Nucleic acid encoding a humanized anti-BCMA chimeric antigen receptor

• US 10,174,095 B2
• Filed: 07/21/2015
• Issued: 01/08/2019
• Est. Priority Date: 07/21/2014
• Status: Active Grant

• Current Assignee: Novartis Ag, Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Original Assignee: Novartis Ag, Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Inventors: Brogdon, Jennifer, Choi, Eugene, Ebersbach, Hilmar Erhard, Glass, David, Huet, Heather, June, Carl H., Mannick, Joan, Milone, Michael C., Murphy, Leon, Plesa, Gabriela, Richardson, Celeste, Ruella, Marco, Singh, Reshma, Wang, Yongqiang, Wu, Qilong
• Primary Examiner(s): Howard, Zachary C
• Application Number:
  US14/805,193
• Publication Number:
  US 20160046724A1
• Time in Patent Office: 1,267 Days
• Field of Search: None
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61K 38/177 Receptors; Cell surface antigens; Cell surface determinants
• CPC Class Codes: A61K 39/395 Antibodies agglutinins A61K38/36 ; as drug carriers A61K47/50; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• CPC Class Codes: A61K 39/3955 against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• CPC Class Codes: A61K 39/39558 against tumor tissues, cells, antigens
• 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: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 14/7051 T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 14/70578 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 NGF-receptor C07K14/71, TNF-receptor C07K14/7151
• CPC Class Codes: C07K 14/7151 for tumor necrosis factor [TNF], for lymphotoxin [LT]
• CPC Class Codes: C07K 16/28 against receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: C07K 16/2878 against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• CPC Class Codes: C07K 19/00 Hybrid peptides , i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C07K 2319/00 : Fusion polypeptide
• CPC Class Codes: C07K 2319/03 : containing a transmembrane segment
• CPC Class Codes: C07K 2319/30 : Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• 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: C07K 2319/70 : containing domain for protein-protein interaction
• CPC Class Codes: C07K 2319/74 : containing a fusion for binding to a cell surface receptor
• CPC Class Codes: C12N 15/63 : Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 5/00 : Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor; plant reproduction by tissue culture techniques A01H4/00

Abstract

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

Claims

1. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an anti-B-cell maturation antigen (BCMA) binding domain, a transmembrane domain, and an intracellular signaling domain, wherein said anti-BCMA binding domain comprises:

(i) a heavy chain complementary determining region 1 (HC CDR1) comprising the amino acid sequence of SEQ ID NO;

394, a heavy chain complementary determining region 2 (HC CDR2) comprising the amino acid sequence of SEQ ID NO;

434, a heavy chain complementary determining region 3 (HC CDR3) comprising the amino acid sequence of SEQ ID NO;

474, a light chain complementary determining region 1 (LC CDR1) comprising the amino acid sequence of SEQ ID NO;

514, a light chain complementary determining region 2 (LC CDR2) comprising the amino acid sequence of SEQ ID NO;

554, and a light chain complementary determining region 3 (LC CDR3) comprising the amino acid sequence of SEQ ID NO;

594;

(ii) a HC CDR1 comprising the amino acid sequence of SEQ ID NO;

634, a HC CDR2 comprising the amino acid sequence of SEQ ID NO;

674, a HC CDR3 comprising the amino acid sequence of SEQ ID NO;

714, a LC CDR1 comprising the amino acid sequence of SEQ ID NO;

754, a LC CDR2 comprising the amino acid sequence of SEQ ID NO;

794, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO;

834;

or

(iii) a HC CDR1 comprising the amino acid sequence of SEQ ID NO;

874, a HC CDR2 comprising the amino acid sequence of SEQ ID NO;

914, a HC CDR3 comprising the amino acid sequence of SEQ ID NO;

954, a LC CDR1 comprising the amino acid sequence of SEQ ID NO;

994, a LC CDR2 comprising the amino acid sequence of SEQ ID NO;

1034, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO;

1074, and wherein;

the intracellular signaling domain comprises a primary signaling domain comprising a functional signaling domain of CD3 zeta or an amino acid sequence with 95-99% identity thereof.

2. The isolated nucleic acid molecule of claim 1, which encodes a CAR comprising:

(i) the amino acid sequence of a light chain variable region comprising the amino acid sequence of SEQ ID NO;

94;

(ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of the amino acid sequence of a light chain variable region comprising the amino acid sequence of SEQ ID NO;

94;

or

(iii) an amino acid sequence with 95-99% identity to the amino acid sequence of a light chain variable region comprising the amino acid sequence of SEQ ID NO;

94.

3. The isolated nucleic acid molecule of claim 1, which encodes a CAR comprising:

(i) the amino acid sequence of a heavy chain variable region comprising the amino acid sequence of SEQ ID NO;

79;

(ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of the amino acid sequence of a heavy chain variable region comprising the amino acid sequence of SEQ ID NO;

79;

or

(iii) an amino acid sequence with 95-99% identity to the amino acid sequence of a heavy chain variable region comprising the amino acid sequence of SEQ ID NO;

79.

4. The isolated nucleic acid molecule of claim 1, which encodes a CAR comprising the amino acid sequence of a light chain variable region comprising the amino acid sequence of SEQ ID NO:

94, and the amino acid sequence of a heavy chain variable region comprising the amino acid sequence of SEQ ID NO;

79.

5. The isolated nucleic acid molecule of claim 1, comprising a nucleic acid sequence encoding the anti-BCMA binding domain, wherein the nucleic acid sequence comprises the nucleotide sequence of SEQ ID NO:

64, or a sequence with 95-99% identity thereof.

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

(i) the encoded CAR comprises a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154;

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

6, an amino acid sequence comprising at least one, two or three modifications but not more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO;

6, or a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO;

6;

or

(iii) the isolated nucleic acid molecule comprises a nucleic acid sequence encoding the transmembrane domain, wherein the nucleic acid sequence comprises the nucleotide sequence of SEQ ID NO;

17, or a nucleotide sequence with 95-99% identity thereof.

7. The isolated nucleic acid molecule of claim 1, wherein the encoded anti-BCMA binding domain comprises:

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

49;

(ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to SEQ ID NO;

49;

or

(iii) an amino acid sequence with 95-99% identity to SEQ ID NO;

49.

8. The isolated nucleic acid molecule of claim 1, wherein the encoded anti-BCMA binding domain is connected to the transmembrane domain by a hinge region, wherein (i) the encoded hinge region comprises the amino acid sequence of SEQ ID NO:

2, or a sequence with 95-99% identity thereof;

or (ii) the isolated nucleic acid molecule comprises a nucleic acid sequence encoding the hinge region, wherein the nucleic acid sequence comprises the nucleotide sequence of SEQ ID NO;

13, or a sequence with 95-99% identity thereof.

9. The isolated nucleic acid molecule of claim 1, wherein the encoded intracellular signaling domain comprises a costimulatory domain, wherein the costimulatory domain comprises a functional signaling domain derived from a protein selected from the group consisting of MHC class I molecules, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, 4-1BB (CD137), B7-H3, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and a ligand that specifically binds with CD83.

10. The isolated nucleic acid molecule of claim 1, wherein the encoded intracellular signaling domain comprises a costimulatory domain, wherein the costimulatory domain comprises the amino acid sequence of SEQ ID NO:

7, or a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO;

7.

11. The isolated nucleic acid molecule of claim 1, wherein the encoded intracellular signaling domain comprises a costimulatory domain, wherein the isolated nucleic acid molecule comprises a nucleic acid sequence encoding the costimulatory domain, wherein the nucleic acid sequence comprises the nucleotide sequence of SEQ ID NO:

18, or a sequence with 95-99% identity thereof.

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

13. The isolated nucleic acid molecule of claim 1, wherein the encoded intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:

7 and the amino acid sequence of SEQ ID NO;

9 or SEQ ID NO;

10;

or a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO;

7 and the amino acid sequence of SEQ ID NO;

9 or SEQ ID NO;

10.

14. The isolated nucleic acid molecule of claim 1, wherein the encoded intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:

7 and the amino acid sequence of SEQ ID NO;

9 or SEQ ID NO;

10, wherein the amino acid sequences comprising the intracellular signaling domain are expressed in the same frame and as a single polypeptide chain.

15. The isolated nucleic acid molecule of claim 1, comprising a nucleic acid sequence encoding the intracellular signaling domain, wherein the nucleic acid sequence comprises the nucleotide sequence of SEQ ID NO:

18, or a sequence with 95-99% identity thereof, and/or the nucleotide sequence of SEQ ID NO;

20 or SEQ ID NO;

21, or a sequence with 95-99% identity thereof.

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

1.

17. The isolated nucleic acid molecule of claim 1, which encodes a CAR comprising:

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

109;

(ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to SEQ ID NO;

109;

or

(iii) an amino acid sequence with 95-99% identity to SEQ ID NO;

109,

with or without a leader sequence comprising the amino acid of SEQ ID NO;

1.

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

124, or a nucleotide sequence with 95-99% identity to SEQ ID NO;

124.

19. A vector comprising the nucleic acid molecule of claim 1, wherein the vector is a DNA vector, a RNA vector, a plasmid, a lentivirus vector, an adenoviral vector, or a retrovirus vector.

20. A method of making an immune effector cell, comprising transducing the immune effector cell with the vector of claim 19.

21. The method of claim 20, wherein the immune effector cell is a T cell.

22. An immune effector cell comprising the nucleic acid molecule of claim 1.

23. The cell of claim 22, further expressing a molecule that comprises a first polypeptide that comprises at least a portion of an inhibitory molecule, associated with a second polypeptide that comprises a positive signal from an intracellular signaling domain.

24. 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.

25. The isolated nucleic acid molecule of claim 1, wherein the encoded intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:

9 or SEQ ID NO;

10, or a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO;

9 or SEQ ID NO;

10.

26. The isolated nucleic acid molecule of claim 1, wherein the encoded CAR is expressed in frame and as a single polypeptide chain.

27. An immune effector cell comprising a CAR encoded by the nucleic acid molecule of claim 1, wherein:

(i) said LC CDR1, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NOs;

514, 554, and 594, respectively; and

(ii) said HC CDR1, HC CDR2 and HC CDR3 comprise the amino acid sequences of SEQ ID NOs;

394, 434, and 474, respectively.

28. The immune effector cell of claim 27, wherein the CAR comprises:

(i) the amino acid sequence of a light chain variable region comprising the amino acid sequence of SEQ ID NO;

94;

(ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of the amino acid sequence of a light chain variable region comprising the amino acid sequence of SEQ ID NO;

94;

(iii) an amino acid sequence with 95-99% identity to the amino acid sequence of a light chain variable region comprising the amino acid sequence of SEQ ID NO;

94;

(iv) the amino acid sequence of a heavy chain variable region comprising the amino acid sequence of SEQ ID NO;

79;

(v) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of the amino acid sequence of a heavy chain variable region comprising the amino acid sequence of SEQ ID NO;

79;

or

(vi) an amino acid sequence with 95-99% identity to the amino acid sequence of a heavy chain variable region comprising the amino acid sequence of SEQ ID NO;

79;

or

(vii) any of (i)-(iii) and any of (iv)-(vi).

29. The immune effector cell of claim 27, wherein the CAR comprises:

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

49;

(ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications SEQ ID NO;

49;

or

(iii) an amino acid sequence with 95-99% identity to SEQ ID NO;

49.

30. The immune effector cell of claim 27, wherein the transmembrane domain comprises a transmembrane domain from a protein selected from the group consisting of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

31. The immune effector cell of claim 27, wherein the transmembrane domain comprises:

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

6;

(ii) an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO;

6;

or

(iii) a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO;

6.

32. The immune effector cell of claim 27, wherein the anti-BCMA binding domain is connected to the transmembrane domain by a hinge region, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:

2 or SEQ ID NO;

36, or a sequence with 95-99% identity thereof.

33. The immune effector cell of claim 27, wherein the encoded intracellular domain comprises a costimulatory domain, wherein the costimulatory domain comprises a functional signaling domain derived from a protein selected from the group consisting of MHC class I molecules, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, 4-1BB (CD137), B7-H3, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and a ligand that specifically binds with CD83.

34. The immune effector cell of claim 27, wherein the costimulatory domain comprises the amino acid sequence of SEQ ID NO:

7, or a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO;

7.

35. The immune effector cell of claim 27, wherein the intracellular signaling domain comprises a functional signaling domain of 4-1BB and a functional signaling domain of CD3 zeta.

36. The immune effector cell of claim 27, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:

7 and the amino acid sequence of SEQ ID NO;

9 or SEQ ID NO;

10, or a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO;

7 and the amino acid sequence of SEQ ID NO;

9 or SEQ ID NO;

10.

37. The immune effector cell of claim 27, wherein the CAR comprises:

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

109;

(ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to SEQ ID NO;

109;

or

(iii) an amino acid sequence with 95-99% identity to SEQ ID NO;

109,

with or without a leader sequence comprising the amino acid of SEQ ID NO;

1.

38. The immune effector cell of claim 27, further expressing a molecule that comprises a first polypeptide that comprises at least a portion of an inhibitory molecule, associated with a second polypeptide that comprises a positive signal from an intracellular signaling domain.

39. The immune effector cell of claim 27, wherein the immune effector cell is a T cell.

40. A pharmaceutical composition comprising an antitumor effective amount of a population of human T cells, wherein the cells of the population include cells that comprise the nucleic acid molecule of claim 1.

41. The pharmaceutical composition of claim 40, further comprising a pharmaceutically acceptable carrier, diluent, or excipient.

42. An immune effector cell comprising a CAR expressed by the nucleic acid molecule of claim 1.

43. The immune effector cell of claim 42, wherein the immune effector cell is a T cell.

44. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an anti-B-cell maturation antigen (BCMA) binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the anti-BCMA binding domain comprises the amino acid sequence of SEQ ID NO:

49, and wherein the intracellular signaling domain comprises a primary signaling domain comprising a functional signaling domain of CD3 zeta or an amino acid sequence with 95-99% identity thereof.

45. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises, from N-terminus to C-terminus:

an anti-B-cell maturation antigen (BCMA) binding domain comprising the amino acid sequence of SEQ ID NO;

49,

a transmembrane domain comprising the amino acid sequence of SEQ ID NO;

6,

a costimulatory domain comprising the amino acid sequence of SEQ ID NO;

7, and

a primary signaling domain comprising the amino acid sequence of SEQ ID NO;

9.

46. A vector comprising the nucleic acid molecule of claim 45.

47. A cell comprising the vector of claim 46.

48. A pharmaceutical composition comprising the cell of claim 47.

49. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an anti-B-cell maturation antigen (BCMA) binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the CAR comprises the amino acid sequence of SEQ ID NO:

109 without a leader sequence comprising the amino acid of SEQ ID NO;

1.

50. An isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:

124.