TREATMENT OF CANCER USING CHIMERIC ANTIGEN RECEPTORS

• US 20180125892A1
• Filed: 10/06/2017
• Published: 05/10/2018
• Est. Priority Date: 10/07/2016
• Status: Active Grant

• Current Assignee: Novartis Ag, Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Original Assignee: Lu Huang
• Inventors: Brannetti, Barbara, Brogdon, Jennifer, Engels, Boris, Granda, Brian, Huang, Lu, Lei, Ming, Li, Na, Zhang, Jimin, Guimaraes, Carla Patricia Pinto
• Granted Patent: US 10,525,083 B2
• Time in Patent Office: 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 one or more of light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2), and light chain complementarity determining region 3 (LCDR3) of any CD20 binding domain of Table 1 or Table 3, and one or more of heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2), and heavy chain complementarity determining region 3 (HCDR3) of any CD20 binding domain of Table 1 or Table 2.

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

(i) the CAR comprises a LCDR1, LCDR2, and LCDR3 according to Table 1 or Table 3,

(ii) the CAR comprises a HCDR1, HCDR2, and HCDR3 according to Table 1 or Table 2,

(iii) the CAR comprises LCDR1, LCDR2, and LCDR3 according to Table 1 or Table 3, and HCDR1, HCDR2, and HCDR3 according to Table 1 or Table 2,

(iv) the LCDR1, LCDR2, and LCDR3, respectively, is chosen from a)-p) of the following;

(a) SEQ ID NOs;

147, 148, and 149;

(b) SEQ ID NOs;

228, 229, and 230;

(c) SEQ ID NOs;

66, 67, and 68;

(d) SEQ ID NOs;

93, 94, and 95;

(e) SEQ ID NOs;

120, 121, and 122;

(f) SEQ ID NOs;

12, 13, and 14;

(g) SEQ ID NOs;

174, 175, and 176;

(h) SEQ ID NOs;

201, 202, and 203;

(i) SEQ ID NOs;

39, 40, and 41;

(i) SEQ ID NOs;

255, 256, and 257;

(k) SEQ ID NOs;

282, 283, and 284;

(l) SEQ ID NOs;

309, 310, and 311;

(m) SEQ ID NOs;

336, 337, and 338;

(n) SEQ ID NOs;

363, 364, and 365;

(o) SEQ ID NOs;

390, 391, and 392;

or

(p) SEQ ID NOs;

417, 418, and 419,

(v) the HCDR1, HCDR2, or HCDR3, respectively, is chosen from a)-p) of the following;

(a) SEQ ID NOs;

136, 137, and 138;

(b) SEQ ID NOs;

217, 218, and 219;

(c) SEQ ID NOs;

55, 56, and 57;

(d) SEQ ID NOs;

82, 83, and 84;

(e) SEQ ID NOs;

109, 110, and 111;

(f) SEQ ID NOs;

1, 2, and 3;

(g) SEQ ID NOs;

163, 164, and 165;

(h) SEQ ID NOs;

190, 191, and 192;

(i) SEQ ID NOs;

28, 29, and 30;

(j) SEQ ID NOs;

244, 245, and 246;

(k) SEQ ID NOs;

271, 272, and 273;

(l) SEQ ID NOs;

298, 299, and 300;

(m) SEQ ID NOs;

325, 326, and 327;

(n) SEQ ID NOs;

352, 353, and 354;

(o) SEQ ID NOs;

379, 380, and 381;

or

(p) SEQ ID NOs;

406, 407, and 408, or

(vi) the encoded amino acid sequence of the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3, respectively, is chosen from a)-p) of the following;

(a) SEQ ID NOs;

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

(b) SEQ ID NOs;

228, 229, 230, 217, 218, and 219;

(c) SEQ ID NOs;

66, 67, 68, 55, 56, and 57;

(d) SEQ ID NOs;

93, 94, 95, 82, 83, and 84;

(e) SEQ ID NOs;

120, 121, 122, 109, 110, and 111;

(f) SEQ ID NOs;

12, 13, 14, 1, 2, and 3;

(g) SEQ ID NOs;

174, 175, 176, 163, 164, and 165;

(h) SEQ ID NOs;

201, 202, 203, 190, 191, and 192;

(i) SEQ ID NOs;

39, 40, 41, 28, 29, and 30;

(i) SEQ ID NOs;

255, 256, 257, 244, 245, and 246;

(k) SEQ ID NOs;

282, 283, 284, 271, 272, and 273;

(l) SEQ ID NOs;

309, 310, 311, 298, 299, and 300;

(m) SEQ ID NOs;

336, 337, 338, 325, 326, and 327;

(n) SEQ ID NOs;

363, 364, 365, 352, 353, and 354;

(o) SEQ ID NOs;

390, 391, 392, 379, 380, and 381;

or

(p) SEQ ID NOs;

417, 418, 419, 406, 407, and 408.

3-7. -7. (canceled)

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

(i) the CAR comprises any light chain variable region listed in Table 1 or Table 5,

(ii) the CAR comprises any heavy chain variable region listed in Table 1 or Table 4,

(iii) the CAR comprises any light chain variable region listed in Table 1 or Table 5 and any heavy chain variable region listed in Table 1 or Table 4,

(iv) the encoded amino acid sequence of the light chain variable region and the heavy chain variable region, respectively, is chosen from a) to p) of the following;

(a) SEQ ID NOs;

156 and 145;

(b) SEQ ID NOs;

237 and 226;

(c) SEQ ID NOs;

21 and 10

(d) SEQ ID NOs;

75 and 64;

(e) SEQ ID NOs;

102 and 91;

(f) SEQ ID NOs;

129 and 118;

(g) SEQ ID NOs;

183 and 172;

(h) SEQ ID NOs;

210 and 199;

(i) SEQ ID NOs;

48 and 37;

(j) SEQ ID NOs;

264 and 253;

(k) SEQ ID NOs;

291 and 280;

(l) SEQ ID NOs;

318 and 307;

(m) SEQ ID NOs;

345 and 334;

(n) SEQ ID NOs;

372 and 361;

(o) SEQ ID NOs;

399 and 388;

or

(p) SEQ ID NOs;

426 and 415,

(v) the CD20 binding domain is an scFv,

(vi) the light chain variable region comprises an amino acid sequence having at least one, two, or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a light chain variable region provided in Table 1 or Table 5, or a sequence with 95-99% identity to an amino acid sequence provided in Table 1 or Table 5,

(vii) the heavy chain variable region comprises an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a heavy chain variable region provided in Table 1 or Table 4, or a sequence with 95-99% identity to an amino acid sequence provided in Table 1 or Table 4,

(viii) the CD20 binding domain comprises an amino acid sequence selected from a group consisting of SEQ ID NO;

159, SEQ ID NO;

240, SEQ ID NO;

24, SEQ ID NO;

51, SEQ ID NO;

78, SEQ ID NO;

105, SEQ ID NO;

132, SEQ ID NO;

186, SEQ ID NO;

213, SEQ ID NO;

267, SEQ ID NO;

294, SEQ ID NO;

321, SEQ ID NO;

348, SEQ ID NO;

375, SEQ ID NO;

402, and SEQ ID NO;

429, or a sequence with 95-99% identity thereof, or having at least one, two or three modifications but not more than 20, 10 or 5 modifications relative to any of the aforesaid amino acid sequences,

(ix) the nucleic acid sequence encoding the CD20 binding domain comprises a nucleotide sequence selected from a group consisting of SEQ ID NO;

160, SEQ ID NO;

241, SEQ ID NO;

25, SEQ ID NO;

52, SEQ ID NO;

79, SEQ ID NO;

106, SEQ ID NO;

133, SEQ ID NO;

187, SEQ ID NO;

214, SEQ ID NO;

268, SEQ ID NO;

295, SEQ ID NO;

322, SEQ ID NO;

349, SEQ ID NO;

376, SEQ ID NO;

403, and SEQ ID NO;

430, or a sequence with 95-99% identity thereof,

(x) the nucleic acid molecule comprises a nucleotide sequence listed in Table 1,

(xi) the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO;

160, SEQ ID NO;

241, SEQ ID NO;

25, SEQ ID NO;

52, SEQ ID NO;

79, SEQ ID NO;

106, SEQ ID NO;

133, SEQ ID NO;

187, SEQ ID NO;

214, SEQ ID NO;

268, SEQ ID NO;

295, SEQ ID NO;

322, SEQ ID NO;

349, SEQ ID NO;

376, SEQ ID NO;

403, and SEQ ID NO;

430, or a sequence with 95-99% identity thereof, or

(xii) the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO;

160, SEQ ID NO;

241, SEQ ID NO;

25, SEQ ID NO;

52, SEQ ID NO;

79, SEQ ID NO;

106, SEQ ID NO;

133, SEQ ID NO;

187, SEQ ID NO;

214, SEQ ID NO;

268, SEQ ID NO;

295, SEQ ID NO;

322, SEQ ID NO;

349, SEQ ID NO;

376, SEQ ID NO;

403, and SEQ ID NO;

430.

9-16. -16. (canceled)

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

(i) the transmembrane domain 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, CD123, CD134, CD137 and CD154,

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

801 or 802, or a sequence with 95-99% identity to an amino acid sequence of SEQ ID NO;

801 or 802,

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

801 or 802,

(iv) the CD20 binding domain is connected to the transmembrane domain by a hinge region,

(v) the CD20 binding domain is connected to the transmembrane domain by a hinge region, wherein the hinge region comprises SEQ ID NO;

799, or SEQ ID NO;

814, or a sequence with 95-99% identity thereof, or

(vi) the CD20 binding domain is connected to the transmembrane domain by a hinge region, wherein the nucleic acid sequence encoding the hinge region comprises a sequence of SEQ ID NO;

800 or SEQ ID NO;

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

18-20. -20. (canceled)

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

(i) the intracellular signaling domain comprises a costimulatory domain,

(ii) 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),

(iii) the intracellular signaling domain comprises a costimulatory domain, wherein the costimulatory domain comprises an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications of an amino acid sequence of SEQ ID NO;

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

803,

(iv) the intracellular signaling domain comprises a costimulatory domain, wherein the costimulatory domain comprises the amino acid sequence of SEQ ID NO;

803,

(v) the intracellular signaling domain comprises a costimulatory domain, wherein the nucleic acid sequence encoding the costimulatory domain comprises the nucleotide sequence of SEQ ID NO;

804, or a sequence with 95-99% identity thereof,

(vi) the intracellular signaling domain comprises a primary signaling domain,

(vii) the intracellular signaling domain comprises a primary signaling domain, wherein the primary signaling domain comprises a functional signaling domain of CD3 zeta,

(viii) the intracellular signaling domain comprises a primary signaling domain, wherein the primary signaling domain comprises the amino acid sequence of SEQ ID NO;

805 or SEQ ID NO;

807,

(ix) the intracellular signaling domain comprises a functional signaling domain of 4-1BB and/or a functional signaling domain of CD3 zeta,

(x) the intracellular signaling domain comprises 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;

803 and/or the amino acid sequence of SEQ ID NO;

805 or SEQ ID NO;

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

803 and/or the amino acid sequence of SEQ ID NO;

805 or SEQ ID NO;

807,

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

803 and/or the amino acid sequence of SEQ ID NO;

805 or SEQ ID NO;

807,

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

803 and the amino acid sequence of SEQ ID NO;

805 or SEQ ID NO;

807,

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

803 and the amino acid sequence of SEQ ID NO;

805 or SEQ ID NO;

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

(xiv) the nucleic acid sequence encoding the intracellular signaling domain comprises the nucleotide sequence of SEQ ID NO;

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

806 or SEQ ID NO;

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

22-29. -29. (canceled)

30. The isolated nucleic acid molecule of claim 1, further comprising:

(i) leader sequence, or

(ii) a leader sequence, wherein the leader sequence encodes a polypeptide comprising the amino acid sequence of SEQ ID NO;

797.

31-33. -33. (canceled)

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

(i) the nucleic acid molecule encodes a CAR comprising an amino acid sequence selected from the group consisting of SEQ ID NO;

161, SEQ ID NO;

242, SEQ ID NO;

26, SEQ ID NO;

53, SEQ ID NO;

80, SEQ ID NO;

107, SEQ ID NO;

134, SEQ ID NO;

188, SEQ ID NO;

215, SEQ ID NO;

269, SEQ ID NO;

296, SEQ ID NO;

323, SEQ ID NO;

350, SEQ ID NO;

377, SEQ ID NO;

404 and SEQ ID NO;

431, or a sequence with 95-99% identity thereof, or an amino acid sequence comprising at least one, two or three modifications but not more than 30, 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO;

161, SEQ ID NO;

242, SEQ ID NO;

26, SEQ ID NO;

53, SEQ ID NO;

80, SEQ ID NO;

107, SEQ ID NO;

134, SEQ ID NO;

188, SEQ ID NO;

215, SEQ ID NO;

269, SEQ ID NO;

296, SEQ ID NO;

323, SEQ ID NO;

350, SEQ ID NO;

377, SEQ ID NO;

404 or SEQ ID NO;

431, wherein the CAR includes or does not include a signal peptide of MALPVTALLLPLALLLHAARP,

(ii) the nucleic acid molecule encodes a CAR comprising an amino acid sequence selected from the group consisting of SEQ ID NO;

161, SEQ ID NO;

242, SEQ ID NO;

26, SEQ ID NO;

53, SEQ ID NO;

80, SEQ ID NO;

107, SEQ ID NO;

134, SEQ ID NO;

188, SEQ ID NO;

215, SEQ ID NO;

269, SEQ ID NO;

296, SEQ ID NO;

323, SEQ ID NO;

350, SEQ ID NO;

377, SEQ ID NO;

404 and SEQ ID NO;

431, wherein the CAR includes or does not include a signal peptide of

35-37. -37. (canceled)

38. An isolated polypeptide molecule encoded by the nucleic acid molecule of claim 1.

39. An isolated CAR molecule comprising a CD20 binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the CD20 binding domain comprises one or more light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2), and light chain complementarity determining region 3 (LCDR3) listed in Table 1 or Table 3, and one or more heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2), and heavy chain complementarity determining region 3 (HCDR3) listed in Table 1 or Table 2.

40. The isolated CAR molecule of claim 39, wherein:

(i) the CAR molecule comprises LCDR1, LCDR2 and LCDR3 of any sequence listed in Table 1 or Table 3,

(ii) the CAR molecule comprises HCDR1, HCDR2, and HCDR3 of any sequence listed in Table 1 or Table 2,

(iii) the CAR molecule comprises LCDR1, LCDR2 and LCDR3 of any sequence listed in Table 1 or Table 3, and HCDR1, HCDR2, and HCDR3 of any sequence listed in Table 1 or Table 2,

(iv) the LCDR1, LCDR2, or LCDR3 comprises an amino acid sequence listed in Table 1, or

(v) the HCDR1, HCDR2, or HCDR3 comprises an amino acid sequence listed in Table 1.

41-44. -44. (canceled)

45. The isolated CAR molecule of claim 39, wherein:

(i) the CAR molecule comprises any light chain variable region listed in Table 1 or Table 5,

(ii) the CAR molecule comprises any heavy chain variable region listed in Table 1 or Table 4,

(iii) the CAR molecule comprises any light chain variable region listed in Table 1 or Table 5 and any heavy chain variable region listed Table 1 or Table 4,

(iv) the CD20 binding domain is an scFv,

(v) the CD20 binding domain comprises a light chain variable region and a heavy chain variable region of an amino acid sequence listed in Table 1,

(vi) the CD20 binding domain comprises;

a light chain variable region comprising an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a light chain variable region provided in Table 1 or Table 5, or a sequence with 95-99% identity with an amino acid sequence provided in Table 1 or Table 5,

(vii) the CD20 binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a heavy chain variable region provided in Table 1 or Table 4, or a sequence with 95-99% identity to an amino acid sequence provided in Table 1 or Table 4, or

(viii) the CD20 binding domain comprises a sequence selected from the group consisting of SEQ ID NO;

159, SEQ ID NO;

240, SEQ ID NO;

24, SEQ ID NO;

51, SEQ ID NO;

78, SEQ ID NO;

105, SEQ ID NO;

132, SEQ ID NO;

186, SEQ ID NO;

213, SEQ ID NO;

267, SEQ ID NO;

294, SEQ ID NO;

321, SEQ ID NO;

348, SEQ ID NO;

375, SEQ ID NO;

402, and SEQ ID NO;

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

46-52. -52. (canceled)

53. The isolated CAR molecule of claim 39, wherein:

(i) the CAR molecule 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, CD123, CD134, CD137 and CD154,

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

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

801,

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

801,

(iv) the CD20 binding domain is connected to the transmembrane domain by a hinge region, or

(v) the CD20 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;

799 or SEQ ID NO;

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

54-55. -55. (canceled)

56. The isolated CAR molecule of claim 39, wherein:

(i) the intracellular signaling domain comprises a costimulatory domain,

(ii) the intracellular signaling domain comprises a costimulatory domain, wherein the costimulatory domain comprises a functional signaling domain of 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),

(iii) the intracellular signaling domain comprises a costimulatory domain, wherein the costimulatory domain comprises 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;

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

803,

(iv) the intracellular signaling domain comprises a costimulatory domain, wherein the costimulatory domain comprises the amino acid sequence of SEQ ID NO;

803,

(v) the intracellular signaling domain comprises a primary signaling domain,

(vi) the intracellular signaling domain comprises a primary signaling domain, wherein the primary signaling domain comprises a functional signaling domain of CD3 zeta,

(vii) the intracellular signaling domain comprises a primary signaling domain, wherein the primary signaling domain comprises the amino acid sequence of SEQ ID NO;

805 or SEQ ID NO;

807,

(viii) the intracellular signaling domain comprises a functional signaling domain of 4-1BB and/or a functional signaling domain of CD3 zeta,

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

803 and/or the amino acid sequence of SEQ ID NO;

805,

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

803 and/or the amino acid sequence of SEQ ID NO;

807,

(xi) the intracellular signaling domain comprises 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;

803 and/or the amino acid sequence of SEQ ID NO;

805 or SEQ ID NO;

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

803 and/or the amino acid sequence of SEQ ID NO;

805 or SEQ ID NO;

807, or

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

803 and the amino acid sequence of SEQ ID NO;

805 or SEQ ID NO;

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

57-63. -63. (canceled)

64. The isolated CAR molecule of claim 39, further comprising:

(i) a leader sequence, or

(ii) a leader sequence, wherein the leader sequence encodes a polypeptide comprising the amino acid sequence of SEQ ID NO;

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

797.

65. The isolated CAR molecule of claim 39, comprising:

(i) an amino acid sequence selected from the group consisting of SEQ ID NO;

161, SEQ ID NO;

242, SEQ ID NO;

26, SEQ ID NO;

53, SEQ ID NO;

80, SEQ ID NO;

107, SEQ ID NO;

134, SEQ ID NO;

188, SEQ ID NO;

215, SEQ ID NO;

269, SEQ ID NO;

296, SEQ ID NO;

323, SEQ ID NO;

350, SEQ ID NO;

377, SEQ ID NO;

404, and SEQ ID NO;

431, or a sequence with 95-99% identity thereof, or an amino acid sequence comprising at least one, two or three modifications but not more than 30, 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO;

161, SEQ ID NO;

242, SEQ ID NO;

26, SEQ ID NO;

53, SEQ ID NO;

80, SEQ ID NO;

107, SEQ ID NO;

134, SEQ ID NO;

188, SEQ ID NO;

215, SEQ ID NO;

269, SEQ ID NO;

296, SEQ ID NO;

323, SEQ ID NO;

350, SEQ ID NO;

377, SEQ ID NO;

404, or SEQ ID NO;

431, wherein the CAR molecule includes or does not include a signal peptide of MALPVTALLLPLALLLHAARP, or

(ii) an amino acid sequence selected from the group consisting of SEQ ID NO;

161, SEQ ID NO;

242, SEQ ID NO;

26, SEQ ID NO;

53, SEQ ID NO;

80, SEQ ID NO;

107, SEQ ID NO;

134, SEQ ID NO;

188, SEQ ID NO;

215, SEQ ID NO;

269, SEQ ID NO;

296, SEQ ID NO;

323, SEQ ID NO;

350, SEQ ID NO;

377, SEQ ID NO;

404 and SEQ ID NO;

431, wherein the CAR molecule includes or does not include a signal peptide of MALPVTALLLPLALLLHAARP.

66. (canceled)

67. A CD20 binding domain comprising one or more light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2), and light chain complementarity determining region 3 (LCDR3) of any CD20 binding domain selected from the group consisting of SEQ ID NO:

159, SEQ ID NO;

240, SEQ ID NO;

24, SEQ ID NO;

51, SEQ ID NO;

78, SEQ ID NO;

105, SEQ ID NO;

132, SEQ ID NO;

186, SEQ ID NO;

213, SEQ ID NO;

267, SEQ ID NO;

294, SEQ ID NO;

321, SEQ ID NO;

348, SEQ ID NO;

375, SEQ ID NO;

402, and SEQ ID NO;

429, and one or more heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2), and heavy chain complementarity determining region 3 (HCDR3) of any CD20 binding domain selected from the group consisting of SEQ ID NO;

159, SEQ ID NO;

240, SEQ ID NO;

24, SEQ ID NO;

51, SEQ ID NO;

78, SEQ ID NO;

105, SEQ ID NO;

132, SEQ ID NO;

186, SEQ ID NO;

213, SEQ ID NO;

267, SEQ ID NO;

294, SEQ ID NO;

321, SEQ ID NO;

348, SEQ ID NO;

375, SEQ ID NO;

402, and SEQ ID NO;

429.

68. The CD20 binding domain of claim 67, wherein:

(i) the LCDR1, LCDR2, and/or LCDR3 comprises an amino acid sequence chosen from Table 1 or Table 3,

(ii) the amino acid sequence of the LCDR1, LCDR2, and LCDR3, respectively, is chosen from a)-p) of the following;

(a) SEQ ID NOs;

147, 148, and 149;

(b) SEQ ID NOs;

228, 229, and 230;

(c) SEQ ID NOs;

66, 67, and 68;

(d) SEQ ID NOs;

93, 94, and 95;

(e) SEQ ID NOs;

120, 121, and 122;

(f) SEQ ID NOs;

12, 13, and 14;

(g) SEQ ID NOs;

174, 175, and 176;

(h) SEQ ID NOs;

201, 202, and 203;

(i) SEQ ID NOs;

39, 40, and 41;

(j) SEQ ID NOs;

255, 256, and 257;

(k) SEQ ID NOs;

282, 283, and 284;

(l) SEQ ID NOs;

309, 310, and 311;

(m) SEQ ID NOs;

336, 337, and 338;

(n) SEQ ID NOs;

363, 364, and 365;

(o) SEQ ID NOs;

390, 391, and 392;

or

(p) SEQ ID NOs;

417, 418, and 419,

(iii) the HCDR1, HCDR2, and/or HCDR3 comprises an amino acid sequence chosen from Table 1 or Table 2,

(iv) the amino acid sequence of the HCDR1, HCDR2, and HCDR3, respectively, is chosen from a)-p) of the following;

(a) SEQ ID NOs;

136, 137, and 138;

(b) SEQ ID NOs;

217, 218, and 219;

(c) SEQ ID NOs;

55, 56, and 57;

(d) SEQ ID NOs;

82, 83, and 84;

(e) SEQ ID NOs;

109, 110, and 111;

(f) SEQ ID NOs;

1, 2, and 3;

(g) SEQ ID NOs;

163, 164, and 165;

(h) SEQ ID NOs;

190, 191, and 192;

(i) SEQ ID NOs;

28, 29, and 30;

(j) SEQ ID NOs;

244, 245, and 246;

(k) SEQ ID NOs;

271, 272, and 273;

(l) SEQ ID NOs;

298, 299, and 300;

(m) SEQ ID NOs;

325, 326, and 327;

(n) SEQ ID NOs;

352, 353, and 354;

(o) SEQ ID NOs;

379, 380, and 381;

or

(p) SEQ ID NOs;

406, 407, and 408, or

(v) the amino acid sequence of the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3, respectively, is chosen from a)-p) of the following;

(a) SEQ ID NOs;

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

(b) SEQ ID NOs;

228, 229, 230, 217, 218, and 219;

(c) SEQ ID NOs;

66, 67, 68, 55, 56, and 57;

(d) SEQ ID NOs;

93, 94, 95, 82, 83, and 84;

(e) SEQ ID NOs;

120, 121, 122, 109, 110, and 111;

(f) SEQ ID NOs;

12, 13, 14, 1, 2, and 3;

(g) SEQ ID NOs;

174, 175, 176, 163, 164, and 165;

(h) SEQ ID NOs;

201, 202, 203, 190, 191, and 192;

(i) SEQ ID NOs;

39, 40, 41, 28, 29, and 30;

(j) SEQ ID NOs;

255, 256, 257, 244, 245, and 246;

(k) SEQ ID NOs;

282, 283, 284, 271, 272, and 273;

(l) SEQ ID NOs;

309, 310, 311, 298, 299, and 300;

(m) SEQ ID NOs;

336, 337, 338, 325, 326, and 327;

(n) SEQ ID NOs;

363, 364, 365, 352, 353, and 354;

(o) SEQ ID NOs;

390, 391, 392, 379, 380, and 381;

or

(p) SEQ ID NOs;

417, 418, 419, 406, 407, and 408.

69-72. -72. (canceled)

73. The CD20 binding domain of claim 67, wherein:

(i) the CD20 binding domain is an scFv comprising a light chain variable region of an amino acid sequence selected from Table 1 or Table 5 and a heavy chain variable region of an amino acid sequence selected from Table 1 or Table 4,

(ii) the CD20 binding domain is an scFv comprising a light chain variable region and a heavy chain variable region, wherein the amino acid sequence of the light chain variable region and the heavy chain variable region, respectively, is chosen from a) to p) of the following;

(a) SEQ ID NOs;

156 and 145;

(b) SEQ ID NOs;

237 and 226;

(c) SEQ ID NOs;

21 and 10

(d) SEQ ID NOs;

75 and 64;

(e) SEQ ID NOs;

102 and 91;

(f) SEQ ID NOs;

129 and 118;

(g) SEQ ID NOs;

183 and 172;

(h) SEQ ID NOs;

210 and 199;

(i) SEQ ID NOs;

48 and 37;

(j) SEQ ID NOs;

264 and 253;

(k) SEQ ID NOs;

291 and 280;

(l) SEQ ID NOs;

318 and 307;

(m) SEQ ID NOs;

345 and 334;

(n) SEQ ID NOs;

372 and 361;

(o) SEQ ID NOs;

399 and 388;

or

(p) SEQ ID NOs;

426 and 415, or

(iii) the CD20 binding domain comprises;

a light chain variable region comprising an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a light chain variable region provided in Table 1 or Table 5, or a sequence with 95-99% identity with an amino acid sequence in Table 1 or Table 5; and

/or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a heavy chain variable region provided in Table 1 or Table 4, or a sequence with 95-99% identity to an amino acid sequence in Table 1 or Table 4.

74-76. -76. (canceled)

77. A nucleic acid comprising:

(a) (i) a first nucleic acid encoding the CAR molecule of claim 39, and

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

(b) (i) a first nucleic acid encoding the CAR molecule of claim 39, and

(ii) a second nucleic acid encoding a CAR molecule that binds a B-cell antigen, wherein the B cell antigen is CD19, CD22, CD10, CD34, CD123, FLT-3, ROR-1, CD79b, CD79a, or CD179b,

(c) (i) a first nucleic acid encoding the CAR molecule of claim 39, and

(ii) a second nucleic acid encoding a CAR molecule that binds a B-cell antigen, wherein the first and the second nucleic acids are disposed on a single nucleic acid molecule, or

(d) (i) a first nucleic acid encoding the CAR molecule of claim 39, and

(ii) a second nucleic acid encoding a CAR molecule that binds a B-cell antigen, wherein the first and the second nucleic acids are disposed on separate nucleic acid molecules.

78. (canceled)

79. The nucleic acid of claim 77, wherein:

(i) the B-cell antigen is CD19 or CD22,

(ii) the CAR molecule that binds a B-cell antigen binds CD19 and comprises a nucleotide sequence encoding a CD19 CAR according to Table 11, or

(iii) the CAR molecule that binds a B-cell antigen binds CD22 and comprises a nucleotide sequence encoding a CD22 CAR according to Table 6.

80-81. -81. (canceled)

82. The nucleic acid of claim 77, wherein:

(i) a nucleotide sequence encoding a cleavable peptide, or a nucleotide sequence encoding an IRES is disposed between the first nucleic acid encoding the CAR molecule of claim 39 and the second nucleic acid encoding the CAR molecule that binds a B-cell antigen,

(ii) the CAR molecule of claim 39 and the CAR that binds a B-cell antigen are encoded by a single promoter,

(iii) the CAR molecule of claim 39 and the CAR that binds a B-cell antigen are encoded by a single promoter, wherein the single promoter is an EF-1α

promoter,

(iv) the CAR molecule of claim 39 is encoded by a first promoter and the CAR that binds a B-cell antigen is encoded by a second promoter, or

(v) the nucleic acid comprises RNA or DNA.

83-86. -86. (canceled)

87. An isolated polypeptide molecule encoded by the nucleic acid of claim 77.

88. A CD22 binding domain, or a CAR molecule, comprising:

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

839; and

/or

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

840.

89. The CD22 binding domain or CAR molecule of claim 88, wherein:

(i) the VH and VL sequences are connected directly,

(ii) the VH and VL sequences are connected without a linker,

(iii) the VH and VL sequences are connected via a (Gly4-Ser)n linker, wherein n is 0, 1, 2, 3, 4, 5, or 6,

(iv) the VH and VL sequences are connected via a (Gly4-Ser)n linker, wherein n is 1,

(v) the CD22 binding domain or CAR molecule comprises the amino acid sequence of SEQ ID NO;

837.

90-91. -91. (canceled)

92. A CD22 binding domain, or a CAR molecule, comprising the amino acid sequence of SEQ ID NO:

835 or SEQ ID NO;

836.

93. A nucleic acid encoding the CD22 binding domain or CAR molecule of claim 88.

94. A multispecific antibody molecule, or CAR molecule, having a first binding specificity for CD20 and a second binding specificity for one or more of CD19, CD22, CD10, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a, wherein the first binding specificity comprises the CD20 binding domain of claim 67.

95. A multispecific antibody molecule, or CAR molecule, having a first binding specificity for CD22 and a second binding specificity for one or more of CD19, CD20, CD10, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a, wherein the first binding specificity comprises the CD22 binding domain of claim 88.

96. A multispecific antibody molecule, or CAR molecule, having a first binding specificity for CD19 and a second binding specificity for one or both of CD20 or CD22, wherein:

(i) the multispecific antibody molecule has the arrangement VH1-VL1-VL2-VH2, from an N- to C-terminal orientation;

(ii) the multispecific antibody molecule has the arrangement VL1-VH1-VH2-VL2, from an N- to C-terminal orientation;

(iii) the multispecific antibody molecule has the arrangement VL1-VH1-VL2-VH2, from an N- to C-terminal orientation;

or

(iv) the multispecific antibody molecule has the arrangement VH1-VL1-VH2-VL2, from an N- to C-terminal orientation, and

wherein a linker is disposed between the two antibodies or antibody fragments.

97-98. -98. (canceled)

99. The multispecific antibody molecule, or CAR molecule, of claim 96, wherein:

(i) the second binding specificity is for CD20, wherein the CD20 binding specificity comprises a VH and VL as depicted in Table 1,

(ii) the second binding specificity is for CD22, wherein the CD22 binding specificity comprises a VH and VL as depicted in Table 6,

(iii) the linker is a (Gly4-Ser)n linker, wherein n is 1, 2, 3, 4, 5, or 6, or the linker comprises, or consists of, the amino acid sequence;

LAEAAAK,

(iv) the CD19 binding specificity comprises a VH and VL as depicted in Table 11,

(v) the multispecific antibody molecule, or CAR molecule comprises a VL1-VH1 binding specificity to CD19, and a VL2-VH2 or VH2-VL2 binding specificity to CD22,

(vi) the multispecific antibody molecule, or CAR molecule is encoded by a nucleic acid comprising, or consisting of, the nucleotide sequence of any of SEQ ID NOs;

844, 846, 848, 850, 852, 854, 856, 860, 895, 859, 861, 863, 865, 867, 869, 871, or 873, or a nucleotide sequence at least 95% identical to any of the aforesaid sequences, or

(vii) the multispecific antibody molecule, or CAR molecule comprises, or consists of, the amino acid sequence of any of SEQ ID NOs;

845, 847, 849, 851, 853, 855, 857, 858, 860, 862, 864, 866, 868, 870, 872, or 874, or an amino acid sequence at least 95% identical to any of the aforesaid sequences or having at least one, two or three modifications but not more than 30, 20 or 10 modifications.

100-104. -104. (canceled)

105. The multispecific antibody molecule, or CAR molecule, of claim 94, wherein the multispecific antibody molecule is present in a CAR molecule, wherein:

(i) the CAR molecule comprises a proximal or distal binding specificity for CD19;

(ii) the CAR molecule comprises a proximal or distal binding specificity for CD22;

or

(iii) the CAR molecule comprises a proximal or distal binding specificity for CD20.

106-123. -123. (canceled)

124. A nucleic acid encoding the multispecific antibody molecule or CAR molecule of claim 94.

125. A nucleic acid encoding a multispecific antibody molecule or a CAR molecule, comprising, or consisting of, the nucleotide sequence of any of SEQ ID NOs:

844, 846, 848, 850, 852, 854, 856, 860, 895, 859, 861, 863, 865, 867, 869, 871, or 873, or a nucleotide sequence at least 95% identical to any of the aforesaid sequences.

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

127-130. -130. (canceled)

131. A cell comprising the nucleic acid molecule of claim 1.

132-135. -135. (canceled)

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

137. 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.

138. 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.

139. The population of claim 138, wherein:

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

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

(iii) the CAR that binds a B-cell antigen binds CD19 and comprises a nucleotide sequence encoding a CD19 CAR according to Table 11, or

(iv) the CAR that binds a B-cell antigen binds CD22 and comprises a nucleotide sequence encoding a CD22 CAR according to Table 6.

140-142. -142. (canceled)

143. A method of providing an anti-tumor immunity in a mammal, wherein said method comprises administering to the mammal an effective amount of a cell comprising the nucleic acid molecule of claim 1.

144-146. -146. (canceled)

147. A method of treating a mammal having a disease associated with expression of CD20, wherein said method comprises administering to the mammal an effective amount of a cell comprising the nucleic acid molecule of claim 1.

148-149. -149. (canceled)

150. A method of treating a cancer, wherein said method comprises administering to the mammal an effective amount of a cell expressing the polypeptide of claim 38.

151. The method of claim 147, wherein:

(i) the disease is acute myeloid leukemia (AML), B-cell acute lymphoid leukemia (BALL), small lymphocytic leukemia (SLL), acute lymphoid leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt'"'"'s lymphoma, diffuse large B cell lymphoma (DLBCL), follicular lymphoma, hairy cell leukemia, small cell-lymphoma, large cell-follicular lymphoma, a malignant lymphoproliferative condition, MALT lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia, or myelodysplastic syndrome, myeloproliferative neoplasm, non-Hodgkin'"'"'s lymphoma, Hodgkin'"'"'s lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, preleukemia, or a combination thereof,

(ii) the disease is lymphoma,

(iii) the disease is diffuse large B cell lymphoma (DLBCL), or

(iv) the disease is a B-cell malignancy.

152-163. -163. (canceled)

164. The cell of claim 131, 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 positive signal from an intracellular signaling domain, or

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

165-167. -167. (canceled)

168. A method of treating a patient who is a non-responder, a partial responder, or a relapser to a CD19 inhibitor, comprising administering to the patient an inhibitor of CD20, wherein the inhibitor of CD20 is encoded by the nucleic acid molecule of claim 1.

169-171. -171. (canceled)