Anti-CLL1 specific single-chain chimeric antigen receptors (scCARs) for cancer immunotherapy

1. An isolated Chimeric Antigen Receptor (CAR) comprising:

an extracellular ligand binding domain that binds to C-type lectin molecule 1 (CCL1) comprising a heavy chain variable (V H ) region and a light chain variable (V L ) region wherein:

(i) the V H region comprises CDRs having the amino acid sequences set forth in SEQ ID NO: 131, SEQ ID NO: 132 and SEQ ID NO: 133, and the V L region comprises CDRs having the amino acid sequences set forth in SEQ ID NO: 134, SEQ ID NO: 135 and SEQ ID NO: 136; or

(ii) the V H region comprises CDRs having the amino acid sequences set forth in SEQ ID NO: 161, SEQ ID NO: 162 and SEQ ID NO: 163, and the V L region comprises CDRs having the amino acid sequences set forth in SEQ ID NO: 164, SEQ ID NO: 165 and SEQ ID NO: 166;

a hinge region comprising an FcγRIIIα hinge having the amino acid sequence set forth in SEQ ID NO: 3, a CD8α hinge having the amino acid sequence set forth in SEQ ID NO: 4 or an IgG1 hinge having the amino acid sequence set forth in SEQ ID NO: 5;

a CD8α transmembrane domain having the amino acid sequence set forth in SEQ ID NO: 6; and

a cytoplasmic signaling domain comprising a CD3 zeta signaling domain having the amino acid sequence set forth in SEQ ID NO: 9, and a 4-1 BB co-stimulatory domain having the amino acid sequence set forth in SEQ ID NO: 8.

2. The CAR according to claim 1 , further comprising at least one amino acid sequence of an epitope recognized by a therapeutic monoclonal antibody, wherein the epitope is located:

(i) between the V H and V L regions of the CAR;

(ii) at the N-terminus of the V H region of the CAR; or

(iii) between the V L region and the hinge region of the CAR;

and wherein the amino acid sequence is selected from the group consisting of: SEQ ID NO: 113, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, and SEQ ID NO: 199.

3. The CAR of claim 1 , wherein:

the V H region comprises the amino acid sequence set forth in SEQ ID NO: 15, and wherein the V L region comprises the amino acid sequence set forth in SEQ ID NO: 16; or

the V H region comprises the amino acid sequence set forth in SEQ ID NO: 25, and wherein the V L region comprises the amino acid sequence set forth in SEQ ID NO: 26.

4. The CAR according to claim 2 , wherein the amino acid sequence of the epitope is the amino acid sequence set forth in SEQ ID NO: 113.

5. An isolated polynucleotide encoding a CAR according to claim 1 .

6. An isolated, engineered lymphoid immune cell expressing the CAR according to claim 1 , wherein the CAR is cell surface membrane expressed.

7. A method of treating cancer in a human patient having cancer, the method comprising:

administering to the patient an isolated, engineered cytotoxic T cell comprising the CAR according to claim 1 ,

wherein the cancer comprises cells expressing C-type lectin like molecule 1 (CCL1), thereby treating the cancer in the patient.

8. The method of treating according to claim 7 , wherein the cancer is a hematological cancer.

9. The CAR according to claim 1 , wherein said CAR comprises the amino acid sequence set forth in SEQ ID NO: 55; or the amino acid sequence set forth in SEQ ID NO: 85.

10. A composition comprising the isolated, engineered lymphoid immune cell according to claim 6 .

11. The CAR according to claim 2 , wherein the CAR comprises the amino acid sequence(s) of one, two, three, or four of said epitopes.

12. The CAR according to claim 11 , wherein the extracellular binding domain comprises one of the following sequences:

V 1 -L 1 -V 2 -(L) x -Epitope1-(L) x -;

V 1 -L 1 -V 2 -(L) x -Epitope1-(L) x -Epitope2-(L) x -;

V 1 -L 1 -V 2 -(L) x -Epitope1-(L) x -Epitope2-(L) x -Epitope3-(L) x -;

(L) x -Epitope1-(L) x -V 1 -L 1 -V 2 -;

(L) x -Epitope1-(L) x -Epitope2-(L) x -V 1 -L 1 -V 2 -;

Epitope1-(L) x -Epitope2-(L) x -Epitope3-(L) x -V 1 -L 1 -V 2 -;

(L) x -Epitope1-(L) x -V 1 -L 1 -V 2 -(L) x -Epitope2-(L) x ;

(L) x -Epitope1-(L) x -V 1 -L 1 -V 2 -(L) x -Epitope2-(L) x -Epitope3-(L) x -;

(L) x -Epitope1-(L) x -V 1 -L 1 -V 2 -(L) x -Epitope2-(L) x -Epitope3-(L) x -Epitope4-(L) x -;

(L) x -Epitope1-(L) x -Epitope2-(L) x -V 1 -L 1 -V 2 -(L) x -Epitope3-(L) x -;

(L) x -Epitope1-(L) x -Epitope2-(L) x -V 1 -L 1 -V 2 -(L) x -Epitope3-(L) x -Epitope4-(L) x -;

V 1 -(L) x -Epitope1-(L) x -V 2 ;

V 1 -(L) x -Epitope1-(L) x -V 2 -(L) x -Epitope2-(L) x ;

V 1 -(L) x -Epitope1-(L) x -V 2 -(L) x -Epitope2-(L) x -Epitope3-(L) x ;

V 1 -(L) x -Epitope1-(L) x -V 2 -(L) x -Epitope2-(L) x -Epitope3-(L) x -Epitope4-(L) x ;

(L) x -Epitope1-(L) x -V 1 -(L) x -Epitope2-(L) x -V 2 ; and

(L) x -Epitope1-(L) x -V 1 -(L) x -Epitope2-(L) x -V 2 -(L) x -Epitope3-(L) x ;

wherein,

V 1 is V L and V 2 is V H or V 1 is V H and V 2 is V L ;

L 1 is a linker suitable to link the V H chain to the V L chain;

L is a linker comprising glycine and serine residues, and each occurrence of L in the extracellular binding domain can be identical or different to other occurrences of L in the same extracellular binding domain; and

x is 0 or 1 and each occurrence of x is selected independently from the others; and

Epitope 1, Epitope 2, Epitope 3 and Epitope 4 are independently selected from monoclonal antibody-specific epitopes comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 113, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, and SEQ ID NO: 199.

13. An isolated, engineered lymphoid immune cell expressing the CAR according to claim 2 , wherein the CAR is cell surface membrane expressed.

14. A method of treating cancer in a human patient having cancer, the method comprising:

administering to the patient an isolated, engineered cytotoxic T cell comprising the CAR according to claim 2 ,

wherein the cancer comprises cells expressing C-type lectin like molecule 1 (CCL1), thereby treating the cancer in the patient.

15. A composition comprising the isolated, engineered lymphoid immune cell expressing the CAR according to claim 13 .

16. The method of claim 7 , wherein the cancer is acute myelogenous leukemia (AML).

17. The method of claim 14 , wherein the cancer is acute myelogenous leukemia (AML).

18. The isolated, engineered lymphoid immune cell of claim 6 , wherein the cell comprises an inactivating mutation in at least one gene encoding a T-Cell Receptor (TCR).

19. The isolated, engineered lymphoid immune cell of claim 6 , wherein the cell comprises an inactivating mutation in a gene encoding a β2 microglobulin (β2m) or a gene encoding a human leukocyte antigen (HLA).

20. The isolated, engineered lymphoid immune cell of claim 6 , wherein the cell comprises an inactivating mutation in a receptor of an immunosuppressive agent or a drug target to confer resistance to the immunosuppressive agent or drug.

21. The isolated, engineered lymphoid immune cell of claim 13 , wherein the cell comprises an inactivating mutation in at least one gene encoding a T-Cell Receptor (TCR).

22. The isolated, engineered lymphoid immune cell of claim 13 , wherein the cell comprises an inactivating mutation in a gene encoding a β2 microglobulin (β2m) or a gene encoding a human leukocyte antigen (HLA).

23. The isolated, engineered lymphoid immune cell of claim 13 , wherein the cell comprises an inactivating mutation in a receptor of an immunosuppressive agent or a drug target to confer resistance to the immunosuppressive agent or drug.

24. A kit comprising the isolated, engineered lymphoid immune cell expressing the CAR according to claim 13 , and a therapeutic monoclonal antibody selected from the group consisting of rituximab, palivizumab, cetuximab, and nivolumab.