Chimeric antigen and t cell receptors and methods of use

Abstract

This invention provides a chimeric antigen receptor (CAR) or T-cell receptor (TCR) comprising one or more antigen-binding motifs disclosed herein. The present invention relates to a polynucleotide encoding a chimeric antigen receptor (CAR) or T-cell receptor (TCR) comprising one or more antigen-binding motifs. This invention provides an antibody and antigen-binding system comprising a CD20-binding motif and, optionally, a CD19-binding motif, as well as methods for its production and use. The antibody and antigen-binding system of this invention comprises a CAR comprising an anti-CD20-binding motif and an anti-CD19-binding motif. This invention provides compositions, such as antibodies and CARs comprising or including the anti-CD20 / anti-CD19 antigen-binding system of this invention, and cell therapies comprising them, suitable for, for example, treating cancer.

Claims

1. A method for preparing engineered immune cells, comprising introducing one or more polynucleotides encoding anti-CD20 chimeric antigen receptors (CARs) into immune cells, the anti-CD20 CAR comprising an anti-CD20 antigen-binding fragment, wherein the anti-CD20 antigen-binding fragment comprises a heavy chain variable domain (VH) containing heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable domain (VL) containing light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively comprise the amino acid sequences of SEQ ID NO: 48, 51, 54, 59, 62, and 65.

2. The method of claim 1, wherein the VH contains the amino acid sequence of SEQ ID NO: 45 and the VL contains the amino acid sequence of SEQ ID NO:

56.

3. The method of claim 1, wherein the one or more polynucleotides further encode an anti-CD19 CAR, the anti-CD19 CAR comprising an anti-CD19 antigen-binding fragment specific to CD19.

4. The method of claim 3, wherein the anti-CD19 antigen binding fragment comprises a VH containing HCDR1, HCDR2 and HCDR3, and a VL containing LCDR1, LCDR2 and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences of SEQ ID NO: 224, 227, 230, 235, 238 and 241.

5. The method of claim 4, wherein the VH of the anti-CD19 antigen-binding fragment contains the amino acid sequence of SEQ ID NO: 221, and the VL of the antigen-binding fragment contains the amino acid sequence of SEQ ID NO:

232.

6. The method of claim 4, wherein the anti-CD20 antigen-binding fragment or the anti-CD19 antigen-binding fragment is a single-stranded fragment (scFv).

7. The method of claim 4, wherein the anti-CD20 CAR and the anti-CD19 CAR each further comprise a transmembrane domain of CD8α or CD28.

8. The method of claim 7, wherein the anti-CD20 CAR and the anti-CD19 CAR each further include a signal transduction domain of 4-1BB or CD28.

9. The method of claim 8, wherein the anti-CD20 CAR and the anti-CD19 CAR each further include a CD3z stimulation domain.

10. The method of claim 4, wherein the one or more polynucleotides further encode a cleavable linker between the anti-CD20 CAR and the anti-CD19 CAR.

11. The method of claim 10, wherein the detachable connector is a 2A connector.

12. The method of claim 1, wherein the immune cell is a T cell.

13. A method for preparing engineered immune cells, comprising introducing one or more polynucleotides encoding anti-CD20 chimeric antigen receptors (CARs) into immune cells, the anti-CD20 CAR comprising an anti-CD20 antigen-binding fragment, wherein the anti-CD20 antigen-binding fragment comprises a heavy chain variable domain (VH) containing heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable domain (VL) containing light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively comprise the amino acid sequences of SEQ ID NO: 70, 73, 76, 81, 84, and 87.

14. The method of claim 13, wherein the VH comprises the amino acid sequence of SEQ ID NO: 67 and the VL comprises the amino acid sequence of SEQ ID NO:

78.

15. The method of claim 14, wherein the one or more polynucleotides further encode an anti-CD19 CAR, the anti-CD19 CAR comprising an anti-CD19 antigen-binding fragment specific to CD19.

16. The method of claim 15, wherein the anti-CD19 antigen binding fragment comprises a VH containing HCDR1, HCDR2 and HCDR3, and a VL containing LCDR1, LCDR2 and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences of SEQ ID NO: 224, 227, 230, 235, 238 and 241.

17. The method of claim 16, wherein the VH of the anti-CD19 antigen-binding fragment contains the amino acid sequence of SEQ ID NO: 221, and the VL of the antigen-binding fragment contains the amino acid sequence of SEQ ID NO:

232.

18. The method of claim 17, wherein the anti-CD20 antigen-binding fragment or the anti-CD19 antigen-binding fragment is a single-stranded fragment (scFv).

19. The method of claim 18, wherein the anti-CD20 scFv and the anti-CD19 scFv are each further included in a connector between the VH and the VL.

20. The method of claim 19, wherein the linker comprises the amino acid sequence of SEQ ID NO:

247.

21. The method of claim 20, wherein the anti-CD20 CAR further comprises a transmembrane domain of CD8α.

22. The method of claim 21, wherein the anti-CD20 CAR further comprises a 4-1BB signal transduction domain.

23. The method of claim 22, wherein the anti-CD19 CAR further comprises a transmembrane domain of CD28.

24. The method of claim 23, wherein the anti-CD19 CAR further includes a CD28 signal transduction domain.

25. The method of claim 24, wherein the anti-CD20 CAR and the anti-CD19 CAR each further include a CD3z stimulation domain.

26. The method of claim 25, wherein the one or more polynucleotides further encode a cleavable linker between the anti-CD20 CAR and the anti-CD19 CAR.

27. The method of claim 26, wherein the spliable connector is a 2A connector.

28. The method of claim 13, wherein the immune cell is a T cell.

29. The method of claim 12 or 28, wherein the introduction is by transduction or transfection.

30. The method of claim 29 further includes activating the T cell.

31. The method of claim 30, wherein the T cell line is activated with a stimulant.

32. The method of claim 31, wherein the stimulant comprises an anti-CD3 antibody, an anti-CD28 antibody, or a combination thereof.

33. The method of claim 30 further includes expanding the T cell.