阅读说明：本技术 针对cd19的人源化抗原结合结构域及其使用方法 (Humanized antigen binding domains against CD19 and methods of use thereof ) 是由 J.J.W.威尔特齐乌斯 S.A.西弗斯 于 2018-04-24 设计创作，主要内容包括：本发明提供了人源化抗CD19抗体或其抗原结合片段,其包含轻链可变(VL)区和重链可变(VH)区,其中人源化VL和VL区衍生自小鼠抗CD19克隆FMC63抗体；人源化VL和/或人源化VH区在框架区中包含一个或多个氨基酸取代。人源化抗CD19抗体或抗原结合片段可以是单链可变片段(scFv)、嵌合抗原受体(CAR)或T细胞受体(TCR)的一部分。本发明的其他方面涉及包含CAR或TCR的细胞及其在T细胞疗法中的用途。(The present invention provides a humanized anti-CD 19 antibody or antigen-binding fragment thereof comprising a light chain Variable (VL) region and a heavy chain Variable (VH) region, wherein the humanized VL and VL regions are derived from a mouse anti-CD 19 cloned FMC63 antibody; the humanized VL and/or humanized VH regions comprise one or more amino acid substitutions in the framework regions. The humanized anti-CD 19 antibody or antigen-binding fragment may be part of a single chain variable fragment (scFv), a Chimeric Antigen Receptor (CAR), or a T Cell Receptor (TCR). Other aspects of the invention relate to cells comprising a CAR or TCR and their use in T cell therapy.)

1. A humanized anti-CD 19 antibody or antigen-binding fragment thereof comprising a light chain Variable (VL) region and a heavy chain Variable (VH) region, wherein the VL region comprises a VL Complementarity Determining Region (CDR)1(VL CDR1), a VL CDR2 and a VL CDR3 and the VH region comprises a VH CDR1, a VL CDR2 and a VL CDR3,

Wherein the VL region is derived from SEQ ID NO 36 and the VH region is derived from SEQ ID NO 37 and wherein the VL and/or VH regions comprise one or more amino acid substitutions in the framework regions.

2. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 1, wherein the VL region comprises up to 5, 10, 15, 20, 25, or 30 amino acid substitutions as compared to SEQ ID No. 36.

3. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the one or more amino acid substitutions in the VL region are at positions corresponding to 7, 8, 10, 15, 22, 41, 42, 43, 44, 49, 71, 72, 77, 79, 80, 83, 87, 100, and/or 107 of SEQ ID No. 36.

4. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the one or more amino acid substitutions in the VL region is selected from Ser at position 7, Pro at position 8, Val at position 15, Thr at position 22, Gln at position 41, Lys at position 42, Ala at position 43, Thr at position 72, Ser at position 77, Gln at position 79, Pro at position 80, and/or Lys at position 107 of SEQ ID NO 36.

5. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the VH region comprises up to 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid substitutions as compared to SEQ ID NO 37.

6. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the one or more amino acid substitutions in the VH region are at positions corresponding to 1, 3, 5, 9, 13, 15, 16, 17, 19, 20, 21, 23, 24, 37, 42, 48, 67, 69, 70, 71, 73, 76, 77, 78, 79, 81, 83, 86, 87, 88, 92 and/or 115 of SEQ ID No. 37.

7. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the one or more amino acid substitutions in the VH region are selected from the group consisting of Gln at position 1, Gln at position 3, Val at position 5, Gly at position 9, Lys at position 13, Gln at position 13, Gly at position 15, Arg at position 16, Thr at position 17, Arg at position 19, Leu at position 20, Ser at position 21, Ala at position 24, Gly at position 42, Ile at position 48, Phe at position 67, Ser at position 70, Arg at position 71, Thr at position 73, Asn at position 76, Thr at position 77, Leu at position 78, Tyr at position 79, Gln at position 81, Ser at position 83, Thr at position 86, Ala at position 87, of SEQ ID No. 37, Glu at position 88, Ala at position 88, Val at position 92 and/or Leu.

8. A humanized anti-CD 19 antibody or antigen-binding fragment thereof comprising a light chain Variable (VL) region and a heavy chain Variable (VH) region, wherein the VL region comprises a VL Complementarity Determining Region (CDR)1(VL CDR1), a VL CDR2 and a VL CDR3 and the VH region comprises a VH CDR1, a VL CDR2 and a VL CDR3,

Wherein the VL region has an amino acid sequence that is at least 85% identical to SEQ ID NO 14, 20, 11 or 17; and/or

Wherein the VH region has an amino acid sequence at least 85% identical to SEQ ID NO 15, 21, 12 or 18.

9. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of: IgG, Fab ', F (ab') 2, Fv, scFv, and single domain antibodies (dAB).

10. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 9, wherein the antibody or antigen-binding fragment thereof is an scFv.

11. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 10, wherein the VL CDR1 is at least 80% identical to SEQ ID No. 27, the VL CDR2 is at least 80% identical to SEQ ID No. 28, and the VL CDR3 is at least 80% identical to SEQ ID No. 29.

12. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 10 or claim 11, wherein the VL CDR1 comprises SEQ ID No. 27, the VL CDR2 comprises SEQ ID No. 28, and the VL CDR3 comprises SEQ ID No. 29.

13. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of any one of claims 10 to 12, wherein the VH CDR1 is at least 80% identical to SEQ ID No. 30 or 33, the VH CDR2 is at least 80% identical to SEQ ID No. 31 or 34, and the VH CDR3 is at least 80% identical to SEQ ID No. 32.

14. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 13, wherein the VH CDR1 comprises SEQ ID No. 30 or 33, the VH CDR2 comprises SEQ ID No. 31 or 34, and the VH CDR3 comprises SEQ ID No. 32.

15. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 10, wherein said VL CDR1 is at least 80% identical to SEQ ID No. 27, said VL CDR2 is at least 80% identical to SEQ ID No. 28, said VL CDR3 is at least 80% identical to SEQ ID No. 29, said VH CDR1 is at least 80% identical to SEQ ID No. 30 or 33, said VH CDR2 is at least 80% identical to SEQ ID No. 31 or 34, and said VH CDR3 is at least 80% identical to SEQ ID No. 32.

16. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 15, wherein the VL CDR1 comprises SEQ ID No. 27, the VL CDR2 comprises SEQ ID No. 28, the VL CDR3 comprises SEQ ID No. 29, the VH CDR1 comprises SEQ ID No. 30 or 33, the VH CDR2 comprises SEQ ID No. 31 or 34, and the VH CDR3 comprises SEQ ID No. 32.

17. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 16, wherein the VL CDR1 comprises SEQ ID No. 27, the VL CDR2 comprises SEQ ID No. 28, the VL CDR3 comprises SEQ ID No. 29, the VH CDR1 comprises SEQ ID No. 30, the VH CDR2 comprises SEQ ID No. 31, and the VH CDR3 comprises SEQ ID No. 32.

18. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 16, wherein the VL CDR1 comprises SEQ ID No. 27, the VL CDR2 comprises SEQ ID No. 28, the VL CDR3 comprises SEQ ID No. 29, the VH CDR1 comprises SEQ ID No. 33, the VH CDR2 comprises SEQ ID No. 34, and the VH CDR3 comprises SEQ ID No. 32.

19. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 16, wherein the VL CDR1 comprises SEQ ID No. 27, the VL CDR2 comprises SEQ ID No. 28, the VL CDR3 comprises SEQ ID No. 29, the VH CDR1 comprises SEQ ID No. 30, the VH CDR2 comprises SEQ ID No. 34, and the VH CDR3 comprises SEQ ID No. 32.

20. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of any one of claims 1 to 19, wherein the VL is at least 85% identical to SEQ ID No. 14.

21. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 20, wherein the VH is at least 85% identical to SEQ ID No. 15.

22. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 21, wherein the VL is at least 85% identical to SEQ ID No. 14 and the VH is at least 85% identical to SEQ ID No. 15.

23. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 22, wherein the VL is at least 90% identical to SEQ ID No. 14 and the VH is at least 90% identical to SEQ ID No. 15.

24. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 23, wherein the VL is at least 95% identical to SEQ ID No. 14 and the VH is at least 95% identical to SEQ ID No. 15.

25. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 24, wherein the VL is at least 99% identical to SEQ ID No. 14 and the VH is at least 99% identical to SEQ ID No. 15.

26. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 25, wherein the VL comprises SEQ ID No. 14 and the VH comprises SEQ ID No. 15.

27. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 26, wherein the polypeptide comprises SEQ ID No. 24.

28. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of any one of claims 1 to 19, wherein the VL is at least 85% identical to SEQ ID No. 20.

29. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 28, wherein the VH is at least 85% identical to SEQ ID No. 21.

30. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 29, wherein the VL is at least 85% identical to SEQ ID No. 20 and the VH is at least 85% identical to SEQ ID No. 21.

31. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 30, wherein the VL is at least 90% identical to SEQ ID No. 20 and the VH is at least 90% identical to SEQ ID No. 21.

32. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 31, wherein the VL is at least 95% identical to SEQ ID No. 20 and the VH is at least 95% identical to SEQ ID No. 21.

33. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 32, wherein the VL is at least 99% identical to SEQ ID No. 20 and the VH is at least 99% identical to SEQ ID No. 21.

34. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 33, wherein the VL comprises SEQ ID No. 20 and the VH comprises SEQ ID No. 21.

35. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 34, wherein the polypeptide comprises SEQ ID No. 26.

36. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of any one of claims 1 to 19, wherein the VL is at least 85% identical to SEQ ID No. 11.

37. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 36, wherein the VH is at least 85% identical to SEQ ID NO 12.

38. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 37, wherein the VL is at least 85% identical to SEQ ID No. 11 and the VH is at least 85% identical to SEQ ID No. 12.

39. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 38, wherein the VL is at least 90% identical to SEQ ID No. 11 and the VH is at least 90% identical to SEQ ID No. 12.

40. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 39, wherein the VL is at least 95% identical to SEQ ID NO 11 and the VH is at least 95% identical to SEQ ID NO 12.

41. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 40, wherein the VL is at least 99% identical to SEQ ID NO. 11 and the VH is at least 99% identical to SEQ ID NO. 12.

42. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 41, wherein the VL comprises SEQ ID NO 11 and the VH comprises SEQ ID NO 12.

43. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 42, wherein the polypeptide comprises SEQ ID NO 23.

44. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of any one of claims 1 to 19, wherein the VL is at least 85% identical to SEQ ID No. 17.

45. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 44, wherein the VH is at least 85% identical to SEQ ID NO 18.

46. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 45, wherein the VL is at least 85% identical to SEQ ID NO 17 and the VH is at least 85% identical to SEQ ID NO 18.

47. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 46, wherein the VL is at least 90% identical to SEQ ID NO 17 and the VH is at least 90% identical to SEQ ID NO 18.

48. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 47, wherein the VL is at least 95% identical to SEQ ID NO 17 and the VH is at least 95% identical to SEQ ID NO 18.

49. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 48, wherein the VL is at least 99% identical to SEQ ID NO 17 and the VH is at least 99% identical to SEQ ID NO 18.

50. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 49, wherein the VL comprises SEQ ID NO 17 and the VH comprises SEQ ID NO 18.

51. The humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 50, wherein the polypeptide comprises SEQ ID NO 25.

52. A polypeptide encoded by the humanized anti-CD 19 antibody or antigen-binding fragment thereof of any one of claims 1 to 51.

53. The polypeptide of claim 52, further comprising a His tag comprising the amino acid sequence of SEQ ID NO 8.

54. The polypeptide of claim 53, wherein said polypeptide is at least 85% identical to SEQ ID NO 10, 13, 16 or 19.

55. The polypeptide of claim 54, wherein the polypeptide is at least 90% identical to SEQ ID NO 10, 13, 16, or 19.

56. The polypeptide of claim 55, wherein the polypeptide is at least 95% identical to SEQ ID NO 10, 13, 16, or 19.

57. The polypeptide of claim 56, wherein said polypeptide is at least 99% identical to SEQ ID NO 10, SEQ ID NO 13, SEQ ID NO 16, or SEQ ID NO 19.

58. The polypeptide of claim 57, wherein the polypeptide comprises SEQ ID NO 10, SEQ ID NO 13, SEQ ID NO 16, or SEQ ID NO 19.

59. The polypeptide of any one of claims 52 to 58, wherein the polypeptide is linked to a therapeutic agent.

60. The polypeptide of claim 59, wherein the therapeutic agent is a chemotherapeutic agent, a cytokine, a radioactive atom, an siRNA, or a toxin.

61. The polypeptide of claim 60, wherein the therapeutic agent is a chemotherapeutic agent.

62. The polypeptide of claim 60, wherein the therapeutic agent is a radioactive atom.

63. A Chimeric Antigen Receptor (CAR) or T Cell Receptor (TCR) comprising: (i) an antigen binding domain, (ii) a co-stimulatory domain, and (iii) an activation domain,

Wherein the co-stimulatory domain comprises an extracellular domain, a transmembrane domain, and an intracellular domain, and

Wherein the antigen binding domain comprises at least the polypeptide of claim 52.

64. A CAR or TCR as claimed in claim 63 wherein the co-stimulatory domain is derived from or derived from CD, CD delta, CD epsilon, CD gamma, CD11 (ITGAL), CD11 (ITGAM), CD11 (ITGAX), CD11 (ITGAD), CD (ITGB), CD (B), CD (TNFRSF), CD (ITGB), CD (TNFRSF), CD (SLAMF), CD49 (ITGA), CD66 (CEACAM), CD (CLEC), CD79 (B cell antigen receptor complex-related alpha chain), CD79 (B cell antigen receptor complex-related beta chain), CD (SLAMF), CD (TaMA), CD100 (SEGAL 4), CD103 (GAE), CD79 (B2B), CD 1-IL 158), CD158 (KIDL 2 (KIDL 158), CD2 (KIDL 158), CD-D), CD (CD-2 (KIDL 158), CD-D), CD2 (KIDL) and CD (KIDL) 2 (KIDL 158) CD158F2(KIR2DL5B), CD158K (KIR3DL2), CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD229(SLAMF3), CD244(SLAMF4), CD247(CD3-zeta), CD258(LIGHT), CD268(BAFFR), CD270(TNFSF14), CD272(BTLA), CD276(B7-H3), CD279(PD-1), CD314(NKG2D), CD319(SLAMF D), CD335(NK-p D), CD336 (P D), CD NK (NK-p D), CD352(SLAMF D), CD353 (AMF 36353 (SLAMF D), CD355(CRTAM), CD357(TNFRSF D), induced T cell co-stimulators (ICOS), SLFA-1 (SLPA 3611/SLPF D), SLAF D, LAMG-X D, LAM D, LAMG-X D, LAM-7, LAM D, LAMMIL-7, LAM-D, LAMMIL-D, LAM-GAMMIL-D, LAM-, MHC class 2 molecules, TNF receptor proteins, immunoglobulin proteins, cytokine receptors, integrins, activating NK cell receptors, Toll ligand receptors, and fragments or combinations thereof.

65. The CAR or TCR of claim 63 or claim 64, wherein the transmembrane domain is derived or derived from 4-1BB/CD137, the alpha chain of a T cell receptor, the beta chain of a T cell receptor, CD delta, CD epsilon, CD gamma, CD α, CD β, CD11 (ITGAL), CD11 (ITGAX), CD11 (ITGAD), CD (ITGB), CD (B), CD (TNFRSF), CD (ITGB), CD (TNFRSF), CD49 (ITGA), CD66 (CEACAM), CD66 (CEACM), CD66 (CEACAM), CD (CLEC), CD79 (B cell antigen receptor associated alpha complex), CD79 (B cell antigen receptor associated complex), CD79 (beta chain antigen receptor), CD 4-GAI), CD-100 (TAC), CD-I, CD-E, CD-103, CD-5 (ITGAX), CD-E, CD-III, CD-, CD150(SLAMF1), CD158A (KIR2DL1), CD158B1(KIR2DL2), CD158B2(KIR2DL3), CD158C (KIR3DP1), CD158D (KIRDL4), CD158F1(KIR2DL5A), CD158F2(KIR2DL5B), CD158K (KIR3DL2), CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD229(SLAMF3), CD244(SLAMF4), CD (CD 3-zezetta), CD258(LIGHT), CD268 (FFR), CD270 (TNFS3672), CD272 (36LA), CD (B3-H3), CD (PD-1), CD314 (CD2 NKG 3), CD3 (NKAMNK 355) NPA-3), CD 3-NKAFP 3, CD-3-NFK 3, CD-363672, CD-induced SLNANK-3665, CD-NFK-3, CD-induced SLNAIL (LAP), CD-3, CD-3665, CD-3, CD-363636363665, CD-3, gads (grpl), SLP-76(LCP2), PAG1/CBP, CD83 ligand, Fc gamma receptor, MHC class 1 molecules, MHC class 2 molecules, TNF receptor proteins, immunoglobulin proteins, cytokine receptors, integrins, activated NK cell receptors, Toll ligand receptors, and combinations thereof.

66. A CAR or TCR as claimed in any of claims 63 to 65 wherein the intracellular domain is derived or derived from 4-1BB/CD137, an activating NK cell receptor, B-H, BAFFR, BLAME (SLAMF), BTLA, CD100(SEMA 4), CD103, CD160 (BY), CD19, CD247, CD276 (B-H), CD delta, CD epsilon, CD gamma, CD49, CD8alpha, CD8beta, CD (Tactile), CDl la, CDl lb, CDl lc, CDl ld, CDS, CEM, ACAM, cytokine receptor, DAP-10, DNAM (CD226), Fc gamma receptor, GADS, GITR, EM (LIGHT), IA, ICAM-1, Ig-alpha, Ig-2, IL2 alpha, IL2, IL 7-like protein (IL-7, IL2, Integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGBl, KIRDS2, LAT, LFA-1, a ligand that specifically binds to CD83, LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), LTBR, Ly9(CD229), lymphocyte function-associated antigen-1 (LFA-1(CDl la/CD18), MHC class I molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80(KLRF1), OX-40, PAG/Cbp, programmed death 1(PD-1), PSGL1, SELPLG (CD162), signal transduction lymphocyte activation molecules (SLAM protein), SLAM (SLAMF 1; CD 150; IPO-3), SLAMF4(CD 244; 2B4) SLAMF6 (NTB-a; lyl08), SLAMF7, SLP-76, TNF receptor protein, TNFR2, Toll ligand receptor, TRANCE/RANKL, VLA1 or VLA-6, or combinations thereof.

67. A CAR or TCR as claimed in any of claims 63 to 66 wherein the extracellular domain is derived or derived from CD, CD delta, CD epsilon, CD gamma, CD α, CD β, CD11 (ITGAL), CD11 (ITGAM), CD11 (ITGAX), CD11 (ITGAD), CD (ITGB), CD (B), CD (TNFRSF), CD (ITGB), CD (TNFRSF), CD (SLAMF), CD49 (ITGA), CD66 (CEACAM), CD79 (B cell antigen receptor complex related chain), CD (SLbetef), CD (TaAMMA) 100 (SEC 4), CD100 (SEC), CD150 (SEDL), CD2 (KIDL 158), CD3 (CD) 2-IL 2(CD 158), CD-D158), CD3 (KIDL 158), CD-D2 (KIDL 158), CD2 (KIDL) 2 (KIDL 158), CD2 (CD-D158), CD-D2 (ITGA), CD-E), CD-D2 (ITGA), CD-D) and CD (ITGA), CD, CD158D (KIRDL D), CD158F D (KIR2DL 5D), CD158D (KIR3DL D), CD160(BY D), CD162(SELPLG), CD226(DNAM D), CD229(SLAMF D), CD244(SLAMF D), CD247(CD D-zeta), CD258(LIGHT), CD268(BAFFR), CD270(TNFSF D), CD272(BTLA), CD276(B D-H D), CD279(PD-1), CD314(NKG2D), CD319(SLAMF D), CD335(NK-p D), CD336(NK-p D), CD337(NK-p D), CD352(SLAMF D), CD353(SLAMF D), CD355 SLLFM (CRNK-p D), CD336 (CRNK-p D), CD D, LAMIC-D, LAMC-D, LAMIC-7, LAMC-D, LAMIC-D, LAMC-D, LAMIC-D, LAMC-7, LAMIC-7, MHC class 1 molecules, MHC class 2 molecules, TNF receptor proteins, immunoglobulin proteins, cytokine receptors, integrins, activating NK cell receptors, Toll ligand receptors, and fragments or combinations thereof.

68. The CAR or TCR of any of claims 63 to 67, wherein the activation domain is from or derived from CD3-zeta or CD 3-episilon.

69. A humanized anti-CD 19 antibody or antigen-binding fragment thereof encoding the CAR or TCR of any one of claims 63-68.

70. A vector comprising the humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 69.

71. The vector of claim 70, wherein said vector is an adenoviral vector, an adeno-associated vector, a DNA vector, a lentiviral vector, a plasmid, a retroviral vector, or an RNA vector.

72. The vector of claim 71, wherein the vector is a retroviral vector.

73. The vector of claim 72, wherein the retroviral vector is a lentiviral vector.

74. A Chimeric Antigen Receptor (CAR) or T Cell Receptor (TCR) encoded by the vector of any one of claims 70 to 73.

75. A cell comprising the humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 69, the vector of any one of claims 70 to 73, or the Chimeric Antigen Receptor (CAR) or T Cell Receptor (TCR) of any one of claims 63 to 67 or claim 74.

76. The cell of claim 75, wherein said cell is a T cell.

77. The cell of claim 76, wherein said T cell is an allogeneic T cell, an autologous T cell, an engineered autologous T cell (eACT), or a tumor-infiltrating lymphocyte (TIL).

78. The cell of claim 77, wherein said T cell is a CD4+ T cell.

79. The cell of claim 77, wherein said T cell is a CD8+ T cell.

80. The cell of any one of claims 75 to 79, wherein the cell is an in vitro cell.

81. The cell of any one of claims 76 to 80, wherein the T cell is an autologous T cell.

82. the cell of any one of claims 75 to 81, wherein said cell produces at least interferon gamma (IFN γ) upon binding to human CD 19.

83. A composition comprising a plurality of the cells of any one of claims 75-82.

84. The composition of claim 83, wherein said composition comprises CD4+ or CD8+ cells.

85. The composition of claim 84, wherein said composition comprises CD4+ and CD8+ cells.

86. The composition of claim 85, wherein between about 20% and 80% of said T cells are CD4+ cells and the remainder of said T cells are CD8+ cells.

87. The composition of claim 86, wherein between about 30% and 70% of said T cells are CD4+ cells and the remainder of said T cells are CD8+ cells.

88. the composition of claim 87, wherein between about 40% and 60% of said T cells are CD4+ cells and the remainder of said T cells are CD8+ cells.

89. The composition of claim 88, wherein about 50% of said T cells are CD4+ cells and about 50% of said T cells are CD8+ cells.

90. The composition of claim 85, wherein between about 20% and 80% of said T cells are CD8+ cells and the remainder of said T cells are CD4+ cells.

91. The composition of claim 90, wherein between about 30% and 70% of said T cells are CD8+ cells and the remainder of said T cells are CD4+ cells.

92. The composition of claim 91, wherein between about 40% and 60% of said T cells are CD8+ cells and the remainder of said T cells are CD4+ cells.

93. The composition of any one of claims 77-79, wherein each cell of said plurality of cells is an autologous T cell.

94. The composition of any one of claims 83 to 92, wherein said composition comprises at least one pharmaceutically acceptable excipient.

95. A composition comprising the humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 69, the vector of any one of claims 70 to 73, or the Chimeric Antigen Receptor (CAR) or T Cell Receptor (TCR) of any one of claims 63 to 67 or claim 74.

96. A method for making a cell expressing a Chimeric Antigen Receptor (CAR) or a T Cell Receptor (TCR), comprising the step of transducing a cell with the humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 69 or the vector of any one of claims 70 to 67.

97. The method of claim 96, wherein the cells are lymphocytes isolated from a patient in need of treatment.

98. The method of claim 97, wherein said lymphocyte is a natural killer cell, a T cell, or a B cell.

99. The method of any one of claims 96 to 98, further comprising the step of culturing the cell under conditions that promote cell proliferation and/or T cell activation.

100. The method of any one of claims 96 to 99, further comprising the step of isolating the desired T cell.

101. The method of claim 100, wherein the step of isolating the desired T cells occurs after about six days of culture.

102. The method of claim 101, wherein the desired T cells express CD4+ and/or CD8 +.

103. A method for treating a B-cell lymphoma comprising administering to a subject in need thereof the cell of any one of claims 75 to 82 or the composition of any one of claims 83 to 95.

104. The method of claim 103, wherein the B cell lymphoma is selected from the group consisting of: acute Lymphoblastic Leukemia (ALL), AIDS-related lymphoma, ALK-positive large B-cell lymphoma, Burkitt's lymphoma, Chronic Lymphocytic Leukemia (CLL), classical Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, intravascular large B-cell lymphoma, HHV 8-related large B-cell lymphoma caused by multicenter Castleman's disease, lymphomatoid granuloma, lymphoplasmacytic lymphoma, Mantle Cell Lymphoma (MCL), marginal zone B cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), lymph node marginal zone B cell lymphoma (NMZL), nodal lymphocyte predominant hodgkin lymphoma, non-hodgkin lymphoma, plasmablatic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, Spleen Marginal Zone Lymphoma (SMZL), and waldenstrom's macroglobulinemia.

105. The method of claim 104, wherein said B cell lymphoma is selected from the group consisting of: acute Lymphoblastic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, Mantle Cell Lymphoma (MCL), marginal zone B-cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), and non-Hodgkin's lymphoma.

106. The method of claim 105, wherein said B cell lymphoma is non-hodgkin's lymphoma.

Background

Human cancers by their very nature comprise normal cells that have undergone genetic or epigenetic transformation to become abnormal cancer cells. At this point, the cancer cells begin to express proteins and other antigens and/or proteins that are different from those expressed by normal cells in amounts significantly greater than those expressed by normal cells.

Chimeric Antigen Receptors (CARs) and engineered T Cell Receptors (TCRs) comprising a binding domain capable of interacting with cancer cell-expressed proteins, allowing T cells to target and kill cancer cells expressing specific proteins. Similarly, antibodies conjugated to therapeutic agents are capable of selectively providing therapeutic agents to and killing cancer cells expressing particular proteins.

There is a need for CARs, TCRs, and antibodies comprising humanized antigen binding domains for targeting and killing cancer cells.

Summary of The Invention

The present invention meets this need by providing compositions and methods comprising engineered immune cells that express a Chimeric Antigen Receptor (CAR) or a T Cell Receptor (TCR) with a humanized antigen binding domain; these CARs and TCRs specifically target and kill cancer cells. The present invention also meets this need by providing compositions and methods that include antibodies that specifically target and kill cancer cells.

One aspect of the invention is a humanized anti-CD 19 antibody or antigen-binding fragment thereof. The antibody comprises a light chain Variable (VL) region and a heavy chain Variable (VH) region, wherein the VL region comprises a VL Complementarity Determining Region (CDR)1(VL CDR1), VL CDR2, and VL CDR3 and the VH region comprises a VH CDR1, VL CDR2, and VL CDR 3. The VL region may be derived from SEQ ID NO 36 and the VH region derived from SEQ ID NO 37, and the VL and/or VH regions comprise one or more amino acid substitutions in the framework regions.

In some embodiments, the humanized anti-CD 19 antibody or antigen-binding fragment thereof of claim 1, wherein the VL region comprises up to 5, 10, 15, 20, 25, or 30 amino acid substitutions as compared to SEQ ID No. 36.

In some embodiments, one or more amino acid substitutions in the VL region are at positions corresponding to 7, 8, 10, 15, 22, 41, 42, 43, 44, 49, 71, 72, 77, 79, 80, 83, 87, 100, and/or 107 of SEQ ID NO: 36.

In some embodiments, the one or more amino acid substitutions in the VL region are selected from Ser at position 7, Pro at position 8, Val at position 15, Thr at position 22, Gln at position 41, Lys at position 42, Ala at position 43, Thr at position 72, Ser at position 77, Gln at position 79, Pro at position 80, and/or Lys at position 107 of SEQ ID NO: 36.

In some embodiments, the VH region comprises up to 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid substitutions as compared to SEQ ID No. 37.

In some embodiments, one or more amino acid substitutions in the VH region are at positions corresponding to 1, 3, 5, 9, 13, 15, 16, 17, 19, 20, 21, 23, 24, 37, 42, 48, 67, 69, 70, 71, 73, 76, 77, 78, 79, 81, 83, 86, 87, 88, 92, and/or 115 of SEQ ID No. 37.

In some embodiments, the one or more amino acid substitutions in the VH region are selected from SEQ ID NO: gln at position 1, Gln at position 3, Val at position 5, Gly at position 9, Lys at position 13, Gln at position 13, Gly at position 15, Arg at position 16, Thr at position 17, Arg at position 19, Leu at position 20, Ser at position 21, Ala at position 24, Gly at position 42, Ile at position 48, Phe at position 67, Ser at position 70, Arg at position 71, Thr at position 73, Asn at position 76, Thr at position 77, Leu at position 78, Tyr at position 79, Gln at position 81, Ser at position 83, Thr at position 86, Arg at position 86, Ala at position 87, Glu at position 88, Ala at position 88, Val at position 92, and/or Leu at position 92 of 37.

Another aspect of the invention is a humanized anti-CD 19 antibody or antigen-binding fragment thereof comprising a light chain Variable (VL) region and a heavy chain Variable (VH) region. The VL region comprises VL Complementarity Determining Regions (CDR)1(VL CDR1), VL CDR2 and VL CDR3 and the VH region comprises VH CDR1, VL CDR2 and VL CDR 3. The VL region has an amino acid sequence which is at least 85% identical to SEQ ID NO 14, 20, 11 or 17; and/or the VH region has an amino acid sequence at least 85% identical to SEQ ID NO 15, 21, 12 or 18.

In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of: IgG, Fab ', F (ab') 2, Fv, scFv, and single domain antibodies (dAB). In some embodiments, the antibody or antigen-binding fragment thereof is an scFv.

In some embodiments, VL CDR1 is at least 80% identical to SEQ ID No. 27, VL CDR2 is at least 80% identical to SEQ ID No. 28, and VL CDR3 is at least 80% identical to SEQ ID No. 29.

In some embodiments, VL CDR1 comprises SEQ ID No. 27, VL CDR2 comprises SEQ ID No. 28, and VL CDR3 comprises SEQ ID No. 29.

In some embodiments, VH CDR1 is at least 80% identical to SEQ ID No. 30 or 33, VH CDR2 is at least 80% identical to SEQ ID No. 31 or 34, and VH CDR3 is at least 80% identical to SEQ ID No. 32.

In some embodiments, the VH CDR1 comprises SEQ ID No. 30 or 33, VH CDR2 comprises SEQ ID No. 31 or 34, and VH CDR3 comprises SEQ ID No. 32.

In some embodiments, VL CDR1 is at least 80% identical to SEQ ID No. 27, VL CDR2 is at least 80% identical to SEQ ID No. 28, VL CDR3 is at least 80% identical to SEQ ID No. 29, VH CDR1 is at least 80% identical to SEQ ID No. 30 or 33, VH CDR2 is at least 80% identical to SEQ ID No. 31 or 34, and VH CDR3 is at least 80% identical to SEQ ID No. 32.

In some embodiments, VL CDR1 comprises SEQ ID No. 27, VL CDR2 comprises SEQ ID No. 28, VL CDR3 comprises SEQ ID No. 29, VH CDR1 comprises SEQ ID No. 30 or 33, VH CDR2 comprises SEQ ID No. 31 or 34, and VH CDR3 comprises SEQ ID No. 32.

In some embodiments, VL CDR1 comprises SEQ ID No. 27, VL CDR2 comprises SEQ ID No. 28, VL CDR3 comprises SEQ ID No. 29, VH CDR1 comprises SEQ ID No. 30, VH CDR2 comprises SEQ ID No. 31, and VH CDR3 comprises SEQ ID No. 32.

In some embodiments, VL CDR1 comprises SEQ ID No. 27, VL CDR2 comprises SEQ ID No. 28, VL CDR3 comprises SEQ ID No. 29, VH CDR1 comprises SEQ ID No. 33, VH CDR2 comprises SEQ ID No. 34, and VH CDR3 comprises SEQ ID No. 32.

In some embodiments, VL CDR1 comprises SEQ ID No. 27, VL CDR2 comprises SEQ ID No. 28, VL CDR3 comprises SEQ ID No. 29, VH CDR1 comprises SEQ ID No. 30, VH CDR2 comprises SEQ ID No. 34, and VH CDR3 comprises SEQ ID No. 32.

In some embodiments, the VL is at least 85% identical to SEQ ID NO: 14.

In some embodiments, the VH is at least 85% identical to SEQ ID NO 15.

In some embodiments, the VL is at least 85% identical to SEQ ID NO. 14 and the VH is at least 85% identical to SEQ ID NO. 15. In some embodiments, the VL is at least 90% identical to SEQ ID NO. 14 and the VH is at least 90% identical to SEQ ID NO. 15. In some embodiments, the VL is at least 95% identical to SEQ ID NO. 14 and the VH is at least 95% identical to SEQ ID NO. 15. In some embodiments, the VL is at least 99% identical to SEQ ID NO. 14 and the VH is at least 99% identical to SEQ ID NO. 15. In some embodiments, the VL comprises SEQ ID NO. 14 and the VH comprises SEQ ID NO. 15.

In some embodiments, the polypeptide comprises SEQ ID NO 24.

In some embodiments, the VL is at least 85% identical to SEQ ID NO: 20.

In some embodiments, the VH is at least 85% identical to SEQ ID NO 21.

In some embodiments, the VL is at least 85% identical to SEQ ID NO:20 and the VH is at least 85% identical to SEQ ID NO: 21. In some embodiments, the VL is at least 90% identical to SEQ ID NO:20 and the VH is at least 90% identical to SEQ ID NO: 21. In some embodiments, the VL is at least 95% identical to SEQ ID NO 20 and the VH is at least 95% identical to SEQ ID NO 21. In some embodiments, the VL is at least 99% identical to SEQ ID NO:20 and the VH is at least 99% identical to SEQ ID NO: 21. In some embodiments, the VL comprises SEQ ID NO 20 and the VH comprises SEQ ID NO 21.

In some embodiments, the polypeptide comprises SEQ ID NO 26.

In some embodiments, the VL is at least 85% identical to SEQ ID NO: 11.

In some embodiments, the VH is at least 85% identical to SEQ ID NO 12.

In some embodiments, the VL is at least 85% identical to SEQ ID NO. 11 and the VH is at least 85% identical to SEQ ID NO. 12. In some embodiments, the VL is at least 90% identical to SEQ ID NO 11 and the VH is at least 90% identical to SEQ ID NO 12. In some embodiments, the VL is at least 95% identical to SEQ ID NO 11 and the VH is at least 95% identical to SEQ ID NO 12. In some embodiments, the VL is at least 99% identical to SEQ ID NO 11 and the VH is at least 99% identical to SEQ ID NO 12. In some embodiments, the VL comprises SEQ ID NO 11 and the VH comprises SEQ ID NO 12.

In some embodiments, the polypeptide comprises SEQ ID NO 23.

In some embodiments, the VL is at least 85% identical to SEQ ID NO 17.

In some embodiments, the VH is at least 85% identical to SEQ ID NO 18.

In some embodiments, the VL is at least 85% identical to SEQ ID NO 17 and the VH is at least 85% identical to SEQ ID NO 18. In some embodiments, the VL is at least 90% identical to SEQ ID NO 17 and the VH is at least 90% identical to SEQ ID NO 18. In some embodiments, the VL is at least 95% identical to SEQ ID NO 17 and the VH is at least 95% identical to SEQ ID NO 18. In some embodiments, the VL is at least 99% identical to SEQ ID NO 17 and the VH is at least 99% identical to SEQ ID NO 18. In some embodiments, the VL comprises SEQ ID NO 17 and the VH comprises SEQ ID NO 18.

In some embodiments, the polypeptide comprises SEQ ID NO 25.

Another aspect of the invention is a polypeptide encoded by the humanized anti-CD 19 antibody of the above aspects or embodiments.

In some embodiments, the polypeptide comprises a His tag comprising the amino acid sequence of SEQ ID NO 8.

In some embodiments, the polypeptide is at least 85% identical to SEQ ID NO 10, 13, 16, or 19. In some embodiments, the polypeptide is at least 90% identical to SEQ ID NO 10, 13, 16, or 19. In some embodiments, the polypeptide is at least 95% identical to SEQ ID NO 10, 13, 16, or 19. In some embodiments, the polypeptide is at least 99% identical to SEQ ID NO 10, 13, 16, or 19. In some embodiments, the polypeptide comprises SEQ ID NO 10, SEQ ID NO 13, SEQ ID NO 16, or SEQ ID NO 19.

In some embodiments, the polypeptide is linked to a therapeutic agent. In some embodiments, the therapeutic agent is a chemotherapeutic agent, a cytokine, a radioactive atom, an siRNA, or a toxin. In some embodiments, the therapeutic agent is a chemotherapeutic agent.

In some embodiments, the therapeutic agent is a radioactive atom.

Yet another aspect of the invention is a Chimeric Antigen Receptor (CAR) or T Cell Receptor (TCR). The CAR or TCR comprises: (i) an antigen binding domain, (ii) a co-stimulatory domain, and (iii) an activation domain. The co-stimulatory domain comprises an extracellular domain, a transmembrane domain, and an intracellular domain, and the antigen-binding domain comprises at least the polypeptide of claim 52.

In some embodiments, the co-stimulatory domain is from or derived from CD, CD delta, CD3epsilon, CD gamma, CD α, CD β, CD11 (ITGAL), CD11 (ITGAM), CD11 (ITGAX), CD11 (ITGAD), CD (ITGB), CD (B), CD (TNFRSF), CD (ITGB), CD (TNFRSF), CD (SLAMF), CD49 (ITGA), CD66 (CEACAM), CD (CLEC), CD79 (B cell antigen receptor complex-related alpha chain), CD79 (B cell antigen receptor complex-related beta chain), CD (SLAMF), CD (tamaf), CD (tama), CD100 (secti 4), CD103 (CD 103), CD150 (B cell antigen receptor complex-related alpha chain), CD 158), CD5 (tdl 158), CD158 (CD 158), CD2 (tdl 158), CD158 (CD 158), CD158 (tdl), CD158 (r 158), CD2 (r 158), CD1 DL), CD (tdl), CD158 (tdl), CD1 (e), CD158 (k 2 (e), CD 158), CD5 (e), CD (k 2 (e), CD 158), CD (e), CD1 (e), CD 158), CD, CD158K (KIR3DL2), CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD229(SLAMF3), CD244(SLAMF4), CD247(CD3-zeta), CD258(LIGHT), CD268(BAFFR), CD270(TNFSF14), CD272(BTLA), CD276(B7-H3), CD279(PD-1), CD314(NKG2D), CD319(SLAMF7), CD335(NK-p46), CD336(NK-p44), CD337(NK-p30), CD352(SLAMF6), CD353(SLAMF8), CD355(CRTAM), CD 36357 (TNFRSF18), inducible T cell costimulator (ICOS), LFA-1(CD 11/a/CD a), NKG2, SLPG 72, LCP 72-a, MHC 14 (LAMMA-a), MHC ligand, SLAMF a-a, MHC ligand, LAMMIL a, SLIP a, SLAMF a, SLIP a-a, SLIL a, SLIP-a, SLIP-a, SLE 7, TNF receptor proteins, immunoglobulin proteins, cytokine receptors, integrins, activating NK cell receptors, Toll ligand receptors, and fragments or combinations thereof.

In some embodiments, the transmembrane domain is derived or derived from 4-1BB/CD137, the alpha chain of a T cell receptor, the beta chain of a T cell receptor, CD delta, CD epsilon, CD gamma, CD alpha, CD beta, CD11 (ITGAL), CD11 (ITGAM), CD11 (ITGAX), CD11 (ITGAD), CD (ITGB), CD (B), CD (TNFRSF), CD (ITGB), CD (TNFRSF), CD (SLAMF), CD49 (ITGA), CD66 (CEACAM), CD66 (acacem), CD66 (acam), CD (CLEC), CD79 (B cell antigen receptor complex-related alpha chain), CD79 (B cell antigen receptor complex-related CD beta), CD137 (taclf), CD150 (tacil), CD 4-slf), CD2 (ITGA), CD2 (tacil), CD2 (ITGA), CD150 (ITGA), CD2 (ITGA), CD16 (ITGA), CD15 (ITGA), CD158B (KIR2 DL), CD158 (KIR3 DP), CD158 (KIRDL), CD158F (KIR2DL 5), CD158 (KIR3 DL), CD160 (BY), CD162(SELPLG), CD226 (DNAM), CD229 (SLAMF), CD244 (SLAMF), CD247 (CD-zeta), CD258(LIGHT), CD268(BAFFR), CD270 (TNFSF), CD272(BTLA), CD276 (B-H), CD279(PD-1), CD314(NKG 2), CD319 (SLAMF), CD335 (NK-p), CD336 (NK-p), CD337 (SLNK-p), CD352 (AMF), CD353 (SLAMF), CD355 (CRM), CD357 (RSF), TNFR, LFT cell co-stimulators (LFT), SLPA-1 (SLPS-OS-1, LAM-11/LAMS-76, LAMG-P, LAMG-7 (LAMG-L), LAMG-7 (LAMG-1, LAMG-7, LAMG-L, LAMG-1, LAMG (LAMG-1, LAMG-7, LAMG-L), CD-1, LAMG (LAMG-1, LAMG-7, LAMG-L, PAG1/CBP, CD83 ligand, Fc gamma receptor, MHC class 1 molecules, MHC class 2 molecules, TNF receptor proteins, immunoglobulin proteins, cytokine receptors, integrins, activated NK cell receptors, Toll ligand receptors, and combinations thereof.

In some embodiments, the intracellular domain is derived or derived from 4-1BB/CD137, activating NK cell receptor, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEA 4D), CD103, CD160(BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276(B7-H3), CD28, CD29, CD3delta, CD3epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8alpha, CD8beta, CD84 (Tactile), CDlb la, CDl, CDlcl, CDld, CDI 36ld, CECRT 72, ACAM 72, CDNA 72, CD8alpha, CD84, GAMMA 84, GAITEM 84, GAI-IL 72, GAI 84, GAI-7, GAI-II receptor, GAI 84, CD84, GAI 7, CD84, GAI 7, CD84, CD 36, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGBl, KIRDS2, LAT, LFA-1, a ligand that specifically binds to CD83, LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), LTBR, Ly9(CD229), lymphocyte function-associated antigen-1 (LFA-1(CDl la/CD18), MHC class I molecules, NKG2C, NKG2D, NKp 6327, NKp44, NKp46, NKp80(KLRF1), OX-40, PAG/Cbp, programmed death 1(PD-1), PSGL1, SELPLG (CD162), signal transduction lymphocyte activation molecules (SLAM protein), SLAM (AMSLAF 1; CD 150; IPO-3), VLAMF-5 (VLAMF-1), VLGL 1, SELL 6324, TNFR 596, TNFR-599, or a receptor combination thereof.

In some embodiments, the extracellular domain is from or derived from CD, CD delta, CD epsilon, CD gamma, CD alpha, CD beta, CD11 (ITGAL), CD11 (ITGAM), CD11 (ITGAX), CD11 (ITGAD), CD (ITGB), CD (B), CD (TNFRSF), CD (ITGB), CD (TNFRSF), CD (SLAMF), CD49 (ITGA), CD66 (CEACAM), CD66 (acacem), CD (CLEC), CD79 (B cell antigen receptor complex-related alpha chain), CD79 (B cell antigen receptor complex-related beta chain), CD (SLAMF), CD (tale), CD100 (secti 4), CD103 (itgabe), CD150 (slb), CD5 (sldl), CD2 (sldl), CD158 (CD 158), CD158 (sldl), CD2 (sldl), CD158 (sldl), CD2 (k), CD158 (k), CD2 (sldl), CD158 (k), CD (k 2 (k), CD158 (k), CD1 (sldl, CD158K (KIR3DL2), CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD229(SLAMF3), CD244(SLAMF4), CD247(CD3-zeta), CD258(LIGHT), CD268(BAFFR), CD270(TNFSF14), CD272(BTLA), CD276(B7-H3), CD279(PD-1), CD314(NKG2D), CD319(SLAMF7), CD335(NK-p46), CD336(NK-p44), CD337(NK-p30), CD352(SLAMF6), CD353(SLAMF8), CD355(CRTAM), CD 36357 (TNFRSF18), inducible T cell costimulator (ICOS), LFA-1(CD 11/a/CD a), NKG2, SLPG 72, LCP 72-a, MHC 14 (LAMMA-a), MHC ligand, SLAMF a-a, MHC ligand, LAMMIL a, SLIP a, SLAMF a, SLIP a-a, SLIL a, SLIP-a, SLIP-a, SLE 7, TNF receptor proteins, immunoglobulin proteins, cytokine receptors, integrins, activating NK cell receptors, Toll ligand receptors, and fragments or combinations thereof.

In some embodiments, the activation domain is from or derived from CD3-zeta or CD 3-episilion.

One aspect of the invention is a humanized anti-CD 19 antibody or antigen-binding fragment thereof that encodes a CAR or TCR of the above aspects or embodiments.

Yet another aspect of the invention is a vector comprising the humanized anti-CD 19 antibody or antigen-binding fragment thereof of the above aspects or embodiments.

In some embodiments, the vector is an adenoviral vector, an adeno-associated vector, a DNA vector, a lentiviral vector, a plasmid, a retroviral vector, or an RNA vector. In some embodiments, the vector is a retroviral vector. In some embodiments, the retroviral vector is a lentiviral vector.

Another aspect of the invention is a Chimeric Antigen Receptor (CAR) or T Cell Receptor (TCR) encoded by the vector of the above aspects or embodiments.

Yet another aspect of the invention is a cell comprising the humanized anti-CD 19 antibody or antigen-binding fragment thereof of the above aspects or embodiments, the vector of the above aspects or embodiments, or the Chimeric Antigen Receptor (CAR) or T Cell Receptor (TCR) of the above aspects or embodiments.

In some embodiments, the cell is a T cell.

In some embodiments, the T cell is an allogeneic T cell, an autologous T cell, an engineered autologous T cell (eACT), or a Tumor Infiltrating Lymphocyte (TIL).

In some embodiments, the T cell is a CD4+ T cell.

In some embodiments, the T cell is a CD8+ T cell.

In some embodiments, the cell is an in vitro cell.

In some embodiments, the T cell is an autologous T cell.

In some embodiments, the cell produces at least interferon gamma (IFN γ) upon binding to human CD 19.

One aspect of the invention is a composition comprising a plurality of cells of the above aspects or embodiments.

In some embodiments, the composition comprises CD4+ or CD8+ cells. In some embodiments, the composition comprises CD4+ and CD8+ cells. In some embodiments, between about 20% and 80% of the T cells are CD4+ cells and the remainder of the T cells are CD8+ cells. In some embodiments, between about 30% and 70% of the T cells are CD4+ cells and the remainder of the T cells are CD8+ cells. In some embodiments, between about 40% and 60% of the T cells are CD4+ cells and the remainder of the T cells are CD8+ cells. In some embodiments, about 50% of the T cells are CD4+ cells and about 50% of the T cells are CD8+ cells.

In some embodiments, each cell of the plurality of cells is an autologous T cell.

In some embodiments, the composition comprises at least one pharmaceutically acceptable excipient.

Another aspect of the invention is a composition comprising the humanized anti-CD 19 antibody or antigen-binding fragment thereof of the above aspects or embodiments, a vector of the above aspects or embodiments, or a Chimeric Antigen Receptor (CAR) or T Cell Receptor (TCR) of the above aspects or embodiments.

Yet another aspect of the invention is a method for making a cell expressing a Chimeric Antigen Receptor (CAR) or a T Cell Receptor (TCR). The method comprises the step of transducing a cell with the humanized anti-CD 19 antibody or antigen-binding fragment thereof of the above aspects or embodiments or the vector of the above aspects or embodiments.

In some embodiments, the cell is a lymphocyte isolated from a patient in need of treatment.

In some embodiments, the lymphocyte is a natural killer cell, a T cell, or a B cell.

In some embodiments, the method further comprises the step of culturing the cells under conditions that promote cell proliferation and/or T cell activation.

In some embodiments, the method further comprises the step of isolating the desired T cell.

In some embodiments, the step of isolating the desired T cells occurs after about six days of culture.

In some embodiments, the desired T cells express CD4+ and/or CD8 +.

Yet another aspect of the invention is a method for treating a B-cell lymphoma comprising administering to a subject in need thereof a cell of the above aspect or embodiment or a composition of the above aspect or embodiment.

In some embodiments, the B cell lymphoma is selected from the group consisting of: acute Lymphoblastic Leukemia (ALL), AIDS-related lymphoma, ALK-positive large B-cell lymphoma, Burkitt's lymphoma, Chronic Lymphocytic Leukemia (CLL), classical Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, intravascular large B-cell lymphoma, HHV 8-related large B-cell lymphoma caused by multicenter Castleman's disease, lymphomatoid granuloma, lymphoplasmacytic lymphoma, Mantle Cell Lymphoma (MCL), marginal zone B cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), lymph node marginal zone B cell lymphoma (NMZL), nodal lymphocyte predominant hodgkin lymphoma, non-hodgkin lymphoma, plasmablatic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, Spleen Marginal Zone Lymphoma (SMZL), and waldenstrom's macroglobulinemia. In some embodiments, the B cell lymphoma is selected from the group consisting of: acute Lymphoblastic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, Mantle Cell Lymphoma (MCL), marginal zone B-cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), and non-Hodgkin's lymphoma. In some embodiments, the B cell lymphoma is non-hodgkin's lymphoma.

In general, the present invention relates to engineered autologous cell therapy (abbreviated "eACTTM", also known as adoptive cell transfer eACTTM), which is a process of collecting the patient's own T cells, which are then genetically engineered to recognize and target one or more antigens expressed on the cell surface of one or more specific cancers. T cells can be engineered to express, for example, a Chimeric Antigen Receptor (CAR) or a T Cell Receptor (TCR). See fig. 1A, 1B and 2. CAR positive (CAR +) T cells are engineered to express CAR. The CAR can comprise, for example, a humanized single-chain variable fragment (scFv) specific for a particular tumor antigen, such as human CD 19. The scFV may be linked directly or indirectly to an intracellular signaling moiety comprising at least one co-stimulatory domain, which in turn is linked directly or indirectly to at least one activating domain. The components of the CAR may be arranged in any order. Examples of CAR T cell therapies and constructs are described in U.S. patent publication nos. 2013/0287748, 2014/0227237, 2014/0099309, and 2014/0050708; U.S. provisional application nos. 62/470,703 and 62/317,258; international patent publication nos. WO2012033885, WO2012079000, WO2014127261, WO2014186469, WO2015080981, WO2015142675, WO2016044745 and WO 2016090369; and Rosenberg and Restifo, Cancer Immunology and Immunotherapy,348:62-68(2015), each of which is incorporated by reference in its entirety.

In addition, the present invention relates generally to humanized single chain variable fragments (scFv) specific for human CD 19. A humanized scFV anti-human CD19scFV can be conjugated (e.g., linked) to a therapeutic agent (e.g., a chemotherapeutic agent and a radioactive atom) to bind to the cancer cells, thereby delivering the therapeutic agent to the cancer cells and killing the cancer cells expressing human CD 19.

Any aspect or embodiment described herein may be combined with any other aspect or embodiment as disclosed herein. While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

The patent and scientific literature referred to herein establishes knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, dictionaries, documents, manuscripts, and scientific literature cited herein are hereby incorporated by reference.

Other features and advantages of the invention will be apparent from the accompanying drawings and from the following detailed description of the invention, including the embodiments, and from the claims.

Brief Description of Drawings

The above described and other features will be more clearly understood from the following detailed description when taken in conjunction with the accompanying drawings. However, these drawings are for illustration purposes only; and are not intended to be limiting.

Fig. 1A and 1B are caricatures depicting features of Chimeric Antigen Receptor (CAR) manufacture and use. Figure 1A shows exemplary polynucleotides encoding a CAR, viral vectors comprising polynucleotides encoding a CAR, transduction of the viral vectors into T cells of a patient, integration into the host genome, and expression of the CAR on the surface of transduced ("CAR-engineered") T cells. Figure 1B shows CAR engineered T cells that have recognized a target antigen located on the surface of cancer cells. Recognition and binding of the target antigen activates mechanisms in T cells, including cytolytic activity, cytokine release, and T cell proliferation; these mechanisms promote killing of cancer cells.

Figure 2 is a cartoon showing the major steps performed during engineered autologous cell therapy (eACTTM).

Fig. 3A and 3B are graphs showing flow cytometry data for seven selected humanized scfvs binding to Raji (CD19+ human burkitt lymphoma) cells. RS, CS, BS, SS, JS, AS-R and NS represent the inventors' internal naming convention for humanized antibodies; WT represents a wild type.

Fig. 4A to 4D are graphs showing Size Exclusion Chromatography (SEC) analysis of NS, SS, JS, and AS humanized scFV. The assay reflects the soluble aggregation propensity of these antibodies.

Fig. 5A and 5B are graphs showing thermal ramping (thermal ramping) to determine the polypeptide stability of CARs comprising NS, SS, JS, and AS humanized scFV. In fig. 5A, the thermostability was determined in the presence of 50mM NaCl and in fig. 5B, NaCl was omitted. The CAR derived from scFVAS ("□"), SS ("Δ"), JS ("), and NS (" X ") is shown.

Fig. 6A and 6B include a series of plots showing detection of the CARs of the invention comprising SS, JS, AS and NS expressed on the surface of T cells obtained from two donor subjects 5244 (fig. 6A) and 5273 (fig. 6B). Donor cells expressing a mimetic CAR and donor cells expressing a CAR comprising a parent antibody ("FMC 63") scFV are also prepared.

Figures 7A-7D include a series of bar graphs showing cytolytic activity of a CAR of the invention in donor cells transduced with a CAR or mimetic from donor 5244. CAR or mock-transduced donor cells were added at a ratio of effector to target of 1:1 or 4: 1. Four target cell types were used: raji (CD19+ human Burkitt lymphoma cells; FIG. 7A), Namalawa (CD19+ human Burkitt lymphoma cells; FIG. 7B), Eol-1(CD 19-human acute myeloid (eosinophilic) leukemia cells; FIG. 7C) and Mv411(CD 19-human bi-epi-B myelomonocytic leukemia cells; FIG. 7D). Staurosporine ("Stauro") was used as a positive control for tumor cell killing.

Figures 8A-8D include a series of bar graphs showing cytolytic activity of a CAR of the invention in donor cells transduced with a CAR or mimetic from donor 5273. CAR or mock-transduced donor cells were added at a ratio of effector to target of 1:1 or 4: 1. Four target cell types were used: raji cells (FIG. 8A), Namalawa cells (FIG. 8B), Eol-1 cells (FIG. 8C), and Mv411 cells (FIG. 8D). Stauro was used as a positive control for tumor cell killing.

Figures 9A and 9B show the growth kinetics of CAR or mock-transduced T cells from additional donors measured within ten days after initial stimulation with OKT 3. The observed growth profile for each CAR is shown in figure 9A. CAR or mock-transduced T cells were then stimulated a second time and differences in growth kinetics were observed, as shown in figure 9B.

Figure 10A shows characterization of T cell populations using anti-CAR antibody and CD3 on day 10. Figure 10B shows CD4/CD8 staining during CAR T cell production.

FIGS. 11A and 11B show cytotoxicity assays performed on CD19+ NAMALWA (FIG. 11A) or CD19-EOL-1 (FIG. 11B) cells.

Detailed Description

The present invention relates to novel polypeptides comprising a humanized antigen binding domain that recognizes and binds human CD19 and polynucleotides encoding the same. Some aspects of the invention relate to polynucleotides encoding a Chimeric Antigen Receptor (CAR) or T Cell Receptor (TCR) comprising a humanized anti-human CD19 antigen binding domain. The invention also provides vectors (e.g., viral vectors) comprising such polynucleotides and compositions comprising such vectors. The invention further provides polynucleotides encoding such CARs or TCRs and compositions comprising such polynucleotides. The invention further provides engineered cells (e.g., T cells) comprising such polynucleotides and/or engineered cells (e.g., T cells) transduced with such viral vectors, and compositions comprising such engineered cells. The invention provides compositions (e.g., pharmaceutical compositions) comprising a plurality of engineered T cells. The invention provides methods for making such engineered T cells and compositions and uses of such engineered T cells and compositions (e.g., in the treatment of B-cell lymphomas). Also, the present invention provides a method of inducing immunity to a tumor comprising administering to a subject an effective amount of a cell comprising a polynucleotide, vector or polypeptide of the present invention. Other aspects of the invention relate to cells comprising a CAR or TCR and their use in T cell therapy, such as autologous cell therapy (eACTTM), for treating a patient having cancer.

Definition of

In order that the invention may be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, the term "or" should be understood to be inclusive and encompass both "or" and "unless the context clearly dictates otherwise.

The term "and/or" as used herein is to be taken as a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and/or" as used in phrases such as "a and/or B" herein is intended to include a and B; a or B; a (alone); and B (alone). Likewise, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass each of the following: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; a (alone); b (alone); and C (alone).

As used herein, the terms "such as" and "i.e.," are used by way of example only and are not intended to be limiting, and should not be construed as referring only to those items explicitly recited in the specification.

The term "or more", "at least", "over", etc., e.g., "at least one" should be understood to include, but not be limited to, at least 1, 2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 1920, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 105, 104, 102, 103, 102, 103, 100, 103, 33, 40, 108. 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more. Any larger numbers or fractions therebetween are also included.

Conversely, the term "not more than" includes every value that is less than the recited value. For example, "no more than 100 nucleotides" includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,8, 7,6, 5, 4, 3, 2, 1 and 0 nucleotides. Any smaller numbers or fractions therebetween are also included.

The terms "plurality", "at least two", "two or more", "at least a second", etc. should be understood to include, but are not limited to, at least 2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 1920, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 103, 101, 104, 105, 106, 107, 109, 108, 106, 109, 108, 105, 106, 109, 103, 45, 47, 48, 110. 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more. Any larger numbers or fractions therebetween are also included.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. It should be understood that the language "comprising" is used herein to describe aspects and also to provide other similar aspects described as "consisting of and/or" consisting essentially of. Unless specifically stated or otherwise apparent from the context, the term "about," as used herein, refers to a value or composition within an acceptable error range for the particular value or composition, as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "consisting essentially of can mean within 1 or over 1 standard deviation as practiced in the art. "about" or "consisting essentially of may mean a range of up to 10% (i.e., ± 10%). Thus, "about" may be understood as being greater than or less than the stated value within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001%. For example, about 5mg may include any amount between 4.5mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the term may mean values up to an order of magnitude or up to 5-fold. When a particular value or composition is provided in the present disclosure, unless otherwise stated, it should be assumed that the meaning of "about" or "consisting essentially of" is within an acceptable error range for that particular value or composition.

As used herein, unless otherwise specified, any concentration range, percentage range, ratio range, or integer range is to be understood as encompassing the value of any integer within the recited range, and where appropriate, including fractions thereof (e.g., tenths and hundredths of integers).

The units, prefixes, and symbols used herein are provided in their international system of units (SI) accepted form. Numerical ranges include the numbers defining the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, Juo, "The circumcise Dictionary of Biomedicine and Molecular Biology", 2nd ed., (2001), CRC Press; "The Dictionary of Cell & Molecular Biology", 5th ed., (2013), Academic Press; and "The Oxford Dictionary Of Biochemistry And Molecular Biology", Cammacack et al.

By "administering" is meant physically introducing the agent into the subject using any of a variety of methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, for example by injection or infusion. As used herein, the phrase "parenteral administration" means modes of administration other than enteral and topical administration, typically by injection and including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, and in vivo electroporation. In some embodiments, the formulation is administered via a non-parenteral route, such as orally. Other non-parenteral routes include topical, epidermal or mucosal routes of administration, such as intranasal, vaginal, rectal, sublingual or topical. Administration may also be performed, for example, once, multiple times, and/or over one or more extended periods.

The term "antibody" (Ab) includes, but is not limited to, glycoprotein immunoglobulins that specifically bind to an antigen. Typically, an antibody may comprise at least two heavy (H) chains and two light (L) chains, or antigen-binding molecules thereof, interconnected by disulfide bonds. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2, and CH 3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises a constant domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR 4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the Ab may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q).

Antibodies can include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain fvs (scfvs), camelized antibodies, affibodies, Fab fragments, F (ab') 2 fragments, disulfide linked fvs (sdfvs), anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies), Minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), and antigen-binding fragments of any of the above. In certain embodiments, an antibody described herein refers to a polyclonal antibody population.

The immunoglobulin may be derived from any commonly known isotype, including but not limited to IgA, secretory IgA, IgG, IgE, and IgM. The IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG 4. "isotype" refers to the Ab class or subclass (e.g., IgM or IgG1) encoded by the heavy chain constant region gene. The term "antibody" includes, for example, both naturally occurring and non-naturally occurring abs; monoclonal and polyclonal Ab; chimeric and humanized abs; human or non-human Ab; ab is fully synthesized; and a single chain Ab. Non-human abs can be humanized by recombinant methods to reduce their immunogenicity in humans. Unless the context indicates otherwise, the term "antibody" also includes antigen-binding fragments or antigen-binding portions of any of the above immunoglobulins, and includes monovalent and divalent fragments or portions, as well as single chain abs.

An "amino acid sequence derived from an antibody" may be physically derived, e.g., expressed from a fragment of a polynucleotide encoding the antibody, or may be computer-derived, e.g., determined as a nucleotide sequence encoding the antibody (or fragment thereof) for use in the synthesis of an artificial polynucleotide sequence (or fragment), and the artificial polynucleotide sequence is expressed as the antibody or fragment thereof.

An "antigen-binding molecule," "antigen-binding portion," or "antibody fragment" refers to any molecule that comprises an antigen-binding portion (e.g., a CDR) of an antibody from which the molecule is derived. The antigen binding molecule may include antigen Complementarity Determining Regions (CDRs). Examples of antibody fragments include, but are not limited to, Fab ', F (ab') 2, and Fv fragments, dabs, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen-binding molecules. Peptibodies (i.e., Fc fusion molecules comprising a peptide binding domain) are another example of suitable antigen binding molecules. In some embodiments, the antigen binding molecule binds to an antigen on a tumor cell. In some embodiments, the antigen binding molecule binds to an antigen on a cell involved in a hyperproliferative disease or binds to a viral or bacterial antigen. In certain embodiments, the antigen binding molecule binds to CD 19. In a further embodiment, the antigen binding molecule is an antibody fragment that specifically binds an antigen, including one or more Complementarity Determining Regions (CDRs) thereof.

In a further embodiment, the antigen binding molecule is a single chain variable fragment (scFv). The scFv polypeptide molecules are covalently linked VH-VL heterodimers that can be expressed from a gene fusion comprising VH and VL encoding genes linked by a peptide-encoding linker. (see Huston et al (1988) Proc Nat Acad Sci USA 85(16): 5879-5883). Linker peptides (e.g., about 10 to about 25 amino acids) are typically rich in glycine for flexibility and serine or threonine for solubility. The linker may link the N-terminus of the VH to the C-terminus of the VL, or the C-terminus of the VH to the N-terminus of the VL. Despite the removal of the constant region and the introduction of the linker, the protein retains the specificity of the original immunoglobulin. The scFv may also include an N-terminal peptide sequence, sometimes referred to as a "signal peptide" or "leader sequence". A number of methods have been described to identify the chemical structures used to convert naturally aggregated but chemically separated light and heavy polypeptide chains from antibody V regions to scFv molecules that will fold into three-dimensional structures that are substantially similar to the structure of the antigen binding site. See, for example, U.S. patent nos. 5,091,513; 5,132, 405; and 4,946,778.

Very large initial scFv libraries have been and can be created to provide a large source of rearranged antibody genes for a large number of target molecules. To isolate disease-specific antibodies, smaller libraries may be constructed from individuals with infectious disease (see Barbas et al, Proc. Natl. Acad. Sci. USA 89:9339-43 (1992); Zebedee et al, Proc. Natl. Acad. Sci. USA 89:3175-79 (1992)).

As used herein, the terms "variable region" or "variable domain" are used interchangeably and are common in the art. The variable region generally refers to a portion of an antibody, typically a light chain or a portion of a heavy chain, typically about the amino terminal 110 to 120 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which differ greatly in sequence between antibodies and are used for binding and specificity of a particular antibody for its particular antigen. The variability of the sequence is concentrated in those regions called Complementarity Determining Regions (CDRs), while the more highly conserved regions in the variable domains are called Framework Regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with the antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises a rodent or murine CDR and a human Framework Region (FR). In particular embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises a rodent or murine CDR and a primate (e.g., non-human primate) Framework Region (FR).

The terms "VL," "VL region," and "VL domain" are used interchangeably to refer to an antigen binding domain, such as the light chain variable region of an antibody or antigen binding fragment thereof, and comprise one, two, or all three CDRs.

The terms "VH", "VH region" and "VH domain" are used interchangeably to refer to an antigen binding domain such as the heavy chain variable region of an antibody or antigen binding fragment thereof, and comprise one, two or all three CDRs.

Many definitions of CDRs are commonly used: kabat numbering, Chothia numbering, AbM numbering, or contact numbering. The AbM definition is a compromise between the two used by Oxford Molecular's AbM antibody modeling software. contact definition is based on analysis of available complex crystal structures.

TABLE 1 CDR numbering

The term "Kabat numbering" and similar terms are art-recognized and refer to the numbering system of amino acid residues in the heavy and light chain variable regions of an antibody or antigen binding molecule thereof. In certain aspects, the CDRs of an antibody may be determined according to the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382 + 391 and Kabat EA et al, (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, CDRs within an antibody heavy chain molecule are typically present at amino acid positions 31 to 35 (which optionally may include one or two additional amino acids (referred to as 35A and 35B in the Kabat numbering scheme)) after 35 (CDR1), amino acid positions 50 to 65(CDR2), and amino acid positions 95 to 102(CDR 3). Using the Kabat numbering system, CDRs within an antibody light chain molecule are typically present at amino acid positions 24 to 34(CDR1), amino acid positions 50 to 56(CDR2), and amino acid positions 89 to 97(CDR 3). In particular embodiments, the CDRs of the antibodies described herein have been determined according to the Kabat numbering scheme.

In certain aspects, the CDRs of an antibody can be determined according to the Chothia numbering scheme, which refers to the location of the structural loops of an immunoglobulin (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-. Typically, when using the Kabat numbering convention, the Chothia CDR-H1 loop is present at heavy chain amino acids 26 to 32, 33, or 34, the Chothia CDR-H2 loop is present at heavy chain amino acids 52 to 56, and the Chothia CDR-H3 loop is present at heavy chain amino acids 95 to 102, while the Chothia CDR-L1 loop is present at light chain amino acids 24 to 34, the Chothia CDR-L2 loop is present at light chain amino acids 50 to 56, and the Chothia CDR-L3 loop is present at light chain amino acids 89 to 97. The ends of the Chothia CDR-HI loops when numbered using the Kabat numbering convention vary between H32 and H34 depending on the length of the loops (since the Kabat numbering scheme places the insertions at H35A and H35B; the loops end at 32 if neither 35A nor 35B are present; the loops end at 33 if only 35A is present; the loops end at 34 if both 35A and 35B are present). In particular embodiments, the CDRs of the antibodies described herein have been determined according to the Chothia numbering scheme.

TABLE 2 CDRs of the light chain Variable (VL) region of FMC63 anti-CD 19 antibody or fragment thereof

TABLE 3 CDRs of the heavy chain Variable (VH) region of FMC63 anti-CD 19 antibody or fragment thereof

As used herein, the terms "constant region" and "constant domain" are interchangeable and have the meaning common in the art. Constant regions are antibody portions, such as the carboxy-terminal portions of light and/or heavy chains, that are not directly involved in binding of the antibody to an antigen, but may exhibit various effector functions, such as interaction with an Fc receptor. The constant regions of immunoglobulin molecules typically have a more conserved amino acid sequence relative to immunoglobulin variable domains.

As used herein, the term "heavy chain" when used in reference to an antibody may refer to any of the different types of amino acid sequences based on the constant domain, such as alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), which produce IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of iggs, such as IgG1, IgG2, IgG3, and IgG 4.

As used herein, the term "light chain" when used in reference to an antibody may refer to any of the different types, e.g., kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In a specific embodiment, the light chain is a human light chain.

"binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise specified, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can be generally expressed by the dissociation constant (KD). Affinity can be measured and/or expressed in a variety of ways known in the art, including but not limited to, equilibrium dissociation constant (KD) and equilibrium association constant (KA). KD is calculated by the quotient koff/kon, while KA is calculated by the quotient kon/koff. kon refers to, for example, the rate constant of binding of an antibody to an antigen, and koff refers to, for example, dissociation of an antibody from an antigen. kon and koff can be determined by techniques known to those of ordinary skill in the art such as KinExA or KinExA.

CD19 (also known as cluster of differentiation 19, B lymphocyte antigen CD19, B lymphocyte surface antigen B4, B4, CVID3, differentiation antigen CD19) is a protein encoded by the CD19 gene in humans. It is found on the surface of B cells. Since CD19 is a B cell marker, it may be a useful antigen for identifying cancer cells caused by this type of B cell (i.e., B cell lymphoma).

As used herein, "conservative amino acid substitution" refers to an amino acid substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having side chains have been defined in the art. These families include amino acids with the following side chains: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, one or more amino acid residues within a CDR or within a framework region of an antibody or antigen binding molecule thereof may be substituted with amino acid residues having similar side chains.

As used herein, the term "heterologous" means from any source other than a naturally occurring sequence. For example, a heterologous sequence included as part of a co-stimulatory protein having the amino acid sequence of SEQ ID NO:1 (e.g., the corresponding human co-stimulatory protein) is an amino acid that does not naturally occur as (i.e., is not aligned with) the wild-type human co-stimulatory protein. For example, a heterologous nucleotide sequence refers to a nucleotide sequence other than the wild-type human co-stimulatory protein coding sequence.

As used herein, the term "epitope" is a term of art and refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be, for example, contiguous amino acids of a polypeptide (linear or contiguous epitope) or an epitope can be, for example, two or more non-contiguous regions from a polypeptide or polypeptides (conformational, non-linear, non-contiguous, or non-contiguous epitopes). In certain embodiments, the epitope to which an antibody binds can be determined by, for example, nuclear magnetic resonance spectroscopy (NMR spectroscopy), X-ray diffraction crystallographic studies, ELISA assays, hydrogen/deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assays, and/or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be accomplished using any method known in the art (e.g., Gieger R et al, (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303). Antibody antigen crystals can be studied using well-known X-ray diffraction techniques and can be refined using computer software known in the art, such as Refmac and Phenix. Mutagenesis mapping studies can be accomplished using any method known to those skilled in the art. For mutagenesis techniques, including alanine scanning mutagenesis techniques, see, e.g., Champe M et al, (1995) J Biol Chem 270:1388-1394 and Cunningham BC & Wells JA (1989) Science 244: 1081-1085.

As used herein, an antigen binding molecule, antibody or antigen binding molecule thereof "cross-competes" with a reference antibody or antigen binding molecule thereof if the interaction between the antigen and the first binding molecule, antibody or antigen binding molecule thereof blocks, limits, inhibits or otherwise reduces the ability of the reference binding molecule, reference antibody or antigen binding molecule thereof to interact with the antigen. The cross-competition may be complete, e.g. binding of the binding molecule to the antigen completely blocks the ability of the reference binding molecule to bind to the antigen, or it may be partial, e.g. binding of the binding molecule to the antigen reduces the ability of the reference binding molecule to bind to the antigen. In certain embodiments, an antigen binding molecule that cross-competes with a reference antigen binding molecule binds to the same or overlapping epitope as the reference antigen binding molecule. Many types of competitive binding assays can be used to determine whether one antigen binding molecule competes with another, for example: solid phase direct or indirect Radioimmunoassay (RIA); solid phase direct or indirect Enzyme Immunoassay (EIA); sandwich competition assays (Stahli et al, 1983, Methods in Enzymology 9: 242-253); solid phase direct biotin-avidin EIA ((Kirkland et al, 1986, J.Immunol.137: 3614-) -3619), solid phase direct labeling assays, solid phase direct labeling sandwich assays (Harlow and Lane,1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press), solid phase direct labeling RIA using 1-125 labels (Morel et al, 1988, mol.Immunol.25: 7-15), solid phase direct biotin-avidin EIA (Cheung, et al, 1990, Virology 176: 546-; 552), and direct labeling RIA (Moldenhauer et al, 1990, Scan.J.32: Immunol.77-82).

As used herein, the terms "immunospecific binding," "immunospecific recognition," "specific binding," and "specific recognition" are similar terms in the context of antibodies and refer to molecules that bind to an antigen (e.g., an epitope or an immune complex) as such binding is understood by those of skill in the art. For example, molecules that specifically bind to an antigen may bind to other peptides or polypeptides, typically with lower affinity, as determined by, for example, immunoassays, the KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art. In particular embodiments, a molecule that specifically binds an antigen binds to the antigen with a KA that is at least 2 log (log), 2.5 log, 3 log, 4 log, or greater than the KA when the molecule binds to another antigen.

In particular embodiments, provided herein are antibodies or antigen binding molecules thereof that bind to a target human antigen (e.g., human CD19) with higher affinity than another target antigen (e.g., non-human CD 19). In certain embodiments, provided herein are antibodies or antigen binding molecules thereof that bind to a target human antigen (e.g., human CD19) with an affinity that is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or higher than another target antigen, as measured by, for example, a radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay. In particular embodiments, an antibody or antigen-binding molecule thereof described herein that binds to a target human antigen will bind to another target antigen with less than 10%, 15%, or 20% of the binding of the antibody or antigen-binding molecule thereof to the human antigen, as measured by, for example, a radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay.

"antigen" refers to any molecule that elicits an immune response or is capable of being bound by an antibody or antigen binding molecule. The immune response may involve antibody production or activation of specific immunocompetent cells or both. One skilled in the art will readily appreciate that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. The antigen may be expressed endogenously, i.e. from genomic DNA, or may be expressed recombinantly. The antigen may be specific for certain tissues, such as cancer cells, or it may be expressed broadly. In addition, larger molecule fragments may serve an antigenic role. In some embodiments, the antigen is a tumor antigen. In a specific embodiment, the antigen is all or a fragment of human CD 19.

The term "neutralizing" refers to an antigen binding molecule, scFv, antibody or fragment thereof that binds to a ligand and prevents or reduces the biological effect of the ligand. In some embodiments, the antigen binding molecule, scFv, antibody or fragment thereof directly blocks a binding site on the ligand or alters the binding capacity of the ligand by an indirect means, such as a structural or energetic change in the ligand. In some embodiments, the antigen binding molecule, scFv, antibody or fragment thereof prevents the protein to which it binds from performing a biological function.

As used herein, the term "autologous" means any substance derived from the same individual into which it is later reintroduced. For example, engineered autologous cell therapy (eACTTM, also known as adoptive cell transfer) is a process in which patient's own T cells are collected, then genetically engineered to express polynucleotides (e.g., polynucleotides encoding a CAR that recognizes and targets one or more antigens expressed on the cell surface of one or more specific tumor cells or malignancies), and then administered back to the same patient.

The term "allogeneic" refers to any substance derived from one individual that is then introduced into another individual of the same species, such as allogeneic T cell transplantation.

The terms "transduction" and "transduced" refer to a process by which foreign DNA is introduced into cells via a viral vector (see Jones et al, "Genetics: printles and analysis," Boston: Jones & Bartlett Publ. (1998)). In some embodiments, the vector is a retroviral vector, a DNA vector, an RNA vector, an adenoviral vector, a baculovirus vector, an EB virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector, a lentiviral vector, or any combination thereof.

As used herein, the terms "genetic engineering" or "engineering" are used interchangeably and refer to methods of modifying the genome of a cell, including, but not limited to, deleting coding or non-coding regions or portions thereof or inserting coding regions or portions thereof. In some embodiments, the modified cell is a lymphocyte, e.g., a T cell, which can be obtained from a patient or donor. The cells can be modified to express an exogenous construct, such as a Chimeric Antigen Receptor (CAR) incorporated into the genome of the cell.

"cancer" refers to a large group of diseases characterized by uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade adjacent tissues and can also metastasize to distant parts of the body through the lymphatic system or blood stream. "cancer" or "cancer tissue" may include tumors. Examples of cancers that can be treated by the methods of the invention include, but are not limited to, cancers of the immune system, including lymphomas, leukemias, myelomas, and other leukocyte malignancies.

Cancers that may be treated include B cell lymphoma, Acute Lymphoblastic Leukemia (ALL), AIDS-related lymphoma, ALK-positive large B cell lymphoma, Burkitt's lymphoma, Chronic Lymphocytic Leukemia (CLL), classical Hodgkin's lymphoma, Diffuse Large B Cell Lymphoma (DLBCL), follicular lymphoma, intravascular large B cell lymphoma, large B cell lymphoma caused by HHV 8-related multicenter Castleman disease, lymphomatoid granuloma, lymphoplasmacytic lymphoma, Mantle Cell Lymphoma (MCL), marginal zone B cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), lymph node B cell marginal zone lymphoma (NMZL), nodal lymphoblastic predominant Hodgkin's lymphoma, non-Hodgkin's lymphoma, plasmablatic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, primary lymphoma, and lymphoma, Splenic Marginal Zone Lymphoma (SMZL) and waldenstrom's macroglobulinemia or a combination thereof. In one embodiment, the B cell lymphoma is Acute Lymphoblastic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL), Diffuse Large B Cell Lymphoma (DLBCL), follicular lymphoma, Mantle Cell Lymphoma (MCL), marginal zone B cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), and non-hodgkin's lymphoma. In one embodiment, the B cell lymphoma is non-hodgkin's lymphoma.

A particular cancer may be responsive to chemotherapy or radiation therapy or the cancer may be refractory. Refractory cancer refers to cancer that is not susceptible to surgical intervention, and that is either initially unresponsive to chemotherapy or radiation therapy, or that becomes unresponsive over time.

As used herein, "anti-tumor effect" refers to a biological effect that can manifest as a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, a decrease in the number of metastases, an increase in overall or progression-free survival, an increase in life expectancy, or an improvement in various physiological symptoms associated with a tumor. An anti-tumor effect may also refer to the prevention of tumorigenesis, e.g. a vaccine.

As used herein, "cytokine" refers to a non-antibody protein released by one cell in response to contact with a particular antigen, where the cytokine interacts with a second cell to mediate a response in the second cell. The cytokine may be expressed endogenously by the cell or administered to the subject. Cytokines can be released by immune cells (including macrophages, B cells, T cells, and mast cells) to spread the immune response. Cytokines can induce a variety of responses in recipient cells. Cytokines may include homeostatic cytokines, chemokines, pro-inflammatory cytokines, effectors, and acute phase proteins. For example, homeostatic cytokines, including Interleukins (IL)7 and IL-15, promote immune cell survival and proliferation, while pro-inflammatory cytokines can promote inflammatory responses. Examples of homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, and Interferon (IFN) gamma. Examples of proinflammatory cytokines include, but are not limited to, IL-1a, IL-1b, IL-6, IL-13, IL-17a, Tumor Necrosis Factor (TNF) -alpha, TNF-beta, Fibroblast Growth Factor (FGF)2, granulocyte macrophage colony stimulating factor (GM-CSF), soluble intercellular adhesion molecule 1(sICAM-1), soluble vascular adhesion molecule 1(sVCAM-1), Vascular Endothelial Growth Factor (VEGF), VEGF-C, VEGF-D, and placental growth factor (PLGF). Examples of effectors include, but are not limited to, granzyme a, granzyme B, soluble Fas ligand (sFasL), and perforin. Examples of acute phase proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid a (saa).

"chemokines" are a class of cytokines that mediate chemotaxis or directed movement of cells. Examples of chemokines include, but are not limited to, IL-8, IL-16, eotaxin-3, macrophage-derived chemokine (MDC or CCL22), monocyte chemotactic protein 1(MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1 alpha (MIP-1 alpha, MIP-1a), MIP-1 beta (MIP-1b), gamma-inducible protein 10(IP-10), and thymus and activation regulated chemokine (TARC or CCL 17).

A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dose" of a therapeutic agent (e.g., an engineered CAR T cell) is any amount that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of a disease or promotes disease regression as evidenced by decreased severity of disease symptoms, increased frequency and duration of asymptomatic phase of the disease, or prevention of injury or disability due to disease affliction. The ability of a therapeutic agent to promote disease regression can be assessed using various methods known to skilled practitioners, for example in human subjects during clinical trials, in animal model systems for predicting efficacy in humans, or by assaying the activity of the agent in an in vitro assay.

As used herein, the term "lymphocyte" includes a Natural Killer (NK) cell, a T cell, or a B cell. NK cells are a class of cytotoxic (cell toxic) lymphocytes that represent a major component of the innate immune system. NK cells reject tumors as well as virus infected cells. It acts through the process of apoptosis or programmed cell death. It is called "natural killing" because it does not require activation to kill cells. T cells play a major role in cell-mediated immunity (no participation of antibodies). Its T Cell Receptor (TCR) distinguishes itself from other lymphocyte types. The thymus, a specialized organ of the immune system, is primarily responsible for the maturation of T cells. There are six known types of T cells, namely: helper T cells (e.g., CD4+ cells), cytotoxic T cells (also known as TC, cytotoxic T lymphocytes, CTL, T killer cells, cytolytic T cells, CD8+ T cells, or killer T cells), memory T cells ((i) stem cell-like memory TSCM cells (e.g., naive cells) are CD45RO-, CCR7+, CD45RA +, CD62L + (L-selectin), CD27+, CD28+, and IL-7 Ra +, but they also express large amounts of CD95, IL-2R β, CXCR3, and LFA-1, and display many of the unique functional attributes of memory cells, (ii) central memory TCM cells express L-selectin and CCR7, which secrete IL-2 but not IFN γ or IL-4, and (iii) effector memory TEM cells, however, do not express L-selectin or 7, but produce effector cytokines, such as IFN γ and IL-4), regulatory T cells (Treg, suppressor T cells or CD4+ CD25+ regulatory T cells), natural killer T cells (NKT), and Gamma Delta T cells. On the other hand, B cells play a major role in humoral immunity (with antibody involvement). B cells produce antibodies, process antigens to perform the role of Antigen Presenting Cells (APCs), and develop into memory B cells upon activation through antigen interactions. In mammals, immature B cells develop in the bone marrow, where the name of the B cell comes from.

The term "genetically engineered" or "engineered" refers to methods of modifying the genome of a cell, including, but not limited to, deletions of coding or non-coding regions or portions thereof, or insertions of coding regions or portions thereof. In some embodiments, the modified cell is a lymphocyte (e.g., a T cell), which can be obtained from a patient or donor. The cells can be modified to express exogenous constructs, such as Chimeric Antigen Receptors (CARs) or T Cell Receptors (TCRs) incorporated into the genome of the cells.

by "immune response" is meant the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, Natural Killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules produced by any of these cells or the liver (including abs, cytokines, and complements) that result in the selective targeting, binding, damaging, destroying, and/or eliminating invading pathogens, pathogen-infected cells or tissues, cancer cells or other abnormal cells in vertebrates, or in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

The term "immunotherapy" refers to the treatment of a subject having a disease or at risk of contracting a disease or of recurrence of a disease by a method that includes inducing, enhancing, suppressing or otherwise modifying an immune response. Examples of immunotherapy include, but are not limited to, T cell therapy. T cell therapies may include adoptive T cell therapy, Tumor Infiltrating Lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACTTM), and allogeneic T cell transplantation. However, one skilled in the art will appreciate that the conditioning methods disclosed herein will enhance the effectiveness of any transplanted T cell therapy. Examples of T cell therapies are described in U.S. patent publication nos. 2014/0154228 and 2002/0006409, U.S. patent No. 5,728,388, and international publication No. WO 2008/081035.

The T cells for immunotherapy may be from any source known in the art. For example, T cells may be differentiated from a population of hematopoietic stem cells in vitro, or T cells may be obtained from a subject. T cells can be obtained from, for example, Peripheral Blood Mononuclear Cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. Furthermore, the T cells may be derived from one or more T cell lines available in the art. T cells may also be obtained from a unit of blood collected from a subject using any number of techniques known to those skilled in the art, such as FICOLLTM isolation and/or apheresis. Additional methods of isolating T cells for use in T cell therapy are disclosed in U.S. patent publication No. 2013/0287748, which is incorporated by reference herein in its entirety.

The term "engineered autologous cell therapy" (which may be abbreviated as "eACTTM", also known as adoptive cell transfer) is a process of collecting the patient's own T cells, which are then genetically altered to recognize and target one or more antigens expressed on the cell surface of one or more specific tumor cells or malignancies. T cells can be engineered to express, for example, a Chimeric Antigen Receptor (CAR) or a T Cell Receptor (TCR), or both. CAR-positive (+) T cells are engineered to express one or more extracellular single-chain variable fragments (scfvs) specific for a particular tumor antigen, linked to an intracellular signaling moiety comprising at least one costimulatory domain and at least one activation domain. The co-stimulatory domain may be derived from a naturally occurring co-stimulatory domain or a variant thereof, and the activation domain may be derived from, for example, CD3-zeta and CD 3-epidilon. In certain embodiments, the CAR is designed to have two, three, four, or more co-stimulatory domains. CAR scFv can be designed to target, for example, human CD19, human CD19 is a transmembrane protein expressed by cells of the B cell lineage (including ALL normal B cell and B cell malignancies, including but not limited to NHL, CLL, and non-T cell ALL). In some embodiments, the CAR is engineered such that the co-stimulatory domains are expressed as separate polypeptide chains. Example CAR T cell therapies and constructs are described in U.S. patent publication nos. 2013/0287748, 2014/0227237, 2014/0099309, and 2014/0050708 and U.S. provisional application nos. 62/470,703 and 62/317,258; these references are incorporated by reference in their entirety.

As used herein, "patient" includes any human having cancer (e.g., lymphoma or leukemia). Herein, the terms "subject" and "patient" are used interchangeably.

As used herein, the term "in vitro cell" refers to any cell cultured ex vivo. In particular, the in vitro cells may comprise T cells.

The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound comprising amino acid residues covalently linked by peptide bonds. There is no limit to the maximum number of amino acids that a protein or peptide contains at least two amino acids and may comprise the sequence of the protein or peptide. A polypeptide includes any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains (which are also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers) and longer chains (which are commonly referred to in the art as proteins, which are of many types). "polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. The polypeptide includes a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.

As used herein, "stimulation" refers to a primary response induced by the binding of a stimulatory molecule to its cognate ligand, wherein the binding mediates a signaling event. A "stimulatory molecule" is a molecule on a T cell, such as the T Cell Receptor (TCR)/CD3 complex that specifically binds to a cognate stimulatory ligand present on an antigen presenting cell. A "stimulatory ligand" is a ligand that, when present on an antigen presenting cell (e.g., APC, dendritic cell, B cell, etc.), can specifically bind to a stimulatory molecule on the T cell, thereby mediating a primary response (including but not limited to activation, initiation of an immune response, proliferation, etc.) of the T cell. Stimulatory ligands include, but are not limited to, anti-CD 3 antibodies (e.g., OKT3), mhc class i molecules loaded with peptides, hyperactivating anti-CD 2 antibodies, and hyperactivating anti-CD 28 antibodies.

As used herein, "co-stimulatory signal" refers to a signal that, when combined with a primary signal (e.g., TCR/CD3 linkage), results in a T cell response (e.g., without limitation, up-or down-regulation of proliferation and/or key molecules).

As used herein, "co-stimulatory ligand" includes molecules on Antigen Presenting Cells (APCs) that specifically bind to homologous co-stimulatory molecules on T cells. Alternatively/additionally, when an antibody (e.g., an anti-CD 28 antibody) is bound to a plate, bead, APC, or in solution, it can be a co-stimulatory ligand. Binding of the co-stimulatory ligand provides a signal that mediates T cell responses including, but not limited to, proliferation, activation, differentiation, etc. The co-stimulatory ligand induces a signal in addition to the primary signal provided by the stimulatory molecule, e.g., provided by the binding of the T Cell Receptor (TCR)/CD3 complex to a Major Histocompatibility Complex (MHC) molecule loaded with a peptide. Costimulatory ligands can include, but are not limited to, 3/TR6, 4-1BB ligand, agonists or antibodies that bind Toll ligand receptors, B7-1(CD80), B7-2(CD86), CD30 ligand, CD40, CD7, CD70, CD83, herpes virus invasion mediator (HVEM), human leukocyte antigen G (HLA-G), ILT4, immunoglobulin-like transcript (ILT)3, inducible costimulatory ligand (ICOS-L), intracellular adhesion molecule (ICAM), ligands that specifically bind B7-H3, lymphotoxin beta receptor, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), OX40 ligand, PD-L2, or Programmed Death (PD) L1. Costimulatory ligands include, but are not limited to, antibodies that specifically bind to costimulatory molecules present on T cells, such as, but not limited to, 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, CD7, ICOS, ligands that specifically bind to CD83, lymphocyte function-associated antigen-1 (LFA-1), natural killer cell receptor C (NKG2C), OX40, PD-1, or tumor necrosis factor superfamily member 14(TNFSF14 or LIGHT).

A "costimulatory molecule" is a cognate binding partner that specifically binds to a costimulatory ligand on a T cell, thereby mediating a costimulatory response (e.g., without limitation, proliferation) of the T cell. Costimulatory molecules include, but are not limited to, 4-1BB/CD137, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD33, CD45, CD100(SEMA4D), CD103, CD134, CD137, CD154, CD16, CD160(BY55), CD18, CD19, CD19a, CD2, CD247, CD2, CD276 (B2-H2), CD2 (alpha; delta; epsilon; gamma; zeta), CD2, CD49 2, CD 368, CD 6372, CD 72, CD2, GAI-L2, CD-I-L2, CD 36III, CD2, CD 36III-L, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGBl, KIRDS2, LAT, LFA-1, LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), LTBR, Ly9(CD229), lymphocyte function-associated antigen-1 (LFA-1(CDl la/CD18), MHC class I molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80(KLRF1), OX40, PAG/Cbp, PD-1, PSGL1, SELPLG (CD162), signaling lymphocyte activating molecule, SLAM (SLAMF 1; CD 150; IPO-3), SLAMF4(CD 244; 2B4), SLAMF6 (VLB-24; VLSLLA 24), SLNL 5976, TNFR 596, TNFR 599, TNFR-9, or a combination thereof.

The terms "reduce" and "reduce" are used interchangeably herein and mean any change less than the original. "reduction" and "decrease" are relative terms and require comparison between before and after measurement. "reduce" and "reduce" include complete consumption.

By "treating" or "treatment" of a subject is meant any type of intervention or procedure performed on the subject, or administration of an active agent to the subject, with the goal of reversing, alleviating, ameliorating, inhibiting, slowing, or preventing the onset, progression, severity, or recurrence of a symptom, complication, or condition, or biochemical indicator associated with the disease. In some embodiments, "treating" or "treatment" includes partial remission. In another embodiment, "treating" or "treatment" includes complete remission.

To calculate percent identity, the compared sequences are typically aligned in a manner that gives the greatest match between the sequences. One example of a Computer program that can be used to determine percent identity is the GCG package, which includes GAP (Devereux et al, 1984, Nucl. acid Res.12: 387; Genetics Computer Group, University of Wisconsin, Madison, Wis.). The computer algorithm GAP is used to align two polypeptides or polynucleotides for which the percentage of sequence identity is to be determined. The sequences are aligned so that their respective amino acids or nucleotides match best ("match span", determined by an algorithm). In certain embodiments, the algorithm also uses a standard comparison matrix (for PAM 250 comparison matrix, see Dayhoff et al, 1978, Atlas of Protein Sequence and Structure 5: 345-.

Various aspects of the invention are described in further detail in the following subsections.

I. Chimeric antigen receptor and T cell receptor

Chimeric antigen receptors (CARs or CAR-T) and T Cell Receptors (TCR) are genetically engineered receptors. These engineered receptors can be readily inserted into and expressed by immune cells (including T cells) according to techniques known in the art. Using CARs, a single receptor can be programmed to recognize a particular antigen and, when bound to that antigen, activate immune cells to attack and destroy cells carrying that antigen. When these antigens are present on tumor cells, the CAR-expressing immune cells can target and kill the tumor cells.

The steps performed in making CAR-expressing cells are shown in figure 1A, and the CAR-mediated killing mechanism mediated via recognition of a target on a tumor cell is shown in figure 1B.

The present invention relates to Chimeric Antigen Receptors (CARs) and T Cell Receptors (TCRs) comprising an antigen binding domain, such as an scFv, that specifically binds to human CD19, and engineered T cells comprising an antigen binding domain that specifically binds to human CD 19. In some embodiments, the antigen binding domain of the invention is an scFv derived from an antibody, e.g., an FMC63 antibody. Other antibodies to human CD19 may be used.

The anti-human CD19 CARs or TCRs of the invention comprise an antigen binding domain that specifically binds to human CD 19. In some embodiments, an anti-human CD19 CAR or TCR further comprises a co-stimulatory domain and/or an extracellular domain (i.e., a "hinge" or "spacer" region) and/or a transmembrane domain and/or an intracellular (signaling) domain and/or a CD3-zeta or CD3-epsilon activation domain. In some embodiments, an anti-human CD19 CAR or TCR comprises a scFv antigen binding domain that specifically binds human CD19, a costimulatory domain, an extracellular domain, a transmembrane domain, and a CD3-zeta or CD3-epsilon activation domain.

In some embodiments, the orientation of the CAR according to the invention comprises an antigen binding domain (e.g., scFv) in tandem with a co-stimulatory domain and an activation domain. The co-stimulatory domain may comprise one or more of an extracellular portion, a transmembrane portion, and an intracellular portion. In other embodiments, multiple co-stimulatory domains may be utilized in tandem.

I.A. antigen binding domains

CARs can be engineered to bind an antigen (e.g., a cell surface antigen) by incorporating an antigen binding molecule that interacts with a particular targeted antigen. In some embodiments, the antigen binding molecule is an antibody fragment thereof, such as one or more single chain antibody fragments ("scFv"). An scFv is a single chain antibody fragment having the variable regions of the heavy and light chains of an antibody linked together. See U.S. Pat. Nos. 7,741,465 and 6,319,494, and Eshhar et al, Cancer Immunol Immunotherapy (1997)45: 131-136. The scFv retains the ability of the parent antibody to specifically interact with the target antigen. scfvs are useful in chimeric antigen receptors, as they can be engineered to be expressed as part of a single chain with other CAR components. As above. See also Krause et al, j.exp.med., Volume 188, No.4,1998 (619-; finney et al, Journal of Immunology,1998,161: 2791-. It will be appreciated that the antigen binding molecule is typically contained within the extracellular portion of the CAR such that it is capable of recognizing and binding the antigen of interest. Bispecific and multispecific CARs having specificity for more than one antigen of interest are contemplated within the scope of the invention.

The present invention relates to anti-human CD19 antibodies, fragments thereof (e.g., scFv), Chimeric Antigen Receptors (CAR), and T Cell Receptors (TCR) having humanized antigen binding domains.

The following nucleotide sequences are shown in the format "leader sequence-VL-linker-VH-His tag-stop codon", i.e. "leader sequence" is shown in bold, linker "is shown in italics," His tag "is shown in bold italics, and VL, VH and" stop codon "are shown in standard font.

WT-FMC63-scFv

SS-scFv

JS-scFv

AS-scFv

NS-scFv

In some embodiments, the polynucleotide sequence may be at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the polynucleotide sequence described above.

Amino acid sequences of four of wild-type scFv and seven selected humanized scFv

The following polypeptide sequences are shown in the format "leader-VL-linker-VH-His-tag", i.e., "leader" is shown in bold, "linker" is shown in italics and "His-tag" is shown in bold italics.

The leader sequence used in each of the following scFv had the amino acid sequence of MEWTWVFLFLLSVTAGVHS (SEQ ID NO: 6). The linker used in each of the following scFv had the amino acid sequence of GSTSGSGKPGSGEGSTKG (SEQ ID NO: 7). The His tag used in each of the following scFv has the amino acid sequence of HHHHHHHHHH (SEQ ID NO: 8).

WT-FMC63-scFv

The VL sequence of WT-FMC63-scFV has

The amino acid sequence of (a).

VH sequence of WT-FMC63-scFV

The amino acid sequence of (a).

SS-scFv

VL sequence of SS-scFV

An amino acid sequence.

The VH sequence of SS-scFV has

The amino acid sequence of (a).

JS-scFv

The VL sequence of JS-scFV has

The amino acid sequence of (a).

VH sequence of JS-scFV

The amino acid sequence of (a).

AS-scFv

The VL sequence of AS-scFV has

The amino acid sequence of (a).

VH sequence of AS-scFV having

The amino acid sequence of (a).

NS-scFv

VL sequences of NS-scFV having

The amino acid sequence of (a).

VH sequence of NS-scFV having

The amino acid sequence of (a).

The WT-FMC63-, SS-, JS-, AS-, and NS-scFv amino acid sequences described above may lack a His tag (of SEQ ID NO: 8). Thus, the scFv has the following amino acid sequence:

WT-FMC63-scFv (without His tag)

SS-scFv (without His tag)

JS-scFv (without His tag)

AS-scFv (without His tag)

NS scFv (without His tag)

In some embodiments, the polypeptide sequence may be at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to any of the above polypeptide sequences.

Humanized antibodies (e.g., scFv) have a VL region that differs from SEQ ID NO:36 by at least one amino acid, e.g., 1, 2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids. For example, a humanized antibody (e.g., scFv) may have a VL region that differs from SEQ ID NO:36 by having a Ser at position 7, a Pro at position 8, a Val at position 15, a Thr at position 22, a Gln at position 41, a Lys at position 42, a Gln at position 42, an Ala at position 43, a Pro at position 44, a Lys at position 49, a Thr at position 72, a Ser at position 77, a Gln at position 79, a Pro at position 80, a Phe at position 83, a Tyr at position 87, a Gln at position 100, and/or a Lys at position 107. In some embodiments, a humanized antibody (e.g., an scFv) may have a VL region that differs from SEQ ID NO:36 by having Ser at position 7, Pro at position 8, Val at position 15, Thr at position 22, Gln at position 42, Ala at position 43, Pro at position 44, Lys at position 49, Thr at position 72, Ser at position 77, Gln at position 79, Pro at position 80, Phe at position 83, Tyr at position 87, Gln at position 100, and/or Lys107 at position. In other embodiments, a humanized antibody (e.g., scFv) may have a VL region that differs from SEQ ID NO:36 by having a Ser at position 7, Pro at position 8, Val at position 15, Thr at position 22, Gln at position 41, Lys at position 42, Ala at position 43, Thr at position 72, Ser at position 77, Gln at position 79, Pro at position 80, and Lys107 at position 107.

Humanized antibodies (e.g., scFv) have a VH region that differs from SEQ ID NO 37 by at least one amino acid, e.g., 1, 2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids. For example, a humanized antibody (e.g., scFv) can have an amino acid sequence that is identical to SEQ ID NO:37 different VH region by having Gln at position 1, Gln at position 3, Val at position 5, Gly at position 9, Lys at position 13, Gln at position 13, Gly at position 15, Arg at position 16, Thr at position 17, Arg at position 19, Leu at position 20, Ser at position 21, Ala at position 24, Gly at position 42, Ile at position 48, Phe at position 67, Ser at position 70, Arg at position 71, Thr at position 73, Asn at position 76, Thr at position 77, Leu at position 78, Tyr at position 79, Gln at position 81, Ser at position 83, Thr at position 86, Arg at position 86, Ala at position 87, Glu at position 88, Ala at position 88, Val at position 92, and/or Leu at position 115. In some embodiments, a humanized antibody (e.g., an scFv) can have a VH region that differs from SEQ ID NO:37 by having a Gln at position 3, a Val at position 5, a Gly at position 9, a Gln at position 13, a Gly at position 15, an Arg at position 16, an Arg at position 19, a Leu at position 20, a Ser at position 21, an Ala at position 24, a Gly at position 42, an Ile at position 48, a Phe at position 67, a Ser at position 70, an Arg at position 71, an Asn at position 76, a Thr at position 77, a Leu at position 78, a Tyr at position 79, a Gln at position 81, an Arg at position 86, an Ala at position 87, a Glu at position 88, a Val at position 92, and/or a Leu at position 115. In other embodiments, a humanized antibody (e.g., an scFv) may have a VH region that differs from SEQ ID NO:37 by having a Gln at position 1, a Gln at position 3, a Lys at position 13, a Thr at position 16, a Thr at position 17, a Gly at position 42, a Ser at position 70, a Thr at position 73, an Asn at position 76, a Ser at position 83, a Thr at position 86, an Ala at position 87, an Ala at position 88, and/or a Leu at position 115.

In some embodiments, the antigen binding molecule binds to a target antigen (e.g., human CD19) with a KD of less than 1x 10-6M, less than 1x 10-7M, less than 1x 10-8M, or less than 1x 10-9M. In some embodiments, the antigen binding molecule binds to a target antigen (e.g., human CD19) with a KD of about 1x 10-8M, about 2x 10-8M, about 3x 10-8M, about 4x 10-8M, about 5x 10-8M, about 6x 10-8M, about 7x 10-8M, about 8x 10-8M, about 9x 10-8M, about 1x 10-9M, about 2x 10-9M, about 3x 10-9M, about 4x 10-9M, about 5x 10-9M, about 6x 10-9M, about 7x 10-9M, about 8x 10-9M, or about 9x 10-9M. In certain embodiments, KD is calculated as the quotient koff/kon, and kon and koff are determined using monovalent antibodies, e.g., Fab fragments, as measured by, e.g., surface plasmon resonance techniques. In other embodiments, KD is calculated as the quotient koff/kon, and kon and koff are determined using a bivalent antibody, e.g., a Fab fragment, as measured by, e.g., surface plasmon resonance techniques.

In some embodiments, the antigen binding molecule is present in an amount less than 1x 10-4M-1s-1, less than 2x 10-4M-1s-1, less than 3x 10-4M-1s-1, less than 4x 10-4M-1s-1, less than 5x 10-4M-1s-1, less than 6x 10-4M-1s-1, less than 7x 10-4M-1s-1, less than 8x 10-4M-1s-1, less than 9x 10-4M-1s-1, less than 1x10-5M-1s-1, less than 2x 10-5M-1s-1, less than 3x 10-5M-1s-1, Less than 4x 10-5M-1s-1, less than 5x 10-5M-1s-1, less than 6x 10-5M-1s-1, less than 7x 10-5M-1s-1, less than 8x 10-5M-1s-1, less than 9x 10-5M-1s-1, less than 1x 10-6M-1s-1, less than 2x 10-6M-1s-1, less than 3x 10-6M-1s-1, less than 4x 10-6M-1s-1, less than 5x 10-6M-1s-1, less than 6x 10-6M-1s-1, less than 7x 10-6M-1s-1, An association rate (kon) of less than 8x 10-6M-1s-1, less than 9x 10-6M-1s-1, or less than 1x 10-7M-1s-1 binds to a target antigen (e.g., human CD 19). In certain embodiments, kon is determined using monovalent antibodies, e.g., Fab fragments, as measured by, e.g., surface plasmon resonance techniques. In other embodiments, a bivalent antibody is used to determine kon as measured by, for example, surface plasmon resonance techniques.

In some embodiments, the antigen binding molecule is present in an amount less than 1x 10-2s-1, less than 2x 10-2s-1, less than 3x 10-2s-1, less than 4x 10-2s-1, less than 5x 10-2s-1, less than 6x 10-2s-1, less than 7x 10-2s-1, less than 8x 10-2s-1, less than 9x 10-2s-1, less than 1x 10-3s-1, less than 2x 10-3s-1, less than 3x 10-3s-1, less than 4x 10-3s-1, less than 5x 10-3s-1, less than 6x 10-3s-1, less than 7x 10-3s-1, less than, An off-rate (koff) of less than 8x 10-3s-1, less than 9x 10-3s-1, less than 1x 10-4s-1, less than 2x 10-4s-1, less than 3x 10-4s-1, less than 4x 10-4s-1, less than 5x 10-4s-1, less than 6x 10-4s-1, less than 7x 10-4s-1, less than 8x 10-4s-1, less than 9x 10-4s-1, less than 1x 10-4s-1, or less than 5x 10-4s-1 binds to a target antigen (e.g., human CD 19). In certain embodiments, koff is determined using a monovalent antibody, e.g., a Fab fragment, as measured by, e.g., surface plasmon resonance techniques. In other embodiments, a bivalent antibody is used to determine koff as measured by, for example, surface plasmon resonance techniques.

In some embodiments, the polynucleotide encodes a TCR, wherein the TCR further comprises a fourth complementarity determining region (CDR 4). In certain embodiments, the polynucleotide encodes a TCR, wherein the TCR further comprises a constant region. In some embodiments, the constant regions are selected from the constant regions of: IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM.

I.B. co-stimulatory domain

Chimeric antigen receptors incorporate a costimulatory (signaling) domain to increase their efficacy. See U.S. Pat. Nos. 7,741,465 and 6,319,494, and Krause et al and Finney et al (supra), Song et al, Blood 119: 696-; kalos et al, Sci Transl. Med.3:95 (2011); porter et al, n.engl.j.med.365:725-33(2011) and Gross et al, annu.rev.pharmacol.toxicol.56: 59-83 (2016). Exemplary costimulatory proteins have the amino acid sequence of the costimulatory protein naturally present on T cells. The complete natural amino acid sequence of this costimulatory protein is described in the NCBI reference sequence: NP-006130.1.

polynucleotide and polypeptide sequences of other costimulatory domains are known in the art. In some embodiments, the polynucleotide encoding the co-stimulatory domain comprises a nucleotide sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a nucleotide sequence known in the art. In some embodiments, the polypeptide sequence of the co-stimulatory domain comprises a polypeptide sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a polypeptide sequence known in the art.

i.B.1 extracellular or "hinge" domains

In one embodiment, the CARs or TCRs of the present disclosure comprise an "extracellular" or "hinge" or "spacer" domain or region, which terms are used interchangeably herein. In another embodiment, the extracellular domain is derived or derived from (e.g., comprises all or a fragment of) CD, CD3delta, CD epsilon, CD gamma, CD α, CD β, CD11 (ITGAL), CD11 (ITGAM), CD11 (ITGAX), CD11 (ITGAD), CD (ITGB), CD (B), CD (TNFRSF), CD28, CD (ITGB), CD (TNFRSF), CD (SLAMF), CD49 (ITGA), CD66 (CEACAM), CD66 (ceec), CD79 (B cell antigen receptor complex-related chain), CD79 (B cell antigen receptor complex-related alpha beta chain), CD (SLAMF), CD (tacitc), CD103 (sekibb), CD4 (sekicd 2 (slb), CD2 (sldl) 158), CD2 (sldl) 158), CD1 (sldp 158), CD3 (slga), CD 158), CD1 (ITGA), CD 158), CD1 (ITGA), CD1 (ITGA), CD 158), CD2 (ITGA), CD2, CD158D (KIRDL D), CD158F D (KIR2DL 5D), CD158D (KIR3DL D), CD160(BY D), CD162(SELPLG), CD226(DNAM D), CD229(SLAMF D), CD244(SLAMF D), CD247(CD D-zeta), CD258(LIGHT), CD268(BAFFR), CD270(TNFSF D), CD272(BTLA), CD276(B D-H D), CD279(PD-1), CD314(NKG2D), CD319(SLAMF D), CD335(NK-p D), CD336(NK-p D), CD337(NK-p D), CD352(SLAMF D), CD353(SLAMF D), CD355 SLLFM (CRNK-p D), CD336 (CRNK-p D), CD D, LAMIC-D, LAMC-D, LAMIC-7, LAMC-D, LAMIC-D, LAMC-D, LAMIC-D, LAMC-7, LAMIC-7, MHC class 1 molecules, MHC class 2 molecules, TNF receptor proteins, immunoglobulin proteins, cytokine receptors, integrins, activating NK cell receptors, Toll ligand receptors, and fragments or combinations thereof. The "extracellular" or "hinge" or "spacer" domains or regions may be derived from natural or synthetic sources. The polynucleotide and polypeptide sequences of these hinge domains are known in the art.

In some embodiments, a polynucleotide encoding a hinge domain comprises a nucleotide sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a nucleotide sequence known in the art. In some embodiments, the polypeptide sequence of the hinge domain comprises a polypeptide sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a polypeptide sequence known in the art.

In some embodiments, the hinge domain is located between the antigen binding domain (e.g., scFv) and the transmembrane domain. In this orientation, the hinge domain provides a distance between the antigen binding domain and the surface of the CAR-expressing cell membrane. In some embodiments, the hinge domain is from or derived from an immunoglobulin. In some embodiments, the hinge domain is selected from the hinge region of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM, or fragments thereof. In other embodiments, the hinge domain comprises, is derived from or derived from a hinge region of CD8 alpha. In some embodiments, the hinge domain comprises, is derived from, or is derived from a hinge region of CD 28. In some embodiments, the hinge domain comprises a fragment of the hinge region of CD8alpha or a fragment of the hinge region of CD28, wherein the fragment is less than the entire hinge region. In some embodiments, a fragment of a CD8alpha hinge region or a fragment of a CD28 hinge region comprises an amino acid sequence of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acids that excludes the N-terminus or the C-terminus or both of a CD8alpha hinge region or a CD28 hinge region.

I.B.2 transmembrane domains

The co-stimulatory domain of the CAR or TCR for use in the invention may further comprise a transmembrane domain and/or an intracellular signaling domain. The transmembrane domain can be designed to fuse with the extracellular domain of the CAR. Similarly, it may be fused to the intracellular domain of the CAR. In one embodiment, a transmembrane domain that is naturally associated with one of the domains in the CAR is used. In some cases, the transmembrane domains may be selected or modified by amino acid substitutions to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize their interaction with other members of the receptor complex. The transmembrane domain may be derived from natural sources or from synthetic sources. When the source is a natural source, the domain may be derived from any membrane-bound or transmembrane protein. The transmembrane domain for particular use in the invention may be derived from (i.e.comprise) 4-1BB/CD137, an activating NK cell receptor, immunoglobulin, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEA 4D), CD103, CD160(BY55), CD18, CD19, CD19 19, CD247, CD19, CD276 (B19-H19), CD19 delta, CD19 epsilon, CD19 gamma, CD19, CD49 19, CD 368, CD8beta, CD19 (Tactile), CDl la, CDl, CDlc, CDL, CACL, ACAM, ACALM, CDNA 72, CD19 alpha-I receptor alpha-IL-7, GAICAM (GAMMA) receptor, CD 72, CD 14-IL-7, GAMMA, CD19, CD14, CD-IL-7-IL receptor (GAICAM), GAMMA, CD-7-IL receptor (GAMMA), CD-IL-7-IL-7, CD-7-IL receptor (GAMMA), GAMMA-IL-7-IL, Integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGBl, KIRDS2, LAT, LFA-1, a ligand that specifically binds to CD83, LIGHT, LTBR, Ly9(CD229), lymphocyte function-associated antigen-1 (LFA-1; CDl-la/CD18), MHC class 1molecule, NKG2C, NKG2, NKp30, NKp44, NKp46, NKp80(KLRF1), OX-40, PAG/ll, programmed death-1 (PD-1), PSGL1, SELPLG (CD 59162), signal transduction lymphocyte activation molecule (SLAM protein), SLAM 1; VLAMF 86150; SLP 8672; SLAPF 8676; TNFAN 08, TNFR 08, TNFAN 08, or a truncated TREB 08, or a receptor. The polynucleotide and polypeptide sequences of these transmembrane domains are known in the art.

In some embodiments, a polynucleotide encoding a transmembrane domain comprises a nucleotide sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a nucleotide sequence known in the art. In some embodiments, the polypeptide sequence of the transmembrane domain comprises a polypeptide sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a polypeptide sequence known in the art.

Optionally, the short linker may form a link between the extracellular, transmembrane and intracellular domains of any or some of the CARs.

I.B.3 intracellular (signalling) Domain

The intracellular (signaling) domain of the engineered T cells of the invention can provide signaling to the activation domain, which then activates at least one normal effector function of the immune cell. The effector function of the T cell may be, for example, cytolytic activity or helper activity, including secretion of cytokines.

In certain embodiments, suitable intracellular signaling domains include (i.e., include) but are not limited to 4-1BB/CD137, activated NK cell receptor, immunoglobulin, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100(SEMA4D), CD103, CD160(BY55), CD18, CD19, CD19 19, CD247, CD19, CD276 (B19-H19), CD19 delta, CD19 epsilon, CD19 gamma, CD19, CD 3649, CD49 19, CD8alpha, CD19 (Tale), CDl la, ctil, CD balb, ACAL 226, ACAL, CDLA, CD 72, CD19 alpha-IL-alpha-7, CD-IL receptor (GAMMAM-21), CD-I receptor alpha-7, CD-I receptor (GAMMAM) CD 72, CD-7, CD-I receptor alpha-II, CD-I7, CD-I receptor (CD-I-II), CD-II receptor CD-II, CD-III), CD-III receptor (CD-III), CD-I, Inducible T cell co-stimulator (ICOS), integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGBl, KIRDS2, LAT, LFA-1, a ligand that specifically binds to CD83, LIGHT, LTBR, Ly9(CD229), Ly 08), lymphocyte function-associated antigen-1 (LFA-1; CDl-la/CD18), MHC class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80(KLRF1), OX-40, PAG/Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecules (SLAM protein), SLAM (SLAMF 1; CD 150; IPO-3), SLAMF4(CD 244; 2B4) SLAMF6(NTB-A, SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE/RANKL, VLA1 or VLA-6, or fragments, truncations or combinations thereof. The polynucleotide and polypeptide sequences of these intracellular signaling domains are known in the art.

In some embodiments, a polynucleotide encoding an intracellular signaling domain comprises a nucleotide sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a nucleotide sequence known in the art. In some embodiments, the polypeptide sequence of an intracellular signaling domain comprises a polypeptide sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a polypeptide sequence known in the art.

I.C activation Domain

CD3 is an element of the T cell receptor on natural T cells and has been shown to be an important intracellular activation element in CARs. In some embodiments, CD3 is CD3-zeta or CD3-epsilon, the respective polynucleotide and polypeptide sequences of which are known in the art.

In some embodiments, the polynucleotide encoding the activation domain comprises a nucleotide sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a nucleotide sequence known in the art. In some embodiments, the polypeptide sequence of the activation domain comprises a polypeptide sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a polypeptide sequence known in the art.

Switch Domain (Switch Domain)

It will be appreciated that adverse events can be minimized by transducing immune cells (containing one or more CARs or TCRs) with a suicide gene. It may also be desirable to incorporate inductive "on" or "accelerator" switches into immune cells. Suitable techniques include the use of inducible caspase-9 (U.S. application 2011/0286980) or thymidine kinase, either before, after, or simultaneously with transduction of cells with the CAR constructs of the invention. Additional methods of introducing suicide genes and/or "on" switches include TALENS, zinc fingers, RNAi, siRNA, shRNA, antisense technologies, and other technologies known in the art.

Additional on-off or other types of control switching techniques may be incorporated herein in accordance with the present invention. These techniques may employ dimerization domains and optionally activators of dimerization of such domains. These techniques include, for example, those described by Wu et al, Science 2014350 (6258), the contents of which are incorporated herein by reference in their entirety, which utilize the FKBP/Rapalog dimerization system in certain cells. Additional dimerization pairs may include cyclosporine-A/cyclophilin receptor, estrogen/estrogen receptor (optionally using tamoxifen), glucocorticoid/glucocorticoid receptor, tetracycline/tetracycline receptor, vitamin D/vitamin D receptor. Further examples of dimerization techniques can be found, for example, in WO 2014/127261, WO 2015/090229, US 2014/0286987, US 2015/0266973, US 2016/0046700, US patent No. 8,486,693, US 2014/0171649, and US 2012/0130076, the contents of which are further incorporated herein by reference in their entirety.

I.E. leader peptide or leader sequence

In some embodiments, a polynucleotide of the invention encodes a CAR or TCR, which may further comprise a leader peptide (also referred to herein as a "signal peptide" or "leader sequence"). In certain embodiments, the leader peptide comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to amino acid sequence MEWTWVFLFLLSVTAGVHS (SEQ ID NO:6) or MALPVTALLLPLALLLHAARP (SEQ ID NO: 35). In some embodiments, the leader peptide comprises the amino acid sequence of SEQ ID NO 6 or SEQ ID NO 35.

Polynucleotide and polypeptide sequences for other leader peptides are known in the art.

In some embodiments, a polynucleotide encoding a leader peptide comprises a nucleotide sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a nucleotide sequence known in the art. In some embodiments, the polypeptide sequence of the leader peptide comprises a polypeptide sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a polypeptide sequence known in the art.

In some embodiments, the polynucleotide of the invention encodes a CAR or TCR, wherein the CAR or TCR comprises a leader peptide (P), an antigen binding molecule (B), an extracellular domain of a costimulatory protein (E), a transmembrane domain (T), a costimulatory region (C), and an activation domain (a), wherein the CAR is configured according to: P-B-E-T-C-A. In some embodiments, the antigen binding molecule comprises a VH and a VL, wherein the CAR is configured according to: P-VH-VL-E-T-C-A or P-VL-VH-E-T-C-A. In some embodiments, the VH and VL are connected by a linker (L), wherein the CAR is configured (from N-terminus to C-terminus) as follows: P-VH-L-VL-E-T-C-A or P-VH-L-VL-E-T-C-A.

In some embodiments, a polynucleotide of the invention encodes a CAR or TCR, wherein the CAR or TCR comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the amino acid sequence of a CAR or TCR known in the art. See, for example, US 62/470,703 and US62/317,258.

Vectors, cells and pharmaceutical compositions

In certain aspects, provided herein are vectors comprising a polynucleotide of the invention. In some embodiments, the invention relates to a vector or set of vectors comprising a polynucleotide encoding a CAR or TCR comprising an antigen binding domain, as described herein.

Any vector known in the art may be suitable for the present invention. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenoviral vector, a baculovirus vector, an EB virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentiviral vector, or any combination thereof.

In other aspects, provided herein are cells comprising a polynucleotide or vector of the invention. In some embodiments, the invention relates to a cell, e.g., an in vitro cell, comprising a polynucleotide encoding a CAR as described herein or comprising an antigen binding domain. In other embodiments, the invention relates to a cell, e.g., an in vitro cell, comprising a polypeptide encoded by a CAR or TCR comprising an antigen binding domain as described herein.

For the polynucleotide, vector or polypeptide of the present invention, any cell may be used as a host cell. In some embodiments, the cell may be a prokaryotic cell, a fungal cell, a yeast cell, or a higher eukaryotic cell such as a mammalian cell. Suitable prokaryotic cells include, but are not limited to, eubacteria, such as gram-negative or gram-positive organisms, for example, bacteria of the family enterobacteriaceae (enterobacteriaceae) such as Escherichia (Escherichia), e.g., Escherichia coli (e.coli); enterobacteria (Enterobacter); erwinia (Erwinia); klebsiella (Klebsiella); proteus (Proteus); salmonella (Salmonella), such as Salmonella typhimurium (Salmonella typhimurium); serratia (Serratia), such as Serratia discolorations (Serratiamarescans) and Shigella (Shigella); bacillus (bacillus) such as bacillus subtilis and bacillus licheniformis (b.licheniformis); pseudomonas (Pseudomonas) such as Pseudomonas aeruginosa (p. aeruginosa); and Streptomyces (Streptomyces). In some embodiments, the cell is a human cell. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is selected from the group consisting of: t cells, B cells, Tumor Infiltrating Lymphocytes (TILs), TCR-expressing cells, Natural Killer (NK) cells, dendritic cells, granulocytes, innate lymphoid cells, megakaryocytes, monocytes, macrophages, platelets, thymocytes, and myeloid cells. In one embodiment, the immune cell is a T cell. In another embodiment, the immune cell is an NK cell. In certain embodiments, the T cell is a Tumor Infiltrating Lymphocyte (TIL), an autologous T cell, an engineered autologous T cell (eACTTM), an allogeneic T cell, a allogeneic T cell, or any combination thereof.

The cells of the invention may be obtained from any source known in the art. For example, T cells may be differentiated from a population of hematopoietic stem cells in vitro, or T cells may be obtained from a subject. T cells can be obtained from, for example, peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. Furthermore, the T cells may be derived from one or more T cell lines available in the art. T cells can also be taken from blood units collected from a subject using any number of techniques known to those skilled in the art, such as FICOLLTM isolation and/or apheresis. In certain embodiments, cells collected by apheresis are washed to remove the plasma fraction and placed in an appropriate buffer or medium for subsequent processing. In some embodiments, the cells are washed with PBS. As should be appreciated, the washing step may be used, for example, by using a semi-automatic non-countercurrent centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMateTM, etc.). In some embodiments, the washed cells are resuspended in one or more biocompatible buffers, or other salt solutions with or without buffers. In certain embodiments, undesired components of the apheresis sample are removed. Additional methods of isolating T cells for use in T cell therapy are disclosed in U.S. patent publication No. 2013/0287748, which is incorporated by reference herein in its entirety.

In certain embodiments, T cells are isolated from PBMCs by lysing erythrocytes and depleting monocytes (e.g., via PERCOLLTM gradient, isolated by using centrifugation). In some embodiments, specific subpopulations of T cells, such as CD4+, CD8+, CD28+, CD45RA +, and CD45RO + T cells, may be further isolated by positive or negative selection techniques known in the art. For example, enrichment of a population of T cells by negative selection can be accomplished using a combination of antibodies directed against surface markers specific to the negatively selected cells. In some embodiments, cell sorting and/or selection via negative magnetic immunoadhesion or flow cytometry (which uses a mixture of monoclonal antibodies directed against cell surface markers present on negatively selected cells) can be used. For example, to enrich for CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD8, CD11b, CD14, CD16, CD20, and HLA-DR. In certain embodiments, flow cytometry and cell sorting are used to isolate cell populations of interest for use in the present invention.

In some embodiments, PBMCs are used directly for genetic modification of immune cells (e.g., CARs or TCRs) using methods as described herein. In certain embodiments, after PBMC isolation, T lymphocytes may be further isolated and both cytotoxic and helper T lymphocytes sorted into naive, memory and effector T cell subsets, either before or after genetic modification and/or expansion.

In some embodiments, CD8+ cells are further sorted into naive, central memory and effector cells by identifying cell surface antigens associated with each of naive, central memory and effector cell types of CD8+ cells. In some embodiments, expression of the phenotypic markers of central memory T cells comprises CCR7, CD3, CD28, CD45RO, CD62L, and CD127 and is granzyme B negative. In some embodiments, the central memory T cells are CD8+, CD45RO +, and CD62L + T cells. In some embodiments, effector T cells are CCR7, CD28, CD62L, and CD127 negative and are granzyme B and perforin positive. In certain embodiments, the CD4+ T cells are further sorted into subpopulations. For example, CD4+ T helper cells can be sorted into naive, central memory and effector cells by identifying cell populations with cell surface antigens.

In some embodiments, immune cells, such as T cells, are genetically modified after isolation using known methods, or immune cells are activated and expanded (or, in the case of progenitor cells, differentiated) in vitro before being genetically modified. In another embodiment, an immune cell, e.g., a T cell, is genetically modified (e.g., transduced with a viral vector comprising one or more nucleotide sequences encoding a CAR) with a chimeric antigen receptor described herein and then activated and/or amplified in vitro. Methods for activating and expanding T cells are known in the art and are described, for example, in U.S. patent nos. 6,905,874; 6,867,041, respectively; and 6,797,514; and PCT publication No. WO 2012/079000, the contents of which are hereby incorporated by reference in their entirety. In general, such methods comprise contacting PBMCs or isolated T cells with stimulating and co-stimulating agents (e.g., anti-CD 3 and anti-CD 28 antibodies, typically adhered to beads or other surfaces) in media with appropriate cytokines (e.g., IL-2). anti-CD 3 and anti-CD 28 antibodies attached to the same beads serve as "replacement" Antigen Presenting Cells (APCs). One example is a system, the CD3/CD28 activator/stimulator system, for physiologically activating human T cells. In other embodiments, T cells are activated and stimulated for proliferation with feeder cells and appropriate antibodies and cytokines using methods such as those described in U.S. patent nos. 6,040,177 and 5,827,642 and PCT publication No. WO 2012/129514 (the contents of which are incorporated herein by reference in their entirety).

In certain embodiments, the T cells are obtained from a donor subject. In some embodiments, the donor subject is a human patient having a cancer or tumor. In other embodiments, the donor subject is a human patient that does not have a cancer or tumor.

Other aspects of the invention relate to compositions comprising a polynucleotide described herein, a vector described herein, a polypeptide described herein, or an in vitro cell described herein. In some embodiments, the composition comprises a pharmaceutically acceptable carrier, diluent, solubilizer, emulsifier, preservative, and/or adjuvant. In some embodiments, the composition comprises an excipient. In one embodiment, the composition comprises a polynucleotide encoding a CAR or TCR comprising an antigen binding domain as described herein. In another embodiment, the composition comprises a CAR or TCR comprising an antigen binding domain as described herein encoded by a polynucleotide of the invention. In another embodiment, the composition comprises a T cell comprising a CAR or a TCR (which comprises an antigen binding domain as described herein).

The process of the invention

Another aspect of the invention relates to a method of making a cell expressing a CAR or TCR comprising transducing a cell with a polynucleotide disclosed herein under suitable conditions. In some embodiments, as disclosed herein, a method comprises transducing a cell with a polynucleotide encoding a CAR or a TCR. In some embodiments, the method comprises transducing a cell with a vector comprising a polynucleotide encoding a CAR or a TCR.

Another aspect of the invention relates to a method of inducing immunity to a tumor comprising administering to a subject an effective amount of a cell comprising a polynucleotide described herein, a vector described herein, or a CAR or TCR described herein. In one embodiment, the method comprises administering to a subject an effective amount of a cell comprising a polynucleotide encoding a CAR or TCR disclosed herein. In another embodiment, the method comprises administering to the subject an effective amount of a cell comprising a vector comprising a polynucleotide encoding a CAR or TCR disclosed herein. In another embodiment, the method comprises administering to the subject an effective amount of a cell comprising a CAR or a TCR encoded by a polynucleotide disclosed herein.

Another aspect of the invention relates to a method of inducing an immune response in a subject comprising administering an effective amount of an engineered immune cell of the present application. In some embodiments, the immune response is a T cell mediated immune response. In some embodiments, the T cell-mediated immune response is directed against one or more target cells. In some embodiments, the engineered immune cell comprises a CAR or a TCR, wherein the CAR or TCR comprises an antigen binding domain as described herein. In some embodiments, the target cell is a tumor cell.

Another aspect of the invention relates to a method for treating or preventing a malignant tumor, the method comprising administering to a subject in need thereof an effective amount of at least one immune cell, wherein the immune cell comprises at least one CAR or TCR, and wherein the CAR or TCR comprises an antigen binding domain as described herein.

Another aspect of the invention relates to a method of treating cancer in a subject in need thereof, comprising administering to the subject a polynucleotide, vector, CAR or TCR, cell or composition disclosed herein. In one embodiment, the method comprises administering a polynucleotide encoding a CAR or a TCR. In another embodiment, the method comprises administering a vector comprising a polynucleotide encoding a CAR or a TCR. In another embodiment, the method comprises administering a CAR or a TCR encoded by a polynucleotide disclosed herein. In another embodiment, the method comprises administering a cell comprising a polynucleotide encoding a CAR or a TCR or a vector comprising a polynucleotide encoding a CAR or a TCR.

In some embodiments, the method of treating cancer in a subject in need thereof comprises T cell therapy. In one embodiment, the T cell therapy of the invention is an engineered autologous cell therapy (eACTTM). According to this embodiment, the method may include collecting blood cells from the patient. The isolated blood cells (e.g., T cells) can then be engineered to express the CARs or TCRs of the invention. In a specific embodiment, the CAR T cells or TCR T cells are administered to a patient. In some embodiments, the CAR T cells or TCR T cells treat a tumor or cancer in the patient. In one embodiment, the CAR T cells or TCR T cells reduce the size of the tumor or cancer.

In some embodiments, donor T cells for use in T cell therapy are obtained from a patient (e.g., for autologous T cell therapy). In other embodiments, donor T cells for use in T cell therapy are obtained from a subject other than a patient.

The T cells may be administered in a therapeutically effective amount. For example, a therapeutically effective amount of T cells can be at least about 104 cells, at least about 105 cells, at least about 106 cells, at least about 107 cells, at least about 108 cells, at least about 109 cells, or at least about 1010 cells. In another embodiment, the therapeutically effective amount of T cells is about 104 cells, about 105 cells, about 106 cells, about 107 cells, or about 108 cells. In a specific embodiment, the therapeutically effective amount of CAR T cells or TCR T cells is about 2X 106 cells/kg, about 3X 106 cells/kg, about 4X 106 cells/kg, about 5X 106 cells/kg, about 6X 106 cells/kg, about 7X 106 cells/kg, about 8X 106 cells/kg, about 9X 106 cells/kg, about 1X 107 cells/kg, about 2X 107 cells/kg, about 3X 107 cells/kg, about 4X 107 cells/kg, about 5X 107 cells/kg, about 6X 107 cells/kg, about 7X 107 cells/kg, about 8X 107 cells/kg, or about 9X 107 cells/kg.

Cancer treatment

The methods of the invention can be used to treat cancer, reduce tumor size, kill tumor cells, prevent tumor cell proliferation, prevent tumor growth, eliminate tumors from a patient, prevent tumor recurrence, prevent tumor metastasis, induce remission in a patient, or any combination thereof in a subject. In certain embodiments, the method induces a complete response. In other embodiments, the method induces a partial response.

Cancers that may be treated include B cell lymphomas. In certain embodiments, the B cell lymphoma is Acute Lymphoblastic Leukemia (ALL), AIDS-related lymphoma, ALK-positive large B cell lymphoma, burkitt's lymphoma, Chronic Lymphocytic Leukemia (CLL), classical hodgkin's lymphoma, Diffuse Large B Cell Lymphoma (DLBCL), follicular lymphoma, intravascular large B cell lymphoma, HHV 8-related large B cell lymphoma caused by multicentric Castleman disease, lymphoma-like granuloma, lymphoplasmacytic lymphoma, Mantle Cell Lymphoma (MCL), marginal zone B cell lymphoma (MZL), mucosa-related lymphoid tissue lymphoma (MALT), lymph node marginal zone B cell lymphoma (NMZL), nodal lymphocyte-predominant hodgkin's lymphoma, non-hodgkin's lymphoma, plasmacytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, lymphoblastic lymphoma, primary effusion lymphoma, lymphomatosis, lymphomas, and lymphomas, Splenic Marginal Zone Lymphoma (SMZL) and waldenstrom's macroglobulinemia or a combination thereof. In one embodiment, the B cell lymphoma is Acute Lymphoblastic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL), Diffuse Large B Cell Lymphoma (DLBCL), follicular lymphoma, Mantle Cell Lymphoma (MCL), marginal zone B cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), and non-hodgkin's lymphoma. In one embodiment, the B cell lymphoma is non-hodgkin's lymphoma.

In some embodiments, the method further comprises administering a chemotherapeutic agent.

In other embodiments, the antigen binding molecule, transduced (or otherwise engineered) cells (e.g., CARs or TCRs), and chemotherapeutic agent are each administered in an amount effective to treat a disease or condition in the subject.

In certain embodiments, a composition comprising an immune effector cell expressing a CAR and/or TCR disclosed herein can be administered before, in combination with, and/or after any number of chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents (alkylating agents), such as thiotepa and Cyclophosphamide (CYTOXANTM); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines (aziridines), such as benzotepa (benzodepa), carboquone (carboquone), metoclopramide (meteredepa) and uretepa (uredepa); ethyleneimines and methylmelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimetylomelamine; nitrogen mustards such as chlorambucil (chlorambucil), chlorambucil (chlorenaphazine), chlorpromazine (chlorpromazine), cholorphamide (cholorophosphamide), estramustine (estramustine), ifosfamide (ifosfamide), mechlorethamine (mechlorethamine), mechlorethamine hydrochloride (mechlorethamine oxide hydrochloride), melphalan (melphalan), neomustard (novembichin), benzene mustard cholesterol (phenosterine), prednimustine (prednimustine), trofosfamide (trofosfamide), uracil mustard (uramustard); nitrosoureas such as carmustine (carmustine), chlorouretocin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine), ramustine (ranimustine); antibiotics such as aclacinomycin (aclacinomycin), actinomycin (actinomycin), anthranomycin (antrramycin), azaserine (azaserine), bleomycin (bleomycin), actinomycin C (cactinomycin), calicheamicin (calicheamicin), karabine (carabicin), carminomycin (carminomycin), carcinomycin (carzinophilin), chromomycin (chromomycin), actinomycin D (dactinomycin), daunorubicin (daunorubicin), ditorexin (detroribin), 6-diaza-5-oxo-L-norleucine, doxorubicin (doxorubicin), epirubicin (epirubicin), elsinomycin (esorubicin), idarubicin (idarubicin), cericin (cericin), mitomycin (mitomycin), mycins (gentamycin), doxorubicin (diphenomycin), diphenomycin (diphenomycin), diphenomycin (, Streptonigrin (streptonigrin), streptozocin (streptozocin), tubercidin (tubicidin), ubenimex (ubenimex), restatin (zinostatin), zorubicin (zorubicin); antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteroyltriglutamic acid (pteropterin), trimetrexate (trimetrexate); purine analogs, such as fludarabine (fludarabine), 6-mercaptopurine (mercaptoprine), thiamiprine (thiamiprine), thioguanine (thioguanine); pyrimidine analogs such as ancitabine (ancitabine), azacitidine (azacitidine), 6-azauridine, carmofur (carmofur), cytarabine (cytarabine), dideoxyuridine (dideoxyuridine), doxifluridine (doxifluridine), enocitabine (enocitabine), floxuridine (floxuridine), 5-FU; androgens such as carotinone (calusterone), dromostanolone propionate, epitioandrostanol (epitiostanol), mepiquitane (mepiquitane), testolactone (testolactone); anti-adrenal agents, such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), trilostane (trilostane); folic acid supplements, such as folinic acid (folinic acid); acetoglucurolactone (acegultone); (ii) an aldophosphamide glycoside; aminolevulinic acid (aminolevulinic acid); amsacrine (amsacrine); tabularil (bestrabucil); bisantrene; edatrexate (edatraxate); desphosphamide (defosfamide); dimecorsine (demecolcine); diazaquinone (diaziqutone); elfornitine; ammonium etitanium acetate; etoglut (etoglucid); gallium nitrate; hydroxyurea (hydroxyurea); lentinan (lentinan); lonidamine (lonidamine); mitoguazone (mitoguzone); mitoxantrone (mitoxantrone); mopidamol (mopidamol); diamine nitracridine (nitrarine); podophyllinic acid (podophyllic acid); methionine mustard (phenamett); pirarubicin (pirarubicin); losoxantrone (losoxantrone); 2-ethyl hydrazide (ethylhydrazide); procarbazine (procarbazine); razoxane (rizoxane); sisofilan (sizofiran); helical germanium (spirogermanium); tenuazonic acid (tenuazonic acid); triimine quinone (triaziquone); 2, 2' -trichlorotriethylamine; urethane (urethan); vindesine (vindesine); dacarbazine (dacarbazine); mannitol mustard (mannomustine); dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); pipobromane (pipobroman); gatifloxacin; cytarabine (arabine) ("Ara-C"); cyclophosphamide; thiotepa (thiotepa); paclitaxel (taxoids), such as paclitaxel (paclitaxel) (taxol, Bristol-Myers Squibb) and docetaxel (doxetaxel) (Rhone-Poulenc Rorer); chlorambucil (chlorambucil); gemcitabine; 6-thioguanine (thioguanine); mercaptopurine (mercaptoprine); methotrexate (methotrexate); platinum analogs, such as cisplatin (cissplatin) and carboplatin (carboplatin); vinblastine (vinblastine); platinum; etoposide (VP-16); ifosfamide (ifosfamide); mitomycin C; mitoxantrone (mitoxantrone); vincristine; vinorelbine; navelbine (navelbine); oncostatin (novantrone); teniposide (teniposide); daunomycin (daunomycin); aminopterin (aminopterin); xeloda; ibandronate (ibandronate); CPT-11; topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); tretinoin (retinic acid) derivatives such as targretin (bexarotene), panretin (alritetinin); ONTAKTM (Denneukindiftitox); esperamicin (esperamicin); capecitabine (capecitabine); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. In some embodiments, a composition comprising a CAR and/or TCR-expressing immune effector cell may be administered in combination with an anti-hormonal agent that acts to modulate or inhibit the effect of hormones on tumors, such as anti-estrogens, including, for example, tamoxifen (tamoxifen), raloxifene (raloxifene), aromatase-inhibiting 4(5) -imidazole, 4-hydroxytamoxifene, trioxifene (trioxifene), woloxifene (keoxifene), LY117018, onapristone (onapristone), and toremifene (toremifene) (Fareston); and antiandrogens such as flutamide (flutamide), nilutamide (nilutamide), bicalutamide (bicalutamide), leuprolide (leuprolide) and goserelin (goserelin); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Combinations of chemotherapeutic agents are also administered where appropriate, including but not limited to CHOP, i.e., cyclophosphamide doxorubicin (hydroxydoxorubicin), vincristine, and prednisone.

In certain embodiments, a composition comprising an immune effector cell expressing a CAR and/or a TCR disclosed herein can be administered before, in conjunction with, and/or after CHOP. CHOP consists of: (C) cyclophosphamide, an alkylating agent that destroys DNA by binding to it and causing cross-links to form; (H) hydroxydaunorubicin (also known as doxorubicin or adriamycin), an intercalator, that disrupts DNA by inserting itself between DNA bases; (O) ncovin (vincristine), which prevents cell replication by binding to the protein tubulin; and (P) prednisone or prednisolone, which is a corticosteroid.

In some embodiments, the chemotherapeutic agent is administered simultaneously with or within one week after the administration of the engineered cell or nucleic acid. In other embodiments, the chemotherapeutic agent is administered 1 to 4 weeks or 1 week to 1 month, 1 week to 2 months, 1 week to 3 months, 1 week to 6 months, 1 week to 9 months, or 1 week to 12 months after administration of the engineered cell or nucleic acid. In some embodiments, the chemotherapeutic agent is administered at least 1 month prior to administration of the cell or nucleic acid. In some embodiments, the method further comprises administering two or more chemotherapeutic agents.

Additional therapeutic agents suitable for use in combination with the present invention include, but are not limited to, ibrutinib (ibrutinib) efamumab (ofatumumab) rituximab (rituximab) bevacizumab (bevacizumab) trastuzumab (trastuzumab) ibrutinib amantansinib (imatinib), cetuximab (cetuximab) panitumumab (panitumumab) katovazumab (catuzumab), tiitumomab (ibritumomab), ofamumab, tositumomab (tositumomab), benitumomab (brentuximab), atramtuzumab (alemtuzumab), erlotinib (erlotinib), gefitinib (gefitinib), vandetanib (vafitinib), atratinib (nilotinib), erlotinib (neratinib), nilotinib (neratinib), neratinib (neratinib), neratinib, Levatinib (lenvatinib), nintedanib (nintedanib), pazopanib (pazopanib), regorafenib (regorafenib), semaxanib (semaxanib), sorafenib (sorafenib), sunitinib (sunitinib), tivaxanib (tivozanib), tosiranib (tocraniib), vandetanib, enretinib (entretinib), cabozatinib (cabozantinib), imatinib (imatinib), dasatinib (dasatinib), nilotinib (nilotinib), panatinib (ponatinib), ramotinib (raditinib), bostatatinib (bosutinib), letatinib (lesuitib), lurosurtinib (prartitinib), tematinib (metrinib), tematinib (blertinib), tematinib (metrinib), temetinib (mTOR), temetinib (toctinib), Temsirolimus (toctinib), tematinib (blertinib), temsirotinib (toctinib), temsirotinib (bletinib), temicinib (temicinib), temib (trotinib (temsirotinib (bletinib), temicinib), temib (trotinib), temib (trotinib), temicinib), temib (trotinib), or (trotinib), or (trotinib), hedgehog inhibitors such as sonchig (sonidegib) and vemuraglib (vismodegib), CDK inhibitors such as CDK inhibitors (palbociclib).

Additional therapeutic agents suitable for use in the present invention include radioactive atoms. Examples of such radioactive atoms include 131I, 90Y, 212Bi, 186Re, 221At, 99 mtc, and mixtures thereof. However, other radioactive atoms may also be used, as is known in the art.

In additional embodiments, a composition comprising a T cell expressing a CAR and/or a TCR, a conjugate comprising a scFV, or the scFV itself is administered with an anti-inflammatory agent. Anti-inflammatory agents or drugs may include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, methylprednisolone, prednisolone, triamcinolone, nonsteroidal anti-inflammatory drugs (NSAIDS), including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide, and mycophenolate mofetil. Exemplary NSAIDs include ibuprofen, naproxen sodium, Cox-2 inhibitors, and sialylate. Exemplary analgesics include paracetamol (acetaminophen), oxycodone (oxycodone), tramadol or propoxyphene hydrochloride (tramadol of prolyphene hydrochloride). Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors such as TNF antagonists, (e.g., etanercept adalimumab (adalimumab) and infliximab (infliximab) biological response modifiers include monoclonal antibodies as well as recombinant forms of the molecules exemplary DMARDs include azathioprine (azathioprine), cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular), and minocycline (minocycline).

In certain embodiments, the compositions described herein are administered in combination with a cytokine. As used herein, "cytokine" means a protein released by one cell population that acts on another cell as an intercellular mediator. Examples of cytokines are lymphokines, monokines, and traditional polypeptide hormones. The cell factor includes growth hormone, such as human growth hormone, N-methionyl human growth hormone and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; (ii) prorelaxin; glycoprotein hormones such as Follicle Stimulating Hormone (FSH), Thyroid Stimulating Hormone (TSH), and Luteinizing Hormone (LH); hepatic Growth Factor (HGF); fibroblast Growth Factor (FGF); prolactin; placental lactogen; mullerian-inhibiting substances (mullerian-inhibiting substance); mouse gonadotropin-related peptides; a statin; an activin; vascular endothelial growth factor; an integrin; thrombopoietin (TPO); nerve Growth Factor (NGF) such as NGF-beta; platelet growth factor; transforming Growth Factors (TGF) such as TGF-alpha and TGF-beta; insulin-like growth factors-I and-II; erythropoietin (EPO); an osteoinductive factor; interferons such as interferon-alpha, beta and-gamma; colony Stimulating Factors (CSFs), such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (IL), such as IL-1, IL-1 α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, tumor necrosis factors such as TNF-alpha or TNF-beta; and other polypeptide factors, including LIF and Kit Ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture, as well as biologically active equivalents of the native sequence cytokines.

V. conjugates

The antigen binding domains of the invention (e.g., anti-human CD19 scFV) can be conjugated (e.g., linked) to a therapeutic agent (e.g., a chemotherapeutic agent and a radioactive atom) as described herein.

Techniques for conjugating such therapeutic agents to antibodies, such as scfvs, are well known; see, e.g., Arnon et al, "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," In Monoclonal Antibodies And Cancer Therapy, Reisfeld et al (eds.),1985, pp.243-56, Alan R.Liss, Inc.); hellstrom et al, "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd Ed.), Robinson et al (eds.),1987, pp.623-53, Marcel Dekker, Inc.); thorpe, "Antibodies Of Cytotoxic Agents In Cancer Therapy: A Review", In Monoclonal Antibodies' 84: Biological And Clinical Applications, Pinchera et al, (eds.),1985, pp.475-506); "Analysis, Results, And d Future productive Of The Therapeutic Use Of radioactive anti In Cancer Therapy", In Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al (eds.),1985, pp.303-16, Academic Press; and Thorpe et al, Immunol.Rev.,62:119-58, 1982. Each of the foregoing references is incorporated by reference in its entirety.

Humanization of antibodies

Antibody humanization is the process of replacing a non-human antibody, e.g., scFv framework, with a human antibody framework. Successful antibody humanization depends on maintaining affinity after residue substitution. In some embodiments, a humanized antibody is an antibody molecule in which at least a portion of the sequence of both the light chain and the heavy chain is from a human gene. Such antibodies are referred to herein as "humanized antibodies", "human antibodies", or "fully human antibodies". Human monoclonal antibodies can be produced by using the trioma (trioma) technique; human B cell hybridoma technology (see Kozbor, et al, 1983Immunol Today 4: 72); and EBV hybridoma technology (see Cole, et al, 1985In: Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp.77-96) for the production of human Monoclonal Antibodies. Human Monoclonal Antibodies can be generated using human hybridomas (see Cote, et al, 1983.Proc Natl Acad Sci USA 80: 2026-.

In addition, human antibodies (e.g., scFv) can be generated using alternative techniques, including phage display libraries (see Hoogenboom and Winter, J.mol.biol.,227:381 (1991); Marks et al, J.mol.biol.,222:581 (1991)). Similarly, human antibodies can be prepared by introducing human immunoglobulin loci into transgenic animals, e.g., mice, in which endogenous immunoglobulin genes have been partially or completely inactivated. After challenge, human antibody production was observed, which is very similar in all respects to that seen in humans, including gene rearrangement, assembly, and antibody repertoire (repotoreire). The process is described in U.S. patent nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126, respectively; 5,633,425, respectively; 5,661,016 and Marks et al, Bio/Technology 10,779-783 (1992); lonberg et al, Nature 368856-; morrison, Nature 368,812-13 (1994); fishwild et al, Nature Biotechnology 14,845-51 (1996); neuberger, Nature Biotechnology 14,826 (1996); and Lonberg and Huszar, Intern.Rev.Immunol.1365-93 (1995).

Methods of humanizing antibodies are provided. This approach follows a combination of CDR grafting, structure-based reverse mutation approach, and rapid screening for expression levels, biophysical properties, and affinity technology (faeba) that generates humanized antibodies, such as scFv, with affinity and optimized properties. The faeba technique is used to optimize the properties of humanized antibodies, such as expression level and thermostability, which are important for optimizing manufacturing and in vivo behavior. Additional details of the method are found at world wide web (www) description. com/Antibody-simulation. html. As available in the disclosure of the present application, the contents of which are incorporated by reference in their entirety.

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. However, citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention. To the extent that any definition or term provided in a reference, which is incorporated by reference, differs from the term and discussion provided herein, the term and definition of the invention shall control.

The invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of all references cited in this application are expressly incorporated herein by reference.

Examples