COMPOSITIONS AND METHODS FOR TCR REPROGRAMMING USING FUSION PROTEINS

• US 20200362011A1
• Filed: 08/10/2020
• Published: 11/19/2020
• 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
• Granted Patent: US 11,028,142 B2
• Time in Patent Office: Days
• 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. AT 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 (i) an extracellular domain, (ii) a transmembrane domain, and (iii) a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain; and

(b) a murine, human or humanized scFv or single domain antibody comprising an antigen binding domain; and

a pharmaceutically acceptable carrier;

wherein the TCR subunit and the antigen binding domain are operatively linked;

wherein the extracellular domain, the transmembrane domain and the intracellular signaling domain are derived from a single subunit, wherein the single subunit is CD3 epsilon or wherein the single subunit is CD3 gamma;

wherein the extracellular domain comprises an extracellular domain of the single subunit;

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

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

2. The T cell of claim 1, wherein the single subunit comprises at least a portion of CD3 epsilon.

3. The T cell of claim 1, wherein the single subunit comprises the extracellular domain of CD3 epsilon.

4. The T cell of claim 2, wherein the single subunit comprises an amino acid sequence that is substantially identical to SEQ ID NO:

56.

5. The T cell of claim 2, wherein the single subunit comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:

56.

6. The T cell of claim 2, wherein the single subunit comprises an amino acid sequence that is at least 89% identical to SEQ ID NO:

56.

7. The T cell of claim 3, wherein the extracellular domain of CD3 epsilon has an amino acid sequence identical to amino acids 23-126 of SEQ ID NO:

56.

8. The T cell of claim 2, wherein the single subunit comprises a transmembrane domain that has an amino acid sequence identical to amino acids 127-152 of SEQ ID NO:

56.

9. The T cell of claim 2, wherein the single subunit comprises an intracellular domain that has an amino acid sequence identical to amino acids 153-207 of SEQ ID NO:

56.

10. The T cell of claim 1, wherein the single subunit comprises an amino acid sequence identical to amino acids 23-207 of SEQ ID NO:

56.

11. The T cell of claim 1, wherein the single subunit consists of an amino acid sequence identical to amino acids 23-207 of SEQ ID NO:

56.

12. The T cell of claim 1, wherein the antigen binding domain comprises an anti-CD19 binding domain.

13. The T cell of claim 12, wherein the anti-CD19 binding domain comprises (i) a light chain (LC) CDR1, LC CDR2 and LC CDR3 amino acid 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 amino acid sequence of SEQ ID NO;

31, SEQ ID NO;

33 and SEQ ID NO;

35, respectively;

or (iii) a combination thereof.

14. The T cell of claim 1, wherein the TFP comprises the murine, human or humanized scFv.

15. The T cell of claim 1, wherein the TFP has an amino acid sequence that is 90% identical to the amino acid sequence of a TFP that is encoded by a vector having SEQ ID NO 17 or SEQ ID NO 18.

16. The T cell of claim 1, wherein the TFP has an amino acid sequence that is 95% identical to the amino acid sequence of a TFP that is encoded by a vector having SEQ ID NO 17 or SEQ ID NO 18.

17. The T cell of claim 1, wherein the TFP comprises the murine, human or humanized single domain antibody.

18. The T cell of claim 17, wherein the murine, human or humanized single domain antibody is a V_HH domain.

19. The T cell of claim 1, wherein the antigen binding domain is connected to the TCR extracellular domain by a linker.

20. The T cell of claim 19, wherein the linker comprises (G₄S)_n, wherein G is glycine, S is serine, and n is an integer from 1 to 4.

21. The T cell of claim 1, wherein the TFP lacks a costimulatory domain.

22. The T cell of claim 1, wherein the TFP lacks a heterologous stimulatory domain.

23. The T cell of claim 13, wherein the TFP functionally interacts with an endogenous TCR complex, at least one endogenous TCR polypeptide, or a combination thereof when expressed in the T cell.

24. The T cell of claim 1, wherein the T cell is formulated as a pharmaceutical composition.

25. The T cell of claim 1, wherein the T cell is a CD8+ human T cell, a CD4+ human T cell, or a combination thereof.

26. The T cell of claim 1, wherein 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 antigen binding domain operatively linked to a CD28 extracellular domain, a CD28 transmembrane domain, a CD28 intracellular domain, and a CD3 zeta intracellular domain in the presence of a human cell expressing an antigen that specifically interacts with the antigen binding domain.

27. The T-cell of claim 26, wherein the pro-inflammatory cytokine is selected from the group consisting of TNFα

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

28. The T-cell claim 27, wherein production of IFNγ

by the T cell is increased compared to a T cell not containing the TFP in the presence of a human cell expressing an antigen that specifically interacts with the antigen binding domain.

29. A pharmaceutical composition comprising 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 (i) an extracellular domain, (ii) a transmembrane domain, and (iii) a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain; and

(b) a murine, human or humanized single domain antibody comprising an antigen binding domain; and

a pharmaceutically acceptable carrier;

wherein the TCR subunit and the antigen binding domain are operatively linked;

wherein the extracellular domain, the transmembrane domain and the intracellular signaling domain are derived from a single subunit, wherein the single subunit is CD3 epsilon;

wherein the single subunit consists of an amino acid sequence with amino acids 23-207 of SEQ ID NO;

56;

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

wherein the T cell exhibits increased cytotoxicity to a human cell expressing an antigen that specifically interacts with the antigen binding domain compared to a T cell not containing the TFP;

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

wherein the antigen binding domain is connected to the TCR extracellular domain by a linker; and

wherein the linker comprises (G₄S)_n, wherein G is glycine, S is serine, and n is an integer from 1 to 4.

30. A pharmaceutical composition comprising 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 (i) an extracellular domain, (ii) a transmembrane domain, and (iii) a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain; and

(b) a murine, human or humanized scFv comprising an antigen binding domain; and

a pharmaceutically acceptable carrier;

wherein the TCR subunit and the antigen binding domain are operatively linked;

wherein the extracellular domain, the transmembrane domain and the intracellular signaling domain are derived from a single subunit, wherein the single subunit is CD3 epsilon;

wherein the single subunit consists of an amino acid sequence with amino acids 23-207 of SEQ ID NO;

56;

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

wherein the T cell exhibits increased cytotoxicity to a human cell expressing an antigen that specifically interacts with the antigen binding domain compared to a T cell not containing the TFP;

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

wherein the antigen binding domain is connected to the TCR extracellular domain by a linker;

wherein the linker comprises (G₄S)_n, wherein G is glycine, S is serine, and n is an integer from 1 to 4;

wherein the antigen binding domain comprises an anti-CD19 binding domain; and

wherein the anti-CD19 binding domain comprises (i) a light chain (LC) CDR1, LC CDR2 and LC CDR3 amino acid 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 amino acid sequence of SEQ ID NO;

31, SEQ ID NO;

33 and SEQ ID NO;

35, respectively.