阅读说明：本技术 与调节细胞内乳酸浓度的反式代谢分子组合的嵌合受体多肽及其治疗用途 (Chimeric receptor polypeptides in combination with trans-metabolic molecules that modulate intracellular lactic acid concentration and therapeutic uses thereof ) 是由 K.麦金尼斯 S.埃坦伯格 L.巴伦 M.弗雷 C.威尔逊 G.莫茨 于 2019-09-06 设计创作，主要内容包括：本文公开了遗传工程化的造血细胞,所述遗传工程化的造血细胞表达一种或多种乳酸调节因子(例如,多肽)和任选的嵌合受体多肽(例如,抗体偶联的T细胞受体(ACTR)多肽或嵌合抗原受体(CAR)多肽),所述嵌合受体多肽能够与感兴趣的靶抗原结合。本文还公开了工程化的造血细胞用于抑制在有需要的受试者中表达靶抗原的细胞的用途。(Disclosed herein are genetically engineered hematopoietic cells that express one or more lactate modulators (e.g., polypeptides) and optionally a chimeric receptor polypeptide (e.g., an antibody-coupled T cell receptor (ACTR) polypeptide or a Chimeric Antigen Receptor (CAR) polypeptide) that is capable of binding to a target antigen of interest. Also disclosed herein is the use of an engineered hematopoietic cell for inhibiting a cell expressing a target antigen in a subject in need thereof.)

1. A genetically engineered hematopoietic cell having an enhanced intracellular lactate concentration as compared to a native hematopoietic cell of the same type.

2. The genetically engineered hematopoietic cell of claim 1, which expresses or overexpresses

(i) A lactate modulator.

3. The genetically engineered hematopoietic cell of claim 2, wherein the lactate modulator is a lactate modulating polypeptide.

4. The genetically engineered hematopoietic cell of claim 3, wherein the lactate modulating polypeptide is a monocarboxylate transporter (MCT), an enzyme involved in lactate synthesis, or a polypeptide that inhibits a pathway that competes for a lactate synthesis substrate.

5. The genetically engineered hematopoietic cell of claim 4, wherein the MCT is MCT1, MCT2, or MCT 4.

6. The genetically engineered hematopoietic cell of claim 4, wherein the enzyme involved in lactate synthesis is lactate dehydrogenase A (LDHA).

7. The genetically engineered hematopoietic cell of claim 4, wherein the polypeptide that inhibits a pathway that competes for lactate synthesis substrate is pyruvate dehydrogenase kinase 1(PDK 1).

8. The genetically engineered hematopoietic cell of any one of claims 1-7, further expressing:

(ii) a chimeric receptor polypeptide, wherein the chimeric receptor polypeptide comprises:

(a) an extracellular target-binding domain;

(b) a transmembrane domain; and

(c) a cytoplasmic signaling domain.

9. The genetically engineered hematopoietic cell of claim 8, wherein the chimeric receptor polypeptide is an antibody-coupled T cell receptor (ACTR) polypeptide in which (a) is an extracellular Fc binding domain.

10. The genetically engineered hematopoietic cell of claim 8, wherein the chimeric receptor polypeptide is a chimeric receptor antigen (CAR) polypeptide in which (a) is an extracellular antigen-binding domain.

11. The genetically engineered hematopoietic cell of any one of claims 8-10, wherein the chimeric receptor polypeptide further comprises at least one costimulatory signaling domain.

12. The genetically engineered hematopoietic cell of any one of claims 8-10, wherein the chimeric receptor polypeptide is optionally an ACTR polypeptide, free of a costimulatory signaling domain.

13. The genetically engineered hematopoietic cell of any one of claims 8-12, wherein the cytoplasmic signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM).

14. The genetically engineered hematopoietic cell of any one of claims 8-13, wherein (C) is located at the C-terminus of the chimeric receptor polypeptide.

15. The genetically engineered hematopoietic cell of any one of claims 8-14, wherein the chimeric receptor polypeptide further comprises a hinge domain located at the C-terminus of (a) and the N-terminus of (b).

16. The genetically engineered hematopoietic cell of any one of claims 8-15, wherein the chimeric receptor polypeptide further comprises a signal peptide at its N-terminus.

17. The genetically engineered hematopoietic cell of any one of claims 8-16, wherein the chimeric receptor polypeptide is an ACTR polypeptide in which the extracellular target-binding domain (a) is an extracellular Fc-binding domain, and wherein the Fc-binding domain is selected from the group consisting of:

(A) an extracellular ligand binding domain of an Fc receptor,

(B) an antibody fragment that binds to said Fc portion of an immunoglobulin,

(C) a naturally occurring protein that binds to said Fc portion of an immunoglobulin or an Fc binding fragment thereof, and

(D) a synthetic polypeptide that binds to said Fc portion of an immunoglobulin.

18. The genetically engineered hematopoietic cell of claim 17, wherein the Fc binding domain is (a), which is an extracellular ligand binding domain of an Fc-gamma receptor, an Fc-alpha receptor, or an Fc-epsilon receptor.

19. The genetically engineered hematopoietic cell of claim 18, wherein the Fc binding domain is an extracellular ligand binding domain of CD16A, CD32A, or CD 64A.

20. The genetically engineered hematopoietic cell of claim 18, wherein the Fc binding domain is the extracellular ligand binding domain of F158 CD16A or V158 CD 16A.

21. The genetically engineered hematopoietic cell of claim 17, wherein the Fc-binding domain is (B) which is a single chain variable fragment (scFv) or a single domain antibody.

22. The genetically engineered hematopoietic cell of claim 17, wherein the Fc-binding domain is (C), which is protein a or protein G, or an Fc-binding fragment thereof.

23. The genetically engineered hematopoietic cell of claim 17, wherein the Fc binding domain is (D) which is a Kunitz peptide, SMIP, avimer, affibody, DARPin, or an antiporter protein.

24. The genetically engineered hematopoietic cell of any one of claims 8-16, wherein the chimeric receptor polypeptide is a CAR polypeptide in which the extracellular target-binding domain of (a) is an antigen-binding domain, and wherein the antigen-binding domain is a single-chain antibody fragment that binds to a tumor antigen, a pathogenic antigen, or an immune cell specific for an autoantigen.

25. The genetically engineered hematopoietic cell of claim 24, wherein the tumor antigen is associated with a hematological tumor.

26. The genetically engineered hematopoietic cell of claim 25, wherein the tumor antigen is selected from the group consisting of: CD19, CD20, CD22, kappa chain, CD30, CD123, CD33, LeY, CD138, CD5, BCMA, CD7, CD40 and IL-1 RAP.

27. The genetically engineered hematopoietic cell of claim 24, wherein the tumor antigen is associated with a solid tumor.

28. The genetically engineered hematopoietic cell of claim 27, wherein the tumor antigen is selected from the group consisting of: GD2, GPC3, FOLR, HER2, EphA2, EFGRVIII, IL13RA2, VEGFR2, ROR1, NKG2D, EpCAM, CEA, mesothelin, MUC1, CLDN18.2, CD171, CD133, PSCA, cMET, EGFR, PSMA, FAP, CD70, MUC16, L1-CAM and CAIX.

29. The genetically engineered hematopoietic cell of claim 24, wherein the pathogenic antigen is a bacterial antigen, a viral antigen, or a fungal antigen.

30. The genetically engineered hematopoietic cell of any one of claims 8-29, wherein the transmembrane domain of (b) is of a single transmembrane protein.

31. The genetically engineered hematopoietic cell of claim 30, wherein the transmembrane domain is a membrane protein selected from the group consisting of: CD8 α, CD8 β, 4-1BB, CD28, CD34, CD4, fcepsilon RI γ, CD16A, OX40, CD3 ζ, CD3 ε, CD3 γ, CD3 δ, TCR α, CD32, CD64, VEGFR2, FAS, and FGFR 2B.

32. The genetically engineered hematopoietic cell of any one of claims 8-29, wherein the transmembrane domain of (b) is a non-naturally occurring hydrophobic protein segment.

33. The genetically engineered hematopoietic cell of any one of claims 8-11 and 13-32, wherein the at least one costimulatory signaling domain is of a costimulatory molecule selected from the group consisting of: 4-1BB, CD28, CD28_LL→GGVariants, OX40, ICOS, CD27, GITR, ICOS, HVEM, TIM1, LFA1, and CD 2.

34. The genetically engineered hematopoietic cell of claim 33, wherein the at least one costimulatory signaling domain is a CD28 costimulatory signaling domain or a 4-1BB costimulatory signaling domain.

35. The genetically engineered hematopoietic cell of any one of claims 8-11 and 13-34, wherein the chimeric receptor polypeptide comprises two costimulatory signaling domains.

36. The genetically engineered hematopoietic cell of claim 35, wherein the two costimulatory domains are:

(i) CD28 and 4-1 BB; or

(ii)CD28_{LL GG}Variants and 4-1 BB.

37. The genetically engineered hematopoietic cell of claim 35, wherein one of the costimulatory signaling domains is a CD28 costimulatory signaling domain; and wherein the further co-stimulatory domain is selected from the group consisting of: a 4-1BB costimulatory signaling domain, an OX40 costimulatory signaling domain, a CD27 costimulatory signaling domain, and an ICOS costimulatory signaling domain.

38. The genetically engineered hematopoietic cell of any one of claims 8-37, wherein the cytoplasmic signaling domain of (c) is a cytoplasmic domain of CD3 ζ or fcepsilonr 1 γ.

39. The genetically engineered hematopoietic cell of any one of claims 15-38, wherein the hinge domain is 1 to 60 amino acids in length.

40. The genetically engineered hematopoietic cell of any one of claims 15-39, wherein the hinge domain is of CD28, CD16A, CD 8a, or IgG.

41. The genetically engineered hematopoietic cell of any one of claims 15-40, wherein the hinge domain is a non-naturally occurring peptide.

42. The genetically engineered hematopoietic cell of claim 41, wherein the hinge domain is an extended recombinant polypeptide (XTEN) or (Gly)₄Ser)_nA polypeptide, wherein n is an integer from 3 to 12, inclusive.

43. The genetically engineered hematopoietic cell of any one of claims 8-14 and 16-38, wherein the chimeric receptor polypeptide is optionally an ACTR polypeptide, without any hinge domain.

44. The genetically engineered hematopoietic cell of any one of claims 8-42, wherein the chimeric receptor is optionally an ACTR polypeptide, lacking a hinge domain from any non-CD 16A receptor.

45. The genetically engineered hematopoietic cell of claim 17, wherein the ACTR polypeptide comprises (i) a CD28 costimulatory domain; and (ii) a CD28 transmembrane domain, a CD28 hinge domain, or a combination thereof.

46. The genetically engineered hematopoietic cell of claim 17, wherein the ACTR polypeptide comprises components (a) - (e) as set forth in table 4.

47. The genetically engineered hematopoietic cell of claim 17, wherein the ACTR polypeptide comprises an amino acid sequence selected from SEQ ID No. 1 to SEQ ID No. 80.

48. The genetically engineered hematopoietic cell of claim 24, wherein the chimeric receptor polypeptide is a CAR polypeptide comprising (i) a combination of a CD28 co-stimulatory domain and a CD28 transmembrane domain, a CD28 hinge domain, or a combination thereof, or (ii) a combination of a 4-1BB co-stimulatory domain and a CD8 transmembrane domain, a CD8 hinge domain, or a combination thereof.

49. The genetically engineered hematopoietic cell of claim 24, wherein the CAR polypeptide comprises the amino acid sequence of SEQ ID No. 97 or SEQ ID No. 98.

50. The genetically engineered hematopoietic cell of any one of claims 1-49, wherein the hematopoiesis is a hematopoietic stem cell or an immune cell, optionally wherein the immune cell is a natural killer cell, a macrophage, a neutrophil, an eosinophil, or a T cell.

51. The genetically engineered hematopoietic cell of claim 50, wherein the immune cell is a T cell in which expression of an endogenous T cell receptor, an endogenous major histocompatibility complex, an endogenous beta-2-microglobulin, or a combination thereof has been inhibited or depleted.

52. The genetically engineered hematopoietic cell of any one of claims 1-51, wherein the hematopoietic cell is an immune cell derived from a Peripheral Blood Mononuclear Cell (PBMC), a Hematopoietic Stem Cell (HSC), or an Induced Pluripotent Stem Cell (iPSC).

53. The genetically engineered hematopoietic cell of any one of claims 1-52, wherein the hematopoietic cell comprises a nucleic acid or set of nucleic acids that collectively comprise:

(A) a first nucleotide sequence encoding the lactate modulator; and optionally

(B) A second nucleotide sequence encoding said chimeric receptor polypeptide.

54. The genetically engineered hematopoietic cell of claim 53, wherein the nucleic acid or the set of nucleic acids is an RNA molecule or a set of RNA molecules.

55. The genetically engineered hematopoietic cell of claim 53 or 54, wherein the hematopoietic cell comprises the nucleic acid comprising both the first nucleotide sequence and the second nucleotide sequence.

56. The genetically engineered hematopoietic cell of claim 55, wherein the nucleic acid further comprises a third nucleotide sequence located between the first nucleotide sequence and the second nucleotide sequence, wherein the third nucleotide sequence encodes a ribosome skip site, an Internal Ribosome Entry Site (IRES), or a second promoter.

57. The genetically engineered hematopoietic cell of claim 55, wherein the third nucleotide sequence encodes a ribosome skip site, which is a P2A peptide.

58. The genetically engineered hematopoietic cell of any one of claims 53-57, wherein the nucleic acid or the set of nucleic acids is comprised within a vector or a set of vectors.

59. The genetically engineered hematopoietic cell of claim 58, wherein the vector or set of vectors is an expression vector or set of expression vectors.

60. The genetically engineered hematopoietic cell of claim 58 or 59, wherein the vector or set of vectors comprises one or more viral vectors.

61. The genetically engineered hematopoietic cell of claim 60, wherein the one or more viral vectors are retroviral vectors, optionally lentiviral vectors or gamma retroviral vectors.

62. A pharmaceutical composition comprising the genetically engineered hematopoietic cell of any one of claims 1-61, and a pharmaceutically acceptable carrier.

63. The pharmaceutical composition of claim 62, wherein the genetically engineered hematopoiesis expresses an ACTR polypeptide, and wherein the composition further comprises an Fc-containing therapeutic agent.

64. The pharmaceutical composition of claim 63, wherein the Fc-containing therapeutic agent is a therapeutic antibody or Fc fusion protein.

65. The pharmaceutical composition of claim 63 or 64, wherein the Fc-containing therapeutic agent binds to a target antigen, which is optionally a tumor antigen, a pathogenic antigen, or an immune cell specific for an autoantigen.

66. The pharmaceutical composition of claim 65, wherein the pathogenic antigen is a bacterial antigen, a viral antigen, or a fungal antigen.

67. The pharmaceutical composition of claim 66, wherein the Fc-containing therapeutic agent is a therapeutic antibody selected from the group consisting of: adalimumab, emmetrotuzumab, alemtuzumab, basiliximab, bevacizumab, belimumab, bretuximab, canazumab, cetuximab, certolizumab, dallizumab, dinolizumab, denituximab, eculizumab, efavirenzumab, epratuzumab, gemtuzumab, golimumab, hu14.18K322A, ibritumomab, infliximab, ipilimumab, labuzumab, ranibizumab, rituximab, toluzumab, trastuzumab, tositumomab, teuxezumab, and vedolizumab.

68. A kit, comprising:

a first pharmaceutical composition comprising the genetically engineered hematopoietic cell of any one of claims 8-61, and a pharmaceutically acceptable carrier; and

a second pharmaceutical composition comprising an Fc-containing therapeutic agent and a pharmaceutically acceptable carrier.

69. The kit of claim 68, wherein the Fc-containing therapeutic agent is an Fc fusion protein or a therapeutic antibody.

70. The kit of claim 68 or claim 69, wherein the Fc-containing therapeutic agent binds to a target antigen, which is optionally a tumor antigen, a pathogenic antigen, or an immune cell specific for an autoantigen.

71. The kit of any one of claims 70, wherein the therapeutic antibody is selected from the group consisting of: adalimumab, emmetrotuzumab, alemtuzumab, basiliximab, bevacizumab, belimumab, bretuximab, canazumab, cetuximab, certolizumab, dallizumab, dinolizumab, denituximab, eculizumab, efavirenzumab, epratuzumab, gemtuzumab, golimumab, hu14.18K322A, ibritumomab, infliximab, ipilimumab, labuzumab, ranibizumab, rituximab, toluzumab, trastuzumab, tositumomab, teuxezumab, and vedolizumab.

72. A method for inhibiting cells expressing a target antigen in a subject, the method comprising administering the genetically engineered hematopoietic cell population of any one of claims 8-61 to a subject in need thereof.

73. The method of claim 72, wherein the genetically engineered hematopoietic cells express an ACTR polypeptide, and wherein the subject has been or is being treated with an Fc-containing therapeutic specific for the target antigen.

74. The method of claim 72, wherein the genetically engineered hematopoietic cell expresses a CAR polypeptide comprising an extracellular antigen-binding domain specific for a target antigen.

75. The method of claim 73 or claim 74, wherein the target antigen is a tumor antigen, a pathogenic antigen, or an immune cell specific for an autoantigen.

76. The method of claim 75, wherein the pathogenic antigen is a bacterial antigen, a viral antigen, or a fungal antigen.

77. The method of any one of claims 73-76, wherein at least some of the cells expressing the target antigen are located in a low glucose environment.

78. The method of any one of claims 72-77, wherein the genetically engineered hematopoietic cells are autologous.

79. The method of any one of claims 72-77, wherein the genetically engineered hematopoietic cells are allogeneic.

80. The method of any one of claims 72-79, wherein the genetically engineered hematopoietic cells are activated, expanded, or both ex vivo.

81. The method of any one of claims 73 and 75-78, wherein the Fc-containing therapeutic agent is a therapeutic antibody or Fc fusion protein.

82. The method of claim 81, wherein the Fc-containing therapeutic agent is a therapeutic antibody selected from the group consisting of: adalimumab, emmetrotuzumab, alemtuzumab, basiliximab, bevacizumab, belimumab, bretuximab, canazumab, cetuximab, certolizumab, dallizumab, dinolizumab, denituximab, eculizumab, efavirenzumab, epratuzumab, gemtuzumab, golimumab, hu14.18K322A, ibritumomab, infliximab, ipilimumab, labuzumab, ranibizumab, molumab, matuzumab, obilizumab, obituzumab ozotakib, obinutuzumab, omalizumab, panitumumab, ranituzumab, ranibizumab, rituximab, tositumumab, tositumomab, trastuzumab, ustuzumab and vedolizumab.

83. The method of any one of claims 72-82, wherein the subject is a human patient with cancer and the target antigen is a tumor antigen.

84. The method of claim 83, wherein the cancer is selected from the group consisting of: carcinomas, lymphomas, sarcomas, blastomas, and leukemias.

85. The method of claim 83 or claim 84, wherein the cancer is selected from the group consisting of: cancers of B cell origin, breast cancer, stomach cancer, neuroblastoma, osteosarcoma, lung cancer, skin cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, mesothelioma, pancreatic cancer, head and neck cancer, retinoblastoma, glioma, glioblastoma, liver cancer, and thyroid cancer.

86. The method of claim 85, wherein the B cell derived cancer is selected from the group consisting of: acute lymphoblastic leukemia of B-line, chronic lymphocytic leukemia of B-cell and non-Hodgkin's lymphoma of B-cell.

87. The method of any one of claims 72-86, wherein the genetically engineered hematopoietic cells comprise T cells activated in the presence of one or more of an anti-CD 3 antibody, an anti-CD 28 antibody, IL-2, phytohemagglutinin, and engineered artificially stimulated cells or particles.

88. The method of claim 72, wherein the genetically engineered hematopoietic cells comprise natural killer cells activated in the presence of one or more of 4-1BB ligand, anti-4-1 BB antibody, IL-15, anti-IL-15 receptor antibody, IL-2, IL-12, IL-21, K562 cells, and engineered artificially stimulated cells or particles.

89. A nucleic acid or group of nucleic acids collectively comprising:

(A) a first nucleotide sequence encoding an antibody-coupled T cell receptor (ACTR) polypeptide of any one of claims 8-49; and

(B) a second nucleotide sequence encoding a lactate modulator.

90. A nucleic acid or set of nucleic acids, wherein the lactate modulator is a lactate modulating polypeptide.

91. The nucleic acid or nucleic acid set of claim 90, wherein the lactate modulating polypeptide is a monocarboxylate transporter (MCT), an enzyme involved in lactate synthesis, or a polypeptide that inhibits a pathway that competes for a substrate for lactate synthesis.

92. The nucleic acid or nucleic acid set of claim 91 wherein the MCT is MCT1, MCT2, or MCT 4.

93. The nucleic acid or nucleic acid set of claim 91, wherein the enzyme involved in lactate synthesis is lactate dehydrogenase A (LDHA).

94. The nucleic acid or nucleic acid set of claim 91, wherein the polypeptide that inhibits a pathway that competes for a lactate synthesis substrate is pyruvate dehydrogenase kinase 1(PDK 1).

95. The nucleic acid or set of nucleic acids of any one of claims 89-94, wherein the nucleic acid or set of nucleic acids is an RNA molecule or set of RNA molecules.

96. The nucleic acid or nucleic acid set of any of claims 89-94, wherein the nucleic acid comprises both the first nucleotide sequence and the second nucleotide sequence, and wherein the nucleic acid further comprises a third nucleotide sequence located between the first nucleotide sequence and the second nucleotide sequence, the third nucleotide sequence encoding a ribosome skip site, an Internal Ribosome Entry Site (IRES), or a second promoter.

97. The nucleic acid or set of nucleic acids of claim 96, wherein the ribosome skip site is the P2A peptide.

98. The nucleic acid or set of nucleic acids of any one of claims 89-97, wherein the nucleic acid or set of nucleic acids is comprised within a vector or set of vectors.

99. The nucleic acid or set of nucleic acids of claim 98, wherein the vector or set of vectors is an expression vector or set of expression vectors.

100. The nucleic acid or set of nucleic acids of claim 98 or claim 99, wherein the vector or set of vectors comprises one or more viral vectors.

101. The nucleic acid or nucleic acid set of claim 100, wherein the one or more viral vectors are retroviral vectors, optionally lentiviral vectors or gammaretrovirus vectors.

102. A method of producing a modified hematopoietic cell in vivo, comprising administering to a subject in need thereof the nucleic acid or set of nucleic acids of any one of claims 89-101.

103. The method of claim 102, further comprising administering to the subject an Fc-containing therapeutic agent specific for the target antigen.

Background

Cancer immunotherapy (including cell-based therapies) is used to stimulate an immune response that attacks tumor cells while retaining normal tissue. It is a promising option for the treatment of various types of cancer, as it has the potential to evade the genetic and cellular mechanisms of resistance and target tumor cells while retaining normal tissue.

Cell-based therapies may involve cytotoxic T cells that are reactive towards cancer cells. Eshhar et al, proc.natl.acad.sci.u.s.a.; 1993; 90(2) 720-724; geiger et al, J Immunol.1999; 162(10) 5931-; brentjens et al, nat. med.2003; 9(3) 279-286; cooper et al, blood.2003; 101(4) 1637-1644; and Imai et al, leukamia.2004; 18:676-684. One approach is to express a chimeric receptor having an antigen binding domain fused to one or more T cell activation signaling domains. Binding of cancer antigens via the antigen binding domain results in T cell activation and triggers cytotoxicity. Recent results from clinical trials conducted by infusion of autologous T lymphocytes expressing chimeric receptors provide convincing evidence for their clinical potential. Pure et al, nat.med.2008; 14(11) 1264-; porter et al, N Engl J Med; 2011; 25; 365(8) 725 and 733; bretjens et al, blood.2011; 118(18) 4817-; till et al, blood.2012; 119(17) 3940-; kochenderfer et al, blood.2012; 119(12) 2709 and 2720; and Bretjens et al, Sci Transl Med.2013; 177ra138 in (5), (177).

Another approach is to express antibody-coupled T cell receptor (ACTR) proteins containing an extracellular Fc binding domain in hematopoietic cells (e.g., hematopoietic stem cells, immune cells, such as NK cells, or T cells). When hematopoietic cells expressing ACTR (e.g., T cells expressing ACTR, also referred to as "ACTR T cells") are administered to a subject with an anti-cancer antibody, they can enhance toxicity to the cancer cells targeted by the antibody by binding them to the Fc domain of the antibody. Kudo et al, Cancer Research, (2014)74: 93-103.

Cell-based immunotherapy, while promising, faces challenges arising from the specific properties of the Tumor Microenvironment (TME), the cellular environment created by the interaction between malignant and non-transformed cells. Therefore, it is very important to develop strategies to improve the efficacy of cell-based immunotherapy based on TME.

Disclosure of Invention

The present disclosure is based on the development of strategies to modulate intracellular lactate concentration in hematopoietic cells, such as Hematopoietic Stem Cells (HSCs) or immune cells, including cells that express chimeric receptor polypeptides, such as antibody-coupled T cell receptor (ACTR) polypeptides or Chimeric Antigen Receptor (CAR) polypeptides, for use in cell-based immunotherapy. Modulation of intracellular lactate concentration can be achieved by expressing (e.g., overexpressing) one or more lactate modulating factors, such as a lactate modulating polypeptide, e.g., a lactate modulating polypeptide described herein, in hematopoietic cells (e.g., HSCs or immune cells (e.g., T cells or natural killer cells)). Such genetically engineered hematopoietic cells (e.g., immune cells) are expected to have enhanced metabolic activity relative to the same type of natural hematopoietic cells (e.g., the same type of immune cells), for example, in a low glucose environment, a low amino acid environment, a low pH environment, and/or a hypoxic environment (e.g., in a tumor microenvironment). Such genetically engineered immune cells may also have modulated epigenetic states (e.g., acetylation states) and/or modulated levels of immunosuppressive metabolites (e.g., kynurenine). Thus, hematopoietic cells (such as HSCs or immune cells) co-expressing one or more lactate modulating factors (e.g., polypeptides) and chimeric receptor polypeptides will exhibit excellent biological activity (e.g., under tumor microenvironments, such as low glucose, low amino acid, low pH and/or hypoxic conditions, optionally in the presence of therapeutic antibodies), such as cell proliferation, activation (e.g., increased cytokine production, such as IL-2 or IFN γ production), cytotoxicity and/or in vivo anti-tumor activity.

Thus, provided herein are modified (e.g., genetically modified) hematopoietic cells (e.g., hematopoietic stem cells, or immune cells such as T cells or natural killer cells) having the ability to have altered regulation of intracellular lactate concentration relative to a wild-type immune cell of the same type. In some cases, the modified immune cells may express or overexpress a lactate modulator factor, such as a lactate modulating polypeptide. The lactate modulating polypeptide can be an enzyme involved in lactate synthesis (e.g., LDHA, which catalyzes the interconversion of lactate and pyruvate), a lactate transporter (e.g., MCT), or a polypeptide that inhibits a pathway that competes for a lactate synthesis substrate (e.g., PDK 1). Exemplary lactate modulating polypeptides include, but are not limited to, L-lactate dehydrogenase a chain (LDHA), monocarboxylate transporter 1(MCT1), monocarboxylate transporter 2(MCT2), monocarboxylate transporter 4(MCT4), and pyruvate dehydrogenase kinase 1(PDK 1).

The modified immune cell may also express a chimeric receptor polypeptide that may comprise (a) an extracellular target-binding domain; (b) a transmembrane domain; and (c) a cytoplasmic signaling domain (e.g., a cytoplasmic domain comprising an immunoreceptor tyrosine-based activation motif (ITAM)). In some embodiments, the chimeric receptor polypeptide is an antibody-coupled T cell receptor (ACTR) comprising an extracellular Fc binding domain (a). In other embodiments, the chimeric receptor is a Chimeric Antigen Receptor (CAR) comprising an extracellular antigen-binding domain (a). In some examples, (C) is located at the C-terminus of the chimeric receptor polypeptide. In some cases, the chimeric polypeptide may further comprise at least one costimulatory signaling domain. In other cases, the chimeric receptor polypeptide may not contain a costimulatory signaling domain.

Any of the chimeric receptor polypeptides (e.g., ACTR polypeptides or CAR polypeptides) described herein can further comprise a hinge domain located at the C-terminus of (a) and the N-terminus of (b). In other examples, the chimeric receptor polypeptide may not contain any hinge domains. In other examples, a chimeric receptor polypeptide, such as an ACTR polypeptide, may not contain a hinge domain from any non-CD 16A receptor. Alternatively or additionally, the chimeric receptor polypeptide further comprises a signal peptide at its N-terminus.

In some embodiments, the chimeric receptor polypeptides disclosed herein can be ACTR polypeptides comprising an Fc binding domain (a). In some examples, the Fc binding domain of (a) can be an extracellular ligand binding domain of an Fc receptor, such as an extracellular ligand binding domain of an Fc-gamma receptor, an Fc-alpha receptor, or an Fc-epsilon receptor. In particular examples, the Fc binding domain is an extracellular ligand binding domain of CD16A (e.g., F158 CD16A or V158 CD16A), CD32A, or CD 64A. In other examples, the Fc binding domain of (a) can be an antibody fragment that binds the Fc portion of an immunoglobulin. For example, the antibody fragment can be a single chain variable fragment (ScFv), a single domain antibody (e.g., nanobody). In addition, the Fc binding domain of (a) may be a naturally occurring protein that binds to the Fc portion of an immunoglobulin or an Fc binding fragment thereof. For example, the Fc binding domain can be protein a or protein G or an Fc binding fragment thereof. In a further example, the Fc binding domain of (a) can be a synthetic polypeptide that binds to the Fc portion of an immunoglobulin. Examples include, but are not limited to, Kunitz peptide, SMIP, avimer, affibody, DARPin, or anti-transporter (anticalin).

In some embodiments, the chimeric receptor polypeptides disclosed herein can be CAR polypeptides comprising an extracellular antigen-binding domain (a). In some examples, the extracellular antigen-binding domain of (a) is a single chain antibody fragment that binds to a tumor antigen, a pathogenic antigen, or an immune cell specific for an autoantigen. In certain examples, the tumor antigen is associated with a hematologic tumor. Examples include, but are not limited to, CD19, CD20, CD22, kappa chain, CD30, CD123, CD33, LeY, CD138, CD5, BCMA, CD7, CD40, and IL-1 RAP. In certain examples, the tumor antigen is associated with a solid tumor. Examples include, but are not limited to, GD2, GPC3, FOLR (e.g., FOLR1 or FOLR2), HER2, EphA2, EFGRVIII, IL13RA2, VEGFR2, ROR1, NKG2D, EpCAM, CEA, mesothelin, MUC1, CLDN18.2, CD171, CD133, PSCA, cMET, EGFR, PSMA, FAP, CD70, MUC16, L1-CAM, B7H3, and CAIX. In certain examples, the pathogenic antigen is a bacterial antigen, a viral antigen, or a fungal antigen, such as those described herein.

In some embodiments, the transmembrane domain of (b) in any one of the chimeric receptor polypeptides (e.g., ACTR or CAR polypeptides) can be a single transmembrane protein, such as CD 8a, CD8 β, 4-1BB, CD28, CD34, CD4, fcsry, CD16A, OX40, CD3 ζ, CD3 epsilon, CD3 γ, CD3 δ, TCR a, CD32, CD64, VEGFR2, FAS, and FGFR 2B. Alternatively, the transmembrane domain of (b) may be a non-naturally occurring hydrophobin segment.

In some embodiments, if applicable, at least one co-stimulatory signaling domain of a chimeric receptor polypeptide described herein (e.g., an ACTR or CAR polypeptide) can belong to a co-stimulatory molecule, which can be 4-1BB, CD28, CD28_LL→GGVariants, OX40, ICOS, CD27, GITR, ICOS, HVEM, TIM1, LFA1, and CD 2. In some examples, the at least one co-stimulatory signaling domain is a CD28 co-stimulatory signaling domain or a 4-1BB co-stimulatory signaling domain. In some cases, the ACTR polypeptide can comprise two costimulatory signaling domains. In some cases, one of the costimulatory signaling domains is a CD28 costimulatory signaling domain; and the other costimulatory domain can be a 4-1BB costimulatory signaling domain, an OX40 costimulatory signaling domain, a CD27 costimulatory signaling domain, or an ICOS costimulatory signaling domainA domain. Specific examples include, but are not limited to, CD28 and 4-1 BB; or CD28_LL→GGVariants and 4-1 BB. Alternatively, any of the chimeric receptor polypeptides may not comprise any co-stimulatory signaling domain.

In some embodiments, the cytoplasmic signaling domain of (c) in any of the chimeric receptor polypeptides described herein (e.g., ACTR or CAR polypeptides) can be the cytoplasmic domain of CD3 ζ or fcepsilonr 1 γ.

In some embodiments, the hinge domain of any of the chimeric polypeptides described herein (e.g., ACTR or CAR polypeptides) can be of CD28, CD16A, CD 8a, or IgG, as applicable. In other examples, the hinge domain is a non-naturally occurring peptide. For example, the non-naturally occurring peptide can be an extended recombinant polypeptide (XTEN) or (Gly)₄Ser)_nA polypeptide, wherein n is an integer from 3 to 12, inclusive. In some examples, the hinge domain is a short segment, which may contain up to 60 amino acid residues.

In particular examples, ACTR polypeptides as described herein can comprise (i) a CD28 co-stimulatory domain; and (ii) a CD28 transmembrane domain, a CD28 hinge domain, or a combination thereof. For example, the ACTR polypeptide comprises components (a) - (e) as shown in table 4. In particular examples, the ACTR polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO 1 through SEQ ID NO 80.

In particular examples, a CAR polypeptide described herein can comprise (i) a CD28 co-stimulatory domain or a 4-1BB co-stimulatory domain; and (ii) a CD28 transmembrane domain, a CD28 hinge domain, or a combination thereof. In further specific examples, the CAR polypeptide described herein can comprise (i) a CD28 co-stimulatory domain or a 4-1BB co-stimulatory domain, (ii) a CD8 transmembrane domain, a CD8 hinge domain, or a combination thereof. For example, the CAR polypeptide can comprise an amino acid sequence selected from SEQ ID NO:97 and SEQ ID NO: 98.

The hematopoietic cells described herein that express a lactate regulatory factor (e.g., a polypeptide) and optionally a chimeric receptor polypeptide can be hematopoietic stem cells or progeny thereof. In some embodiments, the hematopoietic cell can be an immune cell, such as a natural killer cell, monocyte/macrophage, neutrophil, eosinophil, or T cell. The immune cells may be derived from Peripheral Blood Mononuclear Cells (PBMCs), Hematopoietic Stem Cells (HSCs) or induced pluripotent stem cells (ipscs). In some examples, the immune cell is a T cell in which expression of an endogenous T cell receptor, an endogenous major histocompatibility complex, an endogenous beta-2-microglobulin, or a combination thereof has been inhibited or depleted.

Any of the hematopoietic cells (e.g., HSCs or immune cells) described herein can comprise a nucleic acid or set of nucleic acids that collectively comprise: (a) a first nucleotide sequence encoding a lactate modulator (e.g., a polypeptide); and optionally (b) a second nucleotide sequence encoding a Chimeric Antigen Receptor (CAR) polypeptide. The nucleic acid or group of nucleic acids is an RNA molecule or group of RNA molecules. In some cases, the immune cell comprises a nucleic acid comprising both the first nucleotide sequence and the second nucleotide sequence. In some embodiments, the coding sequence for the lactate regulatory factor is upstream of the coding sequence for the CAR polypeptide. In some embodiments, the coding sequence for the CAR polypeptide is upstream of the coding sequence for the lactate regulatory factor. Such nucleic acids can further comprise a third nucleotide sequence positioned between the first nucleotide sequence and the second nucleotide sequence, wherein the third nucleotide sequence encodes a ribosome skip site (e.g., P2A peptide), an Internal Ribosome Entry Site (IRES), or a second promoter.

In some examples, the nucleic acid or set of nucleic acids is contained within a vector or set of vectors, which may be an expression vector or set of expression vectors (e.g., a viral vector, such as a lentiviral vector or a retroviral vector). A nucleic acid set or vector set refers to a set of two or more nucleic acid molecules or two or more vectors, each of which encodes one of the polypeptides of interest (i.e., lactate modulating polypeptide and CAR polypeptide). Any of the nucleic acids described herein are also within the scope of the present disclosure.

In another aspect, the present disclosure provides a pharmaceutical composition comprising any of the immune cells described herein and a pharmaceutically acceptable carrier.

Further, provided herein is a method of inhibiting cells expressing a target antigen (e.g., reducing the number of such cells, blocking cell proliferation, and/or inhibiting cell activity) in a subject, the method comprising administering to a subject in need thereof a population of immune cells described herein that can co-express a lactate modulator (e.g., a polypeptide) and a CAR polypeptide. A subject (e.g., a human patient, such as a human patient having cancer) may have been or is being treated with an anti-cancer therapy (e.g., an anti-cancer agent). In some examples, at least some of the cells expressing the target antigen are located in a low glucose environment, a low amino acid (e.g., low glutamine) environment, a low pH environment, and/or a hypoxic environment (e.g., tumor microenvironment).

In some examples, the immune cells are autologous. In other examples, the immune cells are allogeneic. In any of the methods described herein, the immune cells can be activated, expanded, or both ex vivo. In some cases, the immune cells comprise T cells that are activated in the presence of one or more of an anti-CD 3 antibody, an anti-CD 28 antibody, IL-2, phytohemagglutinin, and engineered artificially stimulated cells or granules. In other cases, the immune cells comprise natural killer cells that are activated in the presence of one or more of 4-1BB ligand, anti-4-1 BB antibody, IL-15, anti-IL-15 receptor antibody, IL-2, IL-12, IL-21, and K562 cells, engineered artificially stimulated cells, or particles.

In some examples, the subject to be treated by the methods described herein can be a human patient having a cancer, e.g., a carcinoma, lymphoma, sarcoma, blastoma, and leukemia. Additional exemplary target cancers include, but are not limited to, B cell derived cancers, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, skin cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, mesothelioma, pancreatic cancer, head and neck cancer, retinoblastoma, glioma, glioblastoma, liver cancer, and thyroid cancer. Exemplary B cell derived cancers are selected from the group consisting of: acute lymphoblastic leukemia of B-line, chronic lymphocytic leukemia of B-cell and non-Hodgkin's lymphoma of B-cell.

Also within the scope of the disclosure is the use of a genetically engineered immune cell described herein, which co-expresses a lactate modulator (e.g., polypeptide) and a CAR polypeptide, for the treatment of a target disease or disorder, such as cancer or an infectious disorder, and its use for the manufacture of a medicament for the intended medical treatment.

The details of one or more embodiments of the disclosure are set forth in the description below. Other features and advantages of the disclosure will be apparent from the detailed description of several embodiments and from the appended claims.

Drawings

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

Fig. 1 is a schematic diagram showing intracellular synthesis and metabolic pathways of lactic acid and the export and import of lactic acid. Exemplary strategies for modulating intracellular lactate concentration include modulating one or more enzymes involved in lactate synthesis, metabolism, and/or transport (e.g., enhancing the interconversion of intracellular lactate and pyruvate by, e.g., overexpressing LDHA, and increasing the cellular transport of lactate by, e.g., overexpressing MCT).

Figure 2 shows the effect of low glucose concentrations on the proliferation of immune cells expressing anti-GPC 3 chimeric antigen receptor in the presence of target cells expressing GPC 3.

FIGS. 3A-3B are graphs showing that co-expression of MCT1(SEQ ID NO:82) and CAR (SEQ ID NO:98) in T cells enhanced cell proliferation relative to CAR alone (SEQ ID NO:97) under tumor-associated (1.25 mM; FIG. 3A) and approximately peripheral blood levels (10 mM; FIG. 3B) glucose conditions.

FIGS. 4A-4B are graphs showing that co-expression of MCT2(SEQ ID NO:83) and CAR (SEQ ID NO:97) in T cells enhanced cell proliferation relative to CAR alone (SEQ ID NO:97) under tumor-associated (1.25 mM; FIG. 4A) and approximately peripheral blood levels (10 mM; FIG. 4B) glucose conditions.

FIGS. 5A-5B are graphs showing that co-expression of MCT4(SEQ ID NO:84) and CAR (SEQ ID NO:98) in T cells enhanced cell proliferation relative to CAR alone (SEQ ID NO:97) under tumor-associated (1.25 mM; FIG. 5A) and approximately peripheral blood levels (10 mM; FIG. 5B) glucose conditions.

FIGS. 6A-6B are graphs showing the change in IL-2 production and proliferation as a function of antibody concentration of T cells expressing the ACTR (SEQ ID NO:57) polypeptide alone or in combination with the ACTR polypeptide and LDHA after incubation of the T cells with FOLR α expressing IGROV-1 cells with anti-FOLR α antibody for about 48 hours to measure IL-2 production (FIG. 6A) or 8 days to measure proliferation by viable T cell count (FIG. 6B).

FIG. 7 is a graph showing proliferation as measured by viable T cell count as a function of media glucose concentration of T cells expressing the ACTR (SEQ ID NO:57) polypeptide alone or in combination with the ACTR polypeptide and LDHA after incubation for 8 days with FOLRa expressing IGROV-1 cells and anti-FOLRa antibody.

FIGS. 8A-8B are graphs showing the solid tumor-associated inhibitory molecule PGE at various concentrations along with FOLRa-expressing IGROV-1 cells and anti-FOLRa antibodies in T cells expressing ACTR (SEQ ID NO:57) polypeptide alone or in combination with LDHA₂Or for 8 days to measure proliferation by viable T cell count (fig. 8B), a graph of IL-2 production and proliferation of the T cells.

FIG. 9 is a graph showing IL-2 production by T cells expressing the ACTR (SEQ ID NO:57) polypeptide alone or in combination with LDHA after incubation of the T cells with fixed IGROV-1 cells expressing FOLR α and an anti-FOLR α antibody for about 48 hours in the presence of different concentrations of the solid tumor-associated inhibitory molecule kynurenine to measure IL-2 production.

FIG. 10 is a graph showing the change in proliferation as a function of antibody concentration of T cells expressing the ACTR (SEQ ID NO:57) polypeptide alone or in combination with ACTR polypeptide and MCT1 after incubation of the T cells with FOLRa-expressing fixed OVCAR8 cells and anti-FOLRa antibody for 8 days to measure proliferation by ATP content.

FIGS. 11A-11C are graphs showing IL-2 production and proliferation of T cells expressing the ACTR (SEQ ID NO:57) polypeptide alone or in combination with an ACTR polypeptide and MCT1 after incubation with FOLRa-expressing immobilized IGROV-1 cells and an anti-FOLRa antibody in the presence of different concentrations of the solid tumor-associated inhibitory molecule, kynurenine. After incubation for about 48 hours, IL-2 production was measured (FIG. 11A). On day 7, cells were divided into two groups. The first group was pulsed with BrdU for approximately 16 hours and a BrdU uptake assay (Millipore Sigma) was performed to assess proliferation (fig. 11B). Proliferation in the second group was measured by ATP content on day 8 (fig. 11C).

FIGS. 12A-12C are graphs depicting T cells expressing ACTR (SEQ ID NO:57) polypeptide alone or in combination with ACTR polypeptide and MCT2 along with fixed OVCAR8 cells expressing FOLRa and anti-FOLRa antibody at different concentrations of the solid tumor-associated inhibitory molecule PGE₂(FIG. 12A), TGF-. beta. (FIG. 12B), and kynurenine (FIG. 12C) for 8 days to measure the proliferation by ATP content.

FIGS. 13A-13C are graphs showing IL-2 production and proliferation of T cells expressing the ACTR (SEQ ID NO:57) polypeptide alone or in combination with an ACTR polypeptide and MCT2 after incubation with FOLRa-expressing immobilized IGROV-1 cells and an anti-FOLRa antibody in the presence of different concentrations of the solid tumor-associated inhibitory molecule, kynurenine. After incubation for about 48 hours, IL-2 production was measured (FIG. 13A). On day 6, cells were divided into two groups. The first group was pulsed with BrdU for approximately 16 hours and a BrdU uptake assay (Millipore Sigma) was performed to assess proliferation (fig. 13B). Proliferation in the second group was measured by ATP content on day 7 (fig. 13C).

FIGS. 14A-14B are graphs showing IL-2 production by T cells expressing the ACTR (SEQ ID NO:57) polypeptide alone or in combination with the ACTR polypeptide and MCT2 after incubation with live (FIG. 14A) or fixed (FIG. 14B) IGROV-1 cells expressing FOLR α and an anti-FOLR α antibody in the presence of various concentrations of the solid tumor-associated inhibitory molecule kynurenine.

FIG. 15 is a graph showing proliferation as a function of antibody concentration of T cells expressing the ACTR (SEQ ID NO:57) polypeptide alone or in combination with the ACTR polypeptide and MCT4 after incubation of the T cells with FOLRa-expressing fixed OVCAR8 cells and anti-FOLRa antibody for 8 days.

FIG. 16 is a graph showing that T cells expressing the ACTR (SEQ ID NO:57) polypeptide alone or in combination with the ACTR polypeptide and MCT4 together with fixed IGROV-1 cells expressing FOLRa and anti-FOLRa antibodies at different concentrations of the solid tumor-associated inhibitory molecule PGE₂Graph of the IL-2 production of said T cells after incubation in the presence of (a).

FIG. 17 is a graph depicting proliferation of T cells expressing an ACTR (SEQ ID NO:57) polypeptide alone or in combination with an ACTR polypeptide and MCT4 after incubation for 8 days with fixed OVCAR8 cells expressing FOLRa and an anti-FOLRa antibody in the presence of different concentrations of the solid tumor-associated inhibitory molecule TGF- β to measure proliferation by ATP content.

FIGS. 18A-18B are graphs showing IL-2 production and proliferation of T cells expressing the ACTR (SEQ ID NO:57) polypeptide alone or in combination with an ACTR polypeptide and MCT4 after incubation with FOLRa-expressing immobilized IGROV-1 cells and an anti-FOLRa antibody in the presence of different concentrations of the solid tumor-associated inhibitory molecule, kynurenine. After incubation for about 48 hours, IL-2 production was measured (FIG. 18A). On day 6, cells were pulsed with BrdU for approximately 16 hours and a BrdU uptake assay (Millipore Sigma) was performed to assess proliferation (fig. 18B).

Detailed Description

The tumor microenvironment has specific characteristics, such as low glucose, low amino acids, low pH, and/or hypoxic conditions, some of which may limit the activity of effector immune cells, such as effector T cells. The present disclosure is based, at least in part, on the development of strategies for enhancing effector immune cell activity in a tumor microenvironment. In particular, the disclosure features methods for enhancing the metabolic activity of effector immune cells via modulating intracellular lactic acid concentrations in the effector immune cells, thereby enhancing the growth and biological activity of the effector immune cells. Intracellular lactate concentration can be modulated in a variety of ways, including increasing cellular transport of lactate (e.g., by expressing or overexpressing a lactate transporter protein and/or by regulating cellular trafficking or activity of such a protein), increasing synthesis of lactate (e.g., by expressing or overexpressing an enzyme involved in lactate synthesis and/or by regulating cellular trafficking or activity of such a protein), and/or inhibiting pathways that compete for substrates in the lactate synthesis pathway (e.g., by expressing or overexpressing a polypeptide that inhibits pathways that compete for substrates in lactate synthesis and/or by regulating cellular trafficking or activity of a protein involved in such pathways). The present disclosure provides various methods of modulating intracellular lactate concentration in immune cells. Some examples are shown in fig. 1, including: overexpresses endogenous enzymes that stimulate the interconversion of lactate and pyruvate (e.g., LDHA) and/or overexpresses lactate transporter (e.g., MCT1, MCT2, or MCT 4).

The studies disclosed herein unexpectedly demonstrate that co-expression of a lactate modulating polypeptide (e.g., LDHA, MCT, or PDK1) and a chimeric receptor polypeptide, such as a CAR (e.g., having a 4-1BB co-stimulatory domain) or ACTR (e.g., having a 4-1BB or CD28 co-stimulatory domain), in immune cells, such as T cells, exhibit superior characteristics, both in vitro and in vivo, relative to immune cells that express only CAR or ACTR. For example, co-expression of LDHA, MCT1, MCT2, or MCT4 with CARs or ACTRs enhances T cell proliferation/expansion and T cell function, particularly under solid tumor microenvironment conditions (e.g., hypoxia, low glucose conditions, and presence of TME inhibitors). For example, co-expression of a lactate modulating polypeptide (e.g., LDHA, MCT, or PDK1) and a chimeric receptor polypeptide (e.g., CAR or ACTR) can reduce tumor growth and/or tumor formation. For example, in tumor microenvironment-like conditions (e.g., low glucose, PGE)₂Kynurenine), co-expression of LDHA and ACTR enhanced T cell activity. In addition, co-expression of MCT1, MCT4, and MCT4 with ACTR or CAR under tumor microenvironment-like conditions (e.g., low glucose, PGE)₂Kynurenine, TGF β or adenosine).

Accordingly, the present disclosure provides modified (e.g., genetically engineered) hematopoietic cells (e.g., HSCs or immune cells) having enhanced metabolic activity relative to native immune cells of the same type. Regulation of intracellular lactate concentration may be achieved by any suitable route. In some embodiments, such modified immune cells may express one or more lactate modulating factors, such as lactate modulating polypeptides. In some cases, the lactate modulator may be a molecule that is directly involved in lactate synthesis, metabolism, and/or transport, such as an enzyme or transporter involved in such processes. In other instances, the lactate modulator may be a molecule that indirectly modulates lactate synthesis, metabolism, and/or transport (e.g., modulates expression, activity, and/or degradation of a polypeptide involved in lactate synthesis, metabolism, and/or transport).

Such genetically engineered immune cells may also express chimeric receptor polypeptides, such as antibody-coupled T cell receptor (ACTR) polypeptides or Chimeric Antigen Receptor (CAR) polypeptides. Also provided herein are uses of genetically engineered immune cells, optionally in combination with an Fc-containing agent, when needed (e.g., when the immune cells express an ACTR polypeptide), for improving immune cell proliferation and/or inhibiting or reducing target cells (e.g., target cancer cells) in a subject (e.g., a human cancer patient), e.g., via ADCC. The disclosure also provides pharmaceutical compositions and kits comprising the genetically engineered immune cells.

Genetically engineered immune cells expressing (e.g., overexpressing) lactate modulators as described herein can confer at least the following advantages. Expression of a lactate modulator (e.g., a polypeptide or nucleic acid) will enhance the metabolic activity of the T cell. Thus, genetically engineered immune cells may proliferate better in a tumor environment (e.g., low glucose, low amino acid, low pH, and/or hypoxic environment), produce more cytokines, exhibit greater anti-tumor cytotoxicity, exhibit fewer immunosuppressive metabolites, and/or exhibit greater T cell survival, resulting in increased cytokine production, survival, cytotoxicity, and/or anti-tumor activity relative to immune cells that do not express (or do not over-express) lactate regulatory factors (e.g., polypeptides or nucleic acids).

I.Lactic acid modulating factor

As used herein, a lactate modulator may be any type of molecule involved in lactate synthesis and/or metabolism (e.g., an enzyme involved in lactate synthesis and/or metabolism, or an enzyme that inhibits a pathway that competes for a substrate used in lactate synthesis), or any type of molecule involved in lactate cell transport (e.g., a cell surface lactate transporter).

In some cases, the lactate modulator can be a lactate modulating polypeptide, which refers to a polypeptide that modulates the intracellular concentration of lactate. Such lactate modulating polypeptides may regulate intracellular lactate concentration via any mechanism.

In some embodiments, and as exemplified in fig. 1, the lactate modulating polypeptide comprises a lactate transporter protein (i.e., a cell membrane protein that facilitates the transport of lactate across a cell membrane) and/or a modulator of the cellular trafficking or activity of such a protein. In some embodiments, the lactate modulating polypeptide may comprise a bidirectional lactate transporter (e.g., MCT1, MCT2, or MCT4, or a functional variant thereof). In some embodiments, the lactate modulating polypeptide comprises a genetically engineered lactate transporter, wherein the lactate transporter is mutated from a native wild-type form to mimic an activated lactate modulating polypeptide (e.g., a phosphorylation mimetic) and/or to affect intracellular trafficking of lactate (e.g., trafficking to the cell surface), such that the activity of the lactate modulating polypeptide is increased.

In other embodiments, as also exemplified in fig. 1, the lactate modulating polypeptide may include an enzyme involved in lactate synthesis (e.g., an enzyme that stimulates lactate synthesis or the conversion of lactate to another molecule). Such enzymes can convert lactate to pyruvate. For example, the lactate modulating polypeptide can include LDHA or a functional variant thereof. In some embodiments, a lactate modulating polypeptide may include a genetically engineered enzyme involved in lactate synthesis, wherein the enzyme is mutated from a native wild-type form to mimic an activated enzyme (e.g., a phosphorylation mimetic) and/or to affect intracellular trafficking of the lactate such that synthesis or conversion of lactate is increased.

In other embodiments, the lactate modulating polypeptide may be a polypeptide that inhibits a pathway that competes for a lactate synthesis substrate and/or a modulator of cellular trafficking or activity of a protein involved in such a pathway. For example, the lactate modulating polypeptide may include PDK1 or a functional variant thereof. In some embodiments, the lactate modulating polypeptide comprises a genetically engineered protein inhibitor, wherein the protein inhibitor is mutated from a native wild-type form to mimic an activated protein inhibitor (e.g., a phosphorylation mimetic) and/or to affect intracellular trafficking of the lactate such that inhibition of a competitive pathway is increased.

Any such regulatory polypeptide, which may be of any suitable species (e.g., mammalian, such as human), is contemplated for use with the compositions and methods described herein.

Exemplary lactate modulating polypeptides may include, but are not limited to, L-lactate dehydrogenase a chain (LDHA), monocarboxylate transporter 1(MCT1), monocarboxylate transporter 2(MCT2), monocarboxylate transporter 4(MCT4), and pyruvate dehydrogenase kinase 1(PDK 1).

LDHA is a dehydrogenase that catalyzes the interconversion of pyruvate, a key molecule in the krebs cycle, with lactate. Overexpression of LDHA may facilitate conversion of lactate to pyruvate, as cellular pyruvate storage is reduced at high metabolic activity. This results in an increase in intracellular pyruvate concentration and a decrease in intracellular lactate concentration, and has the effect of providing flux to the krebs cycle and increasing lactate transport. Thus, increased LDHA expression or activity increases lactate transport, resulting in an eventual increase in intracellular lactate concentration. The amino acid sequence of an exemplary human LDHA enzyme is provided below:

LDHA(SEQ ID NO:81)

MATLKDQLIYNLLKEEQTPQNKITVVGVGAVGMACAISILMKDLADELALVDVIEDKLKGEMMDLQHGSLFLRTPKIVSGKDYNVTANSKLVIITAGARQQEGESRLNLVQRNVNIFKFIIPNVVKYSPNCKLLIVSNPVDILTYVAWKISGFPKNRVIGSGCNLDSARFRYLMGERLGVHPLSCHGWVLGEHGDSSVPVWSGMNVAGVSLKTLHPDLGTDKDKEQWKEVHKQVVESAYEVIKLKGYTSWAIGLSVADLAESIMKNLRRVHPVSTMIKGLYGIKDDVFLSVPCILGQNGISDLVKVTLTSEEEARLKKSADTLWGIQKELQF

MCT proteins (e.g., MCT1, MCT2, or MCT4) are a family of monocarboxylic acid transporters that catalyze the bidirectional transport of lactate as well as pyruvate, ketone bodies, and other structurally related metabolites. MCT2 has a higher affinity for lactic acid than MCT1, while MCT4 has a lower affinity for pyruvic acid than MCT 1. Increased MCT expression or activity results in increased lactate output, which subsequently leads to increased glycolysis. Similarly, increased MCT expression or activity can result in increased metabolic flux of lactate into a biological pathway. The amino acid sequences of exemplary human MCT1, MCT2, and MCT4 proteins are provided below:

MCT1(SEQ ID NO:82)

MPPAVGGPVGYTPPDGGWGWAVVIGAFISIGFSYAFPKSITVFFKEIEGIFHATTSEVSWISSIMLAVMYGGGPISSILVNKYGSRIVMIVGGCLSGCGLIAASFCNTVQQLYVCIGVIGGLGLAFNLNPALTMIGKYFYKRRPLANGLAMAGSPVFLCTLAPLNQVFFGIFGWRGSFLILGGLLLNCCVAGALMRPIGPKPTKAGKDKSKASLEKAGKSGVKKDLHDANTDLIGRHPKQEKRSVFQTINQFLDLTLFTHRGFLLYLSGNVIMFFGLFAPLVFLSSYGKSQHYSSEKSAFLLSILAFVDMVARPSMGLVANTKPIRPRIQYFFAASVVANGVCHMLAPLSTTYVGFCVYAGFFGFAFGWLSSVLFETLMDLVGPQRFSSAVGLVTIVECCPVLLGPPLLGRLNDMYGDYKYTYWACGVVLIISGIYLFIGMGINYRLLAKEQKANEQKKESKEEETSIDVAGKPNEVTKAAESPDQKDTDGGPKEEESPV

MCT2(SEQ ID NO:83)

MPPMPSAPPVHPPPDGGWGWIVVGAAFISIGFSYAFPKAVTVFFKEIQQIFHTTYSEIAWISSIMLAVMYAGGPVSSVLVNKYGSRPVVIAGGLLCCLGMVLASFSSSVVQLYLTMGFITGLGLAFNLQPALTIIGKYFYRKRPMANGLAMAGSPVFLSSLAPFNQYLFNTFGWKGSFLILGSLLLNACVAGSLMRPLGPNQTTSKSKNKTGKTEDDSSPKKIKTKKSTWEKVNKYLDFSLFKHRGFLIYLSGNVIMFLGFFAPIIFLAPYAKDQGIDEYSAAFLLSVMAFVDMFARPSVGLIANSKYIRPRIQYFFSFAIMFNGVCHLLCPLAQDYTSLVLYAVFFGLGFGSVSSVLFETLMDLVGAPRFSSAVGLVTIVECGPVLLGPPLAGKLVDLTGEYKYMYMSCGAIVVAASVWLLIGNAINYRLLAKERKEENARQKTRESEPLSKSKHSEDVNVKVSNAQSVTSERETNI

MCT4(SEQ ID NO:84)

MGGAVVDEGPTGVKAPDGGWGWAVLFGCFVITGFSYAFPKAVSVFFKELIQEFGIGYSDTAWISSILLAMLYGTGPLCSVCVNRFGCRPVMLVGGLFASLGMVAASFCRSIIQVYLTTGVITGLGLALNFQPSLIMLNRYFSKRRPMANGLAAAGSPVFLCALSPLGQLLQDRYGWRGGFLILGGLLLNCCVCAALMRPLVVTAQPGSGPPRPSRRLLDLSVFRDRGFVLYAVAASVMVLGLFVPPVFVVSYAKDLGVPDTKAAFLLTILGFIDIFARPAAGFVAGLGKVRPYSVYLFSFSMFFNGLADLAGSTAGDYGGLVVFCIFFGISYGMVGALQFEVLMAIVGTHKFSSAIGLVLLMEAVAVLVGPPSGGKLLDATHVYMYVFILAGAEVLTSSLILLLGNFFCIRKKPKEPQPEVAAAEEEKLHKPPADSGVDLREVEHFLKAEPEKNGEVVHTPETSV

PDK1 is a kinase that inhibits pyruvate dehydrogenase (such as PDHA1), a component of the pyruvate dehydrogenase complex, via phosphorylation. The pyruvate dehydrogenase complex converts pyruvate to acetyl-CoA by decarboxylation. Increased PDK1 expression or activity-and subsequent inhibition of pyruvate dehydrogenase-increases the amount of pyruvate available for LDHA-mediated conversion to lactate. The amino acid sequence of an exemplary human PDK1 enzyme is provided below:

PDK1(SEQ ID NO:85)

MRLARLLRGAALAGPGPGLRAAGFSRSFSSDSGSSPASERGVPGQVDFYARFSPSPLSMKQFLDFGSVNACEKTSFMFLRQELPVRLANIMKEISLLPDNLLRTPSVQLVQSWYIQSLQELLDFKDKSAEDAKAIYERPRRTWLQVSSLCCMACKMIFTDTVIRIRNRHNDVIPTMAQGVIEYKESFGVDPVTSQNVQYFLDRFYMSRISIRMLLNQHSLLFGGKGKGSPSHRKHIGSINPNCNVLEVIKDGYENARRLCDLYYINSPELELEELNAKSPGQPIQVVYVPSHLYHMVFELFKNAMRATMEHHANRGVYPPIQVHVTLGNEDLTVKMSDRGGGVPLRKIDRLFNYMYSTAPRPRVETSRAVPLAGFGYGLPISRLYAQYFQGDLKLYSLEGYGTDAVIYIKALSTDSIERLPVYNKAAWKHYNTNHEADDWCVPSREPKDMTTFRSA

the lactate modulating polypeptide can be a naturally occurring polypeptide from an appropriate species, e.g., a mammalian lactate modulating polypeptide, such as those derived from a human or non-human primate. Such naturally occurring polypeptides are known in the art and can be obtained, for example, by searching publicly available gene databases (e.g., GenBank) using any of the above amino acid sequences as a query. The lactate modulating polypeptides used in the present disclosure may share at least 85% (e.g., 90%, 95%, 97%, 98%, 99%, or more) sequence identity with any of the above exemplary proteins.

The "percent identity" of two amino acid sequences was determined using the algorithm Karlin and Altschul Proc.Natl.Acad.Sci.USA 87: 2264-. This algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al, J.mol.biol.215: 403-. BLAST protein searches can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecules of the invention. In the case of gaps between two sequences, gapped BLAST can be used as described in Altschul et al, Nucleic Acids Res.25(17):3389-3402, 1997. When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

Alternatively, the lactate modulating polypeptide may be a functional variant of the natural counterpart. Such functional variants may contain one or more mutations outside of one or more functional domains of the natural counterpart. The functional domains of native lactate modulating polypeptides may be known in the art or may be predicted based on their amino acid sequence. Mutations outside of one or more functional domains are not expected to substantially affect the biological activity of the protein. In some cases, the functional variant may exhibit increased activity in lactate transport relative to the native counterpart. Alternatively, the functional variant may exhibit reduced activity in lactate transport relative to the natural counterpart. In addition, functional variants may have increased trafficking to the cell surface. Alternatively, the functional variant may have reduced trafficking to the cell surface.

Alternatively or additionally, a functional variant may contain one or more conservative mutations at one or more positions (e.g., up to 20 positions, up to 15 positions, up to 10 positions, up to 5, 4, 3, 2,1 positions) in the natural counterpart. As used herein, "conservative amino acid substitutions" refer to amino acid substitutions that do not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods known to those of ordinary skill in the art for altering polypeptide sequences, such as those found in references compiling such methods, e.g., Molecular Cloning: A Laboratory Manual, J.Sambrook et al, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York,1989 or Current Protocols in Molecular Biology, F.M.Ausubel et al, John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made between amino acids within the following groups: (a) m, I, L, V, respectively; (b) f, Y, W, respectively; (c) k, R, H, respectively; (d) a, G, respectively; (e) s, T, respectively; (f) q, N, respectively; and (g) E, D.

In some embodiments, the lactate modulator may be a molecule that modulates the expression of an endogenous lactate modulating polypeptide. Such lactate regulatory factors may be transcription factors or micrornas. In some cases, a lactate modulator may be a nucleic acid (e.g., a microrna, an interfering RNA such as an siRNA or shRNA, or an antisense nucleic acid) that modulates the expression of one or more enzymes involved in lactate synthesis and/or metabolism and one or more lactate transporters. In other embodiments, the lactate modulator may be a transcription factor that regulates the expression of one or more enzymes or transporters involved in lactate synthesis, metabolism, and/or transport. In other embodiments, the lactate modulator may be a molecule that mediates degradation of endogenous lactate modulating polypeptides (such as the endogenous lactate modulating polypeptides disclosed herein), for example E3 ligase, which is part of the ubiquitin/proteasome pathway. In addition, the transport of endogenous lactate modulating polypeptides can be regulated, for example, by expressing polypeptides that increase their transport to the cell surface.

II.Chimeric receptor polypeptides

As used herein, a chimeric receptor polypeptide refers to a non-naturally occurring molecule that can be expressed on the surface of a host cell. Chimeric receptor polypeptides comprise an extracellular target-binding domain that can target an antigen of interest (e.g., an antigen associated with a disease such as cancer or an antigen associated with a pathogen; see discussion herein). The extracellular target-binding domain can directly bind an antigen of interest (e.g., an extracellular antigen-binding domain in a CAR polypeptide as disclosed herein). Alternatively, the extracellular target-binding domain may bind to the antigen of interest via an intermediate (e.g., an Fc-containing agent, such as an antibody). The chimeric receptor polypeptide can further comprise a transmembrane domain, a hinge domain, a cytoplasmic signaling domain, one or more costimulatory domains, a cytoplasmic signaling domain, or a combination thereof. In some cases, the chimeric receptor polypeptide may not contain a costimulatory domain. The chimeric receptor polypeptide is configured such that, when expressed on a host cell, the extracellular target-binding domain is located extracellularly for binding, directly or indirectly, to a target antigen. The optional costimulatory signaling domain may be located in the cytoplasm to trigger activation and/or effector signaling.

In some embodiments, the chimeric receptor polypeptides described herein can further comprise a hinge domain that can be located C-terminal to the extracellular target-binding domain and N-terminal to the transmembrane domain. The hinge may have any suitable length. In other embodiments, the chimeric receptor polypeptides described herein may not have a hinge domain at all. In other embodiments, the chimeric receptor polypeptides described herein can have a shortened hinge domain (e.g., comprising up to 25 amino acid residues).

In some embodiments, a chimeric receptor polypeptide as described herein can comprise, from N-terminus to C-terminus, an extracellular target-binding domain, a transmembrane domain, and a cytoplasmic signaling domain. In some embodiments, a chimeric receptor polypeptide as described herein comprises, from N-terminus to C-terminus, an extracellular target-binding domain, a transmembrane domain, at least one costimulatory signaling domain, and a cytoplasmic signaling domain. In other embodiments, a chimeric receptor polypeptide as described herein comprises, from N-terminus to C-terminus, an extracellular target-binding domain, a transmembrane domain, a cytoplasmic signaling domain, and at least one costimulatory signaling domain.

In some embodiments, the chimeric receptor polypeptide can be an antibody-coupled T cell receptor (ACTR) polypeptide. As used herein, an ACTR polypeptide (also referred to as an ACTR construct) refers to a non-naturally occurring molecule that can be expressed on the surface of a host cell and comprises an extracellular domain ("Fc-binding agent" or "Fc-binding domain"), a transmembrane domain, and a cytoplasmic signaling domain with binding affinity and specificity for the Fc portion of an immunoglobulin. In some embodiments, the ACTR polypeptides described herein can further comprise at least one co-stimulatory signaling domain.

In other embodiments, the chimeric receptor polypeptides disclosed herein can be Chimeric Antigen Receptor (CAR) polypeptides. As used herein, a CAR polypeptide (also referred to as a CAR construct) refers to a non-naturally occurring molecule that can be expressed on the surface of a host cell and comprises an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain. The CAR polypeptides described herein can further comprise at least one costimulatory signaling domain.

The extracellular antigen-binding domain can be any peptide or polypeptide that specifically binds to a target antigen, including antigen moieties conjugated to a naturally occurring antigen associated with a medical condition (e.g., a disease) or to a therapeutic agent that targets a disease-associated antigen.

In some embodiments, a CAR polypeptide described herein can further comprise at least one co-stimulatory signaling domain. The CAR polypeptide is configured such that, when expressed on a host cell, the extracellular antigen-binding domain is located extracellularly to bind the target molecule and the cytoplasmic signaling domain. The optional costimulatory signaling domain may be located in the cytoplasm to trigger activation and/or effector signaling.

As used herein, the phrase "protein X transmembrane domain" (e.g., CD8 transmembrane domain) refers to any thermodynamically stable portion of a given protein (i.e., protein X that is transmembrane) in a membrane.

As used herein, the phrase "protein X cytoplasmic signaling domain" (e.g., CD3 ζ cytoplasmic signaling domain) refers to any portion of a protein (protein X) that interacts with the interior of a cell or organelle and is capable of transmitting a primary signal as known in the art, which results in immune cell proliferation and/or activation. The cytoplasmic signaling domain as described herein is distinct from the costimulatory signaling domain, which delivers secondary signals to fully activate immune cells.

As used herein, the phrase "protein X costimulatory signaling domain" (e.g., CD28 costimulatory signaling domain) refers to the portion of a given costimulatory protein (protein X, such as CD28, 4-1BB, OX40, CD27, or ICOS) that can transduce a costimulatory signal (secondary signal) into an immune cell, such as a T cell, resulting in complete activation of the immune cell.

A.Extracellular target binding domains

The chimeric receptor polypeptides disclosed herein comprise an extracellular domain that targets an antigen of interest (e.g., those described herein) by direct binding or indirect binding (via an intermediate, such as an antibody). The chimeric receptor polypeptide can be an ACTR polypeptide comprising an Fc binding domain. Alternatively, the chimeric receptor polypeptide can be a CAR polypeptide comprising an extracellular antigen-binding domain.

Fc binding domain

The ACTR polypeptides described herein comprise an extracellular domain that is an Fc binding domain, i.e., an Fc portion that is capable of binding to an immunoglobulin (e.g., IgG, IgA, IgM, or IgE) of a suitable mammal (e.g., human, mouse, rat, goat, sheep, or monkey). Suitable Fc binding domains can be derived from naturally occurring proteins, such as mammalian Fc receptors or certain bacterial proteins (e.g., protein a, protein G). In addition, the Fc binding domain can be a synthetic polypeptide specifically engineered to bind with high affinity and specificity the Fc portion of any of the antibodies described herein. For example, such an Fc binding domain can be an antibody or antigen binding fragment thereof that specifically binds the Fc portion of an immunoglobulin. Examples include, but are not limited to, single chain variable fragments (scFv), domain antibodies, or single domain antibodies (e.g., nanobody). Alternatively, the Fc binding domain may be a synthetic peptide that specifically binds to an Fc moiety, such as a Kunitz domain, a Small Modular Immunopharmaceutical (SMIP), an adnectin, an avimer, an affibody, a DARPin, or an antiporter protein, which can be identified by screening a combinatorial library of peptides for binding activity to Fc.

In some embodiments, the Fc binding domain is an extracellular ligand binding domain of a mammalian Fc receptor. As used herein, an "Fc receptor" is a cell surface binding receptor that is expressed on the surface of many immune cells, including B cells, dendritic cells, Natural Killer (NK) cells, macrophages, neutrophils, mast cells, and eosinophils, and that exhibits binding specificity for the Fc domain of an antibody. Fc receptors are typically composed of at least two immunoglobulin (Ig) -like domains that have binding specificity for the Fc (fragment crystallizable) portion of an antibody. In some cases, binding of an Fc receptor to the Fc portion of an antibody can trigger an antibody-dependent cell-mediated cytotoxicity (ADCC) effect. The Fc receptors used to construct ACTR polypeptides as described herein can be naturally occurring polymorphic variants (e.g., CD16V158 variants) that can have increased or decreased affinity for Fc compared to the wild-type counterpart. Alternatively, the Fc receptor may be a functional variant of the wild-type counterpart, which functional variant carries one or more mutations (e.g., up to 10 amino acid residue substitutions, including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations) that alter binding affinity to the Fc portion of the Ig molecule. In some cases, the mutation may alter the glycosylation pattern of the Fc receptor, and thus alter the binding affinity to Fc.

The following table lists a number of exemplary polymorphisms in the extracellular domain of the Fc receptor (see, e.g., Kim et al, j.mol. evol.53:1-9,2001) that can be used in any of the methods or constructs described herein:

exemplary polymorphisms of Fc receptors

Fc receptors are classified based on the isotype of the antibody to which they are capable of binding. For example, Fc-gamma receptors (fcyr) typically bind IgG antibodies, such as one or more subtypes of the IgG antibody (i.e., IgG1, IgG2, IgG3, IgG 4); fc-alpha receptors (Fc α R) typically bind IgA antibodies; fc-epsilon receptors (Fcepsilon R) typically bind IgE antibodies. In some embodiments, the Fc receptor is an Fc- γ receptor, an Fc- α receptor, or an Fc- ε receptor. Examples of Fc-gamma receptors include, but are not limited to, CD64A, CD64B, CD64C, CD32A, CD32B, CD16A, and CD 16B. An example of an Fc-alpha receptor is Fc alpha R1/CD 89. Examples of Fc-epsilon receptors include, but are not limited to, Fc epsilon RI and Fc epsilon RII/CD 23. The following table lists exemplary Fc receptors and their binding activity to corresponding Fc domains used to construct the ACTR polypeptides described herein:

TABLE 2 exemplary Fc receptors

The choice of ligand binding domains for the Fc receptors of the ACTR polypeptides described herein will be apparent to those skilled in the art. For example, it may depend on factors such as the isotype of the antibody that the Fc receptor is expected to bind to and the desired affinity of the binding interaction.

Extracellular antigen-binding domain of any of the CAR polypeptides in some examples, the Fc binding domain is an extracellular ligand-binding domain of CD16 that can incorporate naturally occurring polymorphisms that can regulate affinity for Fc. In some examples, the Fc binding domain is an extracellular ligand binding domain of CD16 that incorporates a polymorphism (e.g., valine or phenylalanine) at position 158. In some embodiments, the Fc binding domain is produced under conditions that alter its glycosylation state and its affinity for Fc.

The amino acid sequences of human CD16A F158 and CD16A V158 variants are provided below, in which the F158 and V158 residues are highlighted in bold/bold and underlined (signal peptide in italics):

CD16A F158(SEQ ID NO:86)：

CD16A V158(SEQ ID NO:87)：

in some embodiments, the Fc binding domain is an extracellular ligand binding domain of CD16 that incorporates modifications that make the ACTR polypeptide specific to a subset of IgG antibodies. For example, mutations can be incorporated that increase or decrease the affinity for an IgG subtype (e.g., IgG 1).

Any of the Fc binding domains described herein can have suitable binding affinity for the Fc portion of a therapeutic antibody. As used herein, "binding affinity" refers to the apparent binding constant or K_A。K_AIs the dissociation constant K_DThe reciprocal of (c). Binding affinity K of the extracellular ligand binding domain of the Fc receptor domain of the ACTR polypeptides described herein to the Fc portion of an antibody_dCan be at least 10^-5、10^-6、10^-7、10^-8、10^-9、10^-10M or less. In some embodiments, the Fc binding domain has a high binding affinity for the antibody, one or more isoforms of the antibody, or isoforms thereof, as compared to the binding affinity of the Fc binding domain for another antibody, one or more isoforms of the antibody, or isoforms thereof. In some embodiments, the extracellular ligand-binding domain of the Fc receptor is specific for an antibody, one or more isoforms of an antibody, or subtypes thereof, as compared to the binding of the extracellular ligand-binding domain of the Fc receptor to another antibody, one or more isoforms of an antibody, or subtypes thereof.

Other Fc binding domains known in the art may also be used in the ACTR constructs described herein, including, for example, WO2015058018a1 and PCT application numbers: those Fc binding domains described in PCT/US2018/015999, the relevant disclosure of each of which is incorporated by reference for this purpose and the subject matter cited herein.

Extracellular antigen binding domains

The CAR polypeptides described herein comprise an extracellular antigen-binding domain that can redirect the specificity of an immune cell expressing the CAR polypeptide. As used herein, an "extracellular antigen-binding domain" refers to a peptide or polypeptide having binding specificity for a target antigen of interest, which may be a naturally occurring antigen associated with a medical condition (e.g., a disease), or an antigen moiety conjugated to a therapeutic agent that targets a disease-associated antigen. The extracellular antigen-binding domain described herein does not comprise the extracellular domain of an Fc receptor and may not bind to the Fc portion of an immunoglobulin. An extracellular domain that does not bind to an Fc fragment refers to a binding activity between the Fc fragment and the extracellular domain that is not detectable using conventional assays, or only background or biologically insignificant binding activity is detected using conventional assays.

In some cases, the extracellular antigen-binding domain of any CAR polypeptide described herein is a peptide or polypeptide that is capable of binding to a cell surface antigen (e.g., a tumor antigen) or an antigen (or fragment thereof) that is complexed to a major histocompatibility complex and is presented on the cell surface of an antigen-presenting cell. Such extracellular antigen-binding domains can be single chain antibody fragments (scfvs) that can be derived from antibodies that bind to target cell-surface antigens with high binding affinity. Table 3 below lists exemplary cell surface target antigens and exemplary antibodies that bind to such exemplary cell surface target antigens.

TABLE 3 exemplary cell surface target antigens and exemplary antibodies that bind to the exemplary cell surface target antigens

Depending on the target antigen of interest, the extracellular antigen-binding domain may comprise an antigen-binding fragment (e.g., scFv) derived from any of the antibodies listed in table 3.

In other embodiments, the extracellular antigen-binding domain of any of the CAR polypeptides described herein can be specific for a pathogenic antigen, such as a bacterial antigen, a viral antigen, or a fungal antigen. Some examples are provided below: influenza virus neuraminidase, hemagglutinin or M2 protein, human Respiratory Syncytial Virus (RSV) F glycoprotein or G glycoprotein, herpes simplex virus glycoprotein gB, gC, gD or gE, chlamydia MOMP or PorB protein, dengue virus core protein, matrix protein or glycoprotein E, measles virus hemagglutinin, herpes simplex virus type 2 glycoprotein gB, poliovirus I VP1, envelope glycoprotein of HIV 1, hepatitis b core antigen or surface antigen, diphtheria toxin, streptococcus 24M epitope, gonococcal pilin protein, pseudorabies virus G50(gpD), pseudorabies virus ii (gpb), pseudorabies virus iii (gpc), pseudorabies virus glycoprotein H, pseudorabies virus glycoprotein E, transmissible gastrointestinal glycoprotein 195, infectious inflammatory matrix protein or human hepatitis c virus glycoprotein E1 or E2.

In addition, the extracellular antigen-binding domain of the CAR polypeptides described herein can be specific for a tag conjugated to a therapeutic agent that targets an antigen associated with a disease or condition (e.g., a tumor antigen or a pathogenic antigen described herein). In some cases, the tag conjugated to the therapeutic agent can be antigenic, and the extracellular antigen-binding domain of the CAR polypeptide can be an antigen-binding fragment (e.g., scFv) of an antibody having high binding affinity and/or specificity for the antigen tag. Exemplary antigen tags include, but are not limited to, biotin, avidin, fluorescent molecules (e.g., GFP, YRP, luciferase or RFP), Myc, Flag, His (e.g., polyHis, such as 6XHis), HA (hemagglutinin), GST, MBP (maltose binding protein), KLH (keyhole limpet hemocyanin), trx, T7, HSV, VSV (e.g., VSV-G), Glu-Glu, V5, E-tag, S-tag, KT3, E2, Au1, Au5, and/or thioredoxin.

In other cases, the tag conjugated to the therapeutic agent is a member of a ligand-receptor pair, and the extracellular antigen-binding domain comprises the other member of the ligand-receptor pair or a fragment thereof. For example, the tag conjugated to the therapeutic agent can be biotin, and the extracellular antigen-binding domain of the CAR polypeptide can comprise a biotin-binding fragment of avidin. See, e.g., Urbanska et al, 2012, Lohmueler et al, 2018. Other examples include anti-tag CARs in which the extracellular antigen-binding domain is a scFv fragment specific for a protein tag such as FITC (Tamada et al, 2012; Kim et al, 2015; Cao et al, 2016; and Ma et al, 2016), PNE (Rodgers et al, 2016), La-SS-B (cartelliri et al, 2016), biotin (lohmullulanr et al, 2017), and leucine zipper (Cho et al, 2018). The choice of antigen binding domain for use in the CAR polypeptides described herein will be apparent to those skilled in the art. For example, the selection may depend on factors such as the type of target antigen and the desired affinity of the binding interaction.

The extracellular antigen-binding domain of any of the CAR polypeptides described herein can have suitable binding affinity for a target antigen (e.g., any of the targets described herein) or an epitope thereof. As used herein, "binding affinity" refers to the apparent binding constant or K_A。K_AIs the dissociation constant K_DThe reciprocal of (c). Binding affinity (K) of the extracellular antigen-binding domain to a target antigen or epitope for use in the polypeptide CAR described herein_D) Can be at least 10^-5、10^-6、10^-7、10^-8、10^-9、10^-10M, or lower. Increased binding affinity corresponds to decreased K_D. The higher affinity binding of the extracellular antigen-binding domain of the first antigen relative to the second antigen may be through the K binding to the first antigen_A(or a smaller value K)_D) Higher than K bound to a second antigen_A(or value K)_D) To indicate. In such cases, the extracellular antigen-binding domain has specificity for the first antigen (e.g., the first protein or mimetic thereof in the first conformation) relative to the second antigen (e.g., the same first protein or mimetic thereof in the second conformation; or the second protein). The difference in binding affinity (e.g., for specificity or other comparison) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000, or 10⁵And (4) doubling.

Binding affinity (or binding specificity) can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using fluorescence assays). An exemplary condition for assessing binding affinity is in HBS-P buffer (10mM HEPES pH7.4, 150mM NaCl, 0.005% (v/v) surfactant P20). These techniques can be used to measure the concentration of bound binding protein as a function of the concentration of the target protein. The concentration of bound protein ([ bound ]) is generally related to the concentration of free target protein ([ free ]) by the following equation:

[ bound ] ([ free ]/(Kd + [ free ])

However, it is not always necessary to determine K accurately_AHowever, because it is sometimes sufficient to obtain a quantitative measure of affinity (e.g., determined using methods such as ELISA or FACS analysis), affinity and K are_AProportional and therefore can be used to make comparisons, such as to determine whether a higher affinity is, for example, 2-fold higher to obtain a qualitative measurement of affinity, or to obtain an inference of affinity, for example, by activity in a functional assay (e.g., an in vitro or in vivo assay).

B.Transmembrane domain

The transmembrane domain of the chimeric receptor polypeptides described herein (e.g., ACTR polypeptides or CAR polypeptides) can be in any form known in the art. As used herein, "transmembrane domain" refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane. Suitable transmembrane domains for the chimeric receptor polypeptides used herein may be obtained from naturally occurring proteins. Alternatively, the transmembrane domain may be a synthetic, non-naturally occurring protein segment, such as a thermodynamically stable hydrophobic protein segment in a cell membrane.

Transmembrane domains are classified based on their three-dimensional structure. For example, the transmembrane domain may form an alpha helix, a complex of more than one alpha helix, a beta barrel, or any other stable structure capable of spanning a cellular phospholipid bilayer. In addition, transmembrane domains can also or alternatively be classified based on transmembrane domain topology, including the number of transmembrane passes and the orientation of the protein. For example, a single transmembrane protein spans a cell membrane once, while a multiple transmembrane protein spans a cell membrane at least twice (e.g., 2, 3, 4, 5, 6, 7, or more times).

Membrane proteins can be defined as type I, type II or type III, depending on their terminal and topology of one or more membrane transport segments relative to the interior and exterior of the cell. Type I membrane proteins have a single transmembrane region and are oriented such that the N-terminus of the protein is present on the extracellular side of the cellular lipid bilayer and the C-terminus of the protein is present on the cytoplasmic side. Type II membrane proteins also have a single transmembrane region, but are oriented such that the C-terminus of the protein is located on the extracellular side of the cellular lipid bilayer and the N-terminus of the protein is present on the cytoplasmic side. Type III membrane proteins have multiple transmembrane segments and can be further subdivided based on the number of transmembrane segments and the location of the N-terminus and C-terminus.

In some embodiments, the transmembrane domain of the chimeric receptor polypeptides described herein is derived from a type I single-penetrating protein. Single transmembrane proteins include, but are not limited to, CD8 α, CD8 β, 4-1BB/CD137, CD27, CD28, CD34, CD4, Fc ε RI γ, CD16, OX40/CD134, CD3 ζ, CD3 ε, CD3 γ, CD3 δ, TCR α, TCR β, TCR ζ, CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40, CD40L/CD154, VEGFR2, FAS, and FGFR 2B. In some embodiments, the transmembrane domain is from a membrane protein selected from the group consisting of: CD8 α, CD8 β, 4-1BB/CD137, CD28, CD34, CD4, Fc ε RI γ, CD16, OX40/CD134, CD3 ζ, CD3 ε, CD3 γ, CD3 δ, TCR α, CD32, CD64, VEGFR2, FAS, and FGFR 2B. In some examples, the transmembrane domain belongs to CD8 (e.g., the transmembrane domain belongs to CD8 α). In some examples, the transmembrane domain belongs to 4-1BB/CD 137. In other examples, the transmembrane domain belongs to CD 28. In some cases, the chimeric receptor polypeptides described herein may not contain a hinge domain from any non-CD 16A receptor. In some cases, such chimeric receptor polypeptides may not contain any hinge domains. Alternatively or additionally, such chimeric receptor polypeptides may comprise two or more co-stimulatory regions as described herein. In other examples, the transmembrane domain belongs to CD 34. In other examples, the transmembrane domain is not derived from human CD8 α. In some embodiments, the transmembrane domain of the chimeric receptor polypeptide is a single-pass alpha helix.

Transmembrane domains from multiple transmembrane proteins may also be compatibly employed in the chimeric receptor polypeptides described herein. The multiple penetrating proteins may comprise complex alpha-helical structures (e.g., at least 2, 3, 4, 5, 6, 7, or more alpha helices) or beta sheet structures. Preferably, the N-terminus and C-terminus of the polyperidin are present on opposite sides of the lipid bilayer, e.g., the N-terminus of the protein is present on the cytoplasmic side of the lipid bilayer and the C-terminus of the protein is present on the extracellular side. One or more helical passes from a multiple-penetrating protein can be used to construct the chimeric receptor polypeptides described herein.

The transmembrane domain for chimeric receptor polypeptides described herein may also comprise at least a portion of a synthetic, non-naturally occurring protein segment. In some embodiments, the transmembrane domain is a synthetic, non-naturally occurring alpha helix or beta sheet. In some embodiments, a protein segment is at least about 20 amino acids, e.g., at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids. Examples of synthetic transmembrane domains are known in the art, for example in U.S. patent No. 7,052,906B1 and PCT publication No. WO 2000/032776 a2, the relevant disclosures of each of which are incorporated herein by reference.

In some embodiments, the amino acid sequence of the transmembrane domain does not comprise a cysteine residue. In some embodiments, the amino acid sequence of the transmembrane domain comprises one cysteine residue. In some embodiments, the amino acid sequence of the transmembrane domain comprises two cysteine residues. In some embodiments, the amino acid sequence of the transmembrane domain comprises more than two cysteine residues (e.g., 3, 4, 5, or more).

The transmembrane domain may comprise a transmembrane region and a cytoplasmic region located C-terminal to the transmembrane domain. The cytoplasmic region of the transmembrane domain may comprise three or more amino acids and, in some embodiments, helps orient the transmembrane domain in the lipid bilayer. In some embodiments, one or more cysteine residues are present in a transmembrane region of the transmembrane domain. In some embodiments, one or more cysteine residues are present in the cytoplasmic region of the transmembrane domain. In some embodiments, the cytoplasmic region of the transmembrane domain comprises positively charged amino acids. In some embodiments, the cytoplasmic region of the transmembrane domain comprises the amino acids arginine, serine, and lysine.

In some embodiments, the transmembrane region of the transmembrane domain comprises hydrophobic amino acid residues. In some embodiments, the transmembrane region comprises predominantly hydrophobic amino acid residues, such as alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or valine. In some embodiments, the transmembrane region is hydrophobic. In some embodiments, the transmembrane region comprises a poly leucine-alanine sequence.

The hydrophilicity, hydrophobicity, or hydropathic character of a protein or protein segment can be assessed by any method known in the art, including, for example, Kyte and Doolittle hydropathicity assays.

C.Co-stimulatory signaling domains

In addition to stimulating antigen-specific signals, many immune cells also require co-stimulation to promote cell proliferation, differentiation and survival, as well as to activate effector functions of the cells. In some embodiments, a chimeric receptor polypeptide described herein, such as an ACTR or CAR polypeptide, comprises at least one co-stimulatory signaling domain. In certain embodiments, the chimeric receptor polypeptide may contain a CD28 co-stimulatory signaling domain or a 4-1BB (CD137) co-stimulatory signaling domain. As used herein, the term "co-stimulatory signaling domain" refers to at least one fragment of a co-stimulatory signaling protein that mediates intracellular signaling to induce an immune response, such as effector function (secondary signal). As is known in the art, activation of immune cells such as T cells typically requires two signals: (1) antigen-specific signal (primary signal) triggered by the binding of the T Cell Receptor (TCR) to the antigenic peptide/MHC complex presented by the antigen presenting cell, which is typically driven by CD3 ζ, which is a component of the TCR complex; (ii) costimulatory signals (secondary signals) triggered by the interaction between a costimulatory receptor and its ligand. Costimulatory receptors transduce costimulatory signals (secondary signals) as an addition to TCR-triggered signaling and modulate responses mediated by immune cells such as T cells, NK cells, macrophages, neutrophils, or eosinophils.

Activation of a costimulatory signaling domain in a host cell (e.g., an immune cell) can induce the cell to increase or decrease cytokine production and secretion, phagocytic properties, proliferation, differentiation, survival, and/or cytotoxicity. The costimulatory signaling domain of any costimulatory molecule can be compatible for use with the chimeric receptor polypeptides described herein. The type of co-stimulatory signaling domain is selected based on factors such as: the type of immune cell (e.g., T cell, NK cell, macrophage, neutrophil, or eosinophil) in which the chimeric receptor polypeptide will be expressed and the desired immune effector function (e.g., ADCC). Examples of co-stimulatory signaling domains for use in chimeric receptor polypeptides may be cytoplasmic signaling domains of co-stimulatory proteins, including but not limited to members of the B7/CD28 family (e.g., B7-1/CD80, B7-2/CD86, B7-H1/PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA/CD272, CD28, CTLA-4, Gi24/VISTA/B7-H5, ICOS/CD278, PD-1, PD-L2/B7-DC, and PDCD 6); TNF superfamily members (e.g., 4-1BB/TNFRSF9/CD137, 4-1BB ligand/TNFRSF 9, BAFF/BLyS/TNFRSF 13 9, BAFF R/TNFRSF13 9, CD 9/TNFRSF 9, CD9 ligand/TNFRSF 9, DR 9/TNFRSF 9, GITR ligand/TNFRSF 9, HVEM/TNFRSF 9, LIGHT/TNFRSF 9, lymphotoxin- α/TNF- β, 9/TNFRSF 9, 9 ligand/TNFRSF 9, REL/TNFRSF 3619, TAFRSF TL/9, TNFRSF13, TNFRSF 1/9, TNFRSF 36RIOX-9, TNFRSF 36RIX/9, TNFRSF 36RIF 9, and TNFRSF 36IROX/9); SLAM family members (e.g., 2B4/CD244/SLAMF4, BLAME/SLAMF8, CD2, CD2F-10/SLAMF9, CD48/SLAMF2, CD58/LFA-3, CD84/SLAMF5, CD229/SLAMF3, CRACC/SLAMF7, NTB-A/SLAMF6, and SLAM/CD 150); and any other co-stimulatory molecule such as CD2, CD7, CD53, CD82/Kai-1, CD90/Thy1, CD96, CD160, CD200, CD300a/LMIR1, HLA class I, HLA-DR, Ikaros, integrin alpha 4/CD49d, integrin alpha 4 beta 1, integrin alpha 4 beta 7/LPAM-1, LAG-3, TCL1A, TCL1B, CRTADAP, 12, Dectin-1/CLEC7A, DPPIV/CD26, EphB6, LFhB-1/KIM-1/HAVCR, TIM-4, TSLP R, lymphocyte function-related antigen 1 (TIM A-1), and NKG 2C. In some embodiments, the co-stimulatory signaling domain belongs to 4-1BB, CD28, OX40, ICOS, CD27, GITR, HVEM, TIM1, LFA1(CD11a), or CD2 or any variant thereof.

Also within the scope of the present disclosure are variants of any of the costimulatory signaling domains described herein, such that the costimulatory signaling domain is capable of modulating the immune response of an immune cell. In some embodiments, the co-stimulatory signaling domain comprises up to 10 amino acid residue mutations (e.g., 1, 2, 3, 4, 5, or 8), such as amino acid substitutions, deletions, or additions, as compared to the wild-type counterpart. Such co-stimulatory signaling domains comprising one or more amino acid variants (e.g., amino acid substitutions, deletions, or additions) may be referred to as variants.

Mutations of amino acid residues of the costimulatory signaling domain may result in increased signal transduction and enhanced stimulation of an immune response relative to a costimulatory signaling domain that does not comprise the mutation. Mutations of amino acid residues of the co-stimulatory signaling domain may result in reduced signal transduction and reduced stimulation of an immune response relative to a co-stimulatory signaling domain that does not comprise the mutation. For example, mutations at residues 186 and 187 of the native CD28 amino acid sequence can result in increased costimulatory activity and induction of an immune response by the costimulatory domain of the chimeric receptor polypeptide. In some embodiments, the mutation is a substitution of the lysine at each of positions 186 and 187 with a glycine residue of the CD28 co-stimulatory domain, referred to as CD28_LL→GGVariants. Additional mutations that can be made in the costimulatory signaling domain that either enhance or reduce the costimulatory activity of that domain would be useful in the artAs will be apparent to those of ordinary skill in the art. In some embodiments, the co-stimulatory signaling domain belongs to 4-1BB, CD28, OX40, or CD28_LL→GGVariants.

In some embodiments, the chimeric receptor polypeptide may contain a single co-stimulatory domain, such as a CD27 co-stimulatory domain, a CD28 co-stimulatory domain, a 4-1BB co-stimulatory domain, an ICOS co-stimulatory domain, or an OX40 co-stimulatory domain.

In some embodiments, the chimeric receptor polypeptide may comprise more than one costimulatory signaling domain (e.g., 2, 3, or more). In some embodiments, the chimeric receptor polypeptide comprises two or more identical costimulatory signaling domains, e.g., two copies of the costimulatory signaling domain of CD 28. In some embodiments, the chimeric receptor polypeptide comprises two or more costimulatory signaling domains from different costimulatory proteins (e.g., any two or more of the costimulatory proteins described herein). The selection of the type of co-stimulatory signaling domain may be based on factors such as: the type of host cell (e.g., T cell or NK cell) to be used with the chimeric receptor polypeptide and the desired immune effector function. In some embodiments, the chimeric receptor polypeptide comprises two costimulatory signaling domains, e.g., two copies of the costimulatory signaling domain of CD 28. In some embodiments, the chimeric receptor polypeptide may comprise two or more co-stimulatory signaling domains from different co-stimulatory receptors, such as any two or more of the co-stimulatory receptors described herein, e.g., CD28 and 4-1BB, CD28 and CD27, CD28 and ICOS, CD28_LL→GGVariants and 4-1BB, CD28 and OX40 or CD28_LL→GGVariants and OX 40. In some embodiments, the two co-stimulatory signaling domains are CD28 and 4-1 BB. In some embodiments, the two co-stimulatory signaling domains are CD28_LL→GGVariants and 4-1 BB. In some embodiments, the two co-stimulatory signaling domains are CD28 and OX 40. In some embodiments, the two co-stimulatory signaling domains are CD28_LL→GGVariantsAnd OX 40. In some embodiments, the chimeric receptor polypeptides described herein can contain a combination of CD28 and ICOSL. In some embodiments, the chimeric receptor polypeptides described herein can contain a combination of CD28 and CD 27. In certain embodiments, the 4-1BB co-stimulatory domain is located in CD28 or CD28_LL→GGThe variant co-stimulates the N-terminus of the signaling domain.

In some embodiments, the chimeric receptor polypeptides described herein do not comprise a costimulatory signaling domain.

D.Cytoplasmic signaling domain

Any cytoplasmic signaling domain can be used to produce the chimeric receptor polypeptides (e.g., ACTR polypeptides or CAR polypeptides) described herein. Such cytoplasmic domain may be any signaling domain involved in triggering cell signaling (primary signaling) that results in immune cell proliferation and/or activation. The cytoplasmic signaling domain as described herein is not a costimulatory signaling domain, which delivers costimulatory or secondary signals to fully activate immune cells as is known in the art.

The cytoplasmic domains described herein can comprise an immunoreceptor tyrosine-based activation motif (ITAM) domain (e.g., at least one ITAM domain, at least two ITAM domains, or at least three ITAM domains) or can be ITAM-free. As used herein, an "ITAM" is a conserved protein motif that is typically present in the tail portion of signaling molecules expressed in many immune cells. This motif may comprise two repeats of the amino acid sequence YxxL/I, separated by 6-8 amino acids, where each x is independently any amino acid, resulting in the conserved motif YxxL/Ix_(6-8)YxxL/I. ITAMs within signaling molecules are important for intracellular signal transduction, which is mediated at least in part by phosphorylation of tyrosine residues in ITAMs upon activation of the signaling molecule. ITAMs can also serve as docking sites for other proteins involved in signaling pathways.

In some examples, the cytoplasmic signaling domain is of CD3 ζ or fcepsilonr 1 γ. In other examples, the cytoplasmic signaling domain is not derived from human CD3 ζ. In other examples, when the extracellular Fc-binding domain of the same chimeric receptor polypeptide is derived from CD16A, the cytoplasmic signaling domain is not derived from an Fc receptor.

In a particular embodiment, several signaling domains may be fused together to produce additive or synergistic effects. Non-limiting examples of useful additional signaling domains include some or all of one or more of the following: TCR zeta chain, CD28, OX40/CD134, 4-1BB/CD137, Fc epsilon RIy, ICOS/CD278, IL 2R-beta/CD 122, IL-2R-gamma/CD 132 and CD 40.

In other embodiments, the cytoplasmic signaling domain described herein does not contain an ITAM motif. Examples include, but are not limited to, the cytoplasmic signaling domain of Jak/STAT, Toll-interleukin receptor (TIR), and tyrosine kinase.

E.Hinge domain

In some embodiments, the chimeric receptor polypeptides described herein (such as ACTR polypeptides or CAR polypeptides) further comprise a hinge domain located between the extracellular ligand-binding domain and the transmembrane domain. Hinge domains are amino acid segments typically found between two domains of a protein, and may allow for the flexibility of the protein and the movement of one or both of the domains relative to each other. Any amino acid sequence that provides such flexibility and movement of the extracellular ligand-binding domain relative to the transmembrane domain of the chimeric receptor polypeptide can be used.

The hinge domain of any protein known in the art that comprises a hinge domain is compatible for use in the chimeric receptor polypeptides described herein. In some embodiments, the hinge domain is at least a portion of a hinge domain of a naturally occurring protein and confers flexibility to the chimeric receptor polypeptide. In some embodiments, the hinge domain belongs to CD 8. In some embodiments, the hinge domain is a portion of the hinge domain of CD8, e.g., a fragment containing at least 15 (e.g., 20, 25, 30, 35, or 40) contiguous amino acids of the hinge domain of CD 8. In some embodiments, the hinge domain belongs to CD 28. In some embodiments, the hinge domain is a portion of the hinge domain of CD28, e.g., a fragment containing at least 15 (e.g., 20, 25, 30, 35, or 40) contiguous amino acids of the hinge domain of CD 28. The hinge domain and/or transmembrane domain may be linked to additional amino acids (e.g., 15aa, 10-aa, 8-aa, 6-aa, or 4-aa) at the N-terminal portion, the C-terminal, or both. Examples can be found, for example, in Ying et al, Nature Medicine,25(6): 947-.

In some embodiments, the hinge domain belongs to the CD16A receptor, e.g., the entire hinge domain of the CD16A receptor or a portion thereof, which may consist of up to 40 consecutive amino acid residues (e.g., 20, 25, 30, 35, or 40) of the CD16A receptor. Such chimeric receptor polypeptides (e.g., ACTR polypeptides) may not contain a hinge domain from a different receptor (other than the CD16A receptor).

The hinge domain of an antibody, such as an IgG, IgA, IgM, IgE, or IgD antibody, is also compatible for use in the chimeric receptor polypeptides described herein. In some embodiments, the hinge domain is a hinge domain that links the constant domains CH1 and CH2 of the antibody. In some embodiments, the hinge domain belongs to an antibody and comprises the hinge domain of the antibody and one or more constant regions of the antibody. In some embodiments, the hinge domain comprises the hinge domain of the antibody and the CH3 constant region of the antibody. In some embodiments, the hinge domain comprises the hinge domain of an antibody and the CH2 and CH3 constant regions of an antibody. In some embodiments, the antibody is an IgG, IgA, IgM, IgE, or IgD antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the hinge region comprises the hinge region of an IgG1 antibody and the CH2 and CH3 constant regions. In some embodiments, the hinge region comprises the hinge region and the CH3 constant region of an IgG1 antibody.

Non-naturally occurring peptides may also be used as the hinge domain of the chimeric receptor polypeptides described herein. In some embodiments, the extracellular target-binding structureThe hinge domain between the C-terminus of the domain and the N-terminus of the transmembrane domain is a peptide linker, such as (Gly_xSer)_nA linker, wherein x and n independently can be an integer between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more. In some embodiments, the hinge domain is (Gly)₄Ser)_n(SEQ ID NO:88), wherein n can be an integer between 3 and 60, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 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, or 60. In certain embodiments, n may be an integer greater than 60. In some embodiments, the hinge domain is (Gly)₄Ser)₃(SEQ ID NO: 89). In some embodiments, the hinge domain is (Gly)₄Ser)₆(SEQ ID NO: 90). In some embodiments, the hinge domain is (Gly)₄Ser)₉(SEQ ID NO: 91). In some embodiments, the hinge domain is (Gly)₄Ser)₁₂(SEQ ID NO: 92). In some embodiments, the hinge domain is (Gly)₄Ser)₁₅(SEQ ID NO: 93). In some embodiments, the hinge domain is (Gly)₄Ser)₃₀(SEQ ID NO: 94). In some embodiments, the hinge domain is (Gly)₄Ser)₄₅(SEQ ID NO: 95). In some embodiments, the hinge domain is (Gly)₄Ser)₆₀(SEQ ID NO:96)。

In other embodiments, the hinge domain is an extended recombinant polypeptide (XTEN) that is an unstructured polypeptide consisting of hydrophilic residues of varying length (e.g., 10-80 amino acid residues). The amino acid sequence of XTEN peptides will be apparent to those skilled in the art and can be found, for example, in U.S. patent No. 8,673,860, the relevant disclosure of which is incorporated herein by reference. In some embodiments, the hinge domain is an XTEN peptide and comprises 60 amino acids. In some embodiments, the hinge domain is an XTEN peptide and comprises 30 amino acids. In some embodiments, the hinge domain is an XTEN peptide and comprises 45 amino acids. In some embodiments, the hinge domain is an XTEN peptide and comprises 15 amino acids.

Any of the hinge domains used to prepare the chimeric receptor polypeptides as described herein can contain up to 250 amino acid residues. In some cases, chimeric receptor polypeptides can contain a relatively long hinge domain, such as containing 150-250 amino acid residues (e.g., 150-180 amino acid residues, 180-200 amino acid residues, or 200-250 amino acid residues). In other cases, the chimeric receptor polypeptide can contain a middle-sized hinge domain that can contain 60-150 amino acid residues (e.g., 60-80, 80-100, 100-120, or 120-150 amino acid residues). Alternatively, the chimeric receptor polypeptide can contain a short hinge domain that can contain less than 60 amino acid residues (e.g., 1-30 amino acids or 31-60 amino acids). In some embodiments, the chimeric receptor polypeptides described herein (e.g., ACTR polypeptides) do not contain a hinge domain or a hinge domain from a non-CD 16A receptor.

F.Signal peptide

In some embodiments, a chimeric receptor polypeptide (e.g., an ACTR polypeptide or a CAR polypeptide) can further comprise a signal peptide (also referred to as a signal sequence) at the N-terminus of the polypeptide. Typically, the signal sequence is a peptide sequence that targets the polypeptide to a desired site in a cell. In some embodiments, the signal sequence will target the chimeric receptor polypeptide to the secretory pathway of the cell and will allow the chimeric receptor polypeptide to integrate and anchor in the lipid bilayer. Signal sequences (including those of naturally occurring proteins, or synthetic, non-naturally occurring signal sequences, which are compatible for use in the chimeric receptor polypeptides described herein) will be apparent to those skilled in the art. In some embodiments, the signal sequence is from CD8 a. In some embodiments, the signal sequence is from CD 28. In other embodiments, the signal sequence is from a murine kappa chain. In other embodiments, the signal sequence is from CD 16.

G.Examples of ACTR Polypeptides

Exemplary ACTR constructs for use with the methods and compositions can be found, for example, in the specification and drawings, or can be found in PCT patent publication nos: WO2016040441A1, WO2017/161333 and PCT application numbers: each of these references is incorporated herein by reference for this purpose, as found in PCT/US 2018/015999. The ACTR polypeptides described herein may comprise a CD16A extracellular domain having binding affinity and specificity for the Fc portion of an IgG molecule, a transmembrane domain, and a CD3 ζ cytoplasmic signaling domain. In some embodiments, the ACTR polypeptide may further comprise one or more costimulatory signaling domains, one of which may be a CD28 costimulatory signaling domain or a 4-1BB costimulatory signaling domain. The ACTR polypeptide is configured such that, when expressed on a host cell, the extracellular ligand binding domain is located extracellularly to bind the target molecule and the CD3 ζ cytoplasmic signaling domain. The costimulatory signaling domain may be located in the cytoplasm to trigger activation and/or effector signaling.

In some embodiments, an ACTR polypeptide as described herein can comprise, from N-terminus to C-terminus, an Fc binding domain (such as a CD16A extracellular domain), a transmembrane domain, optionally one or more costimulatory domains (e.g., a CD28 costimulatory signaling domain, a 4-1BB costimulatory signaling domain, an OX40 costimulatory signaling domain, a CD27 costimulatory signaling domain, or an ICOS costimulatory signaling domain), and a CD3 zeta cytoplasmic signaling domain.

Alternatively or additionally, the ACTR polypeptides described herein may contain two or more costimulatory signaling domains, which may be linked to each other or separated by a cytoplasmic signaling domain. The extracellular Fc-binding agent, transmembrane domain, optional costimulatory signaling domain(s), and cytoplasmic signaling domain in the ACTR polypeptide can be linked to each other directly or through a peptide linker. In some embodiments, any of the ACTR polypeptides described herein can comprise a signal sequence at the N-terminus.

Table 4 provides exemplary ACTR polypeptides described herein. These exemplary constructs have, in order from N-terminus to C-terminus, a signal sequence, an Fc binding domain (e.g., the extracellular domain of an Fc receptor), a hinge domain, and a transmembrane domain, while the positions of the optional costimulatory domain and cytoplasmic signaling domain can be switched.

Table 4: exemplary components of ACTR polypeptides.

The amino acid sequence of an exemplary ACTR polypeptide is provided below (signal sequence in italics).

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H.Examples of CAR polypeptides

Exemplary CAR polypeptides for use with the methods and compositions described herein can be found, for example, in the specification and figures or as those known in the art. The CAR polypeptides described herein can comprise an extracellular domain comprising a single chain antibody fragment (scFv) having binding affinity and specificity for an antigen of interest (e.g., those listed in table 3 above); a transmembrane domain; and a CD3 ζ cytoplasmic signaling domain. In some embodiments, the CAR polypeptide may further comprise one or more costimulatory signaling domains, one of which may be a CD28 costimulatory signaling domain or a 4-1BB costimulatory signaling domain. The CAR polypeptide is configured such that, when expressed on a host cell, the extracellular antigen-binding domain is located extracellularly to bind the target molecule and the CD3 ζ cytoplasmic signaling domain. The costimulatory signaling domain may be located in the cytoplasm to trigger activation and/or effector signaling.

In some embodiments, a CAR polypeptide as described herein can comprise, from N-terminus to C-terminus, an extracellular antigen-binding domain, a transmembrane domain, optionally one or more costimulatory domains (e.g., a CD28 costimulatory domain, a 4-1BB costimulatory signaling domain, an OX40 costimulatory signaling domain, a CD27 costimulatory signaling domain, or an ICOS costimulatory signaling domain), and a CD3 zeta cytoplasmic signaling domain.

Alternatively or additionally, a CAR polypeptide described herein may contain two or more costimulatory signaling domains, which may be linked to each other or separated by a cytoplasmic signaling domain. The extracellular antigen-binding domain, transmembrane domain, optional co-stimulatory signaling domain(s), and cytoplasmic signaling domain in the CAR polypeptide can be linked to each other directly or through a peptide linker. In some embodiments, any of the ACTR polypeptides described herein can comprise a signal sequence at the N-terminus.

Table 5 provides exemplary CAR polypeptides described herein. These exemplary constructs have, in order from N-terminus to C-terminus, a signal sequence, an antigen binding domain (e.g., an scFv fragment that targets an antigen (such as a tumor antigen or a pathogenic antigen), a hinge domain, and a transmembrane domain, while the positions of the optional costimulatory and cytoplasmic signaling domains can be switched.

Table 5: exemplary components of CAR polypeptides.

The amino acid sequence of an exemplary CAR polypeptide is provided below (signal sequence is in italics).

SEQ ID NO:97：

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III.Hematopoietic cells expressing lactate regulatory factor and optionally chimeric receptor polypeptide

Provided herein are genetically engineered host cells (e.g., hematopoietic cells, such as HSCs, and immune cells, e.g., T cells or NK cells) that express one or more lactate regulatory factors (e.g., polypeptides or nucleic acids) in a krebs cycle modulating polypeptide as described herein. The genetically engineered host cell can also express a chimeric receptor polypeptide as also described herein (e.g., a cell that expresses an ACTR, e.g., an ACTR T cell, or a cell that expresses a CAR, e.g., a CAR T cell). In some embodiments, the host cell is a hematopoietic cell or progeny thereof. In some embodiments, the hematopoietic cells may be hematopoietic stem cells. In other embodiments, the host cell is an immune cell, such as a T cell or NK cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is an NK cell. In other embodiments, the immune cell can be an established cell line, such as an NK-92 cell.

In some embodiments, genetically engineered hematopoietic cells such as HSCs or immune cells (e.g., T cells or NK cells) can co-express any of the CAR constructs (such as those disclosed herein) and any of the lactate modulators (such as lactate modulating polypeptides (e.g., LDHA, MCT, or PDK 1)). In some embodiments, the CAR construct may comprise a co-stimulatory domain from 4-1BB or CD28, and the lactate modulating polypeptide is LDHA, MCT (e.g., MCT1, MCT2, or MCT4), or PDK 1. The CAR construct may further comprise a hinge and transmembrane domain from CD8 or CD 28.

In other embodiments, genetically engineered hematopoietic cells such as HSCs or immune cells (e.g., T cells or NK cells) can co-express any of the ACTR constructs (such as those ACTR constructs disclosed herein) and any of the lactate modulators (such as lactate modulating polypeptides (e.g., LDHA, MCT, or PDK 1)). In some embodiments, the ACTR construct may comprise a co-stimulatory domain from 4-1BB or CD28, and the lactate modulating polypeptide is LDHA, MCT (e.g., MCT1, MCT2, or MCT4), or PDK 1. ACTR constructs may also comprise hinge and transmembrane domains from CD8 or CD 28.

Alternatively, the genetically engineered host cells disclosed herein may not express any chimeric receptor polypeptides. In some embodiments, genetically engineered immune cells that can overexpress one or more lactate modulators (e.g., polypeptides) as disclosed herein can be derived from Tumor Infiltrating Lymphocytes (TILs). Overexpression of lactate modulators can enhance anti-tumor activity or TIL in the tumor microenvironment. Alternatively or additionally, the genetically engineered immune cells may be T cells, which may also have genetically engineered T cell receptors. The TIL and/or genetically modified TCR may be targeted to a peptide-MHC complex, wherein the peptide may be derived from a pathogen, a tumor antigen, or an autoantigen. Some examples are provided in table 6 below.

Any of the CAR constructs disclosed herein or antibodies to be used with ACTR T cells can also target any peptide in such peptide/MHC complexes.

TABLE 6 exemplary peptide-MHC targets

Target	Indications of
		NY-ESO-1	Sarcoma, MM
MAGE-A10	NSCLC, bladder, HNSCC
		MAGE-A4	Sarcoma, others
PMEL	Melanoma (MEA)
		WT-1	Ovary (LU) of human
AFP	HCC
		HPV-16E6	Cervical neck
HPV-16E7	Cervical neck

In some embodiments, the host cell is an immune cell, such as a T cell or NK cell. In some embodiments, the immune cell is a T cell. For example, the T cell may be a CD4+ helper cell or a CD8+ cytotoxic cell or a combination thereof. Alternatively or additionally, the T cell may be an inhibitory T cell, such as T_regA cell. In some embodiments, the immune cell is an NK cell. In other embodiments, the immune cell can be an established cell line, such as an NK-92 cell. In some examples, the immune cells can be a mixture of different types of T cells and/or NK cells known in the art. For example, the immune cells can be a population of immune cells isolated from a suitable donor (e.g., a human patient). See the disclosure below.

In some cases, the lactate modulator (e.g., polypeptide or nucleic acid) to be introduced into the host cell is the same as the endogenous protein of the host cell. Introduction of additional copies of the coding sequence for the lactate modulator into the host cell will enhance the expression level (i.e., overexpression) of the polypeptide relative to its native counterpart. In some cases, the lactate modulator to be introduced into a host cell is heterologous to the host cell, i.e., absent or not expressed in the host cell. Such heterologous lactate modulators may be naturally occurring proteins (e.g., from a different species) that are not expressed in nature in the host cell. Alternatively, the heterologous lactate modulator may be a variant of a native protein, such as those described herein. In some examples, an exogenous (i.e., not native to the host cell) copy of the encoding nucleic acid can be present extrachromosomally. In other examples, the exogenous copy of the coding sequence may be integrated into the chromosome of the host cell and may be located at a different site than the native locus of the endogenous gene.

Such genetically engineered host cells have enhanced glycolysis rate capabilities, and may, for example, have enhanced ability to take up glucose from the environment. Thus, these genetically engineered host cells may exhibit better growth and/or biological activity under low glucose, low amino acid, low pH, and/or hypoxic conditions (e.g., in a tumor microenvironment). When expressing a chimeric receptor polypeptide as disclosed herein, the genetically engineered cells can recognize and inhibit the target cells directly (e.g., by CAR-expressing immune cells) or by Fc-containing therapeutic agents such as anti-tumor antibodies (e.g., by ACTR-expressing immune cells). Given their expected high proliferation rate, biological activity and/or survival in low glucose, low amino acid, low pH and/or hypoxic environments (e.g., in a tumor microenvironment), genetically engineered cells, such as T cells and NK cells, would be expected to have higher therapeutic efficacy relative to chimeric receptor polypeptide T cells that do not express or express lower levels or lower active forms of lactate modulator.

Alternatively, the population of immune cells can be derived from stem cells, e.g., hematopoietic stem cells and induced pluripotent stem cells (ipscs). sources suitable for obtaining the desired type of host cells will be apparent to those skilled in the art.

To construct immune cells expressing any of the lactate modulator and optional chimeric receptor polypeptides described herein, expression vectors for stable or transient expression of the lactate modulator and/or chimeric receptor polypeptides can be created by conventional methods as described herein and introduced into immune host cells. For example, a nucleic acid encoding a lactate modulator and/or chimeric receptor polypeptide can be cloned into one or two suitable expression vectors (such as a viral vector or a non-viral vector operably linked to a suitable promoter). In some cases, each of the coding sequences for the chimeric receptor polypeptide and lactate regulatory factor are on two separate nucleic acid molecules and can be cloned into two separate vectors, which can be introduced into a suitable host cell simultaneously or sequentially. Alternatively, the coding sequences for the chimeric receptor polypeptide and lactate modulator are on a nucleic acid molecule and can be cloned into a vector. The coding sequences for the chimeric receptor polypeptide and the lactate regulatory factor may be operably linked to two different promoters such that expression of the two polypeptides is controlled by the different promoters. Alternatively, the coding sequences for the chimeric receptor polypeptide and the lactate regulatory factor may be operably linked to one promoter such that expression of the two polypeptides is controlled by a single promoter. Appropriate sequences may be inserted between the coding sequences for the two polypeptides so that two separate polypeptides can be translated from a single mRNA molecule. Such sequences, such as IRES or ribosome skip sites, are well known in the art. Additional description is provided below.

The nucleic acid and the one or more vectors may be contacted with the restriction enzyme under suitable conditions to produce complementary ends on each molecule that may be paired with each other and ligated to the ligase. Alternatively, a synthetic nucleic acid linker may be attached to the end of the nucleic acid encoding the lactate modulator and/or chimeric receptor polypeptide. The synthetic linker may contain nucleic acid sequences corresponding to specific restriction sites in the vector. The choice of expression vector/plasmid/viral vector will depend on the type of host cell used to express the lactate regulatory factor and/or chimeric receptor polypeptide, but should be suitable for integration and replication in eukaryotic cells.

Various promoters including, but not limited to, Cytomegalovirus (CMV) mid-early promoter, viral LTR (such as the Rous sarcoma virus LTR), HIV-LTR, HTLV-1LTR, simian virus 40(SV40) early promoter, human EF 1-alpha promoter, or herpes simplex tk virus promoter may be used to express the lactate regulatory factor and/or chimeric receptor polypeptides described herein. Additional promoters for expression of the lactate regulatory factor and/or chimeric receptor polypeptide include any constitutively active promoter in an immune cell. Alternatively, any regulatable promoter may be used, such that expression of the promoter may be regulated within the immune cell.

Furthermore, the vector may contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene or the kanamycin gene, for selecting stable or transient transfectants in a host cell; an enhancer/promoter sequence from the immediate early gene of human CMV that is used for high levels of transcription; an intron sequence from the human EF 1-a gene; transcriptional termination and RNA processing signals from SV40, which are used for mRNA stability; SV40 polyoma viral origin of replication and ColE1, SV40 polyoma viral origin of replication and ColE1 for proper episomal replication; internal ribosome binding sites (IRESes), multipurpose multiple cloning sites; the T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA; a "suicide switch" or "suicide gene" which, when triggered, results in the death of a cell carrying the vector (e.g., HSV thymidine kinase or an inducible caspase, such as iCasp 9); and a reporter gene for assessing expression of the lactate modulating polypeptide and/or the chimeric receptor polypeptide.

In a particular embodiment, such vectors further comprise a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes the death of a cell expressing the suicide gene. A suicide gene may be a gene that confers sensitivity to an agent (e.g., a drug) to a cell expressing the gene, and causes the cell to die when the cell is contacted with or exposed to the agent. Suicide genes are known in the art (see, e.g., suide Gene Therapy: Methods and Reviews, Springer, Caroline J. (Cancer Research UK Centre for Cancer Therapeutics at the Institute of Cancer Research, Sutton, Surrey, UK), Humana Press,2004) and include, e.g., the Herpes Simplex Virus (HSV) Thymidine Kinase (TK) Gene, cytosine deaminase, purine nucleoside phosphorylase, nitroreductase, and caspase (such as caspase 8).

Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art. Examples of the preparation of vectors for expressing lactate regulatory factor and/or chimeric receptor polypeptides can be found, for example, in US2014/0106449, which is incorporated herein by reference in its entirety.

Any of the vectors comprising a nucleic acid sequence encoding a lactate modulator and/or chimeric receptor polypeptide described herein are also within the scope of the present disclosure. Such vectors or sequences encoding lactate modulator and/or chimeric receptor polypeptides contained therein may be delivered to a host cell, such as a host immune cell, by any suitable method. Methods of delivering vectors to immune cells are well known in the art and may include DNA electroporation, RNA electroporation, transfection using agents such as liposomes, or viral transduction (e.g., retroviral transduction, such as lentiviral transduction).

In some embodiments, the vector for expressing the lactate modulator and/or chimeric receptor polypeptide is delivered to the host cell by viral transduction (e.g., retroviral transduction, such as lentiviral transduction or gamma-retroviral transduction). Exemplary viral methods of delivery include, but are not limited to, recombinant retroviruses (see, e.g., PCT publication Nos. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; and WO 91/02805; U.S. Pat. Nos. 5,219,740 and 4,777,127; GB patent No. 2,200,651 and EP patent No. 0345242), alphavirus-based vectors and adeno-associated virus (AAV) vectors (see, e.g., PCT publications Nos. WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984; and WO 95/00655). In some embodiments, the vector for expressing the lactate modulator and/or chimeric receptor polypeptide is a retrovirus. In some embodiments, the vector for expressing the lactate modulator and/or chimeric receptor polypeptide is a lentivirus.

Examples of references describing retroviral transduction include Anderson et al, U.S. patent nos. 5,399,346; mann et al, Cell 33:153 (1983); temin et al, U.S. patent No. 4,650,764; temin et al, U.S. patent No. 4,980,289; markowitz et al, J.Virol.62:1120 (1988); temin et al, U.S. patent No. 5,124,263; international patent publication No. WO 95/07358 to Dougherty et al, published 3, 16, 1995; and Kuo et al, Blood 82:845 (1993). International patent publication No. WO 95/07358 describes efficient transduction of primary B lymphocytes. See also WO 2016/040441a1, which is incorporated herein by reference for the purposes and subject matter cited herein.

In examples where a vector encoding a lactate modulator and/or chimeric receptor polypeptide is introduced into a host cell using a viral vector, viral particles capable of infecting immune cells and carrying the vector can be produced by any method known in the art, and can be found, for example, in PCT application nos. WO 1991/002805a2, WO 1998/009271 a1, and U.S. patent No. 6,194,191. The viral particles are harvested from the cell culture supernatant and may be isolated and/or purified prior to contacting the viral particles with immune cells.

In some embodiments, RNA molecules encoding any of the lactate regulatory factor and/or chimeric receptor polypeptides described herein can be prepared by conventional methods (e.g., in vitro transcription) and then introduced into suitable host cells, such as those described herein, by known methods, e.g., Rabinovich et al, Human Gene Therapy 17: 1027-1035.

In some cases, the nucleic acid encoding the lactate modulator and the nucleic acid encoding the appropriate chimeric receptor polypeptide may be cloned into separate expression vectors, which may be introduced into the appropriate host cell simultaneously or sequentially. For example, an expression vector (or RNA molecule) for expressing a lactate modulator can first be introduced into a host cell, and the transfected host cell expressing the lactate modulator can be isolated and cultured in vitro. An expression vector (or RNA molecule) for expression of the appropriate chimeric receptor polypeptide can then be introduced into host cells expressing the lactate regulatory factor, and transfected cells expressing both polypeptides can be isolated. In another example, expression vectors (or RNA molecules) each for expressing a lactate modulator and a chimeric receptor polypeptide can be introduced into a host cell simultaneously, and transfected host cells expressing both polypeptides can be isolated by conventional methods.

In other cases, the nucleic acid encoding the lactate modulator and the nucleic acid encoding the chimeric receptor polypeptide may be cloned into the same expression vector. Polynucleotides for expression of chimeric receptor polypeptides and lactate modulators (including vectors in which such polynucleotides are operably linked to at least one regulatory element) are also within the scope of the present disclosure. Non-limiting examples of useful vectors of the present disclosure include viral vectors, such as retroviral vectors, including gamma retroviral vectors and lentiviral vectors, and adeno-associated viral vectors (AAV vectors).

In some cases, one or more nucleic acids encoding a lactate modulator and/or chimeric receptor polypeptide can be delivered into a host cell via a transposon. In some cases, one or more encoding nucleic acids can be delivered into a host cell via gene editing, for example, by CRISPR, TALENs, Zinc Finger Nucleases (ZFNs), or meganucleases.

In some cases, a nucleic acid described herein can comprise two coding sequences, one coding sequence encoding a chimeric receptor polypeptide as described herein and the other coding sequence encoding a polypeptide capable of modulating (e.g., enhancing) intracellular lactate concentration (i.e., a lactate modulator). A nucleic acid comprising two coding sequences described herein can be configured such that the polypeptides encoded by the two coding sequences can be expressed as independent (and physically separate) polypeptides. To achieve this, the nucleic acids described herein can contain a third nucleotide sequence located between the first and second coding sequences. The third nucleotide sequence may, for example, encode a ribosome skip site. The ribosome skip site is a sequence that impairs normal peptide bond formation. This mechanism results in the translation of additional open reading frames from one messenger RNA. The third nucleotide sequence may, for example, encode a P2A, T2A, or F2A peptide (see, e.g., Kim et al, PLoS one.2011; 6(4): e 18556). As a non-limiting example, an exemplary P2A peptide may have an amino acid sequence of ATNFSLLKQAGDVEENPGP SEQ ID No.: 99.

In another embodiment, the third nucleotide sequence may encode an Internal Ribosome Entry Site (IRES). IRES are RNA elements that allow translation initiation in an end-independent manner and also allow translation of additional open reading frames from one messenger RNA. Alternatively, the third nucleotide sequence may encode a second promoter that controls expression of the second polypeptide. The third nucleotide sequence may also encode more than one ribosome skip sequence, IRES sequence, additional promoter sequence, or a combination thereof.

The nucleic acid may further comprise additional coding sequences (including but not limited to fourth and fifth coding sequences) and may be configured such that polypeptides encoded by the additional coding sequences are expressed as otherwise independent and physically separate polypeptides. For this purpose, additional coding sequences can be separated from other coding sequences by one or more nucleotide sequences encoding one or more ribosome skipping sequences, IRES sequences or additional promoter sequences.

In some examples, a nucleic acid (e.g., an expression vector or RNA molecule as described herein) can comprise coding sequences for both a lactate modulator (e.g., a lactate modulator as described herein) and a suitable chimeric receptor polypeptide, separated in any order by a third nucleotide sequence (e.g., ATNFSLLKQAGDVEENPGP; SEQ ID NO:99) encoding a P2A peptide. Thus, two separate polypeptides (lactate modulator and chimeric receptor) can be produced from such nucleic acids, with P2A portion ATNFSLLKQAGDVEENPG (SEQ ID NO:100) linked to the upstream polypeptide (encoded by the upstream coding sequence) and residue P from the P2A peptide linked to the downstream polypeptide (encoded by the downstream coding sequence). In some examples, the chimeric receptor polypeptide is an upstream polypeptide and the lactate modulator is a downstream polypeptide. In other examples, the lactate modulator is an upstream polypeptide and the chimeric receptor polypeptide is a downstream polypeptide.

In some examples, a nucleic acid (e.g., an expression vector or RNA molecule as described herein) can comprise coding sequences for both a lactate regulator (e.g., a lactate regulator as described herein) and a suitable ACTR polypeptide, separated in any order by a third nucleotide sequence (e.g., ATNFSLLKQAGDVEENPGP; SEQ ID NO:99) encoding a P2A peptide. Thus, two separate polypeptides (lactate regulator and ACTR) can be produced from such nucleic acids, with P2A part ATNFSLLKQAGDVEENPG (SEQ ID NO:100) linked to the upstream polypeptide (encoded by the upstream coding sequence) and residue P from the P2A peptide linked to the downstream polypeptide (encoded by the downstream coding sequence). In some examples, the ACTR polypeptide is an upstream polypeptide and the lactate modulator is a downstream polypeptide. In other examples, the lactate modulator is an upstream polypeptide and the ACTR polypeptide is a downstream polypeptide.

In some examples, the nucleic acid may also encode a linker (e.g., a GSG linker) between two segments of the encoded sequence, e.g., between the upstream polypeptide and the P2A peptide.

In particular examples, the nucleic acids described herein are configured such that the nucleic acids express two separate polypeptides in a host cell into which the nucleic acids are transfected: (i) a first polypeptide comprising, from N-terminus to C-terminus, a suitable CAR (e.g., SEQ ID NO:97 or SEQ ID NO:98), a peptide linker (e.g., a GSG linker), and a ATNFSLLKQAGDVEENPG (SEQ ID NO:100) segment derived from the P2A peptide; and (ii) a second polypeptide comprising, from N-terminus to C-terminus, a P residue derived from the P2A peptide and a lactate regulator (e.g., any one of SEQ ID NO:81 to SEQ ID NO: 87).

In particular examples, the nucleic acids described herein are configured such that the nucleic acids express two separate polypeptides in a host cell into which the nucleic acids are transfected: (i) a first polypeptide comprising, from N-terminus to C-terminus, a suitable ACTR (e.g., any one of SEQ ID NO:1 to SEQ ID NO:80, e.g., SEQ ID NO:1 or SEQ ID NO:57, as described herein), a peptide linker (e.g., a GSG linker), and a ATNFSLLKQAGDVEENPG (SEQ ID NO:100) segment derived from the P2A peptide; and (ii) a second polypeptide comprising, from N-terminus to C-terminus, a P residue derived from the P2A peptide and a lactate regulator (e.g., any one of SEQ ID NO:81 to SEQ ID NO: 87).

In some cases, additional polypeptides of interest may also be introduced into the host immune cell.

Following introduction of a vector encoding any of the lactate modulator and/or chimeric receptor polypeptides provided herein or a nucleic acid (e.g., an RNA molecule) encoding the chimeric receptor polypeptides and/or lactate modulator into a host cell, the cell can be cultured under conditions that allow expression of the lactate modulator and/or chimeric receptor polypeptides. In examples where the nucleic acid encoding the lactate regulator and/or chimeric receptor polypeptide is regulated by a regulatable promoter, the host cell can be cultured under conditions where the regulatable promoter is activated. In some embodiments, the promoter is an inducible promoter, and the immune cell is cultured in the presence of an inducing molecule or under conditions that produce the inducing molecule. Determining whether a lactate modulator and/or chimeric receptor polypeptide is expressed will be apparent to one skilled in the art and can be assessed by any known method, such as detecting mRNA encoding the lactate modulator and/or chimeric receptor polypeptide by quantitative reverse transcriptase PCR (qRT-PCR), or detecting lactate modulator and/or chimeric receptor polypeptide protein by methods including western blot, fluorescence microscopy, and flow cytometry.

Alternatively, expression of the chimeric receptor polypeptide can occur in vivo following administration of the immune cell to a subject. As used herein, the term "subject" refers to any mammal, such as a human, monkey, mouse, rabbit, or domestic mammal. For example, the subject may be a primate. In a preferred embodiment, the subject is a human.

Alternatively, expression of a lactate modulator and/or chimeric receptor polypeptide in any of the immune cells disclosed herein can be achieved by introducing an RNA molecule encoding the lactate modulator and/or chimeric receptor polypeptide. Such RNA molecules can be prepared by in vitro transcription or chemical synthesis. The RNA molecule can then be introduced into a suitable host cell, such as an immune cell (e.g., a T cell, an NK cell, or both a T cell and an NK cell), by, for example, electroporation. For example, RNA molecules can be synthesized and introduced into host immune cells according to the methods described by Rabinovich et al, Human Gene Therapy,17: 1027-.

In certain embodiments, one or more vectors or one or more RNA molecules comprising a lactate modulator and/or a chimeric receptor polypeptide can be introduced into a host cell or immune cell in vivo. This may be achieved, as non-limiting examples, by: a vector or RNA molecule encoding one or more lactate modulator and/or one or more chimeric receptor polypeptides described herein is administered directly (e.g., by intravenous administration) to a subject, thereby producing host cells comprising the lactate modulator and/or chimeric receptor polypeptides in vivo.

The methods for making a host cell expressing any of the lactate modulator and/or chimeric receptor polypeptides described herein can further comprise activating the host cell ex vivo. Activating a host cell refers to stimulating the host cell to an activated state in which the cell may be able to perform an effector function. The method of activating the host cell will depend on the type of host cell used to express the lactate modulator and/or chimeric receptor polypeptide. For example, T cells may be activated ex vivo in the presence of one or more molecules including, but not limited to: anti-CD 3 antibody, anti-CD 28 antibody, IL-2, phytohemagglutinin, engineered artificially stimulated cells or particles or combinations thereof. The engineered artificial stimulatory cell may be an artificial antigen presenting cell known in the art. See, e.g., Neal et al, j.immunological. res.ther.2017,2(1):68-79 and Turtle et al, Cancer j.2010,16(4): 374-.

In other examples, NK cells can be activated ex vivo in the presence of one or more molecules such as 4-1BB ligand, anti-4-1 BB antibody, IL-15, anti-IL-15 receptor antibody, IL-2, IL12, IL-21, K562 cells, and/or engineered artificially stimulated cells or particles. In some embodiments, a host cell (a cell expressing ACTR/CAR and/or lactate regulator) expressing any of the lactate regulator and/or chimeric receptor polypeptides described herein is activated ex vivo prior to administration to a subject. Determining whether a host cell is activated will be apparent to those skilled in the art and may include assessing the expression of one or more cell surface markers associated with cell activation, cytokine expression or secretion, and cell morphology.

Methods for making a host cell expressing any of the lactate modulator and/or chimeric receptor polypeptides described herein can include ex vivo expansion of the host cell. Expanding the host cell may involve any method that results in an increase in the number of cells expressing the lactate regulatory factor and/or chimeric receptor polypeptide, such as proliferating the host cell or stimulating the proliferation of the host cell. Methods of stimulating host cell expansion will depend on the type of host cell used to express the lactate modulator and/or chimeric receptor polypeptide, and will be apparent to one of ordinary skill in the art. In some embodiments, a host cell expressing any of the lactate modulator and/or chimeric receptor polypeptides described herein is expanded ex vivo prior to administration to a subject.

In some embodiments, the host cells expressing the lactate regulatory factor and/or chimeric receptor polypeptide are expanded and activated ex vivo prior to administration of the host cells to a subject. Activation and amplification of host cells can be used to allow integration of viral vectors into the genome and expression of genes encoding lactate regulatory factors and/or chimeric receptor polypeptides as described herein. Although electroporation may be more effective when performed on activated cells, if mRNA electroporation is used, activation and/or amplification may not be required. In some cases, the lactate modulator and/or chimeric receptor polypeptide is transiently expressed in a suitable host cell (e.g., for 3-5 days). Transient expression may be advantageous if there is potential toxicity, and should aid in the initial phase of clinical testing for possible side effects.

Any of the host cells expressing the lactate regulatory factor and/or chimeric receptor polypeptide may be mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition, which is also within the scope of the present disclosure.

The phrase "pharmaceutically acceptable" as used in connection with compositions of the present disclosure means that the molecular entities and other ingredients of such compositions are physiologically tolerable and do not typically produce adverse reactions when administered to a mammal (e.g., a human). Generally, as used herein, the term "pharmaceutically acceptable" means approved by a federal regulatory agency or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. By "acceptable" is meant that the carrier is compatible with the active ingredient (e.g., nucleic acid, vector, cell, or therapeutic antibody) of the composition and does not adversely affect the subject to which the composition is administered. Any of the pharmaceutical compositions used in the methods of the invention may comprise a pharmaceutically acceptable carrier, excipient or stabilizer, either in lyophilized form or in aqueous solution.

Pharmaceutically acceptable carriers, including buffers, are well known in the art and may include phosphates, citrates and other organic acids; antioxidants, including ascorbic acid and methionine; a preservative; a low molecular weight polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; an amino acid; a hydrophobic polymer; a monosaccharide; a disaccharide; and other carbohydrates; a metal complex; and/or a nonionic surfactant. See, e.g., Remington, The Science and Practice of Pharmacy, 20 th edition, (2000) Lippincott Williams and Wilkins, eds K.E.Hoover.

The pharmaceutical compositions of the present disclosure may also contain one or more additional active ingredients necessary for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other. Non-limiting examples of possible additional active compounds include, for example, IL-2, as well as various agents known in the art and listed in the discussion of combination therapies below.

IV.Immunotherapy using genetically engineered hematopoietic cells as described herein

Genetically engineered hematopoietic cells (e.g., hematopoietic stem cells, immune cells, such as NK cells or T cells) disclosed herein can be used in immunotherapy for various disorders, such as cancer, infectious diseases, and autoimmune diseases.

(a) Combination immunotherapy of genetically engineered hematopoietic cells expressing ACTR polypeptides with Fc-containing therapeutic agents

Exemplary ACTR polypeptides of the disclosure confer antibody-dependent cellular cytotoxicity (ADCC) ability to T lymphocytes and enhance ADCC in NK cells. When the receptor is bound by the cell-bound antibody, it triggers T cell activation, sustained proliferation, and specific cytotoxicity to the bound cell.

The degree of affinity of CD16 for the Fc portion of Ig is a key determinant of ADCC and, therefore, of clinical response to antibody immunotherapy. CD16 with the V158 polymorphism that has higher binding affinity for Ig and mediates excellent ADCC relative to CD16 with the F158 polymorphism was selected as an example. Although the F158 receptor is less potent than the V158 receptor in inducing T cell proliferation and ADCC, the in vivo toxicity of the F158 receptor may be lower than that of the V158 receptor, making it useful in some clinical settings.

Lactate modulators co-expressed with ACTR polypeptides in immune cells will facilitate cell-based immunotherapy, such as T cell therapy or NK cell therapy, by allowing cells to grow and/or function efficiently in low glucose, low amino acid, low pH, and/or hypoxic environments. Cytotoxicity against the antibody can be stopped by simply abolishing antibody administration, whenever desired. Clinical safety can be further enhanced by: mRNA electroporation is used to transiently express lactate modulating polypeptides and/or ACTR polypeptides in order to limit any potential autoimmune reactivity.

Accordingly, in one embodiment, the present disclosure provides a method for enhancing the efficacy of antibody-based immunotherapy for cancer in a subject in need of treatment, the subject being treated with an Fc-containing therapeutic agent (such as a therapeutic antibody) that can bind to antigen-expressing cells. Fc-containing therapeutics contain an Fc portion, such as a human or humanized Fc portion, that can be recognized and bound by the Fc binding portion of ACTR expressed on engineered immune cells (e.g., the extracellular domain of human CD 16A).

The methods described herein can include introducing into a subject a therapeutically effective amount of an antibody and a therapeutically effective amount of a genetically engineered host cell, such as a hematopoietic cell, e.g., an immune cell (e.g., a T lymphocyte or an NK cell), which co-expresses a lactate regulatory factor and an ACTR polypeptide of the present disclosure.

In the context of the present disclosure, the terms "treat/treat" and the like, as far as any disease condition described herein is concerned, mean to alleviate or alleviate at least one symptom associated with such condition or to slow or reverse the progression of such condition. Within the meaning of the present disclosure, the term "treating" also means inhibiting, delaying the onset of a disease (i.e., a period prior to the clinical manifestation of a disease) and/or reducing the risk of developing or worsening a disease. For example, the term "treating" in relation to cancer may mean eliminating or reducing the tumor burden of a patient, or preventing, delaying or inhibiting metastasis, or the like.

As used herein, the term "therapeutically effective" as applied to a dose or amount refers to an amount of a compound or pharmaceutical composition that is sufficient to result in a desired activity when administered to a subject in need thereof. It should be noted that when a combination of active ingredients is administered (e.g., a first pharmaceutical composition comprising an antibody, and a second pharmaceutical composition comprising a T lymphocyte or NK cell population that expresses a lactate regulatory factor and/or antibody-coupled T cell receptor (ACTR) construct), the effective amount of the combination may or may not include the amount of each ingredient that would be effective if administered alone. In the context of the present disclosure, the term "therapeutically effective" refers to an amount of a compound or pharmaceutical composition sufficient to delay the manifestation, prevent the progression, alleviate, or alleviate at least one symptom of a condition being treated by the methods of the present disclosure.

Host cells (e.g., hematopoietic cells, e.g., immune cells such as T cells and NK cells) expressing the lactate regulatory factor and ACTR polypeptides described herein can be used to enhance ADCC and/or to enhance the efficacy of antibody-based immunotherapy and/or to enhance the growth and/or proliferation of immune cells in a low glucose environment in a subject. In some embodiments, the subject is a mammal, such as a human, monkey, mouse, rabbit, or domestic mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a human cancer patient. In some embodiments, the subject has been or is being treated with any of the therapeutic antibodies described herein.

To practice the methods described herein, an effective amount of a host cell (e.g., an immune cell (e.g., an NK cell and/or a T lymphocyte)) that expresses any of the lactate regulatory factor and ACTR polypeptides described herein and an effective amount of an antibody or composition thereof can be administered to a subject in need of treatment by a suitable route, such as intravenous administration. As used herein, an effective amount refers to the amount of the corresponding agent (e.g., NK cells and/or T lymphocytes that express lactate modulators, ACTR polypeptides, antibodies, or a combination thereof) that, when administered, confers a therapeutic effect on the subject. It will be apparent to one skilled in the art to determine whether the amount of cells or compositions described herein achieves a therapeutic effect. As recognized by one skilled in the art, an effective amount varies depending on: the particular condition being treated; the severity of the condition; individual patient parameters including age, physical condition, size, gender (gender), gender (sex), and weight; the duration of treatment; the nature of concurrent therapy (if any); a particular route of administration; and similar factors within the knowledge and expertise of health practitioners. In some embodiments, the effective amount reduces, alleviates, ameliorates, reduces symptoms, or delays progression of any disease or disorder in the subject. In some embodiments, the subject is a human. In some embodiments, the subject in need of treatment is a human cancer patient. In some embodiments, a subject in need of treatment has one or more pathogenic infections (e.g., viral, bacterial, and/or fungal infections).

The methods of the present disclosure may be used to treat any cancer or any pathogen. Specific non-limiting examples of cancers that can be treated by the methods of the present disclosure include, for example, lymphoma, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, skin cancer, prostate cancer, colorectal cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, mesothelioma, pancreatic cancer, head and neck cancer, retinoblastoma, glioma, glioblastoma, thyroid cancer, hepatocellular carcinoma, esophageal cancer, and cervical cancer. In certain embodiments, the cancer may be a solid tumor.

The methods of the present disclosure may also be used to treat infectious diseases that may be caused by bacterial, viral, or fungal infections. In such cases, genetically engineered immune cells can be used with Fc-containing therapeutics (e.g., antibodies) that target pathogenic antigens (e.g., antigens associated with bacteria, viruses, or fungi that cause the infection). Specific non-limiting examples of pathogenic antigens include, but are not limited to, bacterial, viral, and/or fungal antigens. Some examples are provided below: influenza virus neuraminidase, hemagglutinin or M2 protein, human Respiratory Syncytial Virus (RSV) F glycoprotein or G glycoprotein, herpes simplex virus glycoprotein gB, gC, gD or gE, chlamydia MOMP or PorB protein, dengue virus core protein, matrix protein or glycoprotein E, measles virus hemagglutinin, herpes simplex virus type 2 glycoprotein gB, poliovirus I VP1, envelope glycoprotein of HIV 1, hepatitis b core antigen or surface antigen, diphtheria toxin, streptococcus 24M epitope, gonococcal pilin protein, pseudorabies virus G50(gpD), pseudorabies virus ii (gpb), pseudorabies virus iii (gpc), pseudorabies virus glycoprotein H, pseudorabies virus glycoprotein E, transmissible gastrointestinal glycoprotein 195, infectious inflammatory matrix protein or human hepatitis c virus glycoprotein E1 or E2.

In some embodiments, the immune cells are administered to the subject in an amount effective to enhance ADCC activity by at least 20% and/or at least 2-fold (e.g., enhance ADCC by 50%, 80%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more).

The immune cells are co-administered with an Fc-containing therapeutic agent (such as a therapeutic antibody) to target cells expressing an antigen to which the Fc-containing therapeutic agent binds. In some embodiments, more than one Fc-containing therapeutic agent, such as more than one antibody, may be used in conjunction with immune cells. Antibody-based immunotherapy can be used to treat, alleviate or reduce the symptoms of any disease or disorder in a subject for which the immunotherapy is believed to be useful.

Antibodies (used interchangeably in various forms) are immunoglobulin molecules that are capable of specifically binding to a target (such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses not only intact (i.e., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof comprising an Fc region, mutants thereof, fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, single domain antibodies (e.g., nanobodies), linear antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configuration of an immunoglobulin molecule comprising an antigen recognition site and an Fc region with the desired specificity, including glycosylated variants of an antibody, amino acid sequence variants of an antibody, and covalently modified antibodies. Antibodies include any class of antibody, such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof), and the antibody need not be of any particular class. Immunoglobulins can be assigned to different classes based on the antibody amino acid sequence of the constant domain of the heavy chain of the immunoglobulin. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Antibodies for use in the present disclosure contain an Fc region that can be recognized by co-used immune cells expressing ACTR and/or lactate modulators. The Fc region may be a human Fc region or a humanized Fc region.

Any of the antibodies described herein can be monoclonal or polyclonal. "monoclonal antibody" refers to a homogeneous antibody population, and "polyclonal antibody" refers to a heterogeneous antibody population. These two terms do not limit the source or manner of preparation of the antibody.

In one example, the antibody used in the methods described herein is a humanized antibody. Humanized antibodies refer to forms of non-human (e.g., murine) antibodies that are specifically chimeric immunoglobulins, immunoglobulin chains, or antigen-binding fragments thereof that contain minimal sequence derived from non-human immunoglobulins. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a Complementarity Determining Region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody), such as mouse, rat or rabbit, having the desired specificity, affinity and capacity. In some cases, Fv Framework Region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may comprise residues that are not found in either the recipient antibody or the imported CDR or framework sequences, but which are included to further refine and optimize antibody performance. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. The antibody may have a modified Fc region as described in WO 99/58572. The antibodies used herein can be glycosylated (e.g., fucosylated) or afucosylated. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, six) that have been altered relative to the original antibody, also referred to as one or more CDRs "derived" from one or more CDRs of the original antibody. Humanized antibodies may also be involved in affinity maturation.

In another example, an antibody described herein is a chimeric antibody that can include a heavy constant region and a light constant region from a human antibody. A chimeric antibody is an antibody having a variable region or a portion of a variable region from a first species and a constant region from a second species. Typically, in these chimeric antibodies, the variable regions of both the light and heavy chains mimic those of an antibody derived from one mammalian species (e.g., non-human mammals such as mice, rabbits, and rats), while the constant portions are homologous to sequences in an antibody derived from another mammal such as a human. In some embodiments, amino acid modifications may be made in the variable and/or constant regions.

Hematopoietic cells (e.g., immune cells (e.g., T lymphocytes and/or NK cells) or HSCs) expressing any of the lactate modulators and/or ACTR polypeptides disclosed herein can be administered to a subject who has been or is being treated with an Fc-containing antibody. For example, the immune cells can be administered to the human subject concurrently with the antibody. Alternatively, immune cells may be administered to a human subject during the course of antibody-based immunotherapy. In some examples, the immune cells and antibodies can be administered to the human subject at least 4 hours apart, e.g., at least 12 hours apart, at least 1 day apart, at least 3 days apart, at least one week apart, at least two weeks apart, or at least one month apart.

In some embodiments, an antibody described herein specifically binds to a corresponding target antigen or epitope thereof. Antibodies that "specifically bind" to an antigen or epitope are terms well known in the art. A molecule is said to exhibit "specific binding" if it reacts more frequently, more rapidly, for a longer duration, and/or with greater affinity with a particular target antigen than with an alternative target. An antibody "specifically binds" to a target antigen or epitope if it binds with greater affinity (affinity), avidity (avidity), more readily, and/or for a longer duration than it binds to other substances. For example, an antibody that specifically (or preferentially) binds to an antigen or epitope thereof is an antibody that binds to the target antigen with greater affinity, more readily, and/or for a longer duration than to other antigens or other epitopes in the same antigen. It is also understood using this definition that, for example, an antibody that specifically binds a first target antigen may or may not specifically bind or preferentially bind a second target antigen. Thus, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. In some examples, an antibody that "specifically binds" a target antigen or epitope thereof may not bind to other antigens or other epitopes in the same antigen.

In some embodiments, an antibody as described herein has a suitable binding affinity for a target antigen (e.g., any of the targets described herein) or an epitope thereof. Antibodies for use in the immunotherapeutic methods described herein can bind (e.g., specifically bind) a target antigen of interest, or a specific region or epitope therein. Table 3 above lists exemplary target antigens of interest and exemplary antibodies that bind to such exemplary target antigens of interest.

(b) Immunotherapy of genetically engineered hematopoietic cells expressing CAR polypeptidesMethod of

Genetically engineered hematopoietic cells (e.g., hematopoietic stem cells, immune cells, such as T cells or natural killer cells) co-expressing a lactate regulatory factor and a CAR polypeptide described herein can be used in immunotherapy, such as T cell therapy or NK cell therapy, to inhibit diseased cells expressing an antigen targeted by the CAR polypeptide, either directly or indirectly (e.g., via a therapeutic agent conjugated to a tag to which the CAR polypeptide is bound). Lactate modulators co-expressed with CAR polypeptides in immune cells will facilitate cell-based immunotherapy by allowing cells to effectively grow and/or function in low glucose, low amino acids, low pH, and/or hypoxic environments (e.g., in a tumor microenvironment). Clinical safety can be further enhanced by: mRNA electroporation is used to transiently express lactate modulator and/or CAR polypeptides to limit any potential non-tumor specific reactivity.

The subject (e.g., a human patient, such as a human cancer patient) may additionally have or be being treated with anti-cancer or anti-infection therapy, including but not limited to anti-cancer therapeutics or anti-infectives.

Host cells (e.g., hematopoietic cells, e.g., immune cells such as T cells and NK cells) expressing the lactate regulatory factor and CAR polypeptides described herein can be used to inhibit cells expressing the target antigen and/or to enhance growth and/or proliferation of immune cells in a low glucose environment, a low amino acid environment, a low pH environment, and/or a hypoxic environment (e.g., in a tumor microenvironment). In some embodiments, the subject is a mammal, such as a human, monkey, mouse, rabbit, or domestic mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a human cancer patient. In some embodiments, the subject has additionally been or is being treated with any of the therapeutic antibodies described herein.

To practice the methods described herein, an effective amount of hematopoietic cells (e.g., immune cells (NK cells and/or T lymphocytes)) or compositions thereof expressing any of the lactate regulatory factor and CAR polypeptides described herein can be administered to a subject in need of treatment by a suitable route, such as intravenous administration. As used herein, an effective amount refers to the amount of the corresponding agent (e.g., NK cells and/or T lymphocytes expressing a lactate modulator, CAR polypeptide, or a composition thereof) that, when administered, confers a therapeutic effect on the subject. It will be apparent to one skilled in the art to determine whether the amount of cells or compositions described herein achieves a therapeutic effect. As recognized by one skilled in the art, an effective amount varies depending on: the particular condition being treated; the severity of the condition; individual patient parameters including age, physical condition, size, gender (gender), gender (sex), and weight; the duration of treatment; the nature of concurrent therapy (if any); a particular route of administration; and similar factors within the knowledge and expertise of health practitioners. In some embodiments, the effective amount reduces, alleviates, ameliorates, reduces symptoms, or delays progression of any disease or disorder in the subject. In some embodiments, the subject is a human. In some embodiments, the subject in need of treatment is a human cancer patient. In some embodiments, a subject in need of treatment has one or more pathogenic infections (e.g., viral, bacterial, and/or fungal infections).

The methods of the present disclosure may be used to treat any cancer or any pathogen. Specific non-limiting examples of cancers that can be treated by the methods of the present disclosure include, for example, lymphoma, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, skin cancer, prostate cancer, colorectal cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, mesothelioma, pancreatic cancer, head and neck cancer, retinoblastoma, glioma, glioblastoma, thyroid cancer, hepatocellular carcinoma, esophageal cancer, and cervical cancer. In certain embodiments, the cancer may be a solid tumor.

The methods of the present disclosure may also be used to treat infectious diseases that may be caused by bacterial, viral, or fungal infections. In such cases, genetically engineered immune cells expressing CAR polypeptides specific for pathogenic antigens (e.g., antigens associated with bacteria, viruses, or fungi that cause the infection) can be used to eliminate infected cells. Specific non-limiting examples of pathogenic antigens include, but are not limited to, bacterial, viral, and/or fungal antigens.

In some embodiments, the immune cells are administered to the subject in an amount effective to inhibit cells expressing the target antigen by at least 20% and/or at least 2-fold (inhibit cells expressing the target antigen by 50%, 80%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more).

Additional therapeutic agents (e.g., antibody-based immunotherapeutics) can be used to treat, alleviate, or reduce the symptoms of any disease or disorder in a subject for which the therapeutic agent is believed to be useful.

The efficacy of cell-based immunotherapy as described herein can be assessed by any method known in the art and will be apparent to the skilled medical professional. For example, the efficacy of cell-based immunotherapy can be assessed by the survival rate of the subject or the tumor or cancer burden in the subject or a tissue or sample thereof. In some embodiments, the immune cells are administered to a subject in need of treatment in an amount effective to increase the efficacy of the cell-based immunotherapy by at least 20% and/or at least 2-fold (e.g., increase the efficacy of the antibody-based immunotherapy by 50%, 80%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more) compared to the efficacy in the absence of the immune cells expressing the lactate modulator and/or the CAR polypeptide.

In any of the compositions or methods described herein, the immune cells (e.g., NK and/or T cells) can be autologous to the subject, i.e., the immune cells can be obtained from a subject in need of treatment, genetically engineered for expression of a lactate regulatory factor and/or CAR polypeptide, and then administered to the same subject. In a particular embodiment, the autoimmune cells (e.g., T lymphocytes or NK cells) are activated and/or expanded ex vivo prior to reintroduction into the subject. Administration of autologous cells to a subject may result in reduced host cell rejection compared to administration of non-autologous cells.

Alternatively, the host cell is an allogeneic cell, i.e., the cell is obtained from a first subject, genetically engineered to express a lactate modulator and/or chimeric receptor polypeptide (e.g., an ACTR polypeptide or a CAR polypeptide), and administered to a second subject that is different from the first subject but of the same species. For example, the allogeneic immune cells may be derived from a human donor and administered to a human recipient that is different from the donor. In a particular embodiment, the T lymphocytes are allogeneic T lymphocytes in which expression of endogenous T cell receptors has been inhibited or depleted. In a particular embodiment, the allogeneic T lymphocytes are activated and/or expanded ex vivo prior to introduction into the subject. T lymphocytes can be activated by any method known in the art, for example in the presence of anti-CD 3/CD28, IL-2, phytohemagglutinin, engineered artificially stimulated cells or particles, or a combination thereof.

NK cells may be activated by any method known in the art, e.g. in the presence of one or more agents selected from the group consisting of: CD137 ligand protein, CD137 antibody, IL-15 protein, IL-15 receptor antibody, IL-2 protein, IL-12 protein, IL-21 protein and K562 cell line, and/or engineered artificially stimulated cells or particles. For a description of available methods for expanding NK cells, see, e.g., U.S. patent nos. 7,435,596 and 8,026,097. For example, NK cells for use in the compositions or methods of the present disclosure may be preferentially expanded by exposure to cells lacking or poorly expressing major histocompatibility complex I and/or II molecules and that have been genetically modified to express membrane-bound IL-15 and/or 4-1BB ligand (CDI 37L). Such cell lines include, but are not necessarily limited to, K562[ ATCC, CCL 243; lozzio et al, Blood 45(3):321-334 (1975); klein et al, int.J. cancer 18: 421-. Preferably, the cell lines used lack or poorly express both MHC I and MHC II molecules, such as K562 and HFWT cell lines. Solid phase carriers can be used instead of cell lines. Such a carrier should preferably have attached to its surface at least one molecule capable of binding to NK cells and inducing a primary activation event and/or a proliferative response or capable of binding to a molecule having such an effect so as to act as a scaffold. The carrier may have attached to its surface a CD137 ligand protein, a CD137 antibody, an IL-15 protein or an IL-15 receptor antibody. Preferably, the carrier will have bound to its surface both an IL-15 receptor antibody and a CD137 antibody.

In one embodiment of the described compositions or methods, T lymphocytes, NK cells, or T lymphocytes and NK cells are introduced (or reintroduced) into a subject, and a therapeutically effective amount of IL-2 is then administered to the subject.

According to the present disclosure, may be at about 10 by infusion⁵To 10¹⁰A therapeutically effective dose of immune cells (such as T lymphocytes or NK cells) comprising a CAR polypeptide of the present disclosure, in the range of one or more cells per kilogram body weight (cells/Kg) to treat the patient. The infusion may be repeated as often and as many times as the patient can tolerate until the desired response is achieved. The appropriate infusion dose and schedule will vary from patient to patient, but may be determined by the treating physician for a particular patient. Typically, about 10 infusions will be made⁶Initial dose of individual cells/Kg, escalating to 10⁸One or more cells/Kg. IL-2 may be co-administered to expand the infused cells. The amount of IL-2 may be about 1-5X 10⁶International units per square meter body surface.

The term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limits of the measurement system. For example, "about" may mean within an acceptable standard deviation, according to practice in the art. Alternatively, "about" may represent a range of up to ± 20%, preferably up to ± 10%, more preferably up to ± 5%, and more preferably still up to ± 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within one order of magnitude, preferably within 2-fold of the value. Where a particular value is described in the application and claims, unless otherwise stated, the term "about" is implied and in this context is meant to be within an acceptable error range for the particular value.

The efficacy of the compositions or methods described herein can be assessed by any method known in the art and will be apparent to the skilled medical professional. For example, the efficacy of a composition or method described herein can be assessed by the survival rate of the subject or the cancer or pathogen burden in the subject or a tissue or sample thereof. In some embodiments, the compositions and methods described herein can be based on the safety or toxicity of the therapy (e.g., administration of immune cells expressing a lactate modulator and/or a CAR polypeptide) in the subject, e.g., assessed by the overall health status of the subject and/or the presence of an adverse event or severe adverse event.

(c) Other immunotherapy

In some embodiments, genetically engineered immune cells expressing one or more of the lactate modulators (e.g., LDHA or MCT, such as MCT1, MCT2, or MCT4) can be derived from natural immune cells specific for a diseased cell (e.g., a cancer cell or a pathogen-infected cell). Such genetically engineered immune cells (e.g., tumor infiltrating lymphocytes or TILs) may not co-express any chimeric receptor polypeptides and may be used to destroy target disease cells, such as cancer cells. Genetically engineered TILs expressing one or more lactate modulators without expressing a chimeric receptor may be used with bispecific antibodies capable of binding to target tumor cells and TIL (bite).

In some embodiments, the genetically engineered immune cell expressing one or more lactate modulators (e.g., LDHA or MCT, such as MCT1, MCT2, or MCT4) can be T_regA cell. Such a T_regThe cells may co-express a chimeric receptor polypeptide as disclosed herein. Alternatively, T_regThe cells may not co-express any chimeric receptor polypeptide and may be used for the intended therapy.

V.Combination therapy

The compositions and methods described in this disclosure may be utilized in conjunction with other types of cancer therapies, such as chemotherapy, surgery, radiation, gene therapy, and the like, or anti-infection therapies. Such therapies may be administered simultaneously or sequentially (in any order) with an immunotherapy according to the present disclosure. When co-administered with additional therapeutic agents, the appropriate therapeutically effective dose of each agent may be reduced due to additive or synergistic effects.

In some cases, immune cells (e.g., T lymphocytes and/or NK cells) expressing any of the lactate modulator and/or chimeric receptor polypeptides disclosed herein can be administered to a subject who has been or is being treated with an additional therapeutic agent (e.g., an additional anti-cancer therapeutic agent). For example, the immune cells can be administered to the human subject concurrently with the additional therapeutic agent. Alternatively, the immune cells may be administered to the human subject prior to the addition of the therapeutic agent. Alternatively, the immune cells can be administered to the human subject after the addition of the therapeutic agent.

Genetically engineered immune cells (e.g., T cells or NK cells) that co-express a lactate regulatory factor and a CAR polypeptide specific for a tag can be co-used with a therapeutic agent conjugated to the tag. Such genetically engineered immune cells can bind to and inhibit the growth of diseased cells, such as tumor cells, by a therapeutic agent that is capable of binding to an antigen associated with the diseased cells. Any of the antibodies listed in table 1 above, or other antibodies specific for the same target antigen also listed in table 1, can be conjugated to a suitable tag (e.g., those described herein) and used in conjunction with immune cells that co-express a lactate modulator and a CAR polypeptide specific for the tag.

The treatment of the present disclosure may be combined with other immunomodulatory treatments, such as therapeutic vaccines (including but not limited to GVAX, DC-based vaccines, etc.), checkpoint inhibitors (including but not limited to agents that block CTLA4, PD1, LAG3, TIM3, etc.), or activators (including but not limited to agents that enhance 41BB, OX40, etc.).

Non-limiting examples of other therapeutic agents for use in combination with the immunotherapies of the present disclosure include (I) anti-angiogenic agents (e.g., TNP-470, platelet factor 4, thrombospondin-1, tissue inhibitors of metalloproteinases (TIMP1 and TIMP2), prolactin (16-Kd fragment), angiostatin (38-Kd fragment of plasminogen), endostatin, bFGF soluble receptor, transforming growth factor beta, interferon alpha, soluble KDR and FLT-1 receptor, placental proliferative protein-related proteins, and those listed by Carmeliet and Jain (2000)); (ii) a VEGF antagonist or VEGF receptor antagonist, such as an anti-VEGF antibody, a VEGF variant, a soluble VEGF receptor fragment, an aptamer capable of blocking VEGF or VEGFR, a neutralizing anti-VEGFR antibody, an inhibitor of VEGFR tyrosine kinase, and any combination thereof; and (iii) chemotherapeutic compounds such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine), purine analogs, folic acid antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine (cladribine)); antiproliferative/antimitotic agents, including natural products, such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disrupting agents such as taxanes (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones, and navelbine (navelbine), epipodophyllotoxins (etoposide and teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide (cyclophosphamide), dactinomycin (dactinomycin), daunorubicin, doxorubicin, epirubicin, hexamethamine, oxaliplatin, ifosfamide, melphalan, mechlorethamine (merchrehthahtamine), mitomycin, mitoxantrone, nitrosoureas, plicamycin, procarbazine, paclitaxel, taxotere, teniposide, triethylenethiophosphoramide, and etoposide (16); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin), and mitomycin; enzymes (l-asparaginase, which systemically metabolizes l-asparagine and deprives cells of the ability to synthesize self-asparagine); anti-platelet agents; antiproliferative/antimitotic alkylating agents such as nitrogen mustard (mechlorethamine), cyclophosphamide (cyclophosphamide) and the like, melphalan, chlorambucil, ethylenimine and methyl melamine (hexamethyl pyrimethanil and thiotepa), alkyl sulfonates busulfan, nitrosoureas (carmustine (BCNU) and the like, streptozotocin), triazene-Dacarbazine (DTIC); antiproliferative/antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other thrombin inhibitors); fibrinolytic agents (such as tissue plasminogen activator, streptokinase, and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; anti-migratory agents (antimigrating agents); antisecretory agents (brefeldin); immunosuppressants (cyclosporin, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic compounds (e.g., TNP-470, genistein (genistein), bevacizumab) and growth factor inhibitors (e.g., Fibroblast Growth Factor (FGF) inhibitors); an angiotensin receptor blocker; a nitric oxide donor; an antisense oligonucleotide; antibodies (trastuzumab); cell cycle inhibitors and differentiation inducers (tretinoin); AKT inhibitors (such as MK-22062 HCl, piperacillin (KRX-0401), GSK690693, Iptasertib (GDC-0068), AZD5363, eposertib (uprosetib), afluoroteib (afuresertib) or triciribine); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, geniposide (eniposide), epirubicin, etoposide, idarubicin, mitoxantrone, topotecan, and irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prednisolone); growth factor signal transduction kinase inhibitors; mitochondrial dysfunction inducers and caspase activators; and a chromatin disrupting agent.

See also the physicians' Desk Reference, 59 th edition, (2005), Thomson P D R, Montvale N.J.; gennaro et al, Remington's The Science and Practice of Pharmacy, 20 th edition, (2000), by Lippincott Williams and Wilkins, Baltimore; braunwald et al, Harrison's Principles of Internal Medicine, 15 th edition, (2001), McGraw Hill, NY; berkow et al, The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway N.J.

Administration of the additional therapeutic agent may be performed by any suitable route, including systemic administration as well as direct administration to the site of disease (e.g., to a tumor).

In some embodiments, the method involves administering to the subject an additional therapeutic agent (e.g., an antibody) at one dose. In some embodiments, the method involves administering to the subject additional therapeutic agents (e.g., antibodies) in multiple doses (e.g., at least 2, 3, 4, 5, 6, 7, or 8 doses). In some embodiments, the additional therapeutic agent (e.g., antibody) is administered to the subject in multiple doses, wherein a first dose of the additional therapeutic agent (e.g., antibody) is administered to the subject about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days prior to administration of the immune cells expressing the lactate modulator and/or the CAR polypeptide. In some embodiments, the first dose of the additional therapeutic agent (e.g., antibody) is administered to the subject between about 24-48 hours prior to administration of the immune cells expressing the lactate modulator and/or CAR polypeptide. In some cases, the additional therapeutic agent can be an antibody specific for a target antigen of interest, such as those listed in table 1, as well as other antibodies specific for the same target antigen.

In some embodiments, an additional therapeutic agent (e.g., an antibody) is administered to the subject prior to administration of the immune cells expressing the lactate modulator and/or CAR polypeptide, and then subsequently administered to the subject about once every two weeks. In some embodiments, the first two doses of the additional therapeutic agent (e.g., antibody) are administered about one week apart (e.g., about 6 days, 7 days, 8 days, or 9 days). In certain embodiments, the third and subsequent doses are administered about once every two weeks.

In any of the embodiments described herein, the timing of the administration of the additional therapeutic agent (e.g., antibody) is approximate and includes three days before and three days after the indicated date (e.g., once every three weeks includes administration on day 18, day 19, day 20, day 21, day 22, day 23, or day 24).

The efficacy of the compositions or methods described herein can be assessed by any method known in the art and will be apparent to the skilled medical professional and/or are those described herein. For example, the efficacy of antibody-based immunotherapy can be assessed by the survival rate of the subject or the cancer burden in the subject or a tissue or sample thereof. In some embodiments, antibody-based immunotherapy is based on the safety or toxicity of the therapy in a subject, e.g., assessed by the subject's overall health status and/or the presence of an adverse event or severe adverse event.

VI.Kit for therapeutic use

The present disclosure also provides kits for use of the compositions described herein. For example, the present disclosure also provides kits comprising a population of immune cells (e.g., T lymphocytes or NK cells, constructed in vitro or in vivo) that express a lactate modulator and optionally a chimeric receptor polypeptide for inhibiting the growth of diseased cells (e.g., tumor cells) and/or enhancing the growth and/or proliferation of immune cells in a low glucose environment, a low amino acid environment, a low pH environment, and/or a hypoxic environment (e.g., in a tumor microenvironment). The kit may further comprise a therapeutic agent or a therapeutic agent conjugated to a tag (e.g., those described herein) to which the chimeric receptor polypeptide expressed on the immune cells binds. Such kits can include one or more containers comprising a population of genetically engineered immune cells (e.g., T lymphocytes and/or NK cells) as described herein that co-express a lactate modulator and a chimeric receptor polypeptide (such as those described herein); and optionally a therapeutic agent or a therapeutic agent conjugated to a label.

In some embodiments, the kits described herein comprise immune cells that express a lactate modulator and that express a chimeric receptor expanded in vitro; and antibodies specific for cell surface antibodies present on activated T cells, such as anti-CD 5 antibodies, anti-CD 38 antibodies, or anti-CD 7 antibodies. The immune cells expressing the lactate modulator and expressing the chimeric receptor polypeptide can express any of the chimeric receptor polypeptide constructs known in the art or disclosed herein.

Alternatively, a kit disclosed herein can comprise a nucleic acid or set of nucleic acids described herein that collectively encode any chimeric receptor polypeptide and any lactate modulator also described herein.

In some embodiments, the kit can further comprise instructions for use in any of the methods described herein. Included instructions for use can include a description of administering the first pharmaceutical composition and the second pharmaceutical composition to a subject to achieve a desired activity, e.g., inhibiting growth of a target cell in the subject and/or enhancing growth and/or proliferation of an immune cell in a low glucose environment, a low amino acid environment (e.g., a low glutamine environment), a low pH environment, and/or a hypoxic environment (e.g., a low glucose, low amino acid, low pH, and/or hypoxic tumor microenvironment). The kit can further include a description of selecting a subject suitable for treatment based on identifying whether the subject is in need of treatment. In some embodiments, the instructions for use include a description of administration of a population of genetically engineered immune cells and optionally a description of administration of a tag-conjugated therapeutic agent.

As described herein, instructions for use in connection with the use of immune cells and optionally label-conjugated therapeutic agents generally include information regarding the dosage, dosing regimen, and route of administration of the intended treatment. The container may be a unit dose. Bulk packaging (e.g., multi-dose packaging) or sub-unit dosage. The instructions for use provided in the kits of the present disclosure are typically written instructions for use on a label or package insert. The label or package insert indicates that the pharmaceutical composition is for treating, delaying the onset of, and/or alleviating a disease or condition in a subject.

The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Packaging for use in conjunction with a particular device, such as an inhaler, nasal administration device, or infusion device, is also contemplated. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle). The vessel may also have a sterile access port. At least one active agent in the second pharmaceutical composition is an antibody as described herein. The at least one active agent in the first pharmaceutical composition is a population of immune cells (e.g., T lymphocytes or NK cells) that express a chimeric receptor polypeptide and/or a lactate modulating polypeptide as described herein.

The kit may optionally provide additional components, such as buffers and explanatory information. Typically, a kit includes a container and a label or package insert on or associated with the container. In some embodiments, the present disclosure provides articles of manufacture comprising the above-described kit contents.

General techniques

The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are well explained in the literature, such as Molecular Cloning, A Laboratory Manual, second edition (Sambrook et al, 1989) Cold Spring Harbor Press; oligonucleotide Synthesis (m.j. gait, 1984); methods in Molecular Biology, human Press; cell Biology A Laboratory Notebook (J.E.Cellis, 1989) Academic Press; animal Cell Culture (r.i. freshney, 1987); introduction to Cell and Tissue Culture (J.P.Mather and P.E.Roberts,1998) Plenum Press; cell and Tissue Culture Laboratory Procedures (A.Doyle, J.B.Griffiths and D.G.Newell, 1993-8) J.Wiley and Sons; methods in Enzymology (Academic Press, Inc.); handbook of Experimental Immunology (D.M.Weir and C.C.Blackwell) Gene Transfer Vectors for Mammarian Cells (J.M.Miller and M.P.Calos eds., 1987); current Protocols in Molecular Biology (F.M. Ausubel et al, 1987); PCR The Polymerase Chain Reaction, (Mullis et al, 1994); current Protocols in Immunology (J.E.Coligan et al, 1991); short Protocols in Molecular Biology (Wiley and Sons, 1999); immunobiology (c.a. janeway and p.travers, 1997); antibodies (p.finch, 1997); antibodies a practical proproach (D.Catty. eds., IRL Press, 1988-; monoclonal antigens a practical proproach (P. shepherd and C. dean, Oxford University Press, 2000); useful Antibodies: a Laboratory manual (E.Harlow and D.Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M.Zantetti and J.D.Capra, eds., Harwood Academic Publishers, 1995); DNA Cloning: A practical application, volumes I and II (D.N.Glover, eds., 1985); Nucleic Acid Hybridization (B.D.Hames & S.J.Higgins, eds. (1985); transformation and transformation (B.D.Hames & S.J.Higgins, eds.) (1984; Animal Cell (R.I.shock.; Cell 6; filtration, RL.1986; Cell et al, (1984; catalog et al; filtration, 1986; Cell et al; catalog et al; 1986; plant, Inc.; filtration, 1984; plant, et al; plant, Inc. (1986; plant, 1986).

Without further elaboration, it is believed that one skilled in the art can, based on the description above, utilize the present disclosure to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter of citation herein.

Examples

Example 1: effect of reduced glucose concentration on T cell function

An exemplary GPC 3-targeted CAR expression construct gamma retrovirus encoding SEQ ID NO:97 was generated by recombinant techniques and used to infect primary human T cells to produce cells expressing a GPC 3-targeted CAR polypeptide on their cell surface. GPC 3-targeted CAR-expressing cells were then tested for functionality using a six day flow-based proliferation assay. Specifically, 200,000 untransduced mock T cells or T cells expressing a CAR construct targeting GPC3 were incubated with 50,000 GPC3+ hepatocellular carcinoma JHH7 tumor cells or Hep3B tumor cells at a ratio of 4:1 (effector/CAR expressing T cells to target cells). The CO-cultures were incubated at 37 ℃ in the presence of different concentrations of glucose at 5% CO₂Incubate in incubator for six days. At the end of six days, co-cultures were harvested and stained with anti-CD 3 antibody. The number of CD3 positive cells was assessed by flow cytometry as a measure of T cell proliferation. At lower glucose concentrations, less CAR-T proliferation was observed (figure 2). These experiments demonstrate that a low glucose environment can have a negative impact on CAR-T cell proliferative activity.

Example 2: effect of expression of lactate regulatory factor on T cell function Using CAR-T expression constructs targeting GPC3

Gamma retroviruses encoding the exemplary GPC 3-targeted CAR polypeptide expression construct (SEQ ID NO:97) were generated by recombinant techniques and used to infect primary human T cells to produce cells expressing a GPC 3-targeted CAR polypeptide on their cell surface. In addition, the encoded exemplary GPC 3-targeted CAR polypeptide (SEQ ID NO:97 or 98) and lactate transport polypeptide (MCT1, MCT2 or MCT4) (SEQ ID NO:82 through SEQ ID N4) were generated by recombinant techniquesO:84) and used to infect primary human T cells to produce cells expressing a polypeptide targeted to GPC3 and a lactate regulatory factor (e.g., a polypeptide). In constructs encoding both the CAR polypeptide and the lactate regulatory factor, the two polypeptides are separated by a P2A ribosome skip sequence. The expressed variants are combinations of CAR with lactate modulators as disclosed herein, such as CAR + MCT1(SEQ ID NO:98 and SEQ ID NO:82), CAR + MCT2(SEQ ID NO:97 and SEQ ID NO:83), and CAR + MCT4(SEQ ID NO:98 and SEQ ID NO: 84). GPC 3-targeted CAR-expressing cells were then tested for functionality using a six day flow-based proliferation assay. Specifically, 200,000 untransduced mock T cells, T cells expressing a CAR polypeptide targeting GPC3, or T cells expressing a CAR polypeptide targeting GPC3 and a lactate modulator were incubated with 50,000 GPC3+ hepatocellular carcinoma JHH7 tumor cells at a ratio of 4:1 (effector cells/CAR expressing T cells to target cells). Co-cultures were incubated at 37 ℃ in the presence of 1.25mM glucose (associated with tumors) and 10mM glucose (at approximately peripheral blood levels) at 5% CO₂And (4) incubating for six days. At the end of six days, co-cultures were harvested and stained with anti-CD 3 antibody. The number of CD3 positive cells was assessed by flow cytometry as a measure of T cell proliferation. T cells expressing lactate regulatory factor in addition to the CAR polypeptide showed enhanced T cell proliferation relative to T cells expressing CAR construct alone (fig. 3-5). This enhanced proliferation also occurs at low glucose concentrations associated with tumors. These experiments demonstrate that expression of lactate regulatory factor in T cells has a positive effect on CAR-T cell proliferative activity.

Example 3: effect of expressing a combination of LDHA and ACTR Polypeptides on T cell function

T cells were transduced with viruses encoding the ACTR polypeptide (SEQ ID NO:57) and LDHA (SEQ ID NO:81), separated by the P2A ribosome skip sequence. At 37 ℃ in 5% CO₂In the incubator, T cells were cultured at an E: T ratio of 4:1 in RPMI 1640 medium supplemented with 10% fetal bovine serum, together with FOLR α -expressing IGROV-1 cells and 0-20 μ g/mL titrated anti-FOLR α antibody. After about 48 hours, a sample of the supernatant was taken for cytokineAnd (6) analyzing. Supernatants were analyzed for IL-2 using a homogeneous time-resolved fluorescence (HTRF) assay (Cisbio) according to the manufacturer's protocol. And analyzed using an EnVision multi-label plate reader (Perkin Elmer) to detect fluorescence. IL-2 production was normalized based on T cells of ACTR alone versus transduction efficiency of cells co-expressing ACTR and LDHA. After 8 days, cultures were harvested, stained with live/dead markers and anti-CD 3 antibody, and analyzed by flow cytometry. The number of live CD3 positive cells was used to measure T cell proliferation.

Normalized IL-2 production (fig. 6A) and T cell proliferation (fig. 6B) were plotted as a function of anti-folra antibody concentration. These results indicate that T cells co-expressing ACTR and LDHA enhance T cell function relative to T cells expressing ACTR alone, as measured by IL-2 release or T cell proliferation in the presence of target cells and cognate targeting antibodies.

Example 4: t cells co-expressing ACTR and LDHA showed enhanced proliferation under restricted glucose conditions

T cells were transduced with viruses encoding the ACTR polypeptide (SEQ ID NO:57) and LDHA (SEQ ID NO:81), separated by the P2A ribosome skip sequence. At 37 ℃ in 5% CO₂In the incubator, T cells were cultured at an E: T ratio of 4:1 with IGROV-1 cells expressing folra and 5 μ g/mL anti-folra antibody in glucose-free RPMI 1640 medium supplemented with 10% fetal bovine serum and 0-20mM glucose. After 8 days, cultures were harvested, stained with live/dead markers and anti-CD 3 antibody, and analyzed by flow cytometry. The number of live CD3 positive cells was used to measure T cell proliferation.

T cell proliferation was plotted as a function of glucose concentration (fig. 7). These results indicate that T cells co-expressing ACTR and LDHA enhanced T cell function relative to T cells expressing ACTR alone under restricted glucose conditions, as measured by T cell proliferation in the presence of target cells and cognate targeting antibodies.

Example 5: solid tumor-associated inhibitor PGE₂Shows enhanced function in T cells co-expressing ACTR and LDHA in the presence of

By codingViruses of ACTR polypeptide (SEQ ID NO:57) and LDHA (SEQ ID NO:81), separated by a P2A ribosome skip sequence, transduce T cells. At 37 ℃ in 5% CO₂In an incubator, T cells were mixed with FOLRa-expressing IGROV-1 cells, 5. mu.g/mL anti-FOLRa antibody and 0-16. mu.M PGE at an E: T ratio of 4:1₂Cultured together in RPMI 1640 medium supplemented with 10% fetal bovine serum.

After approximately 48 hours, a sample of the supernatant was removed for cytokine analysis. The supernatants were analyzed for IL-2 using a homogeneous time-resolved fluorescence (HTRF) assay (Cisbio) and analyzed using an EnVision Multi-Label microplate reader (Perkin Elmer) to detect fluorescence according to the manufacturer's protocol. IL-2 production was normalized based on T cells of ACTR alone versus transduction efficiency of cells co-expressing ACTR and LDHA. After 8 days, cultures were harvested, stained with live/dead markers and anti-CD 3 antibody, and analyzed by flow cytometry. The number of live CD3 positive cells was used to measure T cell proliferation.

Normalized IL-2 production (FIG. 8A) or T cell proliferation (FIG. 8B) was plotted as PGE₂As a function of concentration. These results indicate that when exposed to PGE₂(an inhibitor well-established in the solid tumor microenvironment), T cells co-expressing ACTR and LDHA enhance T cell function relative to T cells expressing ACTR alone, as measured by IL-2 release or T cell proliferation in the presence of target cells and cognate targeting antibodies.

Example 6: t cells co-expressing ACTR and LDHA in the presence of the solid tumor-associated inhibitor kynurenine show enhanced IL-2 production

T cells were transduced with viruses encoding the ACTR polypeptide (SEQ ID NO:57) and LDHA (SEQ ID NO:81), separated by the P2A ribosome skip sequence. At 37 ℃ in 5% CO₂In the incubator, T cells were cultured at an E: T ratio of 4:1 with FOLR α -expressing IGROV-1 cells, 5 μ g/mL of anti-FOLR α antibody, and 0-1000 μ M of kynurenine in RPMI 1640 medium supplemented with 10% fetal bovine serum. After approximately 48 hours, a sample of the supernatant was removed for cytokine analysis. According to the manufacturer's protocol, homogeneous time-resolved fluorescence (HTRF) was used) The supernatant was analyzed for IL-2 by assay (Cisbio) and analyzed using an EnVision Multi-Label microplate reader (Perkin Elmer) to detect fluorescence. IL-2 production was normalized based on T cells of ACTR alone versus transduction efficiency of cells co-expressing ACTR and LDHA.

Normalized IL-2 production (FIG. 9) was plotted as a function of kynurenine concentration. These results indicate that T cells co-expressing ACTR and LDHA enhance T cell function when exposed to kynurenine, a well-established inhibitor in the solid tumor microenvironment, relative to T cells expressing ACTR alone, as measured by IL-2 release in the presence of target cells and cognate targeting antibodies.

Example 7: effect of expressing MCT1 in combination with ACTR Polypeptides on T cell function

T cells were transduced with viruses encoding the ACTR polypeptide (SEQ ID NO:57) and MCT1(SEQ ID NO:82), separated by the P2A ribosomal skip sequence from MCT 1. At 37 ℃ in 5% CO₂In the incubator, T cells were cultured at an E: T ratio of 4:1 with fixed OVCAR8 cells expressing folra and 0-30 μ g/mL titrated anti-folra antibody in RPMI 1640 medium supplemented with 10% fetal bovine serum. After 8 days, the cultures were harvested and assayed for ATP content, which is a measure of viable cells, using the ATPlite 1-step luminometric system (Perkin Elmer). ATPlite luminescence signals used as a measure of T cell proliferation were analyzed using an EnVision multi-label plate reader (Perkin Elmer) to detect luminescence according to the manufacturer's instructions.

T cell proliferation (fig. 10) was plotted as a function of anti-FOLR α antibody concentration. These results indicate that T cells co-expressing ACTR and MCT1 enhanced T cell function relative to T cells expressing ACTR alone, as measured by T cell proliferation in the presence of target cells and cognate targeting antibodies.

Example 8: t cells co-expressing ACTR and MCT1 in the presence of the solid tumor-associated inhibitor kynurenine showed enhanced function

T cells were transduced with viruses encoding the ACTR polypeptide (SEQ ID NO:57) and MCT1(SEQ ID NO:82), separated by the P2A ribosomal skip sequence from MCT 1. At 37 deg.CAt 5% CO₂In the incubator, T cells were cultured at an E: T ratio of 4:1 with fixed IGROV-1 cells expressing FOLR α,1 μ g/mL anti-FOLR α antibody, and 0-1000 μ M kynurenine in RPMI 1640 medium supplemented with 10% fetal bovine serum.

After approximately 48 hours, a sample of the supernatant was removed for cytokine analysis. The supernatants were analyzed for IL-2 using a homogeneous time-resolved fluorescence (HTRF) assay (Cisbio) and analyzed using an EnVision Multi-Label microplate reader (Perkin Elmer) to detect fluorescence according to the manufacturer's protocol. IL-2 production was normalized based on the transduction efficiency of ACTR-only T cells versus T cells co-expressing ACTR and MCT 1.

After 7 days, half of the cells were transferred to a new plate for cell proliferation ELISA (Millipore Sigma) and pulsed with BrdU at 37 ℃ in 5% CO₂Incubate in the incubator for about 16 hours and analyze BrdU uptake using EnVision plate reader (Perkin Elmer) to detect chemiluminescence according to the manufacturer's instructions.

After 8 days, the remaining half of the cells were harvested and assayed for ATP content, which is a measure of viable cells, using an ATPlite 1-step luminometric system (Perkin Elmer). ATPlite luminescence signals used as a measure of T cell proliferation were analyzed using an EnVision multi-label plate reader (Perkin Elmer) to detect luminescence according to the manufacturer's instructions.

Normalized IL-2 production (FIG. 11A) or T cell proliferation as measured by BrdU uptake (FIG. 11B) or ATPLite (FIG. 11C) was plotted as a function of kynurenine concentration. These results indicate that T cells co-expressing ACTR and MCT1 enhanced T cell function when exposed to kynurenine, a well-established inhibitor in the solid tumor microenvironment, relative to T cells expressing ACTR alone, as measured by IL-2 release or T cell proliferation in the presence of target cells and cognate targeting antibodies.

Example 9: solid tumor-associated inhibitor PGE₂T cells co-expressing ACTR and MCT2 in the presence of TGF-beta or kynurenine show enhanced proliferation

With viruses encoding ACTR polypeptide (SEQ ID NO:57) and MCT2(SEQ ID NO:83)T cells were transduced and the ACTR polypeptide and MCT2 were separated by a P2A ribosomal skip sequence. At 37 ℃ in 5% CO₂In the incubator, T cells were cultured at an E: T ratio of 4:1 with fixed OVCAR8 cells expressing folra and 1 μ g/mL of anti-folra antibody in RPMI 1640 medium supplemented with 10% fetal bovine serum. The following tumor-associated inhibitors were added individually to the same T cell cultures: 0-16. mu.M PGE₂0-10ng/ml TGF-beta or 0-1000 to 30. mu.M kynurenine. After 7 days, cells were harvested and ATP content, which is a measure of viable cells, was determined using the ATPlite 1-step luminometric system (Perkin Elmer). ATPlite luminescence signals used as a measure of T cell proliferation were analyzed using an EnVision multi-label plate reader (Perkin Elmer) to detect luminescence according to the manufacturer's instructions.

Mapping T cell proliferation as measured by ATP content into PGE₂(FIG. 12A), TGF-. beta. (FIG. 12B), and kynurenine (FIG. 12C). These results indicate that the PGE, a well-established inhibitor in the solid tumor microenvironment, when exposed to₂TGF- β or kynurenine, T cells co-expressing ACTR and MCT2 enhanced T cell function relative to T cells expressing ACTR alone, as measured by T cell proliferation in the presence of target cells and cognate targeting antibodies.

Example 10: t cells co-expressing ACTR and MCT2 in the presence of the solid tumor-associated inhibitor kynurenine showed enhanced function

T cells were transduced with viruses encoding the ACTR polypeptide (SEQ ID NO:57) and MCT2(SEQ ID NO:83), separated by the P2A ribosomal skip sequence from MCT 2. At 37 ℃ in 5% CO₂In the incubator, T cells were cultured at an E: T ratio of 4:1 with immobilized IGROV-1 cells expressing FOLR α,1 μ g/mL anti-FOLR α antibody, and 0-1000 μ M kynurenine or kynurenine-free in RPMI 1640 medium supplemented with 10% fetal bovine serum.

After approximately 48 hours, a sample of the supernatant was removed for cytokine analysis. The supernatants were analyzed for IL-2 using a homogeneous time-resolved fluorescence (HTRF) assay (Cisbio) and analyzed using an EnVision Multi-Label microplate reader (Perkin Elmer) to detect fluorescence according to the manufacturer's protocol. IL-2 production was normalized based on T cells of ACTR alone versus transduction efficiency of cells co-expressing ACTR and MCT 2.

After 6 days, half of the cells were transferred to a new plate for cell proliferation ELISA (Millipore Sigma) and pulsed with BrdU at 37 ℃ in 5% CO₂Incubate in the incubator for about 16 hours and analyze BrdU uptake using EnVision plate reader (Perkin Elmer) to detect chemiluminescence according to the manufacturer's instructions.

After 7 days, the remaining half of the cells were harvested and assayed for ATP content, which is a measure of viable cells, using the ATPlite 1-step luminometric system (Perkin Elmer). ATPlite luminescence signals used as a measure of T cell proliferation were analyzed using an EnVision multi-label plate reader (Perkin Elmer) to detect luminescence according to the manufacturer's instructions.

Normalized IL-2 production (FIG. 13A) and T cell proliferation (FIG. 13B) and ATPLite (FIG. 13C) as measured by BrdU uptake were plotted as a function of kynurenine concentration. These results indicate that T cells co-expressing ACTR and MCT2 enhanced T cell function when exposed to kynurenine, a well-established inhibitor in the solid tumor microenvironment, relative to T cells expressing ACTR alone, as measured by IL-2 release or T cell proliferation in the presence of target cells and cognate targeting antibodies.

Example 11: t cells co-expressing ACTR and MCT2 in the presence of the solid tumor-associated inhibitor adenosine showed enhanced IL-2 production

T cells were transduced with viruses encoding the ACTR polypeptide (SEQ ID NO:57) and MCT2(SEQ ID NO:83), separated by the P2A ribosomal skip sequence from MCT 2. At 37 ℃ in 5% CO₂In the incubator, T cells were cultured at an E: T ratio of 4:1 with FOLR α expressing live or fixed IGROV-1 cells, 1 μ g/mL of anti-FOLR α antibody, and 0-2000 μ M adenosine in RPMI 1640 medium supplemented with 10% fetal bovine serum. After approximately 48 hours, a sample of the supernatant was removed for cytokine analysis. Supernatants were analyzed for IL-2 using a homogeneous time-resolved fluorescence (HTRF) assay (Cisbio) and EnVision Multitag according to the manufacturer's protocolThe assay was performed with a microplate reader (Perkin Elmer) to detect fluorescence. IL-2 production was normalized based on T cells of ACTR alone versus transduction efficiency of cells co-expressing ACTR and MCT 2.

Normalized IL-2 production at live (FIG. 14A) and fixed (FIG. 14B) IGROV-1 targets was plotted as a function of adenosine concentration. These results indicate that T cells co-expressing ACTR and MCT2 enhanced T cell function when exposed to adenosine, a well-established inhibitor in the solid tumor microenvironment, relative to T cells expressing ACTR alone, as measured by IL-2 release in the presence of target cells and cognate targeting antibodies.

Example 12: effect of expressing MCT4 in combination with ACTR Polypeptides on T cell function

T cells were transduced with viruses encoding the ACTR polypeptide (SEQ ID NO:57) and MCT4(SEQ ID NO:84), which are separated from MCT4 by the P2A ribosomal skip sequence. At 37 ℃ in 5% CO₂In the incubator, T cells were cultured at an E: T ratio of 4:1 with fixed OVCAR8 cells expressing folra and 0-30 μ g/mL anti-folra antibody in RPMI 1640 medium supplemented with 10% fetal bovine serum. Cultures were harvested after 8 days and ATP content, which is a measure of viable cells, was determined using the ATPlite 1-step luminometric system (Perkin Elmer). ATPlite luminescence signals used as a measure of T cell proliferation were analyzed using an EnVision multi-label plate reader (Perkin Elmer) to detect luminescence according to the manufacturer's instructions.

T cell proliferation (fig. 15) was plotted as a function of anti-FOLR α antibody concentration. These results indicate that T cells co-expressing ACTR and MCT4 enhanced T cell function relative to T cells expressing ACTR alone, as measured by T cell proliferation in the presence of target cells and cognate targeting antibodies.

Example 13: solid tumor-associated inhibitor PGE₂Shows enhanced IL-2 production by T cells co-expressing ACTR and MCT4 in the presence of

T cells were transduced with viruses encoding the ACTR polypeptide (SEQ ID NO:57) and MCT4(SEQ ID NO:84), which are separated from MCT4 by the P2A ribosomal skip sequence. At 37 ℃ in 5% CO₂Culture boxIn (1), T cells are mixed with FOLRa-expressing IGROV-1 cells, 5. mu.g/mL anti-FOLRa antibody and 0-16. mu.M PGE at an E: T ratio of 2:1₂Cultured together in RPMI 1640 medium supplemented with 10% fetal bovine serum. After approximately 48 hours, a sample of the supernatant was removed for cytokine analysis. The supernatants were analyzed for IL-2 using a homogeneous time-resolved fluorescence (HTRF) assay (Cisbio) and analyzed using an EnVision Multi-Label microplate reader (Perkin Elmer) to detect fluorescence according to the manufacturer's protocol. IL-2 production was normalized based on T cells of ACTR alone versus transduction efficiency of cells co-expressing ACTR and MCT 4.

Normalized IL-2 production (FIG. 16) is plotted as PGE₂As a function of concentration. These results indicate that when exposed to PGE₂(an inhibitor well-established in the solid tumor microenvironment), T cells co-expressing ACTR and MCT4 enhanced T cell function relative to T cells expressing ACTR alone, as measured by IL-2 release in the presence of target cells and cognate targeting antibodies.

Example 14: t cells co-expressing ACTR and MCT4 in the presence of the solid tumor-associated inhibitor TGF-beta show enhanced proliferation

T cells were transduced with viruses encoding the ACTR polypeptide (SEQ ID NO:57) and MCT4(SEQ ID NO:84), which are separated from MCT4 by the P2A ribosomal skip sequence. At 37 ℃ in 5% CO₂In the incubator, T cells were cultured at an E: T ratio of 4:1 with FOLR α -expressing fixed OVCAR8 cells, 1 μ g/mL anti-FOLR α antibody, and 0-10ng/mL TGF- β in RPMI 1640 medium supplemented with 10% fetal bovine serum. Cells were harvested after 8 days and ATP content, which is a measure of viable cells, was determined using the ATPlite 1-step luminometric system (Perkin Elmer). ATPlite luminescence signals used as a measure of T cell proliferation were analyzed using an EnVision multi-label plate reader (Perkin Elmer) to detect luminescence according to the manufacturer's instructions.

T cell proliferation as measured by ATP content was plotted as a function of TGF- β concentration (figure 17). These results indicate that T cells co-expressing ACTR and MCT4 enhanced T cell function relative to T cells expressing ACTR alone when exposed to TGF- β, a well-established inhibitor in the solid tumor microenvironment.

Example 15: t cells co-expressing ACTR and MCT4 in the presence of the solid tumor-associated inhibitor kynurenine showed enhanced function

T cells were transduced with viruses encoding the ACTR polypeptide (SEQ ID NO:57) and MCT4(SEQ ID NO:84), which are separated from MCT4 by the P2A ribosomal skip sequence. At 37 ℃ in 5% CO₂In the incubator, T cells were cultured at an E: T ratio of 4:1 with fixed IGROV-1 cells expressing FOLR α,1 μ g/mL anti-FOLR α antibody, and 0-1000 μ M kynurenine in RPMI 1640 medium supplemented with 10% fetal bovine serum.

After approximately 48 hours, a sample of the supernatant was removed for cytokine analysis. The supernatants were analyzed for IL-2 using a homogeneous time-resolved fluorescence (HTRF) assay (Cisbio) and analyzed using an EnVision Multi-Label microplate reader (Perkin Elmer) to detect fluorescence according to the manufacturer's protocol. IL-2 production was normalized based on T cells of ACTR alone versus transduction efficiency of cells co-expressing ACTR and MCT 4.

After 7 days, half of the cells were transferred to a new plate for cell proliferation ELISA (Millipore Sigma) and pulsed with BrdU at 37 ℃ in 5% CO₂Incubate in the incubator for about 16 hours and analyze BrdU uptake using EnVision plate reader (Perkin Elmer) to detect chemiluminescence according to the manufacturer's instructions.

Normalized IL-2 production (FIG. 18A) and T cell proliferation as measured by BrdU uptake (FIG. 18B) were plotted as a function of kynurenine concentration. These results indicate that T cells co-expressing ACTR and MCT4 enhanced T cell function when exposed to kynurenine, a well-established inhibitor in the solid tumor microenvironment, relative to T cells expressing ACTR alone, as measured by IL-2 release or T cell proliferation in the presence of target cells and cognate targeting antibodies.

Example 16: effect of expression of lactate modulating polypeptides on T cell function on tumor models.

Transgenesis of a lactate modulating polypeptide with a chimeric receptor polypeptide (e.g., an ACTR polypeptide (e.g., ACTR polypeptide) in the same T cellSEQ ID NO:1 to SEQ ID NO:80) or a CAR polypeptide (e.g., SEQ ID NO:97 to SEQ ID NO: 98)). The transgene is, for example, LDHA, MCT1, MCT2, MCT4, or PDK1 (e.g., SEQ ID NO:81 through SEQ ID NO: 85). T cells are transduced with a virus encoding the chimeric receptor polypeptide and the lactate modulating polypeptide, which are separated, for example, by a P2A ribosome skipping sequence. The anti-tumor activity of the transduced T cells was evaluated in a mouse tumor model. For these experiments, a tumor cell line (e.g., IGROV-1) was seeded onto NSG^TM(NOD scidγ，NOD.Cg-Prkdc^scidIL2rg^tm1Wjl/SzJ, strain 005557) mice. The tumor-bearing mice are then dosed with T cells expressing the chimeric receptor polypeptide alone or the chimeric receptor polypeptide and the lactate modulating polypeptide. When the chimeric receptor polypeptide is an ACTR construct, an antibody that targets the tumor is administered.

Tumor growth was monitored throughout the experiment. T cells expressing a lactate modulating polypeptide in addition to the chimeric receptor polypeptide (and optionally an anti-tumor antibody when the chimeric receptor polypeptide is an ACTR construct) are expected to exhibit enhanced anti-tumor activity relative to T cells expressing the chimeric receptor polypeptide alone, e.g., enhanced proliferation, enhanced T cell persistence, and/or enhanced cytokine production relative to T cells expressing the chimeric receptor polypeptide alone. Furthermore, T cells expressing a combination of a lactate modulating polypeptide and a chimeric receptor polypeptide are also expected to exhibit enhanced anti-cancer activity, such as a reduction in tumor growth and/or tumor formation, as compared to T cells expressing the chimeric receptor polypeptide alone.

In summary, the experiments disclosed in this study are intended to demonstrate that expression of exogenous lactate modulating polypeptides in T cells, including those that co-express chimeric receptor polypeptides (e.g., CARs or ACTRs), such as those disclosed herein, will have a positive impact on T cell function and therefore anti-tumor effects in vivo.

Other embodiments

All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Accordingly, other embodiments are within the claims.

Equivalent scheme

Although several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, embodiments of the invention may be practiced otherwise than as specifically described and claimed. Embodiments of the invention disclosed herein relate to each individual feature, system, article, material, kit and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present disclosure.

All definitions, as defined and used herein, should be understood to govern with respect to dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

All references, patents, and patent applications disclosed herein are incorporated by reference herein with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

As used herein in the specification and in the claims, the indefinite articles "a" and "an" should be understood to mean "at least one" unless explicitly indicated to the contrary.

The phrase "and/or" as used in the specification and claims should be understood to mean "one or two" of the elements so combined, i.e., elements that are present in combination in some cases and elements that are present in isolation in other cases. Multiple elements listed with "and/or" should be construed in the same manner, i.e., "one or more" of the elements so combined. In addition to the elements specifically identified by the "and/or" clause, other elements may optionally be present, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, when used in conjunction with open language such as "including," reference to "a and/or B" may refer, in one embodiment, to a alone (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than a); in yet another embodiment, refers to both a and B (optionally including other elements); and so on.

As used in the specification and claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" and/or "should be interpreted as being inclusive, i.e., including at least one but also including more than one quantity or element of the list, and optionally, additional unlisted items. It is only expressly intended that the opposite term, such as "only one" or "exactly one," or, when used in the claims in conjunction with the recitation of "comprising … …, is intended to mean that there is exactly one of the element in the plurality or series of elements. In general, the term "or" as used herein should only be interpreted as referring to an exclusive substitution (i.e., "one or the other, but not both") when preceded by an exclusive term (e.g., "any," "one," "only one," or "exactly one" of "consisting essentially of … …). "consisting essentially of" when used in the claims shall have its ordinary meaning as used in the art of patent law.

As used herein in the specification and claims, the phrase "at least one," when referring to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each element specifically listed in the list of elements, and not excluding any combinations of elements in the list of elements. The definition also allows that elements may optionally be present other than the elements specifically identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of a and B" or, equivalently, "at least one of a or B" or, equivalently "at least one of a and/or B" may refer, in one embodiment, to at least one, optionally including more than one, a, with no B present (and optionally including elements other than B); in another embodiment, refers to at least one, optionally including more than one, B, absent a (and optionally including elements other than a); in yet another embodiment, at least one, optionally including more than one a and at least one, optionally including more than one B (and optionally including other elements); and so on.

It will also be understood that, in any method claimed herein that includes more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are recited, unless clearly indicated to the contrary.