Compositions and methods for inhibiting T cell depletion
阅读说明：本技术 抑制t细胞衰竭的组合物和方法 (Compositions and methods for inhibiting T cell depletion ) 是由 克里斯托·麦考尔 瑞秋·琳恩 埃文·温伯 埃琳娜·索蒂罗 于 2018-12-14 设计创作，主要内容包括：本发明涉及T细胞组合物及其在疗法(therapy)和治疗(treatment)情形下的使用方法。特别地,本发明提供了经修饰的T细胞(例如,遗传和/或功能修饰)以在未经修饰T细胞呈现衰竭的条件下维持功能。本申请公开的组合物和方法可用于预防改造的(例如,嵌合抗原受体(CAR)T细胞)以及未改造的T细胞的衰竭,从而增强T细胞的功能(例如,抗癌症或抗传染病的活性)。(The present invention relates to T cell compositions and methods of use thereof in the context of therapy (therapy) and treatment (treatment). In particular, the invention provides modified T cells (e.g., genetically and/or functionally modified) to maintain function under conditions in which unmodified T cells exhibit depletion. The compositions and methods disclosed herein can be used to prevent the depletion of engineered (e.g., Chimeric Antigen Receptor (CAR) T cells) as well as unmodified T cells, thereby enhancing the function of the T cells (e.g., anti-cancer or anti-infectious disease activity).)
1. A composition comprising an isolated T cell modified to overexpress and/or contain elevated levels of one or more AP-1 transcription factors.
2. The composition of claim 1, wherein the isolated T cell is further modified to express a recombinant receptor.
3. The composition of claim 2, wherein the recombinant receptor is a T Cell Receptor (TCR).
4. The composition of claim 2, wherein the recombinant receptor is a Chimeric Antigen Receptor (CAR).
5. The composition of claim 2, wherein the recombinant receptor is specific for a tumor antigen.
6. The composition of claim 5, wherein the tumor antigen is selected from the group consisting of: CD19, CD20, CD22, ROR1, GD2, EBV proteins or antigens, folate receptor, mesothelin, human carcinoembryonic antigen, CD33/IL3Ra, c-Met, PSMA, glycolipids F77, EGFRvIII, NY-ESO-1, MAGE-A3, MART-1, GP1000 and p 53.
7. The composition of claim 1, wherein the isolated T cell is a natural naturally occurring T cell.
8. The composition of claim 7, wherein the native naturally occurring T cells are obtained from a resected tumor.
9. The composition of claim 7, wherein the natural naturally occurring T cells are obtained by leukapheresis of a blood sample.
10. The composition of claim 9, wherein the T cells are expanded ex vivo.
11. The composition of claim 1, wherein the T cell is selected from the group consisting of: CD3+ T cells, CD8+ T cells, CD4+ T cells, Natural Killer (NK) T cells, gamma T cells, combinations of CD4+ and CD8T + cells, memory T cells, cytokine-induced killer cells, and combinations thereof.
12. The composition of claim 1, wherein the one or more AP-1 transcription factors are selected from the group consisting of: c-Fos, c-Jun, Activated Transcription Factor (ATF), and Jun Dimerization Protein (JDP).
13. The composition of claim 1, wherein the AP-1 transcription factor is c-Jun.
14. The composition of claim 1, wherein the AP-1 transcription factor is a mutated/truncated AP-1 transcription factor.
15. The composition of claim 1, wherein the mutated/truncated AP-1 transcription factor comprises an N-terminal deletion.
16. The composition of claim 1, wherein the mutated/truncated AP-1 transcription factor (i) lacks a transactivation domain or (ii) comprises an inactive transactivation domain.
17. The composition of claim 1, wherein the isolated T cells co-express c-Jun and an engineered receptor.
18. The composition of claim 17, wherein c-Jun and the engineered receptor are expressed from respective expression vector constructs.
19. The composition of claim 17, wherein c-Jun and the engineered receptor are co-expressed from a single expression vector construct.
20. The composition of claim 1, wherein the isolated T cell is a T cell specific and active for a tumor.
21. The composition of claim 20, wherein the T cells are peripheral blood-derived T cells that are genetically modified to express a receptor that recognizes and responds to a tumor.
22. The composition of claim 1, wherein the T cell is further modified to reduce and/or eliminate the expression and/or activity of one or more members of the AP-1 inhibitory complex.
23. The composition of claim 22, wherein the AP-1 inhibitory complex member is selected from the group consisting of: JunB, a BATF family member, IRF4, an ATF family member, or a combination thereof.
24. The composition of claim 22, wherein the member of the AP-1 inhibitory complex is JunB and/or BATF 3.
25. A method of treating a disease or pathological condition in a subject, the method comprising administering to the subject having the disease or pathological condition an effective amount of a composition comprising a T cell modified to express and/or contain elevated levels of one or more AP-1 transcription factors.
26. The method of claim 25, wherein the AP-1 transcription factor is c-Jun.
27. The method of claim 26, wherein the AP-1 transcription factor is a mutated/truncated transcription factor.
28. The method of claim 26, wherein the T cell is further modified to express a recombinant receptor.
29. The method of claim 28, wherein the first and second portions are selected from the group consisting of,
wherein c-Jun and the recombinant receptor are expressed from respective expression vector constructs, or
Wherein c-Jun and the recombinant receptor are co-expressed from a single expression vector construct.
30. The method of claim 25, wherein the T cells modified to express and/or contain elevated levels of one or more AP-1 transcription factors are less susceptible to T cell failure.
31. The method of claim 25, wherein the T cell modified to express and/or contain elevated levels of one or more AP-1 transcription factors has increased reactivity to low antigen density on target cells of the T cell.
32. The method of claim 25, wherein the T cells modified to express and/or contain elevated levels of one or more AP-1 transcription factors are less likely to experience a decrease in memory formation.
33. The method of claim 25, wherein the T cells modified to express and/or contain elevated levels of one or more AP-1 transcription factors are less susceptible to experiencing a decrease in proliferative capacity.
34. The method of claim 25, wherein the disease or disorder is a tumor or cancer.
35. The method of claim 34, wherein the T cell is modified to express a recombinant receptor specific for the tumor or cancer.
36. The method of claim 35, wherein the recombinant receptor is a Chimeric Antigen Receptor (CAR).
37. The method of claim 36, wherein the CAR is specific for a tumor antigen selected from the group consisting of: CD19, CD20, CD22, ROR1, GD2, EBV proteins or antigens, folate receptor, mesothelin, human carcinoembryonic antigen, CD33/IL3Ra, c-Met, PSMA, glycolipids F77, EGFRvIII, NY-ESO-1, MAGE-A3, MART-1, GP1000 and p 53.
38. The method of claim 25, wherein the disease or disorder is a viral, bacterial, and/or parasitic infection.
39. The method of claim 25, wherein the T cell is further modified to reduce and/or eliminate the expression and/or activity of one or more members of the AP-1 inhibitory complex.
40. The method of claim 39, wherein the AP-1 inhibitory complex member is selected from the group consisting of: JunB, a BATF family member, IRF4, an ATF family member, or a combination thereof.
41. The method of claim 40, wherein said AP-1 inhibitory complex member is JunB and/or BATF 3.
42. A method of treating or delaying progression of cancer in a patient, the method comprising administering to the patient a therapeutically effective amount of a composition comprising T cells modified to express and/or contain elevated levels of one or more AP-1 transcription factors and to express a Chimeric Antigen Receptor (CAR) specific for a tumor antigen.
43. The method of claim 42, wherein the administering reduces the number of cancer cells in the patient.
44. The method of claim 42, wherein the administration reduces and/or eliminates tumor burden in the patient.
45. The method of claim 42, further comprising administering to the patient one or more anti-cancer agents and/or one or more chemotherapeutic agents.
46. The method of claim 42, wherein said administering occurs before, concurrently with, and/or after said patient receives radiation therapy.
47. The method of claim 42, wherein the T cells modified to express and/or contain elevated levels of one or more AP-1 transcription factors and modified to express a Chimeric Antigen Receptor (CAR) specific for a tumor antigen are less susceptible to T cell failure.
48. The method of claim 42, wherein the T cell modified to express and/or contain elevated levels of one or more AP-1 transcription factors and modified to express a Chimeric Antigen Receptor (CAR) specific for a tumor antigen has increased reactivity to low antigen density on target cells of the T cell.
49. The method of claim 42, wherein the T cells modified to express and/or contain elevated levels of one or more AP-1 transcription factors and modified to express a Chimeric Antigen Receptor (CAR) specific for a tumor antigen are less susceptible to experiencing a reduction in memory formation.
50. The method of claim 42, wherein the T cells modified to express and/or contain elevated levels of one or more AP-1 transcription factors and modified to express a Chimeric Antigen Receptor (CAR) specific for a tumor antigen are less susceptible to experiencing a decrease in proliferative capacity.
51. Use of a therapeutically effective amount of a composition comprising a T cell modified to express and/or contain elevated levels of one or more AP-1 transcription factors and modified to express a Chimeric Antigen Receptor (CAR) specific for a tumor antigen for treating or delaying the progression of cancer in a subject.
52. The composition for use according to claim 51, further comprising one or more anti-cancer agents and/or one or more chemotherapeutic agents.
53. The composition for use according to claim 51, wherein the composition reduces the number of cancer cells in a patient and reduces or eliminates tumor burden in a patient.
54. The method of claim 51, wherein the T cells modified to express and/or contain elevated levels of one or more AP-1 transcription factors and modified to express a Chimeric Antigen Receptor (CAR) specific for a tumor antigen are less susceptible to T cell failure.
55. The method of claim 51, wherein the T cell modified to express and/or contain elevated levels of one or more AP-1 transcription factors and modified to express a Chimeric Antigen Receptor (CAR) specific for a tumor antigen has increased reactivity to low antigen density on target cells of the T cell.
56. The method of claim 51, wherein the T cells modified to express and/or contain elevated levels of one or more AP-1 transcription factors and to express a Chimeric Antigen Receptor (CAR) specific for a tumor antigen are less susceptible to undergoing a reduction in memory formation.
57. The method of claim 51, wherein the T cells modified to express and/or contain elevated levels of one or more AP-1 transcription factors and modified to express a Chimeric Antigen Receptor (CAR) specific for a tumor antigen are less susceptible to experiencing a decrease in proliferative capacity.
58. A method of treating a disease or disorder in a patient, the method comprising administering to a patient having the disease or disorder an effective amount of a composition comprising a T cell modified to reduce and/or eliminate the expression and/or activity of one or more members of an AP-1 inhibitory complex.
59. The method of claim 58, wherein the disease or disorder is cancer.
60. The method of claim 59, wherein the AP-1 inhibitory complex member is selected from the group consisting of: JunB, BATF3, BATF family member, IRF4, IRF family member, ATF family member, or a combination thereof.
61. The method of claim 58, wherein the AP-1 inhibitory complex member is JunB, BATF3, and/or IRF 4.
62. The method of claim 58, wherein the T cell is modified by CRISPR-Cas9, shRNA, siRNA, RNAi, microRNA, degron, regulatable promoter, or pharmacological inhibition.
63. A composition comprising an isolated T cell modified to reduce and/or eliminate the expression and/or activity of one or more members of an AP-1 inhibitory complex.
64. The composition of claim 63, wherein the AP-1 inhibitory complex member is selected from the group consisting of: JunB, BATF3, BATF family member, IRF4, IRF family member, ATF family member, or a combination thereof.
65. The composition of claim 63, wherein the AP-1 inhibitory complex member is JunB, BATF3, and/or IRF 4.
66. The composition of claim 1, wherein the isolated T cells modified to overexpress and/or contain elevated levels of one or more AP-1 transcription factors are less susceptible to T cell failure.
67. The composition of claim 1, wherein the isolated T cell modified to overexpress and/or contain elevated levels of one or more AP-1 transcription factors has increased reactivity to low antigen density on a target cell of the T cell.
68. The composition of claim 1, wherein the isolated T cells modified to overexpress and/or contain elevated levels of one or more AP-1 transcription factors are less susceptible to a reduction in memory formation.
69. The composition of claim 1, wherein the isolated T cells modified to overexpress and/or contain elevated levels of one or more AP-1 transcription factors are less susceptible to experiencing a decrease in proliferative capacity.
Technical Field
The present invention relates to T cell compositions and methods of use thereof in therapy and treatment settings. In particular, the invention provides modified T cells (e.g., genetically and/or functionally modified) to maintain function under conditions in which unmodified T cells exhibit depletion. The compositions and methods disclosed herein can be used to prevent the depletion of engineered (e.g., Chimeric Antigen Receptor (CAR) T cells) as well as unmodified T cells, thereby enhancing the function of the T cells (e.g., anti-cancer or anti-infectious disease activity). The compositions and methods of the invention are useful in clinical and research fields, such as biology, immunology, medicine and oncology.
Background
T cells are immune cells that are activated by antigen following mobilization via T Cell Receptor (TCR) signaling and co-stimulation. Physiological activation through T cell receptors enables T cells to mediate potent anti-tumor and/or anti-infectious effects. During the resolution of the acute inflammatory response, activated effector T cell subsets differentiate into long-lived memory cells. In contrast, in patients with chronic infections or cancer, pathological differentiation of T cells towards a dysfunctional state often occurs, which is referred to as T cell failure. T cell failure is characterized by a significant change in metabolic function, transcriptional programs, loss of effector functions (e.g., cytokine secretion, killing ability), and co-expression of multiple surface inhibitory receptors. The underlying cause of T cell depletion is that continued antigen exposure results in continued TCR signaling. It has long been sought to prevent or reverse T cell failure as a means of enhancing T cell efficacy (e.g., in patients with cancer or chronic infection).
Chimeric Antigen Receptor (CAR) T cells showed impressive response rates in B cell malignancies, but only long-term disease control in about 50% of B-ALL1 and large B cell lymphoma patients (
Disclosure of Invention
The present invention relates to compositions and methods for preventing the depletion of engineered (e.g., T cells engineered to express synthetic receptors such as engineered T cell receptors or Chimeric Antigen Receptors (CARs)) and unmodified (e.g., naive) T cells. Modified T cells according to the invention (e.g., to prevent T cell failure), compositions comprising the modified T cells, and methods of using the modified T cells to enhance the functionality of T cells (e.g., activity against cancer or infectious disease).
CAR T cells mediate anti-tumor effects in a small class of cancer patients, but dysfunction due to T cell failure greatly impedes its development. To investigate the biological properties of CAR receptor-expressing human T cell failure, during the development of embodiments of the present application, experiments were performed using a model system that employs a complimentary signaling CAR that induces the failure characteristics described in other contexts. The results indicate that failure is associated with a severe defect in IL-2 production with increased accessibility of the AP-1 transcription factor motif to chromatin, and with overexpression of numerous bZIP and IRF transcription factors involved in inhibitory activity. Engineering CAR T cells to overexpress AP-1 factors (e.g., c-Jun) enhances expansion potential, enhances functionality, reduces terminal differentiation and improves antitumor ability in many in vivo tumor models.
Experiments conducted during the development of the embodiments of the present application also demonstrated that functional defects in AP-1 factor (e.g., c-Jun) mediate dysfunction of failing human T cells, and that engineering CAR T cells to overexpress AP-1 factor (e.g., c-Jun) can make them resistant to failure, thus clearing major obstacles to the progress of this emerging therapy approach.
Experiments performed during the development of embodiments of the present application also demonstrated that knockdown of IRF4 significantly improved the functional activity of failing HA-28z CAR T cells, that enhancement of in vivo function of c-Jun modified HA-28z CAR T cells could not be repeated by providing IL-2 ex vivo, that c-Jun could enhance the activity of Her2-BBz CAR T cells in inhibitory solid tumor microenvironments, that overexpression of c-Jun improved resistance to TGF β -mediated inhibition of failing HA-28z CAR T cells, and that transcriptional changes of c-Jun modified cells are consistent with decreased failure and improved memory formation.
As described herein, engineered T cells (e.g., T cells engineered to express synthetic receptors such as engineered T cell receptors or CARs) and unmodified (e.g., naive, native) T cells are provided that are modified to overexpress and/or comprise elevated levels (e.g., to have physiologically elevated levels) of one or more activator-1 (AP-1) transcription factors (e.g., c-Fos, c-Jun, activator-transcription factors (ATFs), and Jun Dimerization Protein (JDP) families) and/or are modified (e.g., genetically modified) to reduce the expression and/or activity of one or more AP-1 inhibitory complex members (e.g., JunB and BATF3 and other BATF family members, IRF4 and ATF family members).
Thus, in one aspect, the invention provides T cells modified to overexpress and/or contain elevated levels of one or more AP-1 transcription factors. For example, in one embodiment, c-Jun is expressed in T cells engineered to express a synthetic receptor (e.g., an engineered T cell receptor or a Chimeric Antigen Receptor (CAR)). In another embodiment, c-Jun is expressed in T cells having an initial native T cell receptor. The present invention is not limited by the manner in which one or more AP-1 transcription factors are expressed. In one embodiment, c-Jun (and/or other AP-1 transcription factors) and the engineered receptor are co-expressed in different viral vectors when co-expressed with the engineered TCR or CAR. In another embodiment, they are expressed in a single vector construct by using a bicistronic vector. C-Jun (and/or other AP-1 transcription factors) can be expressed constitutively or in a regulated manner (e.g., using a system that regulates expression remotely through small molecules or using an endogenous regulation system). In another embodiment, the c-Jun and/or other AP-1 transcription factor genes can be genetically integrated into cellular DNA using retroviral, lentiviral, or other viral vectors or via CRISPR/cas 9-based systems. In yet another embodiment, c-Jun and/or other AP-1 transcription factors are expressed via RNA or oncolytic viruses or other transient expression systems known in the art. C-Jun and/or other AP-1 transcription factors can be delivered to T cells ex vivo for adoptive transfer, or via in vivo genetic transfer.
Similarly, the invention is not limited by the type of T cell that is modified to overexpress and/or contain elevated levels of one or more AP-1 transcription factors and/or that is modified (e.g., genetically modified) to reduce the expression and/or activity of one or more members of the AP-1 inhibitory complex (e.g., JunB and BATF3, as well as other BATF family members, IRF4, and ATF family members). In some embodiments, the T cell is a CD3+ T cell (e.g., a combination of CD4+ and CD8+ T cells). In certain embodiments, the T cell is a CD8+ T cell. In other embodiments, the T cell is a CD4+ T cell. In some embodiments, the T cell is a Natural Killer (NK) T cell. In some embodiments, the T cell is an α β T cell. In some embodiments, the T cell is a gamma T cell. In some embodiments, the T cell is a combination of CD4+ and CD8T + cells (e.g., a CD3+ T cell). In certain embodiments, the T cell is a memory T cell. In certain embodiments, the memory T cell is a central memory T cell. In certain embodiments, the memory T cell is an effector memory T cell. In some embodiments, the T cell is a tumor infiltrating lymphocyte. In certain embodiments, the T cell is a combination of a CD8+ T cell, a CD4+ T cell, a NK T cell, a memory T cell, and/or a gamma T cell. In some embodiments, the T cell is a cytokine-induced killer cell.
In some embodiments, the T cell is an anti-tumor T cell (e.g., a T cell with anti-tumor (e.g., autologous tumor) activity that is activated and expanded in response to an antigen). In one embodiment, anti-tumor T cells (e.g., anti-tumor T cells for adoptive T cell transfer) include peripheral blood-derived T cells genetically modified with receptors that recognize and respond to tumor antigens. Such receptors typically include an extracellular domain comprising a single chain antibody (scFv) specific for a tumor antigen linked to an intracellular T cell signaling motif (see, e.g., Westwood, j.a.et al,2005, proc.natl.acad.sci., USA,102(52): 19051-19056). Other anti-tumor T cells include T cells obtained from resected tumors or tumor biopsy specimens (e.g., tumor-infiltrating lymphocytes (TILs)). In another embodiment, the T cells are polyclonal or monoclonal tumor-reactive T cells (e.g., obtained by apheresis, ex vivo expanded against tumor antigens presented by autologous or artificial antigen presenting cells). In another embodiment, the T cell is engineered to express a human or murine T cell receptor that recognizes a tumor antigen. The present invention is not limited to the type of tumor antigen identified in this manner. Virtually any T cell comprising a receptor that recognizes a tumor antigen can be used in the compositions and methods described herein. Examples include, but are not limited to, T cells expressing a receptor (e.g., an original or naturally occurring receptor, or engineered to express a synthetic receptor (e.g., an engineered T cell receptor or CAR)) that recognizes an antigen selected from the group consisting of: CD19, CD20, CD22, tyrosine kinase-like orphan receptor 1(ROR1), bis-sialylganglioside 2(GD2), epstein-barr virus (EBV) protein or antigen, folate receptor, mesothelin, human carcinoembryonic antigen (CEA), CD33/IL3R α, tyrosine protein kinase Met (c-Met) or Hepatocyte Growth Factor Receptor (HGFR), Prostate Specific Membrane Antigen (PSMA), glycolipid F77, epidermal growth factor receptor variant iii (egfrviii), NY-ESO-1, Melanoma Antigen Gene (MAGE) family member A3(MAGE-A3), T cell recognized melanoma antigen 1(MART-1), GP1000, p53, or other tumor antigens described herein.
In some embodiments, the T cell is engineered to express a CAR. The invention is not limited by the type of CAR. Indeed, any CAR that specifically binds a desired antigen (e.g., a tumor antigen) can be modified to overexpress and/or comprise elevated levels (e.g., to have physiologically elevated levels) of one or more AP-1 transcription factors (e.g., c-Jun) as disclosed and described herein. In certain embodiments, the CAR comprises an antigen binding domain. In certain embodiments, the antigen binding domain is a single chain variable fragment (scFv) comprising heavy and light chain variable regions that specifically bind to the desired antigen. In some embodiments, the CAR further comprises a transmembrane domain (e.g., a T cell transmembrane domain (e.g., CD28 transmembrane domain)) and a signaling domain (e.g., a T cell receptor signaling domain (e.g., a TCR zeta chain)) comprising one or more immunoreceptor tyrosine-based activation motifs (ITAMs). In some embodiments, the CAR comprises one or more co-stimulatory domains (e.g., a domain that provides a second signal to stimulate T cell activation). The invention is not limited by the type of co-stimulatory domain. Indeed, any co-stimulatory domain known in the art may be used, including but not limited to CD28, OX40/CD134, 4-1BB/CD137/TNFRSF9, high affinity immunoglobulin E receptor gamma subunit (FcERI gamma, ICOS/CD278,
In one aspect, the invention provides a method of treating a disease or pathological condition in a subject, the method comprising administering to the subject (e.g., patient) having the disease or disorder an effective amount of T cells modified to express and/or contain elevated levels of one or more AP-1 transcription factors and/or modified (e.g., genetically modified) to reduce the expression and/or activity of one or more AP-1 inhibitory complex members (e.g., JunB and BATF3 and other BATF family members, IRF4 and ATF family members). The invention is not limited by the type of disease or disorder being treated. Indeed, any disease or disorder that can be treated with T cells (e.g., an improvement in an indication or symptom of the disease after treatment) can be treated in an improved and more effective manner using the compositions and methods described herein (e.g., T cells that contain and/or that are modified to express and/or contain elevated levels of one or more AP-1 transcription factors). In one embodiment, the disease or disorder is cancer. In another embodiment, the disease or disorder is an infectious disease. The present invention is not limited by the type of cancer or the type of infectious disease. Virtually any cancer known in the art that can be treated with T cell therapy can be treated using the compositions and methods described herein. In a similar manner, any infectious disease known in the art that can be treated with T cell therapy can be treated using the compositions and methods described herein. In one embodiment, an effective amount of a T cell suppressor T cell depleting modified to express and/or contain elevated levels of one or more AP-1 transcription factors and/or one or more AP-1 inhibitory complex members with reduced expression and/or activity is administered to a subject (e.g., a patient) having a disease or disorder (e.g., as compared to a subject receiving the same amount of an engineered T cell (e.g., a CAR T cell or a T cell comprising a recombinant TCR) that has not been modified to express and/or contain elevated levels of one or more AP-1 transcription factors and/or has one or more AP-1 inhibitory complex members with reduced expression and/or activity).
Thus, in one embodiment, the invention provides a method of inhibiting T cell failure (e.g., maintenance of T cell function upon exposure to an excess of antigen (e.g., in the context of treatment of a disease or disorder) by modifying T cells to express and/or comprise elevated levels of one or more AP-1 transcription factors and/or to reduce expression and/or activity of one or more members of the AP-1 inhibitory complex (e.g., as compared to control T cells that have not been so modified). In one embodiment, T cells modified to express and/or contain elevated levels of one or more AP-1 transcription factors (e.g., c-Jun) exhibit enhanced function and/or activity (e.g., increased antigen-induced cytokine production, enhanced killing capacity (e.g., enhanced recognition of tumor targets with low surface antigens), enhanced memory cell formation, and/or enhanced proliferation of responses to antigens) and/or reduced failure characteristics (e.g., lower levels of markers indicative of failure (e.g., PD-1, TIM-3, LAG-3) and/or lower levels of programmed cell death). In some embodiments, T cells modified to express and/or contain elevated levels of one or more AP-1 transcription factors and/or to reduce the expression and/or activity of one or more members of the AP-1 inhibitory complex described herein can significantly enhance the clinical efficacy (e.g., of engineered T cells (e.g., CAR T cells) and/or unmodified naive T cells).
In certain embodiments, the present invention demonstrates that treating a subject having cancer with a therapeutically effective amount of a composition comprising T cells modified to express and/or contain elevated levels of one or more AP-1 transcription factors is superior to treating a subject having cancer with T cells expressing normal amounts of one or more AP-1 transcription factors. In some embodiments, treatment of an animal (e.g., a human) with cancer with a therapeutically effective amount of an immunotherapeutic composition comprising T cells modified to express and/or contain elevated levels of one or more AP-1 transcription factors can inhibit the development or growth of cancer cells or make these cancer cells a population more susceptible to other treatments (e.g., cell death-inducing activity of cancer treatment drugs or radiation therapy). Thus, the compositions and methods described herein can be used as a monotherapy (e.g., to kill cancer cells and/or reduce or inhibit growth of cancer cells, induce apoptosis of cancer cells, and/or cell cycle arrest), or when used in combination with one or more other agents, such as other anti-cancer agents (e.g., cancer treatment drugs or radiation therapies that induce cell death or disrupt cell cycle), can render a greater proportion of cancer cells susceptible to killing, inhibiting cancer cell growth, inducing apoptosis, and/or cell cycle arrest (as compared to the corresponding proportion of cells in an animal treated with the cancer treatment drug or radiation therapy alone).
Thus, in certain embodiments, the invention provides methods of treating or delaying the progression of cancer in a patient, comprising administering to the patient a therapeutically effective amount of a composition comprising T cells modified (e.g., genetically modified) to express and/or contain elevated levels of one or more AP-1 transcription factors (e.g., c-Jun) and/or modified (e.g., genetically modified) to reduce the expression and/or activity of one or more AP-1 inhibitory complex members (e.g., JunB and BATF3, as well as other BATF family members, IRF4, and ATF family members). In certain embodiments, the therapeutically effective amount of the modified T cell composition reduces the number of cancer cells in the patient following such treatment. In certain embodiments, the therapeutically effective amount of the modified T cell composition reduces and/or eliminates the tumor burden in the patient following such treatment. In certain embodiments, the method further comprises subjecting the patient to radiation therapy. In certain embodiments, radiation therapy is administered before, concurrently with, and/or after the patient receives a therapeutically effective amount of the modified T cell composition. In certain embodiments, the method further comprises administering to the patient one or more anti-cancer agents and/or one or more chemotherapeutic agents. In certain embodiments, one or more anti-cancer agents and/or one or more chemotherapeutic agents are administered before, concurrently with, and/or after the patient receives a therapeutically effective amount of the modified T cell composition. In certain embodiments, treatment of a patient with a therapeutically effective amount of modified T cells in combination with a course of anticancer drug produces a greater tumor response and clinical benefit in such a patient compared to a patient treated with modified T cells or anticancer drug/radiation alone. Since all approved doses of anticancer drugs and radiation therapy are known, the present invention contemplates various combinations thereof with modified T cells.
In certain embodiments, the invention provides a therapeutically effective amount of a composition (e.g., an immunotherapeutic composition) comprising T cells modified according to the invention (e.g., for treating or delaying cancer progression in a subject). As described herein, the composition may further comprise one or more anti-cancer agents, such as one or more chemotherapeutic agents. The invention also provides the use of the composition for inducing cell cycle arrest and/or apoptosis. The invention also relates to the use of said composition for sensitizing cells to other agents, such as inducers of apoptosis and/or cell cycle arrest, and for chemoprotecting normal cells by inducing cell cycle arrest prior to treatment with a chemotherapeutic agent. The compositions of the invention are useful for treating, ameliorating or preventing a disease, such as any type of cancer or infectious disease, and any cell that responds to induction of apoptotic cell death (e.g., diseases characterized by dysregulation of apoptosis, including hyperproliferative diseases such as cancer). In certain embodiments, the compositions can be used to treat, ameliorate or prevent cancer that is otherwise characterized by resistance to cancer therapy (e.g., cancer cells resistant to chemotherapy, radiation, hormones, etc.). The invention also provides pharmaceutical compositions comprising the modified T cells of the invention (e.g., immunotherapeutic compositions) in a pharmaceutically acceptable carrier.
In another embodiment, the invention provides a method of treating or delaying the progression of cancer in a patient, the method comprising administering to the patient a therapeutically effective amount of a composition comprising T cells modified (e.g., genetically modified) to express and/or contain elevated levels of one or more AP-1 transcription factors (e.g., c-Jun) in combination with a therapeutically effective amount of an inhibitor of TCR signaling (e.g., to prevent T cell failure). In certain embodiments, the TCR signaling inhibitor is a tyrosine kinase inhibitor. In another embodiment, the tyrosine kinase inhibitor inhibits Lck kinase. The TCR signaling inhibitors may be administered by any suitable mode of administration, but are typically administered orally. Multiple cycles of treatment may be administered to the subject. In certain embodiments, the inhibitor of TCR signaling is administered according to a standard dosing regimen (e.g., daily or intermittently). In another embodiment, the inhibitor of TCR signaling is administered for a sufficient period of time to restore at least a portion of T cell function, and then terminated.
These and other embodiments of the present invention will readily occur to those skilled in the art in view of the present disclosure.
Drawings
FIGS. 1A-B show that the AP-1 transcription factors c-Fos and c-Jun are down-regulated in GD 2-28Z-depleted CAR T cells.
Figures 2A-E show that enhanced AP-1 expression reduces failure characteristics in CAR T cells.
FIGS. 3A-F show that the functional benefit of AP-1 is derived primarily from the expression of c-Jun.
FIGS. 4A-E show that bicistronic expression of c-Jun together with CAR enhances the functional activity of CAR T.
FIGS. 5A-C show that bicistronic expression of C-Jun together with CAR enhances CAR T functional activity and central memory phenotype.
Figures 6A-F show that bicistronic expression of c-Jun together with CAR enhances CAR T cell pro-inflammatory cytokine production and decreases IL-10.
Figures 7A-E show that bicistronic expression of c-Jun enhances the cellular activity of CD19 and CD22 CAR T in response to tumor cells with low antigen levels.
Figures 8A-D show that knock-out of AP-1 inhibitory family members JunB and BATF3 elevated IL2 production in depleted CAR T cells. (A) JunB in CRISPR gene Knockout (KO) HA-28Z-depleted CAR T cells greatly enhanced production of IL2 (up) and IFNg (down) after exposure to the GD2+ cell lines Nalm6-GD2 (left), 143B osteosarcoma (middle) and kelley neuroblastoma (right). This increase was even more pronounced than just over-expression of (OE) c-Jun. The double JUNB-KO and cJUN-OE T cells did not show any benefit compared to JUNB-KO alone. (B) JunB in CRISPR gene Knockout (KO) GD2-BBZ CAR T cells greatly enhanced IL2 (up) and IFNg (down) production after exposure to GD2+ cell lines Nalm6-GD2 (left), 143B osteosarcoma (middle) and kelley neuroblastoma (right), but GD2-BBZ CAR T cells Overexpressing (OE) c-Jun showed the greatest functional benefit. The double JUNB-KO and cJUN-OE T cells did not show any benefit compared to cJUN-OE alone. (C) JunB in CRISPR gene Knockout (KO) CD19-28Z (left) or CD19-BBZ (right) CAR T cells did not affect IL2 production (supra) after exposure to Nalm6-GD2 leukemia cells, indicating that JunB is a potent inhibitor only in GD2CAR T cells with tonic signaling/failure. (D) BATF3 in CRISPR gene Knockout (KO) HA-28Z-depleted CAR T cells elevated IL2 production (above) after exposure to Nalm6-GD2 (left) and kelly neuroblastoma (right), while IFN γ production was unchanged. HA-28Z-depleted CAR T cells using three independent grnas targeted to BATF3 all showed an increase in IL2 production compared to controls or ZB 2-edited controls.
Figures 9A-B show that c-Jun expressing HA-GD 2CAR T cells exhibit superior, therapeutic in vivo activity compared to unmodified HA-GD 2CAR T cells. After adoptive transfer of 2 × 106 CAR + or mock (untransduced) T cells, the growth of Nalm6-GD2 leukemia cells stably expressing firefly luciferase was followed in vivo using bioluminescence imaging techniques. (A) Quantitative bioluminescence over time. (B) Images of individual mice are shown. Each group of n-5 mice. (all scales are 1X 104-
FIG. 10 shows that c-Jun modified GD2-BBZ CAR T cells show excellent in vivo activity in invasive 143B osteosarcoma solid tumor model.1 1 × 10 was adoptively transferred7After CAR + or mock (untransduced) T cells, the growth of intramuscularly implanted 143B osteosarcoma cells was measured in vivo using a caliper. (A) Quantitative tumor growth over time, n-5 mice per group.
FIGS. 11A-D show that c-Jun modified CD19 CAR T cells show enhanced in vivo activity against CD19 low
FIGS. 12A-J show that HA-28z CAR T cells exhibit phenotypic, functional, transcriptional, and epigenetic signs of T cell failure. a) During primary expansion culture, HA-28z expanded less compared to CD19-28z CAR T cells. D0 ═ bead activation and D2 ═ transduction. Error bars (error bars) represent mean ± SEM from n ═ 10 donors. b) Surface expression of failure-associated markers (D10). c) CD19-28z mainly includes T stem cell memory (CD45RA + CD62L +) and central memory cells (CD45RA-CD62L +, while HA-28z mainly includes CD45RA-CD 62L-effector memory cells (D10). d) IL-2 (left) and IFNg (right) were released after 24 hours of co-culture with CD19+ GD2+ Nalm6-GD2 leukemia cells. Error bars represent mean ± SD of triplicate wells. Each experiment showed one representative donor. e) Principal Component Analysis (PCA) of global transcriptional profiles of naive and CM-derived CD19 or HA CAR T cells at
Figures 13A-E show AP-1 family characteristics in CAR T cells that are depleted. a) The first 25 transcription factor motif deviation scores at
Figures 14A-K show that overexpression of c-Jun enhances the function of depleted CAR T cells. a) Schematic representation of JUN-P2A-CAR expression vector. b) Intracellular flow cytometry showing total c-JUN expression in control and JUN modified CAR T cells (D10). c) Western blot of total c-Jun and phospho-c-
Figures 15A-J show that functional rescue of depleted HA-28z CAR T cells requires the presence of c-Jun during both chronic and acute T cell stimulation, and is independent of JNP. a) Proposed mechanisms for c-Jun mediated repair of T cell failure. AP-1-i represents an AP-1 inhibitory complex. b) Schematic representation of DD regulated JUN expression vector. c) Schematic representation of drug-induced stabilization of JUN-DD expression. Yellow squares-TMP stable molecules. d) Total c-Jun expression in control, JUN-WT and JUN-DD HA-28z CAR T cells (D10) as detected by intracellular flow cytometry (left) and Western blotting (right). e) IL-2 (left) and IFNg (right) production in control, JUN-WT or JUN-DD (OFF, ON) modified HA-28z
Figures 16A-H show that JUN modified CAR T cells have enhanced in vivo activity against leukemia and solid tumors. In a-c, NSG mice were inoculated with 1X Nalm6-GD2 leukemia cells by intravenous injection. 3x106 mock, HA-28z or JUN-HA-28z CAR + T cells were injected intravenously at
Figures 17A-I show that JUN-CAR T cells enhance T cell function under suboptimal stimulation. A) IL-2 and b) IFNg production after 24 hours of stimulation of control or JUN modified HA-28z CAR T cells with immobilized 1A7 anti-CAR idiotypic antibodies. Each curve was fitted with nonlinear dose response kinetics to determine
Figures 18A-G show that High Affinity (HA)14G2a-GD2E101K CAR T cells exhibit expanded failure characteristics compared to the original 14G2a-GD2 CAR. a) Expression of surface inhibitory receptors in CD19, GD2, and HA-GD2E101K CAR T cells on
Fig. 19A-C show that GD2-28z CAR T cells showed failure characteristics at the single cell level. a) Venn plot shows overlapping genes in the differential expression analysis of single cell data (red) and the first 200 genes driving CD19 and HA-28z CAR T cell isolation in bulk RNA-seq. 79 of the first 200 genes from the bulk RNA-seq were differentially expressed in GD2-28z and CD19-28z single cells as analyzed by
FIGS. 20A-D show quality control of ATAC-seq data. a) Insert length b) insert distance from the Transcription Start Site (TSS) for the combined sample (top) and the single sample (bottom). c) Correlation between replicate samples. d) The position of the mapped peak in each sample was determined by the total number of peaks (upper) and the total frequency (lower).
FIGS. 21A-D show that the AP-1 family contains transcription factor motifs that are most significantly enriched in HA-28 z-depleted CAR T cells. a) Differentially accessible chromatin regions in CD4+ CD19 and HA CAR T cells (D10). Each CAR integrates N and CM subclasses. b) PCA in fig. 1h shows that segregation of PC2 is driven by the CM versus N contrast, whereas segregation of PC3 is driven by the CD4 versus CD8 contrast. c) The most abundant transcription factor motifs in the differentially accessible chromatin regions in HA-28z CAR T cells comprise the AP-1/bZIP family of factors in all starting T cell subsets. The CD8+ initial subclass is shown in fig. 2. d) Peaks were clustered by an enrichment heatmap of shared regulatory motifs (left) and transcription factor motifs (right) in each cluster. 10 different clusters, including clusters associated with depleted (EX1-EX4) or healthy (HLT1-HLT2) CAR T cells, CM (CM) or N (naive) starting subclasses, and CD4 or CD8T cell subclasses. The target genes in the respective clusters are highlighted on the right side. (N-initial, CM-Central memory).
FIGS. 22A-C show that AP-1/bZIP family transcription factors are upregulated in HA-28z CAR T cells and form immunoregulatory complexes. a) Fold change in gene expression for the designated AP-1/bZIP and IRF family genes (HA/CD19) was determined from the RNA sequencing data of FIG. 2. Error bars represent mean ± SEM of n ═ 6 samples from 3 independent donors. b) Fold changes in protein expression (HA/CD19) were determined for the indicated AP-1/bZIP and IRF family proteins by densitometric analysis of Western blots. Error bars represent mean ± SEM of 6 experiments out of 3 independent donors. P < - > 05, p < -01, p < -001. c) Western blot analysis of the designated AP-1/bZIP and IRF family member proteins in CD19 and HA-28z CAR T cells after transfusions (left column) or immunoprecipitations of c-Jun (middle column) or JunB (right column). The numbers below represent the fold increase in protein expression of HA compared to CD19 under each condition, and the color shapes represent the complexes identified by size ratio. IP-Western blot demonstrated increased presence of c-Jun/JunB, c-Jun/IRF4, c-Jun/BATF, and c-Jun/BATF3 complexes in HA-28z CAR T cells. The rate of IRF4 binding was also similar to that of JunB binding, while BATF and BATF3 showed preferential complex formation with JunB.
FIGS. 23A-E show that the enhancement of the activity of AP 1-modified CAR T cells is dependent on c-Jun rather than c-Fos. a-c) CAR T cells were co-transduced with (AP1) or without (control) lentiviral vectors encoding AP1 transcription factors Fos and Jun and truncated ngfr (tngfr) surface selection markers. a) Schematic representation of lentivirus constructs. b) Representative transduction efficiency of AP 1-modified CAR T cells determined by surface expression of NGFR in designated CD4+ and CD8+ CAR T cells. c) Production of IL-2 in control or AP 1-modified
Figures 24A-C show expanded function assessment of JUN-modified CAR T cells. a-b) increased fold release of IL-2(a) and IFNg (b) in JUN and control CD19 and HA-28z CAR T cells after 24 hours of co-culture with the indicated target cells. Each point represents an independent experiment from a different donor. c) Expansion of control or JUN-modified CAR T cells in vitro in 3 independent experiments with 3 different healthy donors. At the indicated time points, T cells were re-seeded in fresh T cell culture medium +100IU/
FIGS. 25A-E show that overexpression of c-Jun reduces the prevalence and complexity of the inhibitory AP-1 family members JunB, BATF, and
Figures 26A-E show that JUN-CAR T cells enhance GD2-BBz CAR T cell function in solid tumors. a) Vector schematic for JUN-GD2-BBz retroviral vector constructs. b) IL-2 (left) and IFNg (right) production in JUN-modified (red) or control (blue) GD2-BBz
FIGS. 27A-E show that the N-terminal mutation of c-Jun is able to rescue depleted HA-28z CAR T cells. a) Different c-Jun mutations were cloned into HA-28z CAR T cell vectors. b) Secretion of IL-2 (upper) and IFNg (lower) after 24h of coculture with GD2+143B osteosarcoma target cells. c) 1:1, 1:2 or 1:4 effector cells: ratios of target cells (E: T) in vitro lysis of GFP + Nalm6-GD2 or 143B target cells was measured. At low E: T ratios and later time points, JUN-WT, JUN-AA, JUN-Dd, and JUN-DTAD exhibited better tumor growth control than JUN-De, JUN-Dbasic, JUN-DLeu, and JUN-DbZIP CAR T cells. d) Increased cytokine production was confirmed in both CD4+ and CD8+ HA-28z CAR T cells by intracellular cytokine staining and flow
Figure 28 shows that knock-out of IRF4 significantly improved the functional activity of depleted HA-28z CAR T cells.
Fig. 29A-C show that the transcriptional mutant (JUN-AA) also rescued functional activity and proliferative capacity in CD19 CAR T cells.
Figure 30 shows that the in vivo functional enhancement of c-Jun modified HA-28z CAR T cells cannot be repeated by providing IL-2 ex vivo.
FIGS. 31A-E show that c-Jun enhances Her2-BBz CAR T cell activity in inhibitory solid tumor microenvironments.
FIG. 32 shows that overexpression of c-Jun promotes resistance to TGF-beta mediated inhibition of depleted HA-28z CAR T cells.
FIGS. 33A-D show that transcriptional changes in c-Jun modified cells are consistent with decreased exhaustion and increased memory formation.
Definition of
For the purpose of explaining the present specification, the following definitions will apply and, where appropriate, terms used in the singular will also include the plural and vice versa. In the event that any of the definitions set forth below conflict with any document incorporated by reference into this application, the definitions set forth below control.
As used herein, unless otherwise indicated, the terms "disease" and "pathological condition" are used interchangeably to describe a condition that deviates from what is considered to be a normal or average condition of a species or population member (e.g., a human), and which is deleterious to the affected individual under conditions that are not deleterious to most individuals of that species or population. Such deviation may manifest as a condition, sign and/or symptom associated with any damage or disturbance to the normal state of a normal subject or damage to any organ or tissue that alters the performance of normal functions (e.g., diarrhea, nausea, fever, pain, blisters, burns, rashes, immunosuppression, inflammation, etc.). A disease or pathological condition may be caused or caused by contact with a microorganism (e.g., a pathogen or other infectious agent (e.g., a virus or bacterium)), may be responsive to environmental factors (e.g., malnutrition, industrial hazards, and/or climate), may be responsive to inherent or potential defects in an organism (e.g., genetic abnormalities), or a combination of these and other factors.
The terms "host," "subject," or "patient" are used interchangeably herein and refer to an individual to be treated (e.g., administered) by the compositions and methods described herein. Subjects include, but are not limited to, mammals (e.g., humans, mice, rats, monkeys, horses, cows, pigs, dogs, cats, etc.). In the context of the present invention, the term "subject" generally refers to an individual to whom or to whom one or more compositions described herein (e.g., modified or engineered (e.g., genetically engineered) T cells described herein) are or have been administered.
"T cell failure" refers to a loss of T cell function that may occur as a result of infection (e.g., chronic infection) or disease. T cell failure is associated with increased expression of PD-1, TIM-3 and LAG-3, apoptosis and decreased cytokine secretion. Thus, the terms "reducing T cell failure", "inhibiting T cell failure", "reducing T cell failure" and the like refer to a state of recovery of T cell function characterized by one or more of: reduced expression and/or levels of one or more of PD-1, TIM-3 and LAG-3; increased memory cell formation and/or maintenance of memory markers (e.g., CD 62L); preventing apoptosis; increased antigen-induced cytokine (e.g., IL-2) production and/or secretion; enhanced killing ability; enhanced recognition of tumor targets with low surface antigens; the proliferation should be enhanced against the antigen.
The term "buffer" or "buffering agent" refers to a material that, when added to a solution, causes the solution to resist a change in pH.
As used herein, the terms "cancer" and "tumor" refer to a tissue or growth comprising cells that have lost the ability to control growth and proliferation. Cancer cells and tumor cells are often characterized by loss of contact inhibition, may be invasive, and may exhibit the ability to metastasize (e.g., they have lost the ability to adhere to other cells/tissues). The invention is not limited by the type of cancer or the type of treatment (e.g., prophylactic and/or therapeutic treatment). Indeed, a variety of cancers may be treated with the compositions and methods described herein, including, but not limited to, brain or other cancers of the central nervous system, melanoma, lymphoma, bone, epithelial, breast, ovarian, endometrial, colorectal, lung, kidney, melanoma, kidney, prostate, sarcoma, carcinoma (carcinomas), and/or combinations thereof.
As used herein, "metastasis" refers to the process by which cancer spreads or metastasizes from the site of origin to other areas of the body while developing similar cancerous lesions at new locations. "metastatic" or "metastatic" cells are cells that lose adhesive contact with adjacent cells and migrate from the major part of the disease through blood or lymph to invade tissues in other parts of the body.
As used herein, the term "anti-cancer agent" refers to any therapeutic agent (e.g., chemotherapeutic and/or molecular therapeutic compounds), antisense therapy, radiation therapy, and the like, used to treat hyperproliferative diseases such as cancer (e.g., in mammals such as humans).
An "effective amount" refers to an amount of a pharmaceutical composition, anti-cancer agent, or other drug at a dosage and for a period of time effective to achieve a desired therapeutic or prophylactic effect (e.g., to alleviate some or all of the symptoms of the disease being treated).
As used herein, the term "therapeutically effective amount" refers to an amount of a therapeutic agent sufficient to result in an improvement in one or more symptoms of a disease, or to prevent the progression of a disease or cause regression of a disease. For example, with respect to treatment of cancer, in one embodiment, a therapeutically effective amount refers to an amount of a therapeutic agent that reduces the rate of tumor growth (e.g., reduces and/or eliminates the tumor burden on a patient), reduces tumor mass, reduces the number of metastases, reduces tumor progression, or extends survival by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.
As used herein, the terms "sensitize" and "sensitize" refer to making an animal or a cell within an animal more susceptible to, or responsive to, a biological effect (promoting or delaying an aspect of cellular function, including but not limited to, cell division, cell growth, proliferation, invasion, angiogenesis, necrosis or apoptosis) of a second agent by administering the first agent. Sensitization of a first agent to a target cell can be determined as the difference in the expected biological effect (promotion or delay of an aspect of cellular function, including but not limited to, cell growth, proliferation, invasion, angiogenesis or apoptosis) observed when a second agent is administered with or without administration of the first agent. The response of the sensitized cells may be increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, or at least about 500% over the response in the absence of the first agent.
As used herein, the term "purified" or "purifying" refers to the removal of contaminants or unwanted compounds from a sample or composition. As used herein, the term "substantially purified" refers to a removal of about 70-90%, up to 100%, of contaminants or unwanted compounds from a sample or composition.
As used herein, the terms "administration" and "administering" refer to the act of administering a composition to a subject. Exemplary routes of administration to the human body include, but are not limited to, administration by eye (intraocular), oral (oral), dermal (transdermal), nasal (nasal), lung (inhalation), oral mucosa (buccal tablet), ear, rectal, by injection (e.g., intravenous, subcutaneous, intraperitoneal, intratumoral, etc.), topical, and the like. In one embodiment, administration of the T cells of the invention is via intravenous infusion.
As used herein, the terms "co-administration" and "co-administration" refer to the administration of at least two agents (e.g., a genetically modified immune cell and one or more other agents, such as an anti-cancer agent) or therapies to a subject. In some embodiments, co-administration of two or more agents or therapies is synchronized. In other embodiments, the first agent/therapy is administered before the second agent/therapy. In some embodiments, co-administration may be via the same or different routes of administration. It will be appreciated by those skilled in the art that the formulation and/or route of administration of the various agents or therapies used may vary. Suitable dosages for co-administration are readily determined by one skilled in the art. In some embodiments, when agents or therapies are co-administered, the agents or therapies are each administered at a lower dose than is appropriate for their individual administration. Thus, co-administration is particularly preferred in embodiments where co-administration of the agents or therapies reduces the necessary dosage of potentially harmful (e.g., toxic) agents, and/or where co-administration of two or more agents results in sensitization of the beneficial effects of the subject to one of the agents due to co-administration of the other agent.
As used herein, the term "pharmaceutically acceptable" or "pharmacologically acceptable" refers to a composition that produces substantially no adverse reaction (e.g., toxicity, allergic, or other immunological reaction) when administered to a subject.
As used herein, the term "pharmaceutically acceptable carrier" refers to any standard pharmaceutical carrier, including, but not limited to, phosphate buffered saline solution, water, and various types of wetting agents (e.g., sodium lauryl sulfate), any and all solvents, dispersion media, coatings, sodium lauryl sulfate, isotonic and absorption delaying agents, disintegrants (e.g., potato starch or sodium methyl starch), polyethylene glycol, and the like. The composition may also contain stabilizers and preservatives. Examples of carriers, stabilizers, and adjuvants have been described and are known in the art (see, e.g., Martin, Remington's Pharmaceutical Sciences,15th ed., Mack pub. co., Easton, Pa. (1975), which is incorporated herein by reference).
As used herein, the term "pharmaceutically acceptable salt" refers to any salt of the composition of the present invention that is physiologically tolerated in the subject of interest (e.g., a salt obtained by reaction with an acid or base). The "salts" of the compositions of the present invention may be derived from inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, sulfonic, naphthalene 2-sulfonic, benzenesulfonic acids and the like. Other acids, such as oxalic, while not per se pharmaceutically acceptable, may be used in the preparation of salts and pharmaceutically acceptable acid addition salts thereof which are useful as intermediates in obtaining the compositions of the present invention. Examples of bases include, but are not limited to, hydroxides of alkali metals (e.g., sodium), hydroxides of alkaline earth metals (e.g., magnesium), ammonia, and compounds of formula NW4+ (where W is a C1-4 alkyl group), and the like.
Examples of salts include, but are not limited to: acetate, adipic acid, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, fluoroheptanoate, hexafluoroacetate, disulfate, hemisulfate, hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmitate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecabonate, and the like. Other examples of salts include anions of the compounds of the invention complexed with suitable cations such as Na +, NH4+, NW4+ (wherein W is C1-4 alkyl), and the like. For therapeutic use, salts of the compounds of the present invention are considered to be pharmaceutically acceptable. However, salts of non-pharmaceutically acceptable acids and bases may also be used, for example, in the preparation or purification of pharmaceutically acceptable compounds.
For therapeutic use, salts of the compositions of the present invention are considered pharmaceutically acceptable. However, salts of non-pharmaceutically acceptable acids and bases may also be used, for example, in the preparation or purification of pharmaceutically acceptable compositions.
As used herein, the term "at risk for a disease" refers to a subject predisposed to a particular disease (e.g., an infectious disease). The susceptibility may be genetic (e.g., experiencing a particular genetic predisposition to a disease, such as a genetic disease), or caused by other factors (e.g., environmental conditions, exposure to harmful compounds present in the environment, etc.). Thus, it is not intended that the present invention be limited to any particular risk (e.g., a subject may be "at risk for disease" simply by exposure to and interaction with others) nor that the present invention be limited to any particular disease (e.g., cancer).
As used herein, the term "kit" refers to any delivery system for delivering a material. In the case of immunotherapeutic agents, such delivery systems include systems that allow for the storage, transport, or delivery of immunogenic agents and/or support materials (e.g., written instructions regarding the use of the materials, etc.) from one location to another. For example, a kit includes one or more housings (e.g., cassettes) containing relevant immunotherapeutic agents (e.g., modified T cells and/or support materials). As used herein, the term "separate kit" refers to a delivery system comprising two or more separate containers, each container comprising a portion of the total kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container can contain a composition comprising an immunotherapeutic composition for a particular use, while a second container can contain a second agent (e.g., a chemotherapeutic agent). In fact, any delivery system comprising two or more separate containers, each container comprising a portion of the total kit components, is encompassed by the term "separate kit". In contrast, a "combination kit" refers to a delivery system that contains all of the components required for a particular use in a single container (e.g., in a single box that holds each of the required components). The term "kit" includes both individual and combined kits.
As used herein, the term "immunoglobulin" or "antibody" refers to a protein that binds to one or more epitopes on a particular antigen. Immunoglobulins include, but are not limited to, polyclonal, monoclonal, chimeric and humanized antibodies, as well as the following classes of Fab fragments and F (ab')2Fragment (b): IgG, IgA, IgM, IgD, IgE and secreted immunoglobulin (sigg). An immunoglobulin typically comprises two identical heavy chains and two light chains. However, the terms "antibody" and "immunoglobulin" also encompass single chain antibodies and double chain antibodies.
The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of a heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH". The variable domain of the light chain may be referred to as "VL". These domains are usually the most variable part of an antibody and contain an antigen binding site.
"Single chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Typically, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form a structure for binding to an antigen. For reviews of scFv, see, for example, Pluckthun, in the Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds. (Springer-Verlag, New York,1994), pp 269-315.
As used herein, the term "antigen binding protein" refers to a protein that binds to a particular antigen. "antigen binding proteins" include, but are not limited to, immunoglobulins, including polyclonal, monoclonal, chimeric and humanized antibodies; fab fragments, F (ab')2 fragments and Fab expression libraries; and single chain antibodies.
As used herein, the term "epitope" refers to the portion of an antigen that is in contact with a particular immunoglobulin.
The terms "specifically binds" or "specifically binds" when used in reference to an interaction of an antibody (or portion thereof (e.g., scFv)) and a protein or peptide means that the interaction is dependent on the presence of a particular sequence or structure (e.g., an antigenic determinant or epitope) on the protein; in other words, an antibody (or portion thereof (e.g., scFv)) recognizes and binds to a particular protein sequence or structure, rather than a general protein. For example, if an antibody is specific for epitope "a", the presence of a protein comprising epitope a (or free, unlabeled a) will reduce the amount of labeled a bound to the antibody in a reaction containing labeled "a" and the antibody.
As used herein, the terms "non-specific binding" and "background binding" when used in reference to an antibody interacting with a protein or peptide refers to an interaction that is independent of the presence of a particular structure (i.e., the antibody binds to the protein in general, rather than to a particular structure, e.g., an epitope).
As used herein, the term "subject suspected of having cancer" refers to a subject exhibiting one or more symptoms indicative of cancer (e.g., a significant mass or lump) or being screened for cancer (e.g., during a routine physical examination). A subject suspected of having cancer may also have one or more risk factors for developing cancer. Subjects suspected of having cancer are typically not tested for cancer. However, a "subject suspected of having cancer" includes an individual who has received a preliminary diagnosis (e.g., a CT scan showing a tumor) but has not yet undergone a confirmatory examination (e.g., a biopsy and/or histological examination) or whose type and/or stage of cancer is unknown. The term also includes persons who have previously suffered from cancer (e.g., individuals in remission). A "subject suspected of having cancer" is sometimes diagnosed with cancer and sometimes found to be free of cancer.
As used herein, the term "subject diagnosed with cancer" refers to a subject that has been tested and found to have cancer cells. Cancer may be diagnosed by any suitable method, including, but not limited to, biopsy, X-ray, blood test, and the like.
As used herein, the term "post-operative tumor tissue" refers to cancerous tissue (e.g., organ tissue) that has been removed from a subject (e.g., during surgery).
As used herein, the term "subject at risk for cancer" refers to a subject having one or more risk factors for developing a particular cancer. Risk factors include, but are not limited to, gender, age, genetic susceptibility, environmental exposure and previous carcinogenesis, pre-existing non-cancer diseases and lifestyle.
As used herein, the term "characterizing a cancer in a subject" refers to identifying one or more properties of a cancer sample in a subject, including, but not limited to, the presence of benign, pre-cancerous, or cancerous tissue and the stage of cancer.
As used herein, the term "characterizing a tissue of a subject" refers to identifying one or more properties of a tissue sample (e.g., including, but not limited to, the presence of cancerous tissue, the presence of pre-cancerous tissue that may become cancerous, and the presence of cancerous tissue that may metastasize).
As used herein, the term "cancer stage" refers to a qualitative or quantitative assessment of the level of cancer progression. Criteria for determining the stage of a cancer include, but are not limited to, the size of the tumor, whether the tumor has spread to other parts of the body, and the location of the spread of the cancer (e.g., in the same organ or body part or to another organ).
As used herein, the term "primary tumor cell" refers to a cancer cell that is isolated from a tumor of a mammal and has not been extensively cultured in vitro.
As used herein, the terms "treatment," "therapeutic use," or "medical use" refer to any and all uses of the compositions and methods of the present invention that can correct a disease state or condition, or in whatever manner prevent, hinder, slow or reverse the progression of a disease or other undesirable condition. For example, the terms "treatment of cancer" or "treatment of a tumor" or grammatical equivalents in this application refer to the inhibition, regression, or partial or complete disappearance of the original cancer or tumor. This definition is intended to include any reduction in the size, invasiveness, or growth rate of the original cancer or tumor.
As used herein, the terms "improved therapeutic outcome" and "enhanced therapeutic efficacy" in relation to cancer refer to a slowing or reduction in the growth of cancer cells or solid tumors, or a reduction in the total number of cancer cells or the total tumor burden. By "improved therapeutic outcome" or "enhanced therapeutic efficacy" is meant an improvement in the condition of an individual according to any clinically acceptable criteria, including reversal of established tumors, increased life expectancy, or improved quality of life.
As used herein, the term "gene transfer system" refers to any means of delivering a composition comprising a nucleic acid sequence to a cell or tissue. For example, gene transfer systems include, but are not limited to, vectors (e.g., retrovirus, adenovirus, lentivirus, adeno-associated virus, and other nucleic acid-based delivery systems), microinjection of naked nucleic acid, polymer-based delivery systems (e.g., liposome-based and metal particle-based systems), biolistic injection (biolistic injection), transduction by transposase-based systems for gene integration, Crispr/Cas 9-mediated gene integration, non-integrating vectors (e.g., RNA or adeno-associated virus), and the like.
As used herein, the term "viral gene transfer system" refers to a gene transfer system comprising viral components (e.g., whole viruses, modified viruses, and viral components such as nucleic acids or proteins) to facilitate delivery of a sample to a desired cell or tissue. Non-limiting examples of viral gene transfer systems that can be used in the compositions and methods described herein are lentiviral and retroviral gene transfer systems.
As used herein, the term "site-specific recombination target sequence" refers to a nucleic acid sequence that provides a recognition sequence for a recombination factor and provides a site at which recombination occurs.
As used herein, the term "nucleic acid molecule" refers to any nucleic acid-containing molecule, including but not limited to DNA or RNA. The term encompasses sequences comprising any known base analog of DNA and RNA, including, but not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, azidocytosine, pseudoisocytosine, 5- (carboxyhydroxymethyl) -uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouridine, 1-methylguanine, 1-methylsarcosine, 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, DNA, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β -D-mannosylsarcosine, 5' -methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxoacetic acid methyl ester, uracil-5-oxoacetic acid, oxybutyloximine (oxybutoxosine), pseudouracil, sarcosine, 2-thiocytosine, 5-methyl-2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxoacetic acid methyl ester and 2, 6-diaminopurine.
As used herein, the term "heterologous gene" refers to a gene that is not in its natural environment. For example, a heterologous gene includes a gene from one species introduced into another species. Heterologous genes also include genes that are native to the organism, which have been altered in some way (e.g., mutated, added in multiple copies, linked to non-native regulatory sequences, etc.). Heterologous genes are distinguished from endogenous genes in that the heterologous gene sequence is typically linked to a DNA sequence that is not associated with the gene sequence in the chromosome in its natural state, or is associated with a portion of the chromosome that is not naturally occurring (e.g., a gene that is expressed at a site where the gene is not expressed in its natural state). Cells comprising a heterologous gene are described herein as "modified" or "engineered" cells. For example, T cells containing a heterologous AP-1 transcription factor gene (e.g., a heterologous AP-1 transcription factor gene expression construct (e.g., for overexpressing an AP-1 transcription factor in T cells)) and/or a heterologous receptor gene (e.g., a heterologous T cell receptor gene expression construct or a heterologous chimeric antigen receptor gene expression construct) are described herein as modified and/or engineered T cells.
As used herein, the term "gene expression" refers to the process of converting genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) by "transcription" of the gene (i.e., by the enzymatic action of rnases), and for genes that encode proteins, converting genetic information encoded in a gene into a protein by "translation" of RNA. Gene expression can be regulated at many stages of the process. "Up-regulation" or "activation" refers to regulation that increases the production of a gene expression product (i.e., RNA or protein), while "down-regulation" or "inhibition" refers to regulation that decreases production. Molecules involved in up-regulation or down-regulation (e.g., transcription factors) are commonly referred to as "activators" and "repressors," respectively.
In addition to containing introns, genomic forms of a gene may also include sequences located at the 5 'and 3' ends of sequences on RNA transcripts. These sequences are referred to as "flanking" sequences or regions (which flanking sequences are located 5 'or 3' to the untranslated sequences present in the mRNA transcripts). The 5' flanking region may include regulatory sequences, such as promoters and enhancers which control or influence the transcription of the gene. The 3' flanking region may contain sequences that direct transcription termination, post-transcriptional cleavage, and polyadenylation.
As used herein, the terms "nucleic acid molecule encoding," "DNA sequence encoding," and "DNA encoding" refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids on the polypeptide (protein) chain. Thus, the DNA sequence encodes an amino acid sequence.
As used herein, the terms "oligonucleotide having a nucleotide sequence encoding a gene" and "polynucleotide having a nucleotide sequence encoding a gene" refer to a nucleic acid sequence comprising the coding region of a gene, or in other words, a nucleic acid sequence encoding a gene product. The coding region may be present in the form of cDNA, genomic DNA or RNA. When present in a DNA form, the oligonucleotide or polynucleotide may be single-stranded (i.e., the sense strand) or double-stranded. If desired, appropriate control elements such as enhancers/promoters, splice junctions, polyadenylation signals, and the like may be placed adjacent to the coding region of the gene to allow for proper initiation of transcription and/or proper processing of the primary RNA transcript. Alternatively, the coding region for use in the expression vectors of the invention may comprise endogenous enhancers/promoters, splice junctions, insertion sequences, polyadenylation signals, etc., or a combination of both endogenous and exogenous control elements.
As used herein, the terms "in operable combination," "in operable order," and "operably linked" refer to the joining of nucleic acid sequences in a manner that results in a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule. The term also refers to the linkage of amino acid sequences in such a way that a functional protein is produced.
The term "isolated" when used in reference to a nucleic acid, as in the terms "isolated oligonucleotide" or "isolated polynucleotide" when used in reference to a nucleic acid sequence, refers to a nucleic acid sequence that is identified and isolated from at least one component or contaminant with which it is ordinarily associated in its natural source. Thus, an isolated nucleic acid exists in a form or situation that is different from the form or situation in which it existed in its natural condition. In contrast, non-isolated nucleic acids are nucleic acids such as DNA and RNA that are present in their naturally occurring state. For example, a given DNA sequence (e.g., a gene) is present in the host cell chromosome at a nearby neighboring gene; RNA sequences (e.g., a particular mRNA sequence encoding a particular protein) are present in the cell in admixture with many other mrnas encoding a variety of proteins. However, an isolated nucleic acid encoding a given protein includes, for example, such nucleic acids in cells that normally express the given protein, where the nucleic acid is at a chromosomal location different from that of the native cell, or is flanked by nucleic acid sequences that are different from those found in nature. An isolated nucleic acid, oligonucleotide or polynucleotide may be present in single-stranded or double-stranded form. When an isolated nucleic acid, oligonucleotide or polynucleotide is utilized to express a protein, the oligonucleotide or polynucleotide will comprise at least the sense or coding strand (i.e., the oligonucleotide or polynucleotide may be single-stranded), but may comprise both the sense and antisense strands (i.e., the oligonucleotide or polynucleotide may be double-stranded).
As used herein, the term "native protein" refers to a protein that does not comprise amino acid residues encoded by a vector sequence; that is, a native protein comprises only those amino acids that are naturally present in the protein. The native protein may be produced recombinantly or may be isolated from a naturally occurring source.
As used herein, the term "portion" when referring to a protein (as in "a portion of a given protein") refers to a fragment of that protein. Fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid.
As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA into which other DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). In one embodiment, the present invention provides a method of producing a recombinant DNA construct comprising introducing into a host cell a DNA encoding one or more AP-1 transcription factors and/or CAR constructs, the DNA construct comprising the DNA construct (e.g., a DNA encoding one or more AP-1 transcription factors and/or CAR constructs), the DNA construct comprising the DNA construct(s) and/or the DNA construct(s) into a cell (e.g., a T cell).
As used herein, the term "expression vector" refers to a recombinant DNA molecule comprising a desired coding sequence and appropriate nucleic acid sequences operably linked thereto as necessary for expression of the coding sequence in a particular host organism. The nucleic acid sequences necessary for expression in prokaryotes typically include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.
As used herein, the term "transfection" refers to the introduction of foreign DNA into a eukaryotic cell. Transfection may be accomplished by a variety of methods known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran mediated transfection, polyalkylene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and particle gun methods.
The term "stable transfection" or "stably transfected" refers to the introduction and integration of foreign DNA into the genome of the transfected cell. The term "stable transfectant" refers to a cell that has stably integrated foreign DNA into genomic DNA. The term "transient transfection" or "transient transfection" refers to the introduction of exogenous DNA into a cell in which the exogenous DNA is unable to integrate into the genome of the transfected cell. The foreign DNA persists in the nucleus of the transfected cell (e.g., for several days). During this time, the foreign DNA is under regulatory control which controls the expression of the endogenous gene in the chromosome. The term "transient transfectant" refers to a cell that has taken up exogenous DNA but has failed to integrate the DNA.
As used herein, the term "selectable marker" refers to the use of a gene encoding an enzyme activity capable of conferring the ability to grow in a medium lacking essential nutrients; in addition, a selectable marker may confer resistance to an antibiotic or drug on cells expressing the selectable marker. The selectable marker may be "dominant"; the dominant selectable marker encodes an enzymatic activity that can be detected in any eukaryotic cell line. Examples of dominant selectable markers include the bacterial aminoglycoside 3' -phosphotransferase gene (also known as the neo gene) that confers resistance to drug G418 on mammalian cells, the bacterial hygromycin G phosphotransferase (hyg) gene that confers resistance to the antibiotic hygromycin, and the bacterial xanthine-guanine phosphoribosyltransferase gene (also known as the gpt gene) that confers the ability to grow in the presence of mycophenolic acid. Other selectable markers are not dominant as they must be used in conjunction with cell lines lacking the relevant enzyme activity. Examples of non-dominant selectable markers include the thymidine kinase (tk) gene used in conjunction with tk negative (tk-) cell lines, CAD gene used in conjunction with CAD-deficient cells, and the mammalian hypoxanthine-guanine phosphoribosyltransferase (hprt) gene used in conjunction with hprt negative (hprt-) cell lines. An overview of the use of selectable markers in mammalian cell lines is provided by Sambrook, J.et al, molecular cloning, A Laboratory Manual,2nd ed., Cold Spring Harbor Laboratory Press, New York (1989) pp.16.9-16.15.
As used herein, the term "in vitro" refers to an artificial environment and processes or reactions occurring within an artificial environment. The in vitro environment may consist of, but is not limited to, test tubes and cell cultures. The term "in vivo" refers to the natural environment (e.g., an animal or cell) and processes or reactions that occur in the natural environment.
As used herein, the term "cell culture" refers to any in vitro culture of cells. The term includes continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, transformed cell lines, limited cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro.
As used herein, the term "sample" is used in its broadest sense. In a sense, it is meant to include samples or cultures obtained from any source, as well as biological and environmental samples. Biological samples can be obtained from animals (including humans) and include fluids, solids, tissues, and gases. Biological samples include blood products such as plasma, serum, and the like. However, these examples should not be construed as limiting the type of sample that is suitable for use in the present invention.
Detailed Description
The present invention is based on the following findings: t cells modified (e.g., genetically modified) to overexpress and/or comprise elevated (e.g., greater than physiological) levels of one or more AP-1 transcription factors (e.g., c-Jun) exhibit reduced levels of T cell depletion (e.g., as compared to unmodified T cells expressing normal levels of AP-1 transcription factors). As described in detail herein, the expression of the AP-1 transcription factors fos and c-Jun are low/down-regulated in depleted T cells, and T cells modified to overexpress and/or contain elevated levels of c-Jun or other AP-1 transcription factors exhibit reduced levels of T cell depletion. For example, overexpression of AP-1 transcription factors (particularly c-Jun) prevents the development of T cell failure and maintains T cell functionality (e.g., even after exposure to high levels of antigen) (see examples 1-6). GD2CAR T cells expressing elevated levels of AP-1 transcription factors (especially c-Jun) showed reduced failure characteristics (including lower failure markers, increased memory formation, and increased cytokine production), suggesting that T cells modified to overexpress and/or contain elevated (e.g., over-physiological) levels of one or more AP-1 transcription factors showed enhanced clinical efficacy in multiple malignancies (see, e.g., examples 1-6). Furthermore, both CD19 CAR T cells modified to overexpress c-Jun and CD22 CAR T cells modified to overexpress c-Jun showed enhanced recognition by CAR T cells of leukemic target cells with low levels of surface antigens (see, e.g., example 6). By using cJUN-modified CAR T cells, CAR T cells modified to overexpress AP-1 transcription factors showed reduced T cell failure and enhanced functionality in three different in vivo tumor models (see example 8). Thus, overexpression of AP-1 transcription factors (particularly c-Jun) prevented the development of T cell failure and maintained T cell functionality. Taken together, these findings indicate that the compositions and methods described herein are broadly applicable and address many of the existing obstacles to successful CAR T cell therapy.
Accordingly, the present invention provides modified T cells that maintain functionality (e.g., genetically and/or functionally modified, e.g., to overexpress and/or contain elevated levels of one or more AP-1 transcription factors and/or to reduce the expression and/or activity of one or more members of the AP-1 inhibitory complex) under conditions in which the unmodified T cells exhibit T cell depletion. In this manner, the compositions and methods described herein can be used to prevent the failure of engineered T cells (e.g., engineered to express a particular T cell receptor, or engineered to express a Chimeric Antigen Receptor (CAR)) and to prevent the failure of unmodified T cells (e.g., native or natural T cells (e.g., isolated from a subject)), thereby enhancing engineered as well as unmodified T cell functionality.
Modifying T cells to overexpress and/or contain elevated levels of one or more AP-1 transcription factors can prevent the depletion of AP-1 transcription factors in T cells (e.g., the depletion of AP-1 transcription factors that occurs with T cell exhaustion (see, e.g., example 1)) and/or result in elevated (e.g., more than physiological) levels of AP-1 transcription factors. AP-1 transcription factors (e.g., c-Jun) are induced upon T cell activation and are involved in T cell production and secretion of cytokines (e.g., interleukin-2). CAR-expressing T cells undergo recruitment due to receptor aggregation, are antigen-independent of signaling, and replicate the basic biological properties of T cell depletion, manifested by higher expression levels of PD-1, TIM-3, and LAG-3, reducing antigen-induced cytokine production and excessive apoptosis. AP-1 transcription factors were identified and characterized as decreased in depleted T cells (see, e.g., example 1). Modifying T cells to overexpress and/or contain elevated levels of one or more AP-1 transcription factors significantly enhances T cell functionality under exposure to conditions that induce T cell failure (see, e.g., examples 2-5). Although the mechanism need not be understood to practice the present invention, and although the present invention is not limited to any particular mechanism of action, maintenance and/or elevation of AP-1 transcription factor levels (e.g., levels of c-Jun) within T cells may prevent T cell dysfunction associated with T cell failure (e.g., the observation of a minor effect of AP-1 transcription factor overexpression in non-failing T cells indicates that relative or absolute lack of c-Jun is an important factor in T cell failure-related dysfunction).
In some embodiments, T cells are modified to overexpress and/or contain elevated levels of one or more mutated and/or truncated AP-1 transcription factors to prevent the depletion of AP-1 transcription factors in the T cells. Experiments conducted during the development of the embodiments of the present application have shown that a portion of an AP-1 family protein (e.g., c-Jun) can be mutated and/or truncated without affecting the ability of the mutated/truncated AP-1 factor to mediate the rescue of dysfunctional T cells. For example, a c-Jun polypeptide having N-terminal deletions and mutations (e.g., in the transactivation domain) retains its ability to rescue HA-28 z-depleted CAR T cell function and maintains an equivalent increase in cytokine production compared to c-Jun. In some embodiments, the AP-1 (e.g., c-Jun) polypeptide comprises a mutation or deletion in a transactivation and/or domain. In some embodiments, the AP-1 (e.g., c-Jun) polypeptide comprises a mutation or deletion that inactivates or does not exist the transactivation and/or domain. In some embodiments, the mutant/truncated AP-1 (e.g., C-Jun) polypeptide comprises sequence identity of 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100% or a range therebetween) or more to the C-terminal amino acid residue (e.g., 50 residues, 75 residues, 100 residues, 150 residues, 200 residues, or 250 residues, or a range therebetween), the C-terminal portion (e.g., one-fourth, one-third, one-half), or the C-terminal domain (e.g., the C-terminus of, bZIP, and amino acids thereof) of the wild-type AP-1 transcription factor (e.g., C-Jun). In some embodiments, the N-terminal amino acid residues (e.g., 50 residues, 75 residues, 100 residues, 150 residues, or a range therebetween), the N-terminal portion (e.g., one quarter, one third, one half), or the N-terminal domain (e.g., the N-terminus of, the transactivation domain, and amino acids thereof) of the wild-type AP-1 transcription factor (e.g., c-Jun) are deleted, mutated, or otherwise inactivated. Any of the embodiments described herein with respect to an AP-1 transcription factor can comprise a mutated/truncated AP-1 (e.g., c-Jun) polypeptide, e.g., in accordance with the above.
Although enhanced expression of AP-1 transcription factors (e.g., c-Fos and c-Jun) enhances the function of modified T cells, other inhibitory AP-1 family members are also expressed in depleted activated T cells. Inhibition/knockdown of members of the AP-1 inhibitory complex (e.g., to increase the availability of classical AP-1 factor) also reduces T cell failure (e.g., see example 7). In particular, inhibition/knock-out of members of the AP-1 inhibitory complex results in a significant enhancement of T cell function (e.g., enhanced production and/or expression of cytokines) in depleted T cells, but not in healthy T cells (e.g., see fig. 8A-D). Thus, in certain embodiments, the present invention provides compositions and methods for inhibiting T cell failure by inhibiting the expression and/or activity of AP-1 inhibitory complex members, such as BATF3 and other BATF family members (BATFs 1 and 2), IRF4, IRF8 and other IRF family members (
Accordingly, the present invention provides compositions and methods for reducing T cell depletion comprising T cells modified to overexpress and/or contain elevated levels of one or more AP-1 transcription factors and/or modified to reduce the expression and/or activity of one or more members of the AP-1 inhibitory complex. The invention is not limited by the diseases or conditions that can be treated using the modified T cells described herein. Indeed, the compositions and methods provided herein can be used to treat any disease for which increased T cell viability provides a therapeutic benefit.
Accordingly, the present invention provides a composition comprising a T cell modified to overexpress and/or contain elevated levels of one or more AP-1 transcription factors and/or modified (e.g., genetically modified) to reduce the expression and/or activity of one or more AP-1 inhibitory complex members (e.g., JunB, BATF3 and other BATF family members, IRF4, IRF8 and other IRF family members, and ATF family members). As described in detail herein, the T cell may be an engineered or unmodified T cell (e.g., a Tumor Infiltrating Lymphocyte (TIL)). Furthermore, the invention is not limited to types of T cells modified to overexpress and/or contain elevated levels of one or more AP-1 transcription factors. In some embodiments, the T cell is a CD3+ T cell (e.g., a combination of CD4+ and CD8+ T cells). In certain embodiments, the T cell is a CD8+ T cell. In other embodiments, the T cell is a CD4+ T cell. In some embodiments, the T cell is a Natural Killer (NK) T cell. In some embodiments, the T cell is a gamma T cell. In some embodiments, the T cell is a combination of CD4+ and CD8T + cells (e.g., a CD3+ T cell). In certain embodiments, the T cell is a memory T cell. In certain embodiments, the T cell is a combination of a CD8+ T cell, a CD4+ T cell, a NK T cell, a memory T cell, and/or a gamma T cell. In some embodiments, the T cell is a cytokine-induced killer cell. In some embodiments, the T cell is engineered to express a chimeric antigen receptor. In another embodiment, the T cell is engineered to express a specific T cell receptor (e.g., specific for a tumor antigen or an infectious disease antigen). In some embodiments, the T cell is an anti-tumor T cell. The composition may comprise a pharmaceutically acceptable carrier (e.g., a buffer). The compositions can optionally include one or more other agents, such as other drugs for treating T cell failure (e.g., anti-PD-1 checkpoint inhibitors (e.g., nivolumab) or other drugs for treating infection or disease associated with T cell failure in a subject (e.g., antiviral, antibiotic, antimicrobial, or anticancer drugs).
Antibacterial therapeutic agents may be used as therapeutic agents in the compositions of the present invention. Any agent that kills, inhibits or reduces the function of microorganisms can be used, and any agent that has such activity is contemplated. Antibacterial agents include, but are not limited to, natural and synthetic antibiotics, antibodies, inhibitory proteins (e.g., defensins), antisense nucleic acids, membrane disruptive agents, and the like, which may be used alone or in combination. Virtually any type of antibiotic may be used, including, but not limited to, antibacterial, antiviral, antifungal agents, and the like.
Several strategies for targeting tumor antigens are known in the art. For example, Immunotherapy targeting GD2 is currently in clinical and preclinical investigation for several diseases, including neuroblastoma, osteosarcoma, and melanoma (see, e.g., Thomas et al, PLoS One,2016.11(3): p.e0152196; Long et al, Nature Medicine,2015.21(6): 581-590 page; Long et al, Cancer Immunology Research,2016.4(10): 869-880 page; Yu et al, N Engl J Med,2010.363(14): 1324-34 page; Perez Horta et al, Immunotherapy,2016.8(9): 1097-117 page; Heczey et al, Molecular Therapy). Unlike mabs that do not effectively cross the blood brain barrier, activated CAR T cells can effectively penetrate the CNS after adoptive transfer. Thus, in one embodiment, any CAR T cell can be modified (e.g., modified to overexpress and/or contain elevated levels of one or more AP-1 transcription factors (e.g., to enhance the functionality of the CAR T cell and/or inhibit the failure of the CAR T cell)) according to the compositions and methods described herein.
For example, as described herein, a modified T cell described herein (e.g., modified to overexpress and/or contain elevated levels of one or more AP-1 transcription factors and/or modified (e.g., genetically modified) to reduce the expression and/or activity of one or more AP-1 inhibitory complex members (e.g., JunB and BATF3 and other BATF family members, IRF4 and ATF family members)) can also be engineered to comprise a CAR comprising a target-specific binding element, also referred to as an antigen-binding portion. The choice of moiety depends on the type and amount of ligand that defines the surface of the target cell. For example, the antigen binding domain can be selected to recognize a ligand that serves as a cell surface marker on target cells associated with a particular disease state. Examples of cell surface markers that can act as ligands for the antigenic part domain in a CAR according to the invention include cell surface markers associated with viral, bacterial and parasitic infections, autoimmune diseases and cancer cells.
For example, a CAR can be engineered to target a tumor antigen of interest by engineering a desired antigen-binding moiety that specifically binds to an antigen on a tumor cell. As used herein, "tumor antigen" or "hyperproliferative disease antigen" or "antigen associated with a hyperproliferative disease" or "cancer antigen" refers to antigens that are common in particular hyperproliferative diseases, such as cancer. Exemplary antigens mentioned herein are included as examples. This list is not intended to be exclusive and other examples will be apparent to those skilled in the art.
Tumor antigens are proteins produced by tumor cells that are capable of eliciting an immune response, particularly a T cell mediated immune response. Thus, the antigen-binding moiety may be selected based on the particular type of cancer to be treated. Tumor antigens are well known in the art and include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), β -human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2(AS), intestinal carboxyesterase, mut hsp70-2, M-CSF, Prostate Specific Antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostaglandins, PSMA, Her2/neu, survivin and telomerase, prostate cancer tumor antigen 1(PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, Insulin Growth Factor (IGF) -I, IGF-II, IGF-I receptor, and mesothelin.
The tumor antigen may comprise one or more antigenic cancer antigens/epitopes associated with a malignancy. Malignant tumors express a variety of proteins that can serve as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase and GP100 in melanoma, and Prostate Acid Phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of molecules involved in transformation, for example the oncogene HER-2/Neu/ErbB-2. Another group of target antigens are carcinoembryonic antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotypic immunoglobulins constitute a true tumor-specific immunoglobulin antigen that is unique to a single tumor. B cell differentiation antigens (e.g., CD19, CD20, and CD37) are other candidates as target antigens in B cell lymphomas.
The tumor antigen may also be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). TSA is characteristic of tumor cells and does not occur on other cells in the body. TAAs are not specific to tumor cells, but can also be expressed on certain normal cells under conditions that do not induce an immune-tolerant state to the antigen. Expression of the antigen on the tumor may occur under conditions that allow the immune system to respond to the antigen. TAAs may be antigens expressed on normal cells during embryonic development where the immune system is immature and unresponsive, or they may be antigens that are normally present at very low levels on normal cells but are expressed at very high levels on tumor cells.
Examples of TSA or TAA include, but are not limited to, differentiation antigens such as MART-1/MelanA (MART-1), gp100(Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multiple lineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2,
Depending on the desired antigen to be targeted, the CAR can be engineered to include a specific antigen binding moiety appropriate for the target of the desired antigen. For example, if CD19 is the desired antigen to be targeted, an antibody directed against CD19 can be used as an antigen binding moiety for incorporation into a CAR described herein.
Method of treatment
In another aspect, the application provides a method of treating a disease or disorder in a subject, the method comprising administering to the subject (e.g., patient) suffering from the disease or disorder an effective amount of T cells modified to express and/or comprise elevated levels of one or more AP-1 transcription factors. The invention is not limited by the type of disease or condition being treated. Indeed, any disease or disorder that can be treated by administration of T cells (e.g., the symptoms or symptoms of the disease are ameliorated after treatment) can be treated in an improved and more effective manner using the compositions and methods described herein (containing and/or using T cells modified to express and/or contain elevated levels of one or more AP-1 transcription factors). In one embodiment, the disease or disorder is cancer. In another embodiment, the disease or disorder is an infectious disease. The present invention is not limited by the type of cancer or the type of infectious disease. Indeed, any cancer known in the art for which T cell therapy is useful can be treated using the compositions and methods described herein. Similarly, any infectious disease known in the art for which T cell therapy is useful can be treated using the compositions and methods described herein. In one embodiment, administration of an effective amount of T cells modified to express and/or contain elevated levels of one or more AP-1 transcription factors to a subject (e.g., a patient) having a disease or disorder can inhibit T cell failure in the subject (e.g., T cell failure that occurs when the same amount of unmodified T cells are administered to the subject). For example, in certain embodiments, the methods comprise methods of treating a subject having a disease or condition responsive to treatment with adoptive cell therapy. In certain embodiments, the method of treating a subject having a disease or condition responsive to treatment with adoptive cell therapy comprises the step of administering a composition comprising an effective amount of T cells genetically modified to express elevated levels of one or more AP-1 transcription factors, wherein the T cells genetically modified to express elevated levels of one or more AP-1 transcription factors exhibit reduced levels of T cell depletion compared to T cells expressing normal levels of one or more AP-1 transcription factors. In certain embodiments, the disease or disorder comprises cancer.
In one aspect, the invention provides methods of treating cancer/tumors by using T cells modified to express one or more AP-1 transcription factors. In various aspects of the invention, methods of treating cancer/tumors are provided, the methods comprising administering to a patient having such a cancer or tumor an effective amount of T cells modified to express one or more AP-1 transcription factors. The present invention is not limited by the type of cancer and/or tumor being treated. As used herein, the term "cancer" refers to various types of malignancies, most of which can invade surrounding tissues and can metastasize to different sites (see, e.g., PDR Medical Dictionary, 1 st edition (1995), the entire contents of which are incorporated by reference herein for all purposes). The terms "tumor (neoplasms)" and "tumor (tumor)" refer to abnormal tissue that grows faster than normal by cellular proliferation and continues to grow after removal of the stimulus that initiates proliferation. Such abnormal tissue exhibits a partial or complete loss of structural organization and functional coordination with normal tissue, which may be benign (i.e., benign tumor) or malignant (i.e., malignant tumor). Examples of general classes of cancer include, but are not limited to, carcinomas (e.g., malignancies derived from epithelial cells, such as the common forms of breast, prostate, lung, and colon cancer), sarcomas (e.g., malignancies derived from connective or mesenchymal cells), lymphomas (e.g., malignancies derived from hematopoietic cells), leukemias (e.g., malignancies derived from hematopoietic cells), germ cell tumors (e.g., tumors derived from totipotent cells the most common in adults is in the testis or ovary; in fetuses, infants, and young children the most common in the body midline, especially at the tip of the coccyx), embryonic tumors (e.g., malignancies that typically resemble immature or embryonic tissue), and the like. Other examples of tumors and/or neoplasms (neoplasms) that may be treated using the compositions and methods described herein include, but are not limited to, tumors associated with cancers of neural tissue, blood-forming tissue, breast, skin, bone, prostate, ovary, uterus, cervix, liver, lung, brain, larynx, gall bladder, pancreas, rectum, parathyroid, thyroid, adrenal gland, immune system, head and neck, colon, stomach, bronchi, and/or kidney. In some embodiments, the cancer is a occult cancer, a previously diagnosed primary cancer, or a metastatic cancer.
In certain embodiments, the presence of elevated levels of one or more AP-1 transcription factors in a modified T cell (e.g., a CAR T cell (e.g., as compared to an unmodified T cell)) enables more effective treatment (killing and/or inhibiting progression) of a cancer/tumor as compared to treatment with a T cell that has not been modified to contain elevated levels of one or more AP-1 transcription factors (e.g., a CAR T cell).
In certain embodiments, the invention provides methods of treating (e.g., inhibiting growth and/or killing) cancer and/or tumor using T cells (e.g., CD3+ T cells) genetically engineered to overexpress and/or contain elevated (e.g., over physiological) levels of one or more AP-1 transcription factors and engineered to express receptors that recognize tumor surface antigens (e.g., CD19, CD20, CD22, ROR1, GD2, EBV proteins or antigens, folate receptors, mesothelin, human carcinoembryonic antigen, CD33/IL3Ra, c-Met, PSMA, glycolipid F77, EGFRvIII, NY-ESO-1, MAGE-A3, MART-1, GP1000, and/or p53) and deliver signals that activate T cells to induce T cell expansion and/or tumor killing. One non-limiting example of a receptor is a Chimeric Antigen Receptor (CAR) that incorporates an scFv derived from a mAb that recognizes GD2, as well as a transmembrane domain and one or more intracellular signaling domains. The CAR may further be engineered to incorporate other signaling elements that promote expansion of the engineered cell upon encountering a tumor cell antigen (e.g., GD2 antigen) and elements that enable long-term persistence of the engineered cell. The invention further provides compositions comprising the genetically engineered cells (e.g., immunotherapeutic compositions produced and administered in amounts sufficient to reach a cancer and/or tumor).
Construction of modified T cells.
As described herein, the present invention provides compositions comprising T cells modified (e.g., genetically modified (e.g., transduced)) to express (e.g., heterologously express) one or more AP-1 transcription factors. For example, the invention provides transduced T cells. A "transduced cell" is a cell into which a nucleic acid molecule has been introduced using molecular biology techniques. T cells modified/transduced to express one or more heterologous AP-1 transcription factors can be transduced by any technique capable of introducing nucleic acid molecules into such cells, including, but not limited to, transfection with viral vectors (e.g., retroviruses, lentiviruses, or other viral vectors), by CRISPR/Cas 9-based systems, transformation with plasmid vectors, and/or introduction of naked DNA by electroporation, lipid and particle gun acceleration. Viral and/or plasmid vectors may be used for in vitro, in vivo and/or ex vivo expression. The AP-1 transcription factor can be co-expressed with the engineered TCR or CAR, while the transcription factor and TCR and/or CAR can be co-expressed from different viral vectors. In another embodiment, they are expressed from a single vector construct by using a bicistronic vector. C-Jun (and/or other AP-1 transcription factors) can be constitutively expressed or expressed in a regulated manner (e.g., using a system that remotely regulates expression via small molecules or using an endogenous regulatory system). In another embodiment, the c-Jun and/or other AP-1 transcription factor genes can be genetically integrated into cellular DNA using a retrovirus, lentivirus or other viral vector or via a CRISPR/Cas9 based system. In yet another embodiment, the c-Jun and/or other AP-1 transcription factors are expressed via RNA or oncolytic viruses or other transient expression systems known in the art. The C-Jun and/or other AP-1 transcription factors can be delivered to T cells ex vivo (e.g., where the T cells are used for adoptive transfer), or by in vivo genetic transfer.
The invention is not limited by the Chimeric Antigen Receptor (CAR) expressed in the T cell (e.g., the CAR construct used in the methods of the invention). In one embodiment, the CAR comprises a fusion protein of an immunoglobulin heavy chain (VH) and light chain (VL) variable regions (e.g., single chain variable fragments (scFv)) that specifically binds to a tumor antigen (e.g., GD 2). One of ordinary skill in the art knows that an scFv is a fusion protein of the heavy (VH) and light (VL) chain variable regions of an immunoglobulin, which is linked to a linker peptide (e.g., a linker peptide having about 10 to about 25 amino acids). The invention is not limited by the type of linker peptide. In some embodiments, the linker peptide is glycine-rich (e.g., for flexibility). In some embodiments, the linker peptide comprises a serine and/or a threonine (e.g., for solubility). In some embodiments, the linker peptide comprises a glycine-rich moiety and a serine and/or threonine-containing moiety.
Any antibody/immunoglobulin that specifically binds to a tumor antigen (e.g., GD2) can be used to construct a CAR (e.g., fusion protein scFv using VH and VL regions) for expression in immune cells used in the treatment methods described herein. For GD2, examples of such antibodies/immunoglobulins include, but are not limited to: 14G2a, ch14.18, hu14.18K322A, m3F8, hu3F8-IgG1, hu3F8-IgG4, HM3F8, UNITUXIN, DMAb-20, or any other antibody that specifically binds to GD2 (e.g., known or described in the art, or yet to be identified). Tumor antigen (e.g., GD2) CARs may comprise receptors that incorporate intra-scFv variants of tumor antigen (e.g., GD2) antibodies that are generated to enhance affinity and/or reduce complimentary signaling. A tumor antigen (e.g., GD2) CAR may incorporate a variable length hinge region (e.g., between the scFv and the signaling domain) and/or a varying transmembrane domain. The invention is not limited by the transmembrane domain used. Virtually any transmembrane domain can be used, including, but not limited to, all or part of the transmembrane domains of the TCR zeta chain (CD3 zeta), CD28, OX40/CD134, 4-1BB/CD137/TNFRSF9, FcERI gamma, ICOS/CD278, ILRB/CD122, IL-2RG/CD132, or
The CAR constructs of the invention can include an intracellular signaling domain (e.g., CD3 ζ and/or other signaling domain (e.g., MyD88 signaling domain) of a native T cell receptor complex) that transduces ligand binding to an intracellular signal (e.g., activates (e.g., partially activates) a T cell). In the absence of costimulatory signals, receptor-ligand binding is often insufficient to fully activate and proliferate T cells. Thus, the CAR construct may comprise one or more co-stimulatory domains (e.g., domains that provide a second signal to stimulate full T cell activation). In one embodiment, a co-stimulatory domain is used which promotes the production of CAR immune T cell cytokines. In another embodiment, a co-stimulatory domain is used which promotes T cell replication. In yet another embodiment, a co-stimulatory domain is used which prevents CAR T cell failure. In another embodiment, a co-stimulatory domain is used which promotes the anti-tumor activity of T cells. In another embodiment, a co-stimulatory domain is used that enhances survival of CAR T cells (e.g., following infusion into a patient). Examples of proteins, or domains or portions thereof, that can be used to provide a costimulatory signal include, but are not limited to, B7-1/CD 80; CD 28; B7-2/CD 86; CTLA-4; B7-H1/PD-L1; ICOS/CD 278; ILRB/CD 122; IL-2RG/CD 132; B7-H2; PD-1; B7-H3; PD-L2; B7-H4; PDCD 6; BTLA; 4-1BB/TNFRSF9/CD 137; FcERI γ; CD40 ligand/TNFSF 5; 4-1BB ligand/TNFSF 9; GITR/TNFRSF 18; BAFF/BLyS/TNFSF 13B; GITR ligand/TNFSF 18; BAFF R/TNFRSF 13C; HVEM/TNFRSF 14; CD27/TNFRSF 7; LIGHT/TNFSF 14; CD27 ligand/TNFSF 7; OX40/TNFRSF 4; CD30/TNFRSF 8; OX40 ligand/TNFSF 4; CD30 ligand/TNFSF 8; TACL/TNFRSF 13B; CD40/TNFRSF 5; 2B4/CD244/SLAMF 4; CD84/SLAMF 5; BLAME/SLAMF 8; CD229/SLAMF 3; CD2 CRACC/SLAMF 7; CD2F-10/SLAMF 9; NTB-A/SLAMF 6; CD48/SLAMF 2; SLAM/CD 150; CD 58/LFA-3; CD 2; ikaros; CD 53; integrin α 4/CD49 d; CD 82/Kai-1; integrin α 4 β 1; CD90/Thy 1; integrin α 4 β 7/LPAM-1; CD 96; LAG-3; CD 160; LMIR1/CD 300A; CRTAM; TCL 1A; DAP 12; TIM-1/KIM-1/HAVCR; Dectin-1/CLEC 7A; TIM-4; DPPIV/CD 26; TSLP; EphB 6; TSLP R; and HLA-DR. In one embodiment, the CAR constructs expressed in T cells used in the compositions and methods described herein comprise a CD28 endodomain, a 4-1BB endodomain, and/or an OX40 endodomain. In certain embodiments, a CAR construct specific for a tumor antigen (e.g., GD2) described herein comprises a scFv of an antibody that specifically binds to the tumor antigen (e.g., GD2), a transmembrane domain (e.g., CD8), a T cell receptor intracellular signaling domain (e.g., TCR zeta chain (CD3 zeta)), and at least one costimulatory domain (e.g., 4-1 BB).
The present invention is not limited to means to genetically express a TCR, CAR, and/or one or more AP-1 transcription factors in a T cell. Indeed, any means known in the art and/or described herein may be used. Non-limiting examples of methods of genetically engineering T cells include, but are not limited to, retrovirus or lentivirus mediated transduction, transduction with a transposase-based system for gene integration, criprpr/
Cancer treatments, compositions and combination therapies.
In certain embodiments, the present invention provides a method for treating or delaying the progression of cancer, or for treating or delaying the progression of an infectious disease in an individual, comprising administering to the individual an effective amount of the modified T cells of the invention. In some embodiments, the treatment results in a sustained response in the individual after cessation of treatment. The methods described herein can be used to treat conditions where enhanced immunogenicity is desired, for example to boost tumor immunogenicity for the treatment of cancer. The present application also provides a method of enhancing immune function in an individual having cancer, the method comprising administering to the individual an effective amount of a modified T cell of the invention. Any type of T cell genetically modified to express a CAR and/or TCR known in the art or described herein can be used in these methods.
In some embodiments, the individual has a cancer that is resistant (e.g., has been demonstrated to be resistant) to one or more other forms of anti-cancer therapy (e.g., chemotherapy, immunotherapy, etc.). In some embodiments, the resistance comprises a recurrence of cancer or refractory cancer. Recurrence may refer to the reappearance of cancer at the original site or new site after treatment. In some embodiments, the resistance comprises progression of the cancer during treatment with chemotherapy. In some embodiments, the resistance comprises a cancer that is unresponsive to traditional or conventional therapy with a chemotherapeutic agent. Cancer may be resistant at the beginning of treatment, or may become resistant during treatment. In some embodiments, the cancer is at an early or late stage.
In certain embodiments, the present invention provides that exposing an animal (e.g., a human) having a cancer/tumor to a therapeutically effective amount of an immunotherapeutic composition comprising T cells modified to express and/or contain elevated levels of one or more AP-1 transcription factors can drastically inhibit the growth of such cancer cells and/or render such cells into a population more susceptible to (e.g., to the cell death-inducing activity of) a cancer therapeutic drug or radiation therapy. The immunotherapeutic compositions and methods described herein are useful for treating, ameliorating, or preventing a disease, such as any type of cancer.
In certain embodiments, immunotherapeutic compositions comprising T cells modified to express and/or contain elevated levels of one or more AP-1 transcription factors are used to treat, ameliorate or prevent cancer characterized by resistance to one or more conventional cancer therapies (e.g., cancer cells that are chemically resistant, radioresistant, hormone resistant, etc.). As described herein, any T cell genetically modified to express a tumor-specific CAR can be used in the immunotherapeutic compositions and methods described herein.
Immunotherapeutic compositions described herein (e.g., comprising T cells modified to express and/or contain elevated levels of one or more AP-1 transcription factors) and methods described herein are useful for inducing cytotoxic activity against tumor cells and/or promoting cell survival and function (e.g., survival and function of modified immune cells). For example, the immunotherapeutic compositions and methods described herein may be used to induce interleukin 2(IL-2) to promote T cell survival; for inducing Fas ligand (FasL) and/or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (e.g., to induce apoptosis of tumor cells); and/or for inducing Interferon (IFN) - γ (e.g., to activate an innate immune response (e.g., against cancer)). In some embodiments, the compositions and methods described herein are used to induce cell cycle arrest and/or apoptosis, and also to enhance induction of cell cycle arrest and/or apoptosis, either alone or in response to other apoptosis-inducing signals. In some embodiments, T cells modified to express and/or contain elevated levels of one or more AP-1 transcription factors sensitize cancer cells to the induction of cell cycle arrest and/or apoptosis, including cells that are generally resistant to such induction stimuli.
In some embodiments, the compositions and methods described herein can be used to treat diseased cells, tissues, organs, or pathological conditions and/or disease states in animals (e.g., mammalian patients, including but not limited to humans and companion animals). In this regard, various diseases and pathologies can be treated or prevented using the methods and compositions described herein. In some embodiments, the cancer cells being treated are metastatic. In other embodiments, the cancer cell being treated is resistant to an anticancer agent.
Some embodiments of the invention provide methods of administering an effective amount of T cells modified to express and/or contain elevated levels of one or more AP-1 transcription factors and at least one other therapeutic agent (including but not limited to chemotherapeutic antineoplastics, apoptosis modulators, antimicrobials, antivirals, antifungals, and anti-inflammatory agents) and/or therapeutic techniques (e.g., surgical intervention and/or radiation therapy). In a particular embodiment, the additional therapeutic agent is an anti-cancer agent.
A number of suitable anti-cancer agents may be contemplated for use in the methods of the invention. Indeed, the present invention contemplates, but is not limited to, administration of multiple anti-cancer agents, such as: an agent that induces apoptosis; polynucleotides (e.g., antisense, ribozyme, siRNA); polypeptides (e.g., enzymes and antibodies); a biological mimetic; an alkaloid; an alkylating agent; an anti-tumor antibiotic; an antimetabolite; a hormone; a platinum compound; monoclonal or polyclonal antibodies (e.g., antibodies conjugated to anti-cancer drugs, toxins, defensins), toxins; a radionuclide; biological response modifiers (e.g., interferons (e.g., IFN- α) and interleukins (e.g., IL-2)); adoptive immunotherapies; a hematopoietic growth factor; drugs that induce tumor cell differentiation (e.g., all-trans retinoic acid); gene therapy agents (e.g., antisense therapeutic agents and nucleotides); a tumor vaccine; an angiogenesis inhibitor; proteasome inhibitors: NF- κ B modulator; an anti-CDK compound; HDAC inhibitors, and the like. Many other examples of chemotherapeutic compounds and anti-cancer therapies suitable for co-administration with the disclosed compounds are known to those skilled in the art.
In certain embodiments, the anti-cancer agent comprises an agent that induces or stimulates apoptosis. Agents that induce apoptosis include, but are not limited to, radiation (e.g., X-rays, gamma rays, UV); tumor Necrosis Factor (TNF) -related factors (e.g., TNF family receptor proteins, TNF family ligands, TRAIL-R1, or antibodies to TRAIL-R2); kinase inhibitors (e.g., Epidermal Growth Factor Receptor (EGFR) kinase inhibitors, Vascular Growth Factor Receptor (VGFR) kinase inhibitors, Fibroblast Growth Factor Receptor (FGFR) kinase inhibitors, platelet-derived growth factor receptor (PDGFR) kinase inhibitors, and Bcr-Abl kinase inhibitors (e.g., GLEEVEC)); an anti-sense molecule antibody; antibodies (e.g., HERCEPTIN, RITUXAN, ZEVALIN and AVASTIN); antiestrogens (e.g., raloxifene and tamoxifen); antiandrogens (e.g., flutamide, bicalutamide, finasteride, aminoglutamine, ketoconazole, and corticosteroids); cyclooxygenase 2(COX-2) inhibitors (e.g., celecoxib, meloxicam, NS-398, and non-steroidal anti-inflammatory drugs (NSAIDs)); anti-inflammatory agents (e.g., flutriadine, decaduron, DELTASONE, dexamethasone enhancer alcohol, DEXONE, hexdol, hydroxychloroquine, METICORTEN, oradex, orasone, oxybenzone, PEDIAPRED, phenylbutanone, PLAQUENIL, prednisolone, prednisone, PRELONE, and TANDEARIL); and cancer chemotherapeutic drugs (e.g., irinotecan (CAMPTOSAR), CPT-11, Fludarabine (FLUDARA), Dacarbazine (DTIC), dexamethasone, mitoxantrone, MYLOTARG, VP-16, cisplatin, carboplatin, oxaliplatin, 5-FU, doxorubicin, gemcitabine, bortezomib, gefitinib, bevacizumab, TAXOTERE, or TAXOL); a cell signaling molecule; ceramides and cytokines; staurosporine, and the like.
In other embodiments, the compositions and methods of the invention are used with at least one anti-hyperproliferative or anti-neoplastic agent selected from the group consisting of: alkylating agents, antimetabolites, and natural products (e.g., herbal and other plant and/or animal derived compounds).
Alkylating agents suitable for use in the compositions and methods of the present invention include, but are not limited to: 1) nitrogen mustards (e.g., methylethylamine, cyclophosphamide, ifosfamide, melphalan (L-myolysin), and chlorambucil); ) Acetyleneimine and methyl melamine (e.g., hexamethylmelamine and tiatepa); 3) alkyl sulfonates (e.g., busulfan); 4) nitrosoureas (e.g., carmustine (BCNU); lomustine (CCNU); sematine (methyl-CCNU); and streptozotocin (streptozotocin)); and 5) triazenes (e.g., dacarbazine (DTIC; dimethyltriazenimide-Azole carboxamide)
In some embodiments, antimetabolites suitable for use in the compositions and methods of the present invention include, but are not limited to: 1) folic acid analogs (e.g., methotrexate); 2) pyrimidine analogs (e.g., fluorouracil (5-fluorouracil; 5-FU), fluorouridine (fluorodeoxyuridine; FudR), and cytarabine (cytarabine)); and 3) purine analogs (e.g., mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG) and pentostatin (2' -deoxymetamycin).
In other embodiments, chemotherapeutic agents suitable for use in the compositions and methods of the invention include, but are not limited to: 1) vinca alkaloids (e.g., Vinblastine (VBL), vincristine); 2) epipodophyllotoxins (e.g., etoposide and teniposide); 3) antibiotics (e.g., actinomycin (actinomycin D), daunorubicin (daunomycin; erythromycin), doxorubicin, bleomycin, plicamycin (mercerizing mycin), and mitomycin (mitomycin C)); 4) enzymes (e.g., L-asparaginase); 5) biological response modifiers (e.g., interferon- α); 6) platinum coordination complexes (e.g., cisplatin (cis-DDP) and carboplatin); 7) anthracenedione (e.g., mitoxantrone); 8) substituted ureas (e.g., hydroxyurea); 9) methylhydrazine derivatives (e.g., procarbazine (N-methylhydrazine; MIH)); 10) adrenocortical hormone inhibitors (e.g., misalane (o, p' -DDD) and aminoglutamine); 11) adrenal corticosteroids (e.g., prednisone); 12) progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and progesterone acetate); 13) estrogens (e.g., diethylstilbestrol and ethinylestradiol); 14) antiestrogens (e.g., tamoxifen); 15) androgens (e.g., testosterone propionate and fluoxymesterone); 16) anti-androgens (e.g., flutamide): and 17) gonadotropin-releasing hormone analogues (e.g., leuprolide).
Any oncolytic agent conventionally used in the treatment of cancer may also be used in the compositions and methods described herein. For example, the united states food and Drug Administration (us.food and Drug Administration) has a collection of oncolytic formulations approved for use in the united states. The international counterpart of U.S. f.d.a. possesses a similar formulation.
Anticancer agents also include compounds that have been identified as having anticancer activity. Examples include, but are not limited to, 3-AP, 12-O-tetradecanoylphorbol-13-acetate, 17AAG, 852A, ABI-007, ABR-217620, ABT-751, ADI-PEG 20, AE-941, AG-013736, AGRO100, Alanoxin, AMG 706, antibody G250, an antineoplastic agent, AP23573, apaquone, APC8015, altimod, ATN-161, atrazine (atrasenten), azacitidine, BB-10901, BCX-1777, bevacizumab, BG00001, bicalutamide, BMS 247550, bortezomib, bryostatin-1, buserelin (buserelin), calcitriol, CCI-779, CDB-2914, cefixime (CpG), cetuximab (cetuximab), 0070, cetirizine peptide (citalopidine), Clogiline (CP), 7983, ofarabine, curcumin-724,714, curcumin-366309, curcumin, Ab-D, Ab-3, Abamectin, Ab-D, Abamectin, Ab-1, Abiracine, Decitabine (decitabine), DENSPM, doxercalciferol (doxercalciferol), E7070, E7389, ecteinascidin 743, ethacryloxil (efaproxil), eflornithine (efloronitine), EKB-569, enzastarin (enzastaurin), erlotinib (erlotinib), etoxisuline (exisulinds), fenretinide (fenretinide), flazadol (flazopiridide), fludarabine, flutamide, fotemustine (fotemustine), FR901228, G17DT, galiximab (galiximatinimab), gefitinib, genistein (genistein), glufosfamide (glufosfamide), GTI-2040, histamine (histrelin), HKI-272, homoharringtonine (moxiderite), interleukin (HSPiqinoline), imipenem-96, IME-14, IMEI-E (IMEI), Hizilin (HK) Lenalidomide (lenalidomide), lestaurtinib (lestaurtinib), leuprorelin, LMB-9 immunotoxin, lonafarnib (lonafarnib), luracizumab (luniliximab), macsfamide (mafosfamide), MB07133, MDX-010, MLN2704, monoclonal antibody 3F8, monoclonal antibody J591, motoxafin (motexafin), MS-275, MVA-MUC1-IL2, nilutamide (nilutamide), nitrocamptothecin (nitrocamptothecin), noratrixate dihydrochloride (nolatrexed dihydrochloride), navadex (nolvadex), NS-9, O6-benzyl guanine, pimelison sodium (oblin sodium), onymab-monodentate, yox-gol 015 (oregovit), nethianib 774-774, pimelitin (OSI), pimela-PD-59906, pimelane (OSI), pimela-PD (OSI), pimelane (OSI), pezil (p-b), pimelane (p-b) and fosamicin (OSI), pimela-g-PD), pimelane (p-b) and fospindol), pimelane (p-b) and (p-b), naproxb-b-g), pimelf), and (p-g-b) of the like, naphalofugine, naproxb, naproxan, PXD101, pyrazoline acridine (pyrazoloacridine), R115777, RAD001, ranpirnase (ranpirnase), butterfly mycin (rebeccamycin) analogues, rhu angiostatin protein, rhuMab 2C4, rosiglitazone (rosiglitazone), rubitecan (rubitecan), S-1, S-8184, satraplatin (satraplatin), SB-15992, SGN-0010, SGN-40, sorafenib (sorafenib), SR31747A, ST1571, SU011248, suberoylishydroxamic acid, suramin (suramin), talasol (talarobot), talapamide (talampanel), tacoquinate (tariquadar), temsirolimus (teiolimus), TGFa-PE 36 immunotoxin, thalidomide (thalidomide), valsartan (tlaxon), valacilin (valacilin), valacitinib (valacilin), Valacilin (VN), valacitinib (valacitinib) 286, valacitinib (valacitinib) and valacilin (VN-4011), valacilin (valacilin), valacilin (troxin (valacilin), valacilin (valtrex) and valtrex (valacilin (valtrex) 2, valacilin (valacilin), valacilin (valaci, Vorinostat (vorinostat), VX-680, ZD1839, ZD6474, zileuton (zileuton), and levosuquinate trihydrochloride.
The invention provides methods of administering the compositions and methods described herein with (e.g., before, during, or after) radiation therapy. The present invention is not limited by the type, amount, or delivery and administration system used to deliver the therapeutic dose of radiation to the animal. For example, the animal may receive photon radiation therapy, particle beam radiation therapy, other types of radiation therapy, and combinations thereof. In some embodiments, the radiation is delivered to the animal using a linear accelerator. In other embodiments, gamma knife is used to deliver the radiation.
The radiation source may be external or internal to the animal. External radiotherapy is the most common and involves directing a high-energy radiation beam through the skin to the tumor site using, for example, a linear accelerator. When the radiation beam is positioned at the tumor site, it is almost impossible to avoid exposure of normal healthy tissue. However, animals are generally well-tolerated by external radiation. Internal radiation therapy involves implanting a radiation-emitting source (e.g., beads, wires, pellets, capsules, particles, etc.) into the body at or near the tumor site, including the use of delivery systems that specifically target cancer cells (e.g., using particles attached to cancer cell binding ligands). Such implants may be removed after treatment or left in the body in an inactive state. Types of internal radiation therapy include, but are not limited to, brachytherapy, interstitial radiation, intracavitary radiation, radioimmunotherapy, and the like.
The animal may optionally receive a radiosensitizer (e.g., metronidazole, misonidazole, intraarterial Budr, intravenous iododeoxyuridine (IudR), nitroimidazole, 5-substituted-4-nitroimidazole, 2H-isoindoledione, [ ((2-bromoethyl) -amino ] methyl ] -nitro-1H-imidazole-1-ethanol, nitroaniline derivatives, DNA affinity hypoxia-selective cytotoxins, halogenated DNA ligands, 1,2,4 benzotriazine oxide, 2-nitroimidazole derivatives, fluoronitrozole derivatives, benzamide, nicotinamide, acridine-intercalator, 5-thiot-oxazole derivatives, 3-nitro-1, 2, 4-triazole, 4, 5-dinitroimidazole derivatives, hydroxylated tesamolin, hydroxytexaphyrin, Cisplatin, mitomycin, tilipamine, nitrosourea, mercaptopurine, methotrexate, fluorouracil, bleomycin, vincristine, carboplatin, epirubicin, doxorubicin, cyclophosphamide, vinblastine, etoposide, paclitaxel, pyretic (hyperthermia), etc.), radioprotectants (e.g., cysteamine, aminoalkyl dihydrothiophosphate, amifostine (WR 2721), IL-1, IL-6, etc.). Radiosensitizers enhance killing of tumor cells. Radioprotectors may protect healthy tissue from the harmful effects of radiation.
Any type of radiation can be administered to the animal as long as the animal is able to tolerate the radiation dose without unacceptable negative side effects. Suitable types of radiation therapy include, for example, ionizing (electromagnetic) radiation therapy (e.g., X-rays or gamma rays) or particle beam radiation therapy (e.g., high-line-energy radiation). Ionizing radiation is defined as radiation that includes particles or photons having energy (i.e., gain or loss of electrons) sufficient to produce ionization (e.g., as described in U.S. patent No. 5,770,581, which is incorporated herein by reference in its entirety). The effect of the radiation may be at least partially controlled by the clinician. In one embodiment, the radiation dose is divided to achieve maximum target cell exposure and reduced toxicity.
In one embodiment, the total dose of radiation administered to the animal is from about 0.01 gray (Gy) to about 100 Gy. In another embodiment, about 10Gy to about 65Gy (e.g., about 15Gy, 20Gy, 25Gy, 30Gy, 35Gy, 40Gy, 45Gy, 50Gy, 55Gy, or 60Gy) is administered during treatment. While in some embodiments, the entire radiation dose may be administered over the course of a day, it is preferred that the total dose be divided and administered over a period of several days. Ideally, radiation therapy is administered over the course of at least about 3 days, such as at least 5,7, 10, 14, 17, 21, 25, 28, 32, 35, 38, 42, 46, 52, or 56 days (about 1-8 weeks). Thus, daily radiation doses should include about 1-5Gy (e.g., about 1Gy, 1.5Gy, 1.8Gy, 2Gy, 2.5Gy, 2.8Gy, 3Gy, 3.2Gy, 3.5Gy, 3.8Gy, 4Gy, 4.2Gy, or 4.5Gy) or 1-2Gy (e.g., 1.5-2 Gy). The daily radiation dose should be sufficient to cause damage to the target cells. In one embodiment, if the time is extended, radiation is not administered daily, thereby allowing the animal to rest and achieve a therapeutic effect. For example, for weekly treatments, it is desirable to have radiation therapy delivered for 5 consecutive days, rather than being administered on 2 days, allowing a2 day rest per week. However, radiation therapy can be administered 1 day/week, 2 days/week, 3 days/week, 4 days/week, 5 days/week, 6 days/week, or all 7 days/week depending on the responsiveness and any potential side effects of the animal. Radiation therapy can be initiated at any time during the treatment. In one embodiment, the radiation is initiated at
In some embodiments of the invention, T cells modified to express and/or contain elevated levels of one or more AP-1 transcription factors and one or more therapeutic or anti-cancer agents are administered to an animal under one or more of the following conditions: in different cycles, in different durations, in different concentrations, by different routes of administration, etc. In some embodiments, T cells modified to express and/or contain elevated levels of one or more AP-1 transcription factors are administered prior to a therapeutic or anti-cancer agent, e.g., 0.5, 1,2, 3, 4,5, 10, 12, 18 hours or more, 1,2, 3, 4,5, 6 or more days, or1, 2, 3, 4,5, 6 or more weeks prior to administration of the therapeutic or anti-cancer agent. In some embodiments, T cells modified to express and/or contain elevated levels of one or more AP-1 transcription factors are administered after a therapeutic or anti-cancer agent, e.g., 0.5, 1,2, 3, 4,5, 10, 12, 18 or more hours, 1,2, 3, 4,5, 6 or more days, or1, 2, 3, 4,5, 6 or more weeks after administration of the anti-cancer agent. In some embodiments, a T cell modified to express and/or comprise an elevated level of one or more AP-1 transcription factors and a therapeutic or anti-cancer agent may be administered simultaneously but on a different schedule, e.g., the modified immune cell is administered daily and the therapeutic or anti-cancer agent is administered weekly, biweekly, every three weeks, every four weeks, or more weekly. In other embodiments, T cells modified to express and/or contain elevated levels of one or more AP-1 transcription factors are administered once a week, while the therapeutic or anti-cancer agent is administered daily, weekly, biweekly, every three weeks, every four weeks or more weekly.
Compositions within the scope of the present invention include all compositions wherein T cells modified to express and/or contain elevated levels of one or more AP-1 transcription factors are included in an amount effective to achieve their intended purpose. Although individual requirements vary, the determination of the optimum range for effective amounts of each component is within the skill of the art. In one non-limiting example, T cells modified to express and/or contain elevated levels of one or more AP-1 transcription factors can be administered to a mammal, such as a human, to provide 1000 to 10 per day of the human10T cells (e.g., for treating cancer). In another embodiment, administration is 1000 to 1010The modified T cell to treat, ameliorate, or prevent cancer (e.g., prevent metastasis, recurrence, and/or progression of cancer). Unit doses can be administered in one or more administrations per day (e.g., 1,2, 3, 4,5, 6 or more days or weeks in a row).
The T cells may be administered as part of a pharmaceutical formulation comprising a suitable pharmaceutically acceptable carrier including excipients and auxiliaries that facilitate processing and/or administration of the modified cells into pharmaceutically acceptable formulations. The T immune cells and/or pharmaceutical preparations comprising the same may be administered intravenously, intramuscularly, subcutaneously, intratumorally, intraperitoneally, intrathecally, or intraventricularly. An effective amount of T cells and/or pharmaceutical preparations comprising the same may be administered to prevent or treat diseases. The appropriate dosage can be determined based on the type of disease to be treated, the type of modified T cells, the severity and course of the disease, the clinical status of the individual, the clinical history and response to treatment of the individual, and the discretion of the attending physician.
The efficacy of any of the methods described herein can be tested in various models known in the art, such as clinical or preclinical models (e.g., a T cell modified to express and/or contain elevated levels of one or more AP-1 transcription factors is treated in combination with one or more chemotherapeutic agents described herein). Suitable preclinical models are exemplified in this application. For any of the exemplary models, after tumor generation, mice were randomly recruited into the treatment group receiving treatment or the control treatment group. Tumor size (e.g., tumor volume) is measured during treatment, and overall survival is also monitored.
In some embodiments, the sample is obtained prior to treatment with T cells (e.g., alone or in combination with another therapy described herein) as a baseline for measuring response to treatment. In some embodiments, the sample is a tissue sample (e.g., formalin-fixed and paraffin-embedded (FFPE), archived, fresh, or frozen). In some embodiments, the sample is whole blood. In some embodiments, the whole blood comprises immune cells, circulating tumor cells, and any combination thereof.
Responsiveness to treatment may refer to any one or more of the following: extending survival (including overall survival and progression-free survival); results in objective responses (including complete responses or partial responses); or ameliorating the signs or symptoms of cancer. In some embodiments, responsiveness may refer to improvement, i.e., response, stability, or progression, of one or more factors according to a set of RECIST guidelines that have been published for determining tumor status in cancer patients. For a more detailed discussion of these criteria, see Eisenhauer et al, Eur J Cancer 2009; 45: 228-47; topallian et al, N EnglJ Med 2012; 366: 2443-54; wolchok et al, Clin Can Res 2009; 15: 7412-20; and therase, p., et al, j.natl.cancer inst.92:205-16 (2000). A responsive subject may refer to a subject whose cancer shows improvement, e.g., based on one or more factors based on RECIST criteria. A non-responsive subject refers to a subject whose cancer does not show improvement, e.g., based on one or more factors based on RECIST criteria.
Traditional response criteria may not be sufficient to characterize the anti-tumor activity of immunotherapeutics, which may result in a delayed response that may begin with an initial apparent radiological progression, including the appearance of new lesions. Thus, modified response criteria have been developed that take into account the possible appearance of new lesions and confirm radiological progress in subsequent evaluations. Thus, in some embodiments, responsiveness may refer to improvement in one or more factors according to immune-related response standard 2 (irRC). See, e.g., Wolchok et al, Clin Can Res 2009; 15:7412-20. In some embodiments, new lesions are added to the determined tumor burden and then tracked, e.g., for tracking of radiologic progression in subsequent assessments. In some embodiments, the presence of a non-target lesion is included in the assessment of a complete response, but not in the assessment of radiologic progression. In some embodiments, radiologic progression may be determined based solely on measurable disease and/or may be confirmed by continuous assessment >4 weeks from the date of first recording.
This description is deemed sufficient to enable one skilled in the art to practice the invention. Various modifications of the invention in addition to those shown and described in this application will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. All publications, patents, and patent applications cited in this application are herein incorporated by reference in their entirety for all purposes.
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