are non-malignant human cells.

11. CD20-specific T cells of claim 10 which are CD4³⁰ and which produce IL-2 when co-cultured in vitro with CD20⁺ lymphoma cells.

12. CD20-specific T cells of claim 10 which are CD8⁺ and which lyse CD20⁺ lymphoma target cells when co-cultured in vitro with the target cells.

13. CD20-specific T cells of claim 10 which comprise a mixed population of CD4⁺ and CD8⁺ cells.

14. CD20-specific T cells of claim 10 wherein the CD20-specific receptor comprises the V_H and V_L chains of a single chain monoclonal antibody (scFv) to CD20.

15. CD20-specific T cells of claim 14 wherein the intracellular signaling domain is the intracellular signaling domain of the zeta chain of human CD3.

16. CD20-specific T cells of claim 15 wherein the CD20-specific chimeric receptor comprises scFvFc:

ζ

, wherein scFv designates the V_H and V_L chains of a single chain monoclonal antibody to CD20, Fc represents at least part of a constant region of an IgG₁, and ζ

represents the intracellular signaling domain of the zeta chain of human CD3.

17. CD20-specific T cells of claim 16 wherein the extracellular domain scFvFc and the intracellular signaling domain ζ

are linked by the transmembrane domain of human CD4.

18. CD20-specific T cells of claim 17 wherein the chimeric receptor is amino acids 21-633 of Seq. ID No. 2.

19. A method of making and expanding the CD20-specific T cells of claim 9 which comprises transfecting T cells with an expression vector containing a DNA construct encoding the CD20-specific chimeric receptor, then stimulating the cells with CD20⁺ cells, recombinant CD20, or an antibody to the receptor to cause the cells to proliferate.

20. The method of claim 19 wherein the transfected T-cells are cloned and a clone demonstrating presence of a single integrated unrearranged plasmid and expression of the chimeric receptor is expanded ex vivo.

21. The method of claim 20 wherein the clone selected for ex vivo expansion is CD8⁺ and demonstrates the capacity to specifically recognize and lyse CD20⁺ target cells.

22. The method of claim 21 wherein the chimeric receptor is amino acids 21-633 of Seq. ID No. 2.