Nucleic acid molecules encoding chimeric antigen receptors comprising a CD20 binding domain

• US 10,525,083 B2
• Filed: 10/06/2017
• Issued: 01/07/2020
• Est. Priority Date: 10/07/2016
• Status: Expired

• Current Assignee: Novartis Ag, Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Original Assignee: Novartis Ag
• Inventors: Brannetti, Barbara, Brogdon, Jennifer, Engels, Boris, Granda, Brian, Huang, Lu, Lei, Ming, Li, Na, Zhang, Jimin, Guimaraes, Carla
• Primary Examiner(s): Landsman, Robert S
• Application Number:
  US15/727,402
• Publication Number:
  US 20180125892A1
• Time in Patent Office: 823 Days
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 2039/804 Blood cells [leukemia, lymphoma]
• 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 35/17 Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes when activated by a specific antigen A61K39/00
• CPC Class Codes: A61K 38/1774 Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
• 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 39/464412 CD19 or B4
• CPC Class Codes: A61K 39/464413 CD22, BL-CAM, siglec-2 or sialic acid binding Ig-related lectin 2
• CPC Class Codes: A61K 39/464424 CD20
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 14/7051 T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 16/2803 against the immunoglobulin superfamily
• CPC Class Codes: C07K 16/2887 against CD20
• CPC Class Codes: C07K 16/2896 against molecules with a "CD"-designation, not provided for elsewhere
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/31 multispecific
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C07K 2319/03 : containing a transmembrane segment
• CPC Class Codes: C07K 2319/33 : fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
• CPC Class Codes: C12N 15/85 : for animal cells
• CPC Class Codes: C12N 5/0636 : T lymphocytes

Abstract

The invention provides compositions and methods for treating diseases associated with expression of CD20 or CD22. The invention also relates to chimeric antigen receptor (CAR) specific to CD20 or CD22, vectors encoding the same, and recombinant T or natural killer (NK) cells comprising the CD20 CAR or CD22 CAR. The invention also includes methods of administering a genetically modified T cell or NK cell expressing a CAR that comprises a CD20 or CD22 binding domain.

Claims

1. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises a CD20 binding domain, a transmembrane domain, and an intracellular signaling domain, wherein said CD20 binding domain comprises a light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2), light chain complementarity determining region 3 (LCDR3) heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2), and heavy chain complementarity determining region 3 (HCDR3), wherein the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprise:

(i) SEQ ID NOs;

147, 148, 149, 136, 137, and 138, respectively;

(ii) SEQ ID NOs;

150, 151, 152, 139, 140, and 141, respectively;

(iii) SEQ ID NOs;

153, 154, 155, 142, 143, and 144, respectively;

or

(iv) SEQ ID NOs;

929, 930, 931, 926, 927, and 928, respectively.

2. The isolated nucleic acid molecule of claim 1, wherein the CD20 binding domain comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein:

(i) the VL comprises the amino acid sequence of SEQ ID NO;

156, 129, or 439, or an amino acid sequence with at least 95% identity thereto;

(ii) the VH comprises the amino acid sequence of SEQ ID NO;

145, 118, or 437, or an amino acid sequence with at least 95% identity thereto;

or

(iii) the VL and VH comprise;

(a) SEQ ID NOs;

156 and 145, respectively;

(b) SEQ ID NOs;

129 and 118, respectively;

or

(c) SEQ ID NOs;

439 and 437, respectively.

3. The isolated nucleic acid molecule of claim 1, wherein:

(i) the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO;

157, 130, or 440, or a nucleotide sequence with at least 95% identity thereto;

(ii) the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO;

146, 119, or 438, or a nucleotide sequence with at least 95% identity thereto;

or

(iii) the nucleic acid molecule comprises;

(a) SEQ ID NOs;

157 and 146;

(b) SEQ ID NOs;

130 and 119;

or

(c) SEQ ID NOs;

440 and 438.

4. The isolated nucleic acid molecule of claim 1, wherein the CD20 binding domain comprises the amino acid sequence of SEQ ID NO:

159 or 132, or an amino acid sequence with at least 95% identity thereto.

5. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:

160 or 133, or a nucleotide sequence with at least 95% identity thereto.

6. The isolated nucleic acid molecule of claim 1, wherein the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD123, CD134, CD137 and CD154.

7. The isolated nucleic acid molecule of claim 1, wherein:

(i) the transmembrane domain comprises the amino acid sequence of SEQ ID NO;

801, or an amino acid sequence with at least 95% identity thereto;

or

(ii) the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO;

802 or 1072, or a nucleotide sequence with at least 95% identity thereto.

8. The isolated nucleic acid molecule of claim 1, wherein the CD20 binding domain is connected to the transmembrane domain by a hinge region.

9. The isolated nucleic acid molecule of claim 8, wherein:

(i) the hinge region comprises the amino acid sequence of SEQ ID NO;

799 or SEQ ID NO;

814, or an amino acid sequence with at least 95% identity thereto;

or

(ii) the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO;

800 or SEQ ID NO;

815, or a nucleotide sequence with at least 95% identity thereto.

10. The isolated nucleic acid molecule of claim 1, wherein the intracellular signaling domain comprises a costimulatory domain, wherein the costimulatory domain is a functional signaling domain obtained from a protein selected from the group consisting of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278) and 4-1BB (CD137).

11. The isolated nucleic acid molecule of claim 1, wherein the intracellular signaling domain comprises a costimulatory domain, wherein:

(i) the costimulatory domain comprises the amino acid sequence of SEQ ID NO;

803, or an amino acid sequence with at least 95% identity thereto;

or

(ii) the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO;

804, or a nucleotide sequence with at least 95% identity thereto.

12. The isolated nucleic acid molecule of claim 1, wherein the intracellular signaling domain comprises a primary signaling domain, wherein the primary signaling domain comprises a functional signaling domain of CD3 zeta.

13. The isolated nucleic acid molecule of claim 1, wherein the intracellular signaling domain comprises a primary signaling domain, wherein:

(i) the primary signaling domain comprises the amino acid sequence of SEQ ID NO;

807 or SEQ ID NO;

805, or an amino acid sequence with at least 95% identity thereto;

or

(ii) the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO;

893, 808, 806, or 1074, or a nucleotide sequence with at least 95% identity thereto.

14. The isolated nucleic acid molecule of claim 1, wherein the intracellular signaling domain comprises a functional signaling domain of 4-1BB and/or a functional signaling domain of CD3 zeta.

15. The isolated nucleic acid molecule of claim 1, wherein:

(i) the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO;

803, or an amino acid sequence with at least 95% identity thereto, and the amino acid sequence of SEQ ID NO;

807 or SEQ ID NO;

805, or an amino acid sequence with at least 95% identity thereto;

(ii) the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO;

803 and the amino acid sequence of SEQ ID NO;

807 or SEQ ID NO;

805;

(iii) the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO;

803 and the amino acid sequence of SEQ ID NO;

807 or SEQ ID NO;

805, wherein the sequences comprising the intracellular signaling domain are expressed in the same frame and as a single polypeptide chain, or

(iv) the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO;

804, or a nucleotide sequence with at least 95% identity thereto, and/or the nucleotide sequence of SEQ ID NO;

893, 808, 806, or 1074, or a nucleotide sequence with at least 95% identity thereto.

16. The isolated nucleic acid molecule of claim 1, further comprising a leader sequence, wherein the leader sequence encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:

797, or an amino acid sequence with at least 95% identity thereto.

17. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule encodes a CAR comprising the amino acid sequence of SEQ ID NO:

161 or 134, or an amino acid sequence with at least 95% identity thereto, wherein the CAR comprises or does not comprise a signal peptide comprising the amino acid sequence of SEQ ID NO;

797.

18. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:

162 or 135, or a nucleotide sequence with at least 95% identity thereto, wherein the nucleic acid molecule comprises or does not comprise the nucleotide sequence of SEQ ID NO;

798.

19. A nucleic acid comprising:

(i) a first nucleic acid comprising the nucleic acid molecule of claim 1, and

(ii) a second nucleic acid encoding a CAR molecule that binds a B-cell antigen.

20. The nucleic acid of claim 19, wherein the B cell antigen is CD19, CD22, CD10, CD34, CD123, FLT-3, ROR-1, CD79b, CD79a, or CD179b.

21. The nucleic acid of claim 19, wherein:

(i) the first and the second nucleic acids are disposed on a single nucleic acid molecule, or

(ii) the first and the second nucleic acids are disposed on separate nucleic acid molecules.

22. The nucleic acid of claim 19, wherein the B-cell antigen is CD19 or CD22.

23. The nucleic acid of claim 19, wherein the CAR molecule that binds a B-cell antigen binds CD19 and comprises:

(i) a HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising SEQ ID NOs;

773, 774, 775, 776, 777, and 778, respectively;

(ii) a scFv comprising the amino acid sequence of SEQ ID NO;

765;

or

(iii) a CAR comprising the amino acid sequence of SEQ ID NO;

764, wherein the CAR molecule comprises or does not comprise a signal peptide comprising the amino acid sequence of SEQ ID NO;

797.

24. The nucleic acid of claim 19, wherein the CAR molecule that binds a B-cell antigen binds CD19 and comprises:

(i) a HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising SEQ ID NOs;

785, 786, 787, 788, 789, and 790, respectively;

(ii) a scFv comprising the amino acid sequence of SEQ ID NO;

1047;

or

(iii) a CAR comprising the amino acid sequence of SEQ ID NO;

1049, wherein the CAR molecule comprises or does not comprise a signal peptide comprising the amino acid sequence of SEQ ID NO;

797.

25. The nucleic acid of claim 19, wherein a nucleotide sequence encoding a cleavable peptide, or a nucleotide sequence encoding an IRES is disposed between the first nucleic acid and the second nucleic acid.

26. The nucleic acid of claim 19, wherein:

(i) the first nucleic acid and the second nucleic acid comprise a single promoter, wherein the single promoter is an EF-1α

promoter;

or

(ii) the first nucleic acid comprises a first promoter and the second nucleic acid comprises a second promoter.

27. A vector comprising the nucleic acid molecule of claim 1.

28. A method of making a cell, comprising transducing a T cell or an NK cell with the nucleic acid molecule of claim 1.

29. A method of generating a population of RNA-engineered cells comprising introducing an in vitro transcribed RNA or synthetic RNA into a cell, wherein the RNA comprises the nucleic acid molecule of claim 1.

30. An isolated cell comprising the nucleic acid molecule of claim 1.

31. The isolated cell of claim 30, further expressing:

(i) an inhibitory molecule that comprises a first polypeptide that comprises at least a portion of an inhibitory molecule, associated with a second polypeptide that comprises a costimulatory domain and primary signaling domain, or

(ii) an inhibitory molecule that comprises a first polypeptide that comprises at least a portion of Programmed Death 1 (PD1) and a second polypeptide comprising a costimulatory domain and primary signaling domain.

32. A population of immune effector cells, comprising:

(i) a first cell population comprising the nucleic acid molecule of claim 1; and

(ii) a second cell population comprising a nucleic acid encoding a CAR that binds a B-cell antigen.

33. The population of claim 32, wherein:

(i) the B-cell antigen is chosen from CD19, CD22, CD10, CD34, CD123, FLT-3, ROR-1, CD79b, CD79a, or CD179b, or

(ii) the B-cell antigen is CD19 or CD22.