Anti-entpd3 chimeric antigen receptor

Abstract

A chimeric antigen receptor (CAR), a cell (particularly an immune cell such as a regulatory T cell) expressing said CAR, a nucleic acid or vector encoding said CAR and various uses of said CAR, cell, nucleic acid or vector is disclosed herein. Particularly, a chimeric antigen receptor (CAR) comprising an antigen recognition domain that specifically binds to ENTPD3, is provided.

Description

This article discloses chimeric antigen receptors (CARs), cells expressing said CARs (particularly immune cells, such as regulatory T cells), nucleic acids or vectors encoding said CARs, and various uses of said CARs, cells, nucleic acids, or vectors. In particular, chimeric antigen receptors (CARs) comprising an antigen recognition domain that specifically binds to ENTPD3 are provided. Abstract

Claims

CLAIMS 1. A chimeric antigen receptor (CAR) comprising an antigen recognition domain that specifically binds to ENTPD3.

2. The CAR according to claim 1, comprising: a. an exodomain comprising the antigen recognition domain; b. a transmembrane domain; and c. an endodomain comprising an intracellular signalling domain.

3. The CAR according to claim 2, further comprising a hinge domain and / or one or more co- stimulatory domains.

4. The CAR according to claim 3, wherein the hinge domain is selected from the hinge regions of CD28, CD8α, CD4, CD7, CH2CH3, an immunoglobulin, or a part or variant thereof, optionally wherein the CAR comprises a CD8α or CH2CH3 hinge region; and / or wherein the co-stimulatory domain is selected from the intracellular domains of CD28, ICOS, CD134 (OX40), CD137 (4-1BB), CD27, or TNFRSF25, or a part or variant thereof, optionally wherein the CAR comprises a CD28 co-stimulatory domain.

5. The CAR according to any of claims 2 to 4, wherein the CAR comprises one or more transmembrane domains selected from the transmembrane domains of CD28, ICOS, CD8α, CD4, CD134 (OX40), CD137 (4-1BB), CD3 zeta, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD154, CH2CH3, or a part or variant thereof, optionally wherein the CAR comprises a CD4, CD28, CD8α or CH2CH3 transmembrane domain; and / or wherein the CAR comprises one or more intracellular signalling domains selected from the group consisting of the CD3 zeta signalling domain or any of its homologs, a CD3 polypeptide, a syk family tyrosine kinase, a src family tyrosine kinase, CD2, CD5, CD28, or a part or variant thereof, optionally wherein the CAR comprises the CD3 zeta signalling domain.

6. The CAR according to any of claims 2 to 5, wherein the CAR comprises: a CD8α or CH2CH3 hinge domain, a CD28, CD8α or CH2CH3 transmembrane domain, a CD28 co-stimulatory domain, and the CD3zeta signalling domain, wherein when the hinge domain is CD8α, the transmembrane domain is CD8α, and when the hinge domain is CH2CH3, the transmembrane domain is CD28 or CH2CH3.

7. The CAR according to any preceding claims, wherein the antigen recognition domain is an antibody, an antibody fragment, or derived from an antibody, optionally wherein the antigen recognition domain is a single chain antibody (scFv).

8. The CAR of any preceding claim, wherein the antigen recognition domain comprises: i. VH CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 7, 8 and 9 respectively and VL CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 10, 11 and 12 respectively; ii. VH CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 19, 20 and 21 respectively and VL CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 22, 23 and 24 respectively; iii. VH CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 73, 74 and 75 respectively and VL CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 76, 77 and 78 respectively; iv. VH CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 25, 26 and 27 respectively and VL CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 28, 29 and 30 respectively; v. VH CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 37, 38 and 39 respectively and VL CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 40, 41 and 42 respectively; vi. VH CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 1, 2 and 3 respectively and VL CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 4, 5 and 6 respectively; vii. VH CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 13, 14 and 15 respectively and VL CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 16, 17 and 18 respectively; viii. VH CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 31, 32 and 33 respectively and VL CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 34, 35 and 36 respectively; ix. VH CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 43, 44 and 45 respectively and VL CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 46, 47 and 48 respectively; x. VH CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 49, 50 and 51 respectively and VL CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 52, 53 and 54 respectively; xi. VH CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 55, 56 and 57 respectively and VL CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 58, 59 and 60 respectively; xii. VH CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 61, 62 and 63 respectively and VL CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 64, 65 and 66 respectively;xiii. VH CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 67, 68 and 69 respectively and VL CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 70, 71 and 72 respectively; or xiv. VH CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 79, 80 and 81 respectively and VL CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 82, 83 and 84 respectively; wherein one or more of said CDR sequences of (i) to (xiv) may optionally comprise 1 to 3 amino acid modifications relative to an aforementioned CDR sequence, particularly wherein one or more of said CDR sequences may optionally be modified by substitution, addition or deletion of 1 to 3 amino acids.

9. The CAR of any preceding claim, wherein the antigen recognition domain comprises: (i) a VH domain comprising the sequence set forth in SEQ ID NO: 87, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 88, or a sequence having at least 70% identity thereto; (ii) a VH domain comprising the sequence set forth in SEQ ID NO: 91, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 92, or a sequence having at least 70% identity thereto; (iii) a VH domain comprising the sequence set forth in SEQ ID NO: 109, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 110, or a sequence having at least 70% identity thereto; (iv) a VH domain comprising the sequence set forth in SEQ ID NO: 93, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 94, or a sequence having at least 70% identity thereto; (v) a VH domain comprising the sequence set forth in SEQ ID NO: 97, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 98, or a sequence having at least 70% identity thereto; (vi) a VH domain comprising the sequence set forth in SEQ ID NO: 85, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 86, or a sequence having at least 70% identity thereto; (vii) a VH domain comprising the sequence set forth in SEQ ID NO: 89, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 90, or a sequence having at least 70% identity thereto; (viii) a VH domain comprising the sequence set forth in SEQ ID NO: 95, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 96, or a sequence having at least 70% identity thereto; (ix) a VH domain comprising the sequence set forth in SEQ ID NO: 99, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 100, or a sequence having at least 70% identity thereto;(x) a VH domain comprising the sequence set forth in SEQ ID NO: 101, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 102, or a sequence having at least 70% identity thereto; (xi) a VH domain comprising the sequence set forth in SEQ ID NO: 103, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 104, or a sequence having at least 70% identity thereto; (xii) a VH domain comprising the sequence set forth in SEQ ID NO: 105, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 106, or a sequence having at least 70% identity thereto; (xiii) a VH domain comprising the sequence set forth in SEQ ID NO: 107, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 108, or a sequence having at least 70% identity thereto; or (xiv) a VH domain comprising the sequence set forth in SEQ ID NO: 111, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 112, or a sequence having at least 70% identity thereto.

10. The CAR according to any preceding claim, wherein the antigen recognition domain comprises or consists of: (i) the sequence set forth in SEQ ID NO: 114 or a sequence having at least 80% sequence identity thereto; (ii) the sequence set forth in SEQ ID NO: 116 or a sequence having at least 80% sequence identity thereto; (iii) the sequence set forth in SEQ ID NO: 125 or a sequence having at least 80% sequence identity thereto; (iv) the sequence set forth in SEQ ID NO: 117 or a sequence having at least 80% sequence identity thereto; (v) the sequence set forth in SEQ ID NO: 119 or a sequence having at least 80% sequence identity thereto; (vi) the sequence set forth in SEQ ID NO: 113 or a sequence having at least 80% sequence identity thereto; (vii) the sequence set forth in SEQ ID NO: 115 or a sequence having at least 80% sequence identity thereto; (viii) the sequence set forth in SEQ ID NO: 118 or a sequence having at least 80% sequence identity thereto; (ix) the sequence set forth in SEQ ID NO: 120 or a sequence having at least 80% sequence identity thereto; (x) the sequence set forth in SEQ ID NO: 121 or a sequence having at least 80% sequence identity thereto; (xi) the sequence set forth in SEQ ID NO: 122 or a sequence having at least 80% sequence identity thereto;(xii) the sequence set forth in SEQ ID NO: 123 or a sequence having at least 80% sequence identity thereto; (xiii) the sequence set forth in SEQ ID NO: 124 or a sequence having at least 80% sequence identity thereto; or (xiv) the sequence set forth in SEQ ID NO: 126 or a sequence having at least 80% sequence identity thereto.

11. The CAR of any preceding claim, wherein the CAR comprises or consists of the sequence set out in any one of SEQ ID NOs: 237 to 262, particularly wherein the CAR comprises or consists of the sequence of SEQ ID NO: 238, 240, 249, 241 or 243.

12. A nucleic acid molecule comprising a nucleotide sequence encoding the CAR of any preceding claim.

13. A vector comprising the nucleic acid molecule of claim 12, optionally further comprising a nucleic acid molecule encoding a FOXP3 polypeptide.

14. A cell comprising the CAR of any of claims 1 to 11, the nucleic acid molecule of claim 12, or the vector of claim 13.

15. The cell of claim 14, wherein the cell is a regulatory T cell (Treg), or a precursor thereof, or an iPSC cell, particularly wherein the cell further comprises an exogenous nucleic acid comprising a nucleotide sequence encoding a FOXP3 polypeptide.

16. A cell population comprising a cell of claim 14 or claim 15.

17. The cell population of claim 16, wherein the plurality of cells is a plurality of T cells, particularly Tregs, and the plurality of T cells have polyclonal endogenous TCRs.

18. A pharmaceutical composition comprising a cell of claim 14 or claim 15, a cell population of claim 16 or 17, or a vector of claim 13.

19. The cell of any one of claim 14 or claim 15, a cell population of claim 16 or 17, or the pharmaceutical composition of claim 18, for use in therapy.

20. The cell, cell population or pharmaceutical composition for use of claim 19, wherein the therapy is adoptive cell transfer therapy.

21. The cell of claim 14 or claim 15, a cell population of claim 16 or 17, or pharmaceutical composition of claim 18, for use in treating or preventing an autoimmune or inflammatory disease, or for use in inducing immunosuppression, or for use in promoting tissue repair and / or tissue regeneration, particularly wherein the cell is a Treg.

22. The cell, cell population or pharmaceutical composition for use of claim 21, wherein the autoimmune or inflammatory disease is type 1 diabetes, for example recent-onset type 1 diabetes.

23. A method of making a cell of claim 14 or claim 15, which comprises the step of introducing into the cell the nucleic acid molecule of claim 12 or the vector of claim 13.

24. A cell obtainable by the method of claim 23.

25. An antibody or antibody fragment that specifically binds to ENTPD3, optionally a single chain antibody (scFv), comprising: (i) VH CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 7, 8 and 9 respectively and VL CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 10, 11 and 12 respectively; (ii) VH CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 19, 20 and 21 respectively and VL CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 22, 23 and 24 respectively; (iii) VH CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 73, 74 and 75 respectively and VL CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 76, 77 and 78 respectively; (iv) VH CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 25, 26 and 27 respectively and VL CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 28, 29 and 30 respectively; (v) VH CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 37, 38 and 39 respectively and VL CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 40, 41 and 42 respectively; (vi) VH CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 1, 2 and 3 respectively and VL CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 4, 5 and 6 respectively; (vii) VH CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 13, 14 and 15 respectively and VL CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 16, 17 and 18 respectively;(viii) VH CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 31, 32 and 33 respectively and VL CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 34, 35 and 36 respectively; (ix) VH CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 43, 44 and 45 respectively and VL CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 46, 47 and 48 respectively; (x) VH CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 49, 50 and 51 respectively and VL CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 52, 53 and 54 respectively; (xi) VH CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 55, 56 and 57 respectively and VL CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 58, 59 and 60 respectively; (xii) VH CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 61, 62 and 63 respectively and VL CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 64, 65 and 66 respectively; (xiii) VH CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 67, 68 and 69 respectively and VL CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 70, 71 and 72 respectively; or (xiv) VH CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 79, 80 and 81 respectively and VL CDR1, 2 and 3 sequences set forth in SEQ ID NOs: 82, 83 and 84 respectively; wherein one or more of said CDR sequences of (i) to (xiv) may optionally comprise 1 to 3 amino acid modifications relative to an aforementioned CDR sequence, particularly wherein one or more of said CDR sequences may optionally be modified by substitution, addition or deletion of 1 to 3 amino acids.

26. The antibody or antibody fragment of claim 25, wherein the antibody or antibody fragment comprises: (i) a VH domain comprising the sequence set forth in SEQ ID NO: 87, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 88, or a sequence having at least 70% identity thereto; (ii) a VH domain comprising the sequence set forth in SEQ ID NO: 91, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 92, or a sequence having at least 70% identity thereto; (iii) a VH domain comprising the sequence set forth in SEQ ID NO: 109, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 110, or a sequence having at least 70% identity thereto;(iv) a VH domain comprising the sequence set forth in SEQ ID NO: 93, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 94, or a sequence having at least 70% identity thereto; (v) a VH domain comprising the sequence set forth in SEQ ID NO: 97, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 98, or a sequence having at least 70% identity thereto; (vi) a VH domain comprising the sequence set forth in SEQ ID NO: 85, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 86, or a sequence having at least 70% identity thereto; (vii) a VH domain comprising the sequence set forth in SEQ ID NO: 89, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 90, or a sequence having at least 70% identity thereto; (viii) a VH domain comprising the sequence set forth in SEQ ID NO: 95, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 96, or a sequence having at least 70% identity thereto; (ix) a VH domain comprising the sequence set forth in SEQ ID NO: 99, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 100, or a sequence having at least 70% identity thereto; (x) a VH domain comprising the sequence set forth in SEQ ID NO: 101, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 102, or a sequence having at least 70% identity thereto; (xi) a VH domain comprising the sequence set forth in SEQ ID NO: 103, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 104, or a sequence having at least 70% identity thereto; (xii) a VH domain comprising the sequence set forth in SEQ ID NO: 105, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 106, or a sequence having at least 70% identity thereto; (xiii) a VH domain comprising the sequence set forth in SEQ ID NO: 107, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 108, or a sequence having at least 70% identity thereto; or (xiv) a VH domain comprising the sequence set forth in SEQ ID NO: 111, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 112, or a sequence having at least 70% identity thereto.

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