Compositions and methods for TCR reprogramming using fusion proteins

• US 10,358,474 B2
• Filed: 04/27/2018
• Issued: 07/23/2019
• Est. Priority Date: 05/18/2015
• Status: Active Grant

• Current Assignee: TCR2 Therapeutics Inc.
• Original Assignee: TCR2 Therapeutics Inc.
• Inventors: Baeuerle, Patrick, Sieczkiewicz, Gregory, Hofmeister, Robert
• Primary Examiner(s): Skelding, Zachary S
• Application Number:
  US15/965,739
• Publication Number:
  US 20180251514A1
• Time in Patent Office: 452 Days
• Field of Search: None
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 2039/5158 Antigen-pulsed cells, e.g. T-cells
• CPC Class Codes: A61K 2239/31 characterized by the route of administration
• CPC Class Codes: A61K 2239/38 characterised by the dose, timing or administration schedule
• CPC Class Codes: A61K 2239/48 Blood cells, e.g. leukemia or lymphoma
• CPC Class Codes: A61K 39/0011 Cancer antigens
• 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 39/4631 Chimeric Antigen Receptors [CAR]
• CPC Class Codes: A61K 39/4632 T-cell receptors [TCR]; antibody T-cell receptor constructs
• CPC Class Codes: A61K 39/464412 CD19 or B4
• CPC Class Codes: A61K 39/464417 Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/7051 T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 14/70578 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 NGF-receptor C07K14/71, TNF-receptor C07K14/7151
• CPC Class Codes: C07K 16/2803 against the immunoglobulin superfamily
• CPC Class Codes: C07K 16/2878 against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• CPC Class Codes: C07K 16/30 from tumour cells
• CPC Class Codes: C07K 16/40 against enzymes
• CPC Class Codes: C07K 2317/24 : containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/56 : variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/622 : Single chain antibody (scFv)
• CPC Class Codes: C07K 2317/73 : Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• CPC Class Codes: C07K 2319/00 : Fusion polypeptide
• CPC Class Codes: C07K 2319/02 : containing a signal sequence
• CPC Class Codes: C07K 2319/03 : containing a transmembrane segment
• CPC Class Codes: C07K 2319/10 : containing a tag for extracellular membrane crossing, e.g. TAT or VP22
• CPC Class Codes: C12N 2510/00 : Genetically modified cells
• CPC Class Codes: C12N 5/0636 : T lymphocytes

Abstract

Provided herein are T-cell receptor (TCR) fusion proteins (TFPs), T-cells engineered to express one or more TFPs, and methods of use thereof for the treatment of diseases, including cancer.

Claims

1. A pharmaceutical composition comprising

(I) a T cell from a human subject, wherein the T cell comprises a recombinant nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP) comprising

(a) a TCR subunit comprising a full-length CD3 epsilon sequence or a full-length CD3 gamma sequence;

and

(b) a mammalian scFv or single domain antibody comprising an anti-CD19 binding domain; and

(II) a pharmaceutically acceptable carrier;

wherein the TCR subunit and the anti-CD19 binding domain are operatively linked;

wherein the TFP functionally interacts with an endogenous TCR when expressed in the T cell; and

wherein the T cell exhibits increased cytotoxicity to a cell expressing an antigen that specifically interacts with the anti-CD19 binding domain compared to a T cell not containing the TFP.

2. The pharmaceutical composition of claim 1, wherein the anti-CD19 binding domain is connected to an extracellular domain of the TCR subunit by a linker.

3. The pharmaceutical composition of claim 2, wherein the linker comprises (G₄S)_n, wherein G is glycine, S is serine, and n is an integer from 1 to 4.

4. The pharmaceutical composition of claim 1, wherein the anti-CD19 binding domain comprises

(i) a light chain (LC) CDR1, LC CDR2 and LC CDR3 sequence of SEQ ID NO:

25, SEQ ID NO;

27 and SEQ ID NO;

29, respectively;

(ii) a heavy chain (HC) CDR1, HC CDR2 and HC CDR3 sequence of SEQ ID NO;

31, SEQ ID NO;

33 and SEQ ID NO;

35, respectively;

or

(iii) a combination thereof.

5. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is substantially free of serum.

6. The pharmaceutical composition of claim 1, wherein the TFP comprises the scFv.

7. The pharmaceutical composition of claim 1, wherein the TFP comprises the single domain antibody.

8. The pharmaceutical composition of claim 7, wherein the single domain antibody is a V_H domain.

9. The pharmaceutical composition of claim 1, wherein in the presence of a human cell expressing an antigen that specifically interacts with the anti-CD19 binding domain the T cell has greater than or more efficient cytotoxic activity than a T cell comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising the anti-CD19 binding domain operatively linked to a CD28 extracellular domain, a CD28 transmembrane domain, a CD28 intracellular domain, and a CD3 zeta intracellular domain.

10. The pharmaceutical composition of claim 1, wherein the TFP molecule functionally interacts with an endogenous TCR complex, at least one endogenous TCR polypeptide, or a combination thereof when expressed in the T cell.

11. The pharmaceutical composition of claim 1, wherein the T cell is a primary T cell.

12. The pharmaceutical composition of claim 4, wherein in the presence of a human cell expressing CD19 production of IL-2 by the T cell is lower than production of the IL-2 by a T cell comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising the anti-CD19 binding domain operatively linked to a CD28 extracellular domain, a CD28 transmembrane domain, a CD28 intracellular domain, and a CD3 zeta intracellular domain.

13. The pharmaceutical composition of claim 1, wherein the T cell is a human CD8+ T cell or a human CD4+ T cell.

14. The pharmaceutical composition of claim 1, wherein the TCR subunit comprises a sequence as set forth in SEQ ID NO:

56.

15. The pharmaceutical composition of claim 1, wherein the TCR subunit comprises a sequence as set forth in SEQ ID NO:

57.

16. The pharmaceutical composition of claim 1, wherein production of IFNγ

by the T cell is increased in the presence of a cell expressing CD19 compared to a T cell not containing the TFP.

17. The pharmaceutical composition of claim 1, wherein the T cell is a population of human CD8+ or CD4+ T cells, wherein an individual T cell of the population comprises at least two TFP molecules, or at least two T cells of the population collectively comprise at least two TFP molecules;

wherein the at least two TFP molecules comprise a mammalian anti-CD19 binding domain, a TCR extracellular domain, a TCR transmembrane domain, and a TCR intracellular domain; and

wherein at least one of the at least two TFP molecules functionally interacts with an endogenous TCR complex, at least one endogenous TCR polypeptide, or a combination thereof.

18. The pharmaceutical composition of claim 1, wherein the TCR subunit comprises an intracellular domain derived only from CD3 gamma.

19. The pharmaceutical composition of claim 1, wherein the TCR subunit comprises an intracellular domain derived only from CD3 epsilon.

20. The pharmaceutical composition of claim 1, wherein the TFP lacks a heterologous stimulatory domain.

21. The pharmaceutical composition of claim 1, wherein the TFP lacks a costimulatory domain.

22. The pharmaceutical composition of claim 1, wherein in the presence of a human cell expressing CD19 production of a pro-inflammatory cytokine by the T cell is lower compared to production of the pro-inflammatory cytokine by a T cell comprising a nucleic acid encoding a CAR comprising the anti-CD19 binding domain operatively linked to a CD28 extracellular domain, a CD28 transmembrane domain, a CD28 intracellular domain, and a CD3 zeta intracellular domain.

23. The pharmaceutical composition of claim 22, wherein the pro-inflammatory cytokine is TNFα

.

24. The pharmaceutical composition of claim 22, wherein the pro-inflammatory cytokine is IL-2.

25. The pharmaceutical composition of claim 22, wherein the pro-inflammatory cytokine is GM-CSF.

26. A pharmaceutical composition comprising

(I) a T cell from a human subject, wherein the T cell comprises a recombinant nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP) comprising

(a) a TCR subunit comprising a full-length CD3 epsilon sequence or a full-length CD3 gamma sequence;

and

(b) a mammalian scFv or single domain antibody comprising an anti-CD19 binding domain; and

(II) a pharmaceutically acceptable carrier;

wherein the TCR subunit and the anti-CD19 binding domain are operatively linked;

wherein the TFP functionally interacts with an endogenous TCR when expressed in the T cell;

wherein the TFP lacks a costimulatory domain and lacks a heterologous stimulatory domain; and

wherein the T cell exhibits increased cytotoxicity to a cell expressing an antigen that specifically interacts with the anti-CD19 binding domain compared to a T cell not containing the TFP.

27. The pharmaceutical composition of claim 26, wherein in the presence of a human cell expressing CD19 production of a pro-inflammatory cytokine by the T cell is lower compared to production of the pro-inflammatory cytokine by a T cell comprising a nucleic acid encoding a CAR comprising the anti-CD19 binding domain operatively linked to a CD28 extracellular domain, a CD28 transmembrane domain, a CD28 intracellular domain, and a CD3 zeta intracellular domain.

28. The pharmaceutical composition of claim 27, wherein the pro-inflammatory cytokine is selected from the group consisting of TNFα

, GM-CSF, IL-2 and combinations thereof.

29. The pharmaceutical composition of claim 26, wherein the TCR subunit comprises a sequence as set forth in SEQ ID NO:

56.

30. The pharmaceutical composition of claim 26, wherein the TCR subunit comprises a sequence as set forth in SEQ ID NO:

57.