GUCY2C-binding molecules and uses thereof

Abstract

The present disclosure provides single-domain antibodies that bind to GUCY2C and chimeric antigen receptors containing the same. Additionally, engineered immune effector cells (such as T cells) containing the chimeric antigen receptor are provided. Pharmaceutical compositions, kits, and methods for treating diseases or disorders are also provided.

Claims

1. An anti-GUCY2C single domain antibody (sdAb), (i) X １ YGMX ２ CDR1 comprising the amino acid sequence １ is A, I or V; X ２ is D or G) (SEQ ID NO:64); (ii) X ３ IX ４ LSGRX ５ X ６ YX ７ DAVX ８ CDR2 comprising an amino acid sequence of X ３ is A, S or T, and X ４ is F, W or Y; X ５ is S or T, and X ６ is E, N or T, and X ７ is A, S or T, and X ８ is K or Q) (SEQ ID NO:65); (iii) GX ９ X １０ TAX １１ S.X. １２ CDR3 comprising the amino acid sequence of RQY (wherein ９ is A, E or P, and X １０ is P or T, and X １１ is S or T, and X １２ is G or V) (SEQ ID NO:66) The anti-GUCY2C sdAb comprising:

2. 2. The anti-GUCY2C sdAb of claim 1, wherein said CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-26, said CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-34, and said CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO:

10.

3. (i) a CDR1 comprising the amino acid sequence of SEQ ID NO: 19, a CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 35; (ii) a CDR1 comprising the amino acid sequence of SEQ ID NO: 20, a CDR2 comprising the amino acid sequence of SEQ ID NO: 28, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 36; (iii) a CDR1 comprising the amino acid sequence of SEQ ID NO:21, a CDR2 comprising the amino acid sequence of SEQ ID NO:29, and a CDR3 comprising the amino acid sequence of SEQ ID NO:37; (iv) a CDR1 comprising the amino acid sequence of SEQ ID NO: 22, a CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 38; (v) a CDR1 comprising the amino acid sequence of SEQ ID NO:23, a CDR2 comprising the amino acid sequence of SEQ ID NO:31, and a CDR3 comprising the amino acid sequence of SEQ ID NO:39; (vi) a CDR1 comprising the amino acid sequence of SEQ ID NO: 24, a CDR2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 40; (vii) a CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a CDR2 comprising the amino acid sequence of SEQ ID NO: 33, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 41; or (viii) a CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and a CDR3 comprising the amino acid sequence of SEQ ID NO:

42.

3. The anti-GUCY2C sdAb of claim 1 or claim 2, comprising:

4. An anti-GUCY2C single domain antibody (sdAb), (i) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3, respectively, set forth in SEQ ID NO:3; (ii) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3, respectively, set forth in SEQ ID NO:4; (iii) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3, respectively, set forth in SEQ ID NO:5; (iv) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO:6, respectively; (v) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO:7, respectively; (vi) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO:8, respectively; (vii) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO:9, respectively; or (viii) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 10, respectively. The anti-GUCY2C sdAb comprising:

5. The anti-GUCY2C sdAb of claim 4, wherein the CDR1, CDR2 or CDR3 is determined according to the Kabat numbering scheme, the IMGT numbering scheme, the AbM numbering scheme, the Chothia numbering scheme, the Contact numbering scheme, or a combination thereof.

6. 6. The anti-GUCY2C sdAb of any one of claims 1 to 5, further comprising one or more FR regions set forth in any one of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:

10.

7. 7. The anti-GUCY2C sdAb of any one of claims 1 to 6, comprising the amino acid sequence of any one of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:

10.

8. The anti-GUCY2C sdAb according to any one of claims 1 to 5, wherein said anti-GUCY2C sdAb is a camelid sdAb or a humanised sdAb.

9. The anti-GUCY2C sdAb of any one of claims 1 to 8, wherein said anti-GUCY2C sdAb is genetically fused or chemically conjugated to a drug.

10. A chimeric antigen receptor (CAR), (a) an extracellular antigen binding domain comprising an anti-GUCY2C sdAb according to any one of claims 1 to 8; (b) a transmembrane domain; and (c) an intracellular signaling domain; The CAR comprising:

11. The CAR of claim 10, wherein the extracellular antigen binding domain further comprises one or more additional antigen binding domains.

12. The CAR of claim 10 or claim 11, wherein the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1.

13. The CAR of any one of claims 10 to 12, wherein the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell.

14. The CAR of claim 13, wherein the primary intracellular signaling domain is derived from CD3ζ.

15. The CAR of claim 13 or claim 14, wherein the intracellular signaling domain further comprises a costimulatory signaling domain.

16. The CAR of claim 15, wherein the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137 (4-1BB), OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and combinations thereof.

17. The CAR according to any one of claims 10 to 16, further comprising a hinge domain positioned between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain, and / or a signal peptide positioned at the N-terminus of the polypeptide.

18. The CAR of claim 17, wherein the hinge domain and / or the signal peptide is derived from CD8α.

19. A chimeric antigen receptor (CAR) comprising: (i) an amino acid sequence selected from the group consisting of SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:55; or (ii) an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to any one of the sequences of SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:

55.

20. 20. An isolated nucleic acid comprising a nucleic acid sequence encoding the anti-GUCY2C sdAb of any one of claims 1 to 8, or a nucleic acid sequence encoding the CAR of any one of claims 10 to 19.

21. 21. The isolated nucleic acid of claim 20, wherein the nucleic acid sequence encoding the anti-GUCY2C sdAb comprises the nucleic acid sequence of SEQ ID NOs: 11-18.

22. 21. The isolated nucleic acid of claim 20, wherein the nucleic acid sequence encoding the CAR comprises a nucleic acid sequence of SEQ ID NO: 56-63.

23. A vector comprising the isolated nucleic acid according to any one of claims 20 to 22.

24. 24. An engineered immune effector cell comprising the CAR of any one of claims 10 to 19, the isolated nucleic acid of any one of claims 20 to 22, or the vector of claim 23.

25. 25. The engineered immune effector cell of claim 24, wherein the immune effector cell is a T cell.

26. A pharmaceutical composition comprising an anti-GUCY2C sdAb according to any one of claims 1 to 8, an engineered immune effector cell according to claim 24 or claim 25, or a vector according to claim 23.

27. 27. A pharmaceutical composition for treating a disease or disorder in a subject, comprising an anti-GUCY2C sdAb according to any one of claims 1 to 8, an engineered immune effector cell according to claim 24 or claim 25, or a vector according to claim 23.

28. 28. The pharmaceutical composition of claim 27, wherein the disease or disorder is a GUCY2C-associated disease or disorder, or cancer.

29. The pharmaceutical composition of claim 28 , wherein the cancer is colon cancer, gastric cancer, or esophageal cancer.

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