阅读说明：本技术 嵌合细胞因子受体 (Chimeric cytokine receptors ) 是由 迈克尔·C·延森 克里斯托弗·P·萨克斯比 于 2020-02-06 设计创作，主要内容包括：本文提供的方法和组合物的一些实施方式涉及嵌合细胞因子受体。在一些实施方式中,嵌合细胞因子受体可包括栓系至胞外IL-7受体结构域的IL-7和连接至胞外IL-7受体结构域的胞内IL-21受体结构域。在一些实施方式中,在不存在外源细胞因子的情况下,包含嵌合细胞因子受体的T细胞可以容易地被激活和/或扩增。(Some embodiments of the methods and compositions provided herein relate to chimeric cytokine receptors. In some embodiments, the chimeric cytokine receptor may include IL-7 tethered to an extracellular IL-7 receptor domain and an intracellular IL-21 receptor domain linked to the extracellular IL-7 receptor domain. In some embodiments, T cells comprising a chimeric cytokine receptor can be readily activated and/or expanded in the absence of exogenous cytokines.)

1. A polynucleotide encoding a chimeric cytokine receptor polypeptide, wherein the chimeric cytokine receptor polypeptide comprises:

IL-7 tethered to the extracellular IL-7 receptor domain;

a transmembrane domain; and

an intracellular IL-21 receptor domain, wherein the transmembrane domain connects the extracellular IL-7 receptor domain to the intracellular IL-21 receptor domain.

2. The polynucleotide of claim 1, comprising:

a first nucleic acid encoding said IL-7;

a second nucleic acid encoding a tether;

a third nucleic acid encoding the extracellular IL-7 receptor domain, wherein the IL-7 is linked to the extracellular IL-7 receptor domain by the tether;

a fourth nucleic acid encoding the transmembrane domain; and

a fifth nucleic acid encoding an intracellular IL-21 receptor domain.

3. The polynucleotide of claim 1 or 2, wherein the tether has a length of 3 amino acids to 30 amino acids.

4. The polynucleotide of any one of claims 1-3, wherein the transmembrane domain comprises an IL-7 receptor transmembrane domain.

5. The polynucleotide of any one of claims 1-4, wherein the first nucleic acid comprises a nucleotide sequence identical to SEQ ID NO: 02 nucleotide sequences having at least 95% identity.

6. The polynucleotide of any one of claims 1-5, wherein the second nucleic acid comprises a nucleotide sequence identical to SEQ ID NO: 03 having at least 95% identity thereto.

7. The polynucleotide of any one of claims 1-6, wherein the third nucleic acid comprises a nucleotide sequence identical to SEQ ID NO: 04 has a nucleotide sequence of at least 95% identity.

8. The polynucleotide of any one of claims 1-7, wherein the third nucleic acid and the fourth nucleic acid together comprise a nucleotide sequence identical to SEQ ID NO: 04 has a nucleotide sequence of at least 95% identity.

9. The polynucleotide of any one of claims 1-8, wherein the fifth nucleic acid comprises a nucleotide sequence identical to SEQ ID NO: 05 nucleotide sequence having at least 95% identity.

10. The polynucleotide of any one of claims 1-9, wherein the first nucleic acid comprises the nucleotide sequence of SEQ ID NO: 02 nucleotide sequence.

11. The polynucleotide of any one of claims 1-10, wherein the second nucleic acid comprises the nucleotide sequence of SEQ ID NO: 03.

12. The polynucleotide of any one of claims 1-11, wherein the third nucleic acid comprises the nucleotide sequence of SEQ ID NO: 04.

13. The polynucleotide of any one of claims 1-12, wherein the third nucleic acid and the fourth nucleic acid together comprise the nucleotide sequence of SEQ ID NO: 04.

14. The polynucleotide of any one of claims 1-13, wherein the fifth nucleic acid comprises the nucleotide sequence of SEQ ID NO: 05.

15. The polynucleotide of any one of claims 1-14, further comprising an inducible promoter.

16. The polynucleotide of any one of claims 1-15, further comprising an inducible cytotoxic gene.

17. The polynucleotide of claim 16, wherein the cytotoxic gene encodes a protein selected from the group consisting of: thymidine kinase, thymidine kinase fused to thymidylate kinase, oxidoreductase, deoxycytidine kinase, uracil phosphoribosyltransferase, cytosine deaminase, or cytosine deaminase fused to uracil phosphoribosyltransferase.

18. The polynucleotide of claim 16 or 17, wherein the cytotoxic gene comprises thymidine kinase.

19. The polynucleotide of claim 18, wherein the thymidine kinase enzyme comprises SR39 TK.

20. The polynucleotide of any one of claims 1-19, further comprising a ribosome skipping sequence.

21. The polynucleotide of claim 20, wherein said ribosome skipping sequence comprises a T2A skipping sequence.

22. The polynucleotide of any one of claims 1-21, wherein the polynucleotide encodes a transduction marker.

23. The polynucleotide of claim 22, wherein said marker comprises truncated CD19(CD19 t).

24. A vector comprising the polynucleotide of any one of claims 1-23.

25. The vector of claim 24, wherein the vector comprises a viral vector.

26. The vector of claim 24 or 25, wherein the vector is selected from a lentiviral vector, an adeno-associated viral vector, or an adenoviral vector.

27. The vector of claim 26, wherein the vector comprises a lentiviral vector.

28. A polypeptide encoded by the polynucleotide of any one of claims 1-23.

29. A cell comprising the polynucleotide of any one of claims 1-23 or a protein encoded by said polynucleotide.

30. The cell of claim 29, further comprising a polynucleotide encoding a Chimeric Antigen Receptor (CAR), or a CAR protein.

31. The cell of claim 29 or 30, wherein the cell is a T cell.

32. The cell of any one of claims 29-31, wherein the cell is a CD8+ T cell or a CD4+ T cell.

33. The cell of any one of claims 29-32, wherein the cell is derived from a CD8+ T cell or a CD4+ T cell.

34. The cell of any one of claims 29-33, wherein the cell is a primary cell.

35. The cell of any one of claims 29-34, wherein the cell is mammalian.

36. The cell of any one of claims 29-35, wherein the cell is human.

37. The cell of any one of claims 29-36, wherein the cell is ex vivo.

38. A method of treating or ameliorating cancer in a subject, the method comprising:

administering the cell of any one of claims 29-37 to the subject in need thereof.

39. The method of claim 38, wherein the treatment or amelioration of cancer lacks co-administration of a cytokine to the subject.

40. The method of claim 38, further comprising co-administering a cytokine to the subject, wherein the dose of the cytokine administered is reduced compared to the dose of the cytokine co-administered to a subject administered cells comprising a CAR and lacking the polynucleotide of any one of claims 1-23 or the protein encoded by the polynucleotide.

41. The method of any one of claims 38-40, wherein the cytokine is selected from IL-7, IL-15, or IL-21.

42. The method of any one of claims 38-41, wherein the cytokine comprises IL-21.

43. The method of any one of claims 38-42, wherein the cells are autologous to the subject.

44. The method of any one of claims 38-43, wherein the cancer comprises a solid tumor, such as colon cancer, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, melanoma, renal cancer, pancreatic cancer, brain cancer, glioblastoma, neuroblastoma, medulloblastoma, sarcoma, bone cancer, or liver cancer, or a non-solid tumor, such as leukemia or multiple myeloma.

45. The method of any one of claims 38-44, wherein the cancer comprises brain cancer.

46. The method of any one of claims 38-45, wherein the subject is a mammal.

47. The method of any one of claims 38-46, wherein the subject is a human.

48. A method of making a population of cells comprising a Chimeric Antigen Receptor (CAR), the method comprising:

(a) transducing a T cell with a polypeptide encoding a chimeric receptor, wherein the polypeptide comprises the polynucleotide of any one of claims 1-23;

(b) transducing the T cell with a polynucleotide encoding a CAR; and

(c) culturing the transduced T cells under conditions sufficient to stimulate activation and expansion of said T cells, wherein the culture medium comprises a reduced amount of exogenous cytokine as compared to an amount sufficient to stimulate activation and expansion of T cells lacking the chimeric receptor-encoding polynucleotide.

49. A method of making a population of cells comprising a Chimeric Antigen Receptor (CAR), the method comprising:

(a) transducing a T cell with the polynucleotide of any one of claims 1-23;

(b) transducing the T cell with a polynucleotide encoding a CAR; and

(c) culturing the transduced T cells under conditions that stimulate activation and expansion of said T cells, wherein the culture medium is devoid of exogenous cytokines.

50. A method according to claim 48 or 49, wherein step (b) is carried out before step (a).

51. A method according to claim 48 or 49, wherein step (a) and step (b) are carried out simultaneously.

52. The method of any one of claims 48-51, wherein the cytokine is selected from IL-7, IL-15, or IL-21.

53. The method of any one of claims 48-52, wherein the cytokine comprises IL-21.

54. The method of any one of claims 48-53, wherein the cell is a CD8+ T cell or a CD4+ T cell.

55. The method of any one of claims 48-54, wherein the cell is derived from a CD8+ T cell or a CD4+ T cell.

56. The method of any one of claims 48-55, wherein the cell is a primary cell.

57. The method of any one of claims 48-56, wherein the cell is mammalian.

58. The method of any one of claims 48-57, wherein the cell is human.

59. The method of any one of claims 48-58, wherein the cell is ex vivo.

Technical Field

Some embodiments of the methods and compositions provided herein relate to chimeric cytokine receptors. In some embodiments, a chimeric cytokine receptor may include IL-7 tethered to an extracellular IL-7 receptor domain and an intracellular IL-21 receptor domain linked to the extracellular IL-7 receptor domain. In some embodiments, T cells comprising a chimeric cytokine receptor can be readily activated and/or expanded in the absence of an exogenous cytokine.

Background

In cell-based adoptive immunotherapy, T cells isolated from a patient may be modified to express synthetic proteins that enable the cells to perform new therapeutic functions after subsequent transfer back into the patient. Examples of such synthetic proteins are Chimeric Antigen Receptors (CARs) and engineered T Cell Receptors (TCRs). Examples of currently used CARs are fusions of an extracellular recognition domain (e.g., an antigen binding domain), a transmembrane domain, and one or more intracellular signaling domains. Upon antigen engagement, the intracellular signaling portion of the CAR can initiate a response in the immune cell associated with activation, e.g., release of a cytolytic molecule to induce tumor cell death.

CAR T cell therapy can be improved in preclinical models by supplementation with gamma chain cytokines (soluble factors that promote T cell growth and survival). However, systemic administration of cytokines to patients is not a therapeutically viable solution, as clinical trials have shown that this approach leads to toxic side effects.

Disclosure of Invention

Some embodiments of the methods and compositions provided herein include polynucleotides encoding a chimeric cytokine receptor polypeptide, wherein the chimeric cytokine receptor polypeptide comprises: IL-7 tethered to the extracellular IL-7 receptor domain; a transmembrane domain; and an intracellular IL-21 receptor domain, wherein the transmembrane domain connects the extracellular IL-7 receptor domain to the intracellular IL-21 receptor domain.

Some embodiments include: a first nucleic acid encoding IL-7; a second nucleic acid encoding a tether (tether); a third nucleic acid encoding an extracellular IL-7 receptor domain, wherein the IL-7 is linked to the extracellular IL-7 receptor domain by the tether; a fourth nucleic acid encoding a transmembrane domain; and, a fifth nucleic acid encoding an intracellular IL-21 receptor domain.

In some embodiments, the tether has a length of from 3 amino acids to 30 amino acids.

In some embodiments, the transmembrane domain comprises an IL-7 receptor transmembrane domain.

In some embodiments, the first nucleic acid comprises a nucleotide sequence identical to SEQ ID NO: 02 nucleotide sequences having at least 95% identity. In some embodiments, the first nucleic acid comprises SEQ ID NO: 02 nucleotide sequence.

In some embodiments, the second nucleic acid comprises a nucleotide sequence identical to SEQ ID NO: 03 having at least 95% identity thereto. In some embodiments, the second nucleic acid comprises SEQ ID NO: 03.

In some embodiments, the third nucleic acid comprises a nucleotide sequence identical to SEQ ID NO: 04 has a nucleotide sequence of at least 95% identity. In some embodiments, the third nucleic acid comprises SEQ ID NO: 04.

In some embodiments, the third nucleic acid and the fourth nucleic acid together comprise a nucleotide sequence identical to SEQ ID NO: 04 has a nucleotide sequence of at least 95% identity. In some embodiments, the third nucleic acid and the fourth nucleic acid together comprise SEQ ID NO: 04.

In some embodiments, the fifth nucleic acid comprises a nucleotide sequence identical to SEQ ID NO: 05 nucleotide sequence having at least 95% identity. In some embodiments, the fifth nucleic acid comprises SEQ ID NO: 05.

Some embodiments further comprise an inducible promoter. Some embodiments further comprise an inducible cytotoxic gene. In some embodiments, the cytotoxic gene encodes a protein selected from the group consisting of: thymidine kinase, thymidine kinase fused to thymidylate kinase, oxidoreductase, deoxycytidine kinase, uracil phosphoribosyltransferase, cytosine deaminase, or cytosine deaminase fused to uracil phosphoribosyltransferase. In some embodiments, the cytotoxic gene comprises thymidine kinase. In some embodiments, the thymidine kinase enzyme comprises SR39 TK.

Some embodiments further comprise a ribosome skipping sequence. In some embodiments, the ribosome skipping sequence comprises a T2A skipping sequence.

In some embodiments, the polynucleotide encodes a transduction marker. In some embodiments, the marker comprises truncated CD19(CD19 t).

Some embodiments of the methods and compositions provided herein include a vector comprising any one of the foregoing polynucleotides. In some embodiments, the vector comprises a viral vector. In some embodiments, the vector is selected from a lentiviral vector, an adeno-associated viral vector, or an adenoviral vector. In some embodiments, the vector comprises a lentiviral vector.

Some embodiments of the methods and compositions provided herein include polypeptides encoded by any of the foregoing polynucleotides.

Some embodiments of the methods and compositions provided herein include cells comprising any of the foregoing polynucleotides or proteins encoded by any of the foregoing polynucleotides.

Some embodiments further include a polynucleotide encoding a Chimeric Antigen Receptor (CAR) or a CAR protein.

In some embodiments, the cell is a T cell. In some embodiments, the cell is a CD8+ T cell or a CD4+ T cell. In some embodiments, the cell is derived from a CD8+ T cell or a CD4+ T cell. In some embodiments, the cell is a primary cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is ex vivo.

Some embodiments of the methods and compositions provided herein include methods of treating or ameliorating cancer in a subject, the methods comprising: administering any of the foregoing cells to a subject in need thereof.

In some embodiments, the treatment or amelioration of cancer lacks co-administration of a cytokine to the subject.

Some embodiments further comprise co-administering a cytokine to the subject, wherein the dose of the cytokine administered is reduced compared to the dose of the cytokine co-administered to a subject that has been administered a cell comprising a CAR but lacking the polynucleotide of any one of claims 1-23 or the protein encoded by the polynucleotide.

In some embodiments, the cytokine is selected from IL-7, IL-15 or IL-21. In some embodiments, the cytokine comprises IL-21.

In some embodiments, the cells are autologous to the subject.

In some embodiments, the cancer comprises a solid tumor, such as colon cancer, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, melanoma, renal cancer, pancreatic cancer, brain cancer, glioblastoma, neuroblastoma, medulloblastoma, sarcoma, bone cancer, or liver cancer, or a non-solid tumor, such as leukemia or multiple myeloma. In some embodiments, the cancer comprises a brain cancer.

In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

Some embodiments of the methods and compositions provided herein include a method of making a population of cells comprising a Chimeric Antigen Receptor (CAR), the method comprising: (a) transducing a T cell with a polypeptide encoding a chimeric receptor, wherein the polypeptide comprises any one of the polynucleotides described above; (b) transducing the T cell with a polynucleotide encoding a CAR; and (c) culturing the transduced T cells under conditions sufficient to stimulate activation and expansion of said T cells, wherein the culture medium comprises a reduced amount of exogenous cytokine as compared to an amount sufficient to stimulate activation and expansion of T cells lacking the polynucleotide encoding the chimeric receptor.

Some embodiments of the methods and compositions provided herein include a method of making a population of cells comprising a Chimeric Antigen Receptor (CAR), the method comprising: (a) transducing T cells with any of the foregoing polynucleotides; (b) transducing the T cell with a polynucleotide encoding a CAR; and (c) culturing the transduced T cells under conditions that stimulate activation and expansion of said T cells, wherein the culture medium lacks exogenous cytokines.

In some embodiments, step (b) is performed before step (a).

In some embodiments, step (a) and step (b) are performed simultaneously.

In some embodiments, the cytokine is selected from IL-7, IL-15 or IL-21. In some embodiments, the cytokine comprises IL-21.

In some embodiments, the cell is a CD8+ T cell or a CD4+ T cell. In some embodiments, the cell is derived from a CD8+ T cell or a CD4+ T cell. In some embodiments, the cell is a primary cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is ex vivo.

Drawings

FIG. 1 depicts a schematic of a chimeric cytokine receptor (CCRIL21) for IL-21 signaling in T cells. Fig. 1 (left panel) depicts the protein design of CCRIL21, while fig. 1 (right panel) depicts exemplary functions of CCRIL 21.

Figure 2A depicts a schematic of a lentiviral construct encoding CCRIL 21.

Fig. 2B depicts FACS analysis of CCRIL21 expressing CD8+ T cells purified after transduction using CD 19-specific magnetic sorting.

Figure 3 depicts an analysis of CD8+ T cells for the presence of phosphorylated STAT3(pSTAT3) or phosphorylated STAT5(pSTAT 5).

Figure 4A depicts a graph showing the percentage of T cells contacted with exogenous IL-21 or cells that contain the chimeric cytokine receptor CCRIL21 that progress through the cell cycle, with error bars illustrating standard deviations.

Figure 4B depicts a graph showing the percentage of cells that undergo apoptosis when T cells are contacted with exogenous IL-21 or contain the chimeric cytokine receptor CCRIL 21.

Figure 5A depicts a graph showing the percentage of tumor cells lysed by T cells containing 806CAR, or T cells containing 806CAR and the chimeric cytokine receptor CCRIL 21.

Fig. 5B depicts FACS analysis for the presence of LAMP-1, GZMB, or PRF.

Figure 6 depicts a graph showing the percentage of on-target and off-target (off-target) tumor cells by T cells containing 806CAR, or T cells containing 806CAR and the chimeric cytokine receptor CCRIL 2.

FIG. 7 depicts an analysis of the presence of BATF or T-beta.

Figure 8A depicts a graph of tumor burden (as measured by luminescence) over time for mice administered with 806 CAR-containing T cells or 806CAR and CCRIL 21-containing T cells.

Figure 8B depicts a Kaplan Meier graph showing the percent survival of tumor-bearing mice administered cells containing 806CAR or T cells containing 806CAR and CCRIL21 as a function of time.

Figure 9A shows a data plot demonstrating T-cell priming (primed) of B7H3CAR expressing CCRIL21 to increase cytotoxicity against tumor cells (example 8).

Figure 9B shows a bar graph of data demonstrating that CAR T cells expressing CCRIL21 were primed to increase cytotoxicity due to increased effector protein expression (example 9).

Fig. 10 shows a bar graph of data demonstrating that CCRIL21 regulates effector function through a key transcription factor (example 10).

Detailed Description

Successful adoptive T cell therapy requires robust expansion and sustained persistence of the T cells administered, and environmental signals received by T cells contribute greatly to these behaviors. In preclinical models, Chimeric Antigen Receptor (CAR) T cell therapy can be improved by supplementing therapy with gamma chain cytokines, which are soluble factors that promote T cell growth and survival. However, systemic administration of cytokines to patients is not a therapeutically viable solution, as clinical trials indicate that such interventions result in toxic side effects (Jeught v, et al, (2014) Oncotarget 6: 1359-81). To confer the benefits of cytokine supplementation to CAR T cell therapy without causing systemic toxicity, a panel of chimeric cytokine receptors that provide constitutive interleukin signaling intrinsic to T cells has been engineered. Chimeric cytokine receptors reproduce the signaling events of specific gamma chain cytokines. In addition, CAR T cells containing certain chimeric cytokine receptors have unexpectedly enhanced activity.

CAR T cells expressing chimeric cytokine receptors were subjected to continuous tumor challenge in vitro and the T cell group was examined for the ability to survive, proliferate and eliminate tumor cells. It was found that CAR T cells expressing chimeric cytokine receptors exhibit increased proliferation and survival in the absence of exogenous cytokines. In addition, specific chimeric cytokine receptors have distinct effects on T cell mitosis and cytotoxicity, and these effects are traced back to cytokine-specific regulation of key transcription factors. In addition, a glioblastoma in situ xenograft mouse model was used to demonstrate that CAR T cells expressing these chimeric cytokine receptors showed significantly improved efficacy in vivo.

Three signals are required for optimal T cell activation and expansion: t cell receptor activation, co-stimulation and stimulatory cytokines. In CAR T cells, CARs can provide the first two signals, but the third signal is still dependent on environmental/exogenous cytokines, which may be rare in the tumor microenvironment. Although cytokine supplementation may improve the efficacy of CAR T cell therapy, systemic cytokine administration results in toxicity in clinical trials. It would be of clinical value to develop methods of selectively providing a third signal to CAR T cells without or with reduced systemic toxicity.

In some embodiments of the methods and compositions provided herein, the CAR T cells are engineered to comprise a chimeric receptor for interleukin 21 (CCRIL 21). CCRIL21 provides stimulatory cytokine signaling to CAR T cells and improves the efficacy of CAR T cell therapy. Figure 1 depicts CCRIL21 in T cells and shows the protein design (left panel) comprising a chimeric receptor protein comprising the extracellular and transmembrane domains of the IL-7 receptor and the intracellular domain of the IL-21 receptor, linked to a tethered IL-7 cytokine by a flexible linker. Figure 1 (right panel) shows an exemplary function of CCRIL21, where tethered IL-7 complexes with endogenous gamma chain receptors and receptor dimerization activates intracellular signaling through the IL-21 receptor domain.

In vitro primary human T cell data demonstrated CD8+ T cells expressing CCRIL 21: induce signaling downstream of IL-21 signaling, promote cytokine-independent proliferation and survival, and confer increased cytotoxicity and effector function. In vivo glioblastoma in situ data from mice demonstrate that CCRIL21 CAR T cells significantly improved tumor clearance and promoted overall survival compared to CAR T cells without CCRIL 21.

Term(s) for

Terms in the present disclosure should be given their ordinary and customary meaning when read in light of the specification. The terms used will be understood by those skilled in the art based on the entire specification.

As used herein, "a" or "an" (a and an) may mean one or more than one.

As used herein, the term "about" means a value that includes inherent variations in error of the method used to determine the value, or variations that exist between experiments.

As used herein, a "chimeric receptor" may include a receptor designed to be synthetic, comprising a ligand binding domain of an antibody or other protein sequence that binds a molecule associated with a disease or disorder, and is preferably linked to one or more intracellular signaling domains (e.g., a co-stimulatory domain) of a T cell or other receptor through a spacer domain. Chimeric receptors may also be referred to as artificial T cell receptors, chimeric immunoreceptors, or Chimeric Antigen Receptors (CARs).

As used herein, a "chimeric cytokine receptor" may include a receptor designed to be synthetic, comprising a cytokine tethered to the extracellular domain of a cytokine receptor polypeptide, a transmembrane domain, and an intracellular cytokine receptor domain linked to the extracellular domain of the cytokine receptor polypeptide by the transmembrane domain. In some embodiments, the cytokine may be selected from a type I cytokine receptor, such as IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, or IL-21. In some embodiments, the extracellular domain of a cytokine receptor polypeptide can be derived from a type I cytokine receptor, such as IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, or IL-21. In some embodiments, the transmembrane domain may be derived from a type I cytokine receptor, such as IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, or IL-21. In some embodiments, the intracellular domain of a cytokine receptor polypeptide can be derived from a type I cytokine receptor, such as IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, or IL-21.

As used herein, "chimeric antigen receptor" (CAR), also referred to as a chimeric T cell receptor, may refer to an artificial T cell receptor that is an engineered receptor that grafts any specificity to immune effector cells. For example, these receptors can be used to graft the specificity of monoclonal antibodies onto T cells; transfer of its coding sequence is facilitated by a retroviral vector or any other suitable gene delivery system. The structure of the CAR may comprise a single chain variable fragment (scFv) derived from a monoclonal antibody fused to the CD 3-zeta transmembrane and endodomain. Such molecules cause transmission of zeta signals in response to recognition of their targets by the scFv. Some alternatives utilize gene delivery vectors with self-inactivating transposase systems. In some alternatives, the gene delivery vector further comprises a sequence of at least one protein. In some alternatives, the protein is a chimeric antigen receptor. The chimeric receptor may also be referred to as an artificial T cell receptor, a chimeric immunoreceptor, and/or a CAR. These CARs are engineered receptors that can graft any specificity onto immune recipient cells. The CAR can include an antibody or antibody fragment, a spacer, a signaling domain, and/or a transmembrane region. However, due to the surprising effect of modifying different components or domains of the CAR, such as an epitope binding region (e.g., an antibody fragment, scFv, or portion thereof), a spacer region, a transmembrane domain, and/or a signaling domain, components of the CAR are described herein in some contexts to include these features as separate elements. For example, changes in different elements of a CAR can result in stronger binding affinity for a particular epitope.

Artificial T cell receptors or CARs can be used as therapies for cancer or viral infection using a technique known as adoptive cell transfer. T cells are removed from a subject and modified so that they express receptors specific for molecules displayed on cancer cells or viruses or virus-infected cells. Genetically engineered T cells are reintroduced into a subject and can then recognize and kill cancer cells or virus-infected cells or promote clearance of the virus. In some alternatives, the gene delivery vector may comprise a sequence for a chimeric antigen receptor. In some alternatives, methods of generating engineered multiplexed T-cells (multiplexed T-cells) for adoptive T cell immunotherapy are provided. In its broadest sense, the method may comprise: providing a gene delivery vector according to any one of the alternatives described herein, introducing the gene delivery vector into a T cell, and selecting cells comprising the gene delivery vector, wherein selecting comprises isolating the T cell expressing the phenotype under selection pressure.

T cell co-stimulation is desirable for the development of an effective immune response, and this event occurs during lymphocyte activation. The costimulatory signal is antigen-nonspecific and is provided by the interaction between costimulatory molecules expressed on the membranes of the T cells and antigen-bearing cells. Costimulatory molecules may include, but are not limited to, CD28, CD80, or CD 86. In some alternatives, methods of generating engineered multiplexed T cells for adoptive T cell immunotherapy are provided. In some alternatives, the T cell is a chimeric antigen receptor-bearing T cell. In some alternatives, the T cell bearing the chimeric antigen receptor is engineered to express a co-stimulatory ligand. In some alternatives, methods for treating, inhibiting, or ameliorating cancer or a viral infection in a subject are provided. In the broadest sense, the method may comprise administering to the subject T cells according to any of the alternatives described herein. In some of these alternatives, the subject is an animal, such as a livestock or companion animal, while in other alternatives, the subject is a human. In some of these alternatives, T cells carrying the chimeric antigen are engineered to express a costimulatory molecule. In some alternatives, the gene delivery vector comprises a sequence for at least one co-stimulatory molecule. In some alternatives, the gene delivery vector is at least 1kB to 20 kB.

As used herein, "genetic modification" and "genetically modified" can include processes of modifying an organism or cell, such as a bacterium, T cell, bacterial cell, eukaryotic cell, insect, plant, or mammal having genetic material (e.g., nucleic acid) that is altered using genetic engineering techniques. For example, a nucleic acid (e.g., DNA) can be inserted into a host genome by: the genetic material of interest is first isolated and copied using molecular cloning methods, or by synthesizing DNA to generate a DNA sequence, and then inserting the construct into a host organism. Nucleases can also be used to remove or "knock out" genes. Gene targeting is a different technique that uses homologous recombination to alter endogenous genes, and can be used to delete genes, remove exons, add genes, or introduce point mutations.

Genetic modification by transduction is described herein. "transduction" refers to a method of transferring genetic material (e.g., DNA or RNA) to, for example, a cell by means of a vector. Commonly used techniques use viral vectors, electroporation and chemical agents to increase cell permeability. The DNA may be transferred by virus or by viral vectors. Described herein are methods for modifying immune CD4+ and/or CD8+ T cells. For example, to achieve high expression of therapeutic genes and/or to increase the amount of chimeric antigen receptors on the cell surface, T cells are transduced with genetic material encoding a protein or chimeric antigen receptor. For example, T cells can be genetically modified using viruses. For example, viruses commonly used for gene therapy are adenoviruses, adeno-associated viruses (AAV), retroviruses or lentiviruses.

A variety of transduction techniques have been developed that utilize recombinant infectious viral particles to deliver nucleic acids encoding chimeric antigen receptors. This represents the currently preferred mode of T lymphocyte transduction. As described herein, viral vectors for transduction may include viral vectors derived from monkey virus 40, adenovirus, AAV, lentiviral vectors or retrovirus. Thus, gene transfer and expression methods are numerous, but essentially play a role in introducing and expressing genetic material in mammalian cells. Various of the above techniques can be used to transduce hematopoietic or lymphoid cells, including: calcium phosphate transfection, protoplast fusion, electroporation, or infection with recombinant adenoviral, adeno-associated viral, lentiviral, or retroviral vectors. Primary T lymphocytes have been successfully transduced by electroporation as well as by retroviral or lentiviral infection. In this regard, retroviral and lentiviral vectors provide an efficient means of gene transfer into eukaryotic cells (e.g., T cells). Moreover, retroviral or lentiviral integration occurs in a controlled manner and results in the stable integration of one or several copies of new genetic information per cell. Cells can be transduced in situ as described herein.

As used herein, a "vector" or "construct" may include a nucleic acid for introducing a heterologous nucleic acid into a cell, which may also have regulatory elements to provide for expression of the heterologous nucleic acid in the cell. Vectors include, but are not limited to, plasmids, minicircles, yeast and viral genomes. In some embodiments, the vector is a plasmid, a minicircle, a viral vector, DNA, or mRNA. In some embodiments, the vector is a lentiviral vector or a retroviral vector. In some embodiments, the vector is a lentiviral vector. As used herein, "Vpx" may include virion-associated proteins encoded by HIV2 type as well as in some simian immunodeficiency virus strains. Vpx can enhance HIV-2 replication in humans. When used for transfection, lentiviral vectors packaged with Vpx protein can cause increased infection of myeloid cells. In some embodiments, the lentiviral vector is packaged with Vpx protein. As used herein, the "Vpr" protein may refer to the viral protein R, which is a 14kDa protein that plays an important role in regulating nuclear import of HIV-1 pre-integration complexes and is essential for viral replication in non-dividing cells. Non-dividing cells may include, for example, macrophages. In some embodiments, the lentiviral vector may be packaged with a Vpr protein or a Vpr protein portion thereof. In some embodiments, the lentiviral vector is packaged with a viral accessory protein. In some embodiments, the viral accessory protein is selected from the group consisting of Vif, Vpx, Vpu, Nef, and Vpr. These accessory proteins (e.g. vif, Vpx, vpu or nef) interact for example with cellular ligands to act as adapter molecules to redirect the normal function of host factors for virus-specific purposes. HIV Accessory Proteins are described in Strebel et al ("HIV Access Proteins cover Host reactions fans, Curr Opin virol.2013 Dec; 3(6): 10.1016/j.coviro.2013.08.004; which is expressly incorporated herein by reference in its entirety).

As used herein, a "promoter" may include a nucleotide sequence that directs transcription of a structural gene. In some embodiments, the promoter is located in the 5' non-coding region of the gene, near the transcription start site of the structural gene. Sequence elements within a promoter that function in initiating transcription are often characterized by a consensus nucleotide sequence. Without limitation, these promoter elements may include RNA polymerase binding sites, TATA sequences, CAAT sequences, or differentiation specific elements (DSE; McGehe et al, mol. Endocrinol.7:551 (1993); explicitly incorporated herein in their entirety by reference), cyclic AMP response elements (CRE), serum response elements (SRE; Treisman et al, seminer in Cancer biol.1:47 (1990); explicitly incorporated herein in their entirety by reference), Glucocorticoid Response Elements (GRE), or binding sites for other transcription factors, such as CRE/ATF (O' Reilly et al, J.biol.Chem.267:19938 (1992); explicitly incorporated herein by reference), AP2(Ye et al, J.biol.Chem.269:25728 (MRE); incorporated herein by reference in their entirety by reference), SP1, cAMP elements (cAMP et al; see, Inc.; overall accession numbers 3: 1993; see [ see: Octagers et al, CRE et al, Invert et al; Genencor.3; incorporated herein by reference), molecular Biology of The Gene, 4 th edition (The Benjamin/Cummings Publishing Company, Inc. 1987; which is expressly incorporated herein by reference in its entirety), and Lemaigre and Rousseau, biochem.J.303:1 (1994); which is expressly incorporated herein by reference in its entirety). As used herein, a promoter may be constitutively active, repressible, or inducible. If the promoter is an inducible promoter, the rate of transcription increases in response to an inducing agent. Conversely, if the promoter is a constitutive promoter, the rate of transcription is not regulated by an inducing agent. Repressible promoters are also known.

As used herein, "treatment" may refer to both therapeutic treatment and prophylactic or preventative treatment, depending on the context.

As used herein, "ameliorating" with respect to a disorder can refer to alleviating symptoms of the disorder, stabilizing a disease, or preventing the progression of the disorder. For disorders such as cancer, this may include reducing the size of the tumor, reducing the growth or proliferation of cancer cells or the number of cancer cells, removing the tumor completely or partially (e.g., in complete or partial response), stabilizing the disease, preventing the progression of the cancer (e.g., progression free survival), or any other effect on the cancer that a physician deems to be therapeutic.

As used herein, "administering" may refer to a method of introducing a compound or a pharmaceutically acceptable salt thereof or a modified cellular composition into a patient or subject, including but not limited to oral, intravenous, intramuscular, subcutaneous, or transdermal.

As used herein, a "subject" or "patient" can refer to any organism to which embodiments described herein can be used or administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. The subject or patient includes, for example, an animal. In some embodiments, the subject is a mouse, rat, rabbit, non-human primate, or human. In some embodiments, the subject is a bovine, ovine, porcine, equine, canine, feline, primate, or human.

As used herein, "co-administration" may refer to the administration of more than one therapeutic agent in combination with another. Each agent may be administered sequentially or simultaneously such that the agents may be present in the bloodstream of the organism at the same time.

Certain polynucleotides

Some embodiments of the methods and compositions provided herein include polynucleotides encoding chimeric cytokine receptor polypeptides. In some embodiments, the chimeric cytokine receptor polypeptide can comprise: cytokines (e.g., IL-7) tethered to the extracellular and transmembrane domains of type I cytokine receptors (e.g., IL-7 receptor extracellular and transmembrane domains); and the intracellular domain of a type I cytokine receptor (e.g., the IL-21 receptor intracellular domain). In some such embodiments, the transmembrane domain connects the extracellular receptor domain to the intracellular receptor domain. In some embodiments, the chimeric cytokine receptor polypeptide can comprise: IL-7 tethered to the extracellular IL-7 receptor domain; a transmembrane domain; and an intracellular IL-21 receptor domain, wherein the transmembrane domain connects the extracellular IL-7 receptor domain to the intracellular IL-21 receptor domain. Exemplary embodiments of such chimeric cytokine receptors are depicted in fig. 2A. In some embodiments, the IL-7 receptor domain is derived from an IL-7 alpha chain domain.

In some embodiments, the polynucleotide may include a first nucleic acid encoding IL-7. In some embodiments, the polynucleotide may include a second nucleic acid encoding a tether. In some embodiments, the polynucleotide may include a third nucleic acid encoding an extracellular IL-7 receptor domain, wherein IL-7 is linked to the extracellular IL-7 receptor domain by the tether. In some embodiments, the polynucleotide may include a fourth nucleic acid encoding an IL-7 receptor transmembrane domain. In some embodiments, the polynucleotide may include a fifth nucleic acid encoding an intracellular IL-21 receptor domain.

In some embodiments, the polynucleotide may include a first nucleic acid encoding IL-7. In some embodiments, the IL-7 is mammalian, such as human. In some embodiments, the first nucleic acid comprises a nucleotide sequence identical to SEQ ID NO: 02 has a percent identity nucleotide sequence. In some such embodiments, the percent identity is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or is 100%, or is within a range defined by any two of the aforementioned percentages. In some such embodiments, the percent identity is greater than about 95%. In some such embodiments, the percent identity is greater than 95%.

In some embodiments, the polynucleotide may include a second nucleic acid encoding a tether. In some embodiments, the tether may have a length as follows: greater than or equal to 2 amino acids and less than or equal to 50 amino acids, greater than or equal to 5 amino acids and less than or equal to 40 amino acids, greater than or equal to 10 amino acids and less than or equal to 35 amino acids, greater than or equal to 10 amino acids and less than or equal to 30 amino acids, or greater than or equal to 15 amino acids and less than or equal to 25 amino acids. In some embodiments, the second nucleic acid comprises a nucleotide sequence identical to SEQ ID NO: 03 has a percentage identity of the nucleotide sequence. In some such embodiments, the percent identity is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or is 100%, or is within a range defined by any two of the aforementioned percentages. In some such embodiments, the percent identity is greater than about 95%. In some such embodiments, the percent identity is greater than 95%.

In some embodiments, the polynucleotide may include a third nucleic acid encoding an extracellular IL-7 receptor domain, wherein IL-7 is linked to the extracellular IL-7 receptor domain by the tether. In some embodiments, the extracellular IL-7 receptor domain is mammalian, e.g., human. In some embodiments, the third nucleic acid comprises a nucleotide sequence identical to SEQ ID NO: 04 has a nucleotide sequence with a percentage of identity. In some such embodiments, the percent identity is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or is 100%, or is within a range defined by any two of the aforementioned percentages. In some such embodiments, the percent identity is greater than about 95%. In some such embodiments, the percent identity is greater than 95%.

In some embodiments, the polynucleotide may include a fourth nucleic acid encoding a transmembrane domain. In some embodiments, the transmembrane domain comprises an IL-7 receptor transmembrane domain or an IL-21 receptor transmembrane domain. In some embodiments, the transmembrane domain comprises an IL-7 receptor transmembrane domain. In some embodiments, the IL-7 receptor transmembrane domain is mammalian, e.g., human. In some embodiments, the third nucleic acid and the fourth nucleic acid together encode the extracellular IL-7 receptor domain and the IL-7 receptor transmembrane domain, and comprise a sequence identical to SEQ ID NO: 04 has a nucleotide sequence with a percentage of identity. In some such embodiments, the percent identity is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or is 100%, or is within a range defined by any two of the aforementioned percentages. In some such embodiments, the percent identity is greater than about 95%. In some such embodiments, the percent identity is greater than 95%.

In some embodiments, the polynucleotide may include a fifth nucleic acid encoding an intracellular IL-21 receptor domain. In some embodiments, the intracellular IL-21 receptor domain is mammalian, e.g., human. In some embodiments, the fifth nucleic acid comprises a nucleotide sequence identical to SEQ ID NO: 05 has a nucleotide sequence with a percentage of identity. In some such embodiments, the percent identity is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or is 100%, or is within a range defined by any two of the aforementioned percentages. In some such embodiments, the percent identity is greater than about 95%. In some such embodiments, the percent identity is greater than 95%.

In some embodiments, the polynucleotide may further comprise an inducible promoter. In some such embodiments, transcription of the chimeric cytokine receptor can be induced in the presence of certain drugs that interact with an inducible promoter.

In some embodiments, the polynucleotide may also encode an inducible cytotoxic gene. In some such embodiments, transcription of a cytotoxic gene can be induced to induce killing of cells containing the polynucleotide. Examples of cytotoxic genes include genes encoding, for example, the following proteins: thymidine kinase, thymidine kinase fused to thymidylate kinase, oxidoreductase, deoxycytidine kinase, uracil phosphoribosyltransferase, cytosine deaminase, or cytosine deaminase fused to uracil phosphoribosyltransferase. In some embodiments, thymidine kinase is preferred. In some such embodiments, the thymidine kinase enzyme comprises SR39 TK. Examples of nucleotide sequences comprising SR39TK include SEQ ID NO: 07, or a pharmaceutically acceptable salt thereof.

In some embodiments, a polynucleotide may comprise more than one protein-encoding sequence. In some such embodiments, the polynucleotide may comprise a ribosome skipping sequence. Examples of ribosome skipping sequences include the T2A skipping sequence. Examples of nucleotide sequences comprising the T2A hopping sequence include SEQ ID NO: 06.

In some embodiments, the polynucleotide may comprise a nucleic acid encoding a transduction marker. Such markers are useful for identifying and obtaining cells that have been successfully transduced with a polynucleotide and successfully express the encoded marker. Examples of transduction markers include truncated CD19(CD19t) molecules.

Some embodiments of the methods and compositions provided herein include polypeptides encoded by the polynucleotides provided herein.

Carrier

Some embodiments of the methods and compositions provided herein relate to vectors comprising the polynucleotides described herein. In some embodiments, the vector is suitable for or configured for transduction into a cell. In some embodiments, the vector comprises a viral vector. Examples of viral vectors include lentiviral vectors, adeno-associated viral vectors, or adenoviral vectors. In some embodiments, the vector comprises a lentiviral vector.

In some embodiments, the vector can comprise a polynucleotide provided herein encoding a chimeric cytokine receptor and a polynucleotide encoding a CAR.

Cells

Some embodiments of the methods and compositions provided herein include cells comprising a polynucleotide provided herein or a polypeptide encoded by a polynucleotide provided herein. In some embodiments, the cell further contains a polynucleotide encoding a CAR, or a CAR protein. In some embodiments, the cell is a T cell. In some embodiments, the cell is a CD8+ T cell or a CD4+ T cell. In some embodiments, the cell is derived from a CD8+ T cell or a CD4+ T cell. In some embodiments, the cell is a primary cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

Method of treatment

Some embodiments of the methods and compositions provided herein relate to methods of treating or ameliorating cancer in a subject. Some such methods comprise administering a cell provided herein to a subject in need thereof. In some embodiments, the cell comprises a chimeric cytokine receptor or a polynucleotide encoding the chimeric cytokine receptor, and a CAR or a polynucleotide encoding a CAR.

In some embodiments, the cancer may comprise a solid tumor, such as colon cancer, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, melanoma, renal cancer, pancreatic cancer, brain cancer, glioblastoma, neuroblastoma, medulloblastoma, sarcoma, bone cancer, or liver cancer, or a non-solid tumor, such as leukemia or multiple myeloma. In some embodiments, the cancer comprises a brain cancer.

Some of the chimeric cytokine receptors provided herein can reduce or eliminate the need for supplemental therapy to a subject by co-administration of cytokines. For example, administering to a subject CAR T cells that do not contain a chimeric cytokine receptor can include co-administering an exogenous cytokine to further stimulate or activate CAR T cells already administered to the subject. As described herein, T cells comprising a chimeric cytokine receptor provided herein can be provided in a sufficiently stimulated and/or activated state in the absence of an exogenous cytokine or at a significantly reduced dose of an exogenous cytokine compared to T cells not comprising a chimeric cytokine receptor provided herein. In some embodiments, the treatment or amelioration of cancer lacks co-administration of a cytokine to the subject. Some embodiments may further comprise co-administering a cytokine to the subject, wherein the dose of the cytokine is reduced compared to the dose of the cytokine co-administered to a subject administered cells comprising the CAR and lacking the chimeric cytokine receptor provided herein. In some such embodiments, the dose of cytokine co-administered to a subject comprising a CAR and a chimeric cytokine receptor can be reduced by an amount that is greater than or greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or within a range defined by any two of the aforementioned percentages, as compared to the dose of cytokine co-administered to a subject administered cells comprising a CAR and lacking a chimeric cytokine receptor.

In some embodiments, administration of a CAR T cell comprising a chimeric cytokine receptor provided herein improves removal of a tumor from a subject compared to removal of a tumor in a subject administered a CAR T cell lacking a chimeric cytokine receptor provided herein.

In some such embodiments, administration of a CAR T cell comprising a chimeric cytokine receptor provided herein reduces tumor volume in a subject compared to the tumor volume in a subject administered a CAR T cell lacking the chimeric cytokine receptor provided herein. In some such embodiments, the tumor volume in a subject administered CAR T cells comprising a chimeric cytokine receptor provided herein can be reduced by an amount greater than or greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or within a range defined by any two of the aforementioned percentages, as compared to the tumor volume in a subject administered CAR T cells lacking the chimeric cytokine receptor provided herein.

In some such embodiments, administration of a CAR T cell comprising a chimeric cytokine receptor provided herein reduces tumor volume in a subject at a greater rate than the reduction in tumor volume in a subject administered a CAR T cell lacking a chimeric cytokine receptor provided herein.

In some embodiments, administration of a CAR T cell containing a chimeric cytokine receptor provided herein increases the overall survival of a subject compared to the overall survival of a subject administered a CAR T cell lacking the chimeric cytokine receptor provided herein.

In some of the foregoing embodiments, the cytokine is selected from IL-7, IL-15, or IL-21. In some embodiments, the cytokine comprises IL-21. In some embodiments, the cells are autologous to the subject. In some embodiments, the subject is a mammal, e.g., a human.

Method for preparing cell population

Some embodiments of the methods and compositions provided herein relate to methods of making a population of cells comprising a CAR. Some such methods include: (a) transducing a T cell with a polynucleotide encoding a chimeric cytokine receptor provided herein; (b) transducing the T cell with a polynucleotide encoding a CAR; and, (c) culturing the transduced T cells under conditions sufficient to stimulate activation and expansion of said T cells, wherein the culture medium comprises a reduced amount of exogenous cytokine as compared to an amount sufficient to stimulate activation and expansion of T cells lacking the chimeric cytokine receptor provided herein. In some embodiments, step (b) is performed before step (a). In some embodiments, step (a) and step (b) are performed simultaneously.

In some embodiments, the amount of exogenous cytokine sufficient to stimulate activation and expansion of T cells comprising a chimeric cytokine receptor and a CAR provided herein is reduced by more than or more than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or an amount within a range defined by any two of the above percentages, as compared to the amount of exogenous cytokine sufficient to stimulate activation and expansion of T cells comprising a CAR lacking the chimeric cytokine receptor provided herein. In some embodiments, the cytokine is selected from IL-7, IL-15 or IL-21. In some embodiments, the cytokine comprises IL-21. In some embodiments, the cell is a CD8+ T cell or a CD4+ T cell. In some embodiments, the cell is derived from a CD8+ T cell or a CD4+ T cell. In some embodiments, the cell is a primary cell. In some embodiments, the cell is a mammalian cell, e.g., a human cell.

Some embodiments include a method of making a population of cells comprising a Chimeric Antigen Receptor (CAR), the method comprising: (a) transducing a T cell with a polynucleotide encoding a chimeric cytokine receptor provided herein; (b) transducing the T cell with a polynucleotide encoding a CAR; and, (c) culturing the transduced T cells under conditions that stimulate activation and expansion of said T cells. In some embodiments, T cells are grown using media lacking exogenous cytokines. In some embodiments, step (b) is performed before step (a). In some embodiments, step (a) and step (b) are performed simultaneously. In some embodiments, the cytokine is selected from IL-7, IL-15 or IL-21. In some embodiments, the cytokine comprises IL-21. In some embodiments, the cell is a CD8+ T cell or a CD4+ T cell. In some embodiments, the cell is derived from a CD8+ T cell or a CD4+ T cell. In some embodiments, the cell is a primary cell. In some embodiments, the cell is a mammalian cell, e.g., a human cell.

Kit and system

Some embodiments of the methods and compositions provided herein relate to kits for preparing T cell populations. In some embodiments, a kit can comprise a polynucleotide or vector provided herein.

Some embodiments of the methods and compositions provided herein include systems for preparing a population of T cells. In some embodiments, a system can comprise a polynucleotide or vector provided herein. In some embodiments, the system can further comprise a cell provided herein.

Examples

EXAMPLE 1 Process

Chimeric cytokine receptor (CCRIL21) construct design for IL21 signaling. The CCRIL21 construct included a single reading frame encoding the CD19 marker protein, followed by the T2A ribosome skip sequence and the CCRIL21 protein (fig. 2A). Expression is driven by the human elongation factor 1 α (EF1 α) promoter. The CCRIL21 protein includes an extracellular domain that includes the extracellular domain of endogenous IL7R and a transmembrane domain that is tethered to the IL7 cytokine by a flexible linker sequence. On the intracellular side of the transmembrane domain of IL7R is the intracellular domain of endogenous IL 21R. Table 1 lists the nucleic acid elements of the chimeric cytokine receptor for IL21 signaling (CCRIL 21). Each sequence in table 1 was concatenated in the order listed to form the complete CCRIL21 sequence. Table 2 lists additional nucleic acid elements useful with certain embodiments provided herein.

Production and culture of T cells. CD8+ T cells were isolated from human Peripheral Blood Mononuclear Cells (PBMCs) by magnetic activated cell sorting and subjected to bead-based CD3/CD28 stimulation using dynabeads (life technologies). After two days, cells were transduced with lentiviruses to introduce CCRIL21 and/or CAR transgenes. Methotrexate (MTX) was added to CAR-transduced T cell cultures between days 10 and 24 post-stimulation to select CAR-expressing populations. Cells expressing CCRIL21 were enriched by magnetic sorting for CD19 expression 24 days after stimulation (fig. 2B). The sorted population is subjected to a rapid amplification protocol (REP). See, e.g., Wang, x, et al, j.immunother.35,689-701 (2012). MTX was replenished again between day 5 and day 14 of REP. At 14 days post REP, flow cytometry confirmed a purified population of T cells expressing CCRIL21 and/or the CAR construct, and the T cells were frozen for later assay. T cells are thawed and immediately dropped into a second REP and the assay is started after 14 days.

And (4) determining the apoptosis state. Cells were stained with Annexin (Annexin) V and 7-aad (biolegend) to determine viability and apoptotic status. Cells were removed from culture and placed in 96-well round bottom plates, washed twice with BioLegend annexin V staining buffer, stained with annexin V and 7-AAD, washed twice with annexin V buffer, and analyzed by flow cytometry over 12 hours.

Cell cycle assay. The Millipore Muse cell cycle assay kit was used to quantify T cell mitogenic activity. The kit is used for flow cytometry to determine the cell cycle phase of individual cells. Cells were gated into the cell cycle phase based on DNA content by propidium iodide staining.

And (4) determining cytotoxicity. Chromium for target cells⁵¹Loaded and co-cultured with T cell groups at variable rates for 4 hours. By measuring chromium released into the supernatant⁵¹To quantify the lysis of target cells (Cooper, L.J.N., et al, Blood 101,1637-1644 (2003)).

Intracellular cytokine staining. T cells were co-cultured with K562 cells expressing EGFRvIII (K562-vIII) at an effector to target ratio of 2:1 for 6 hours. Two hours after the start of co-culture, a transport inhibitor cocktail was added to prevent release of secreted proteins by T cells. At the end of the co-culture, the cells were stained for surface markers, fixed and permeabilized using the CytoFix/CytoPerm kit (BD), and finally stained intracellularly for secreted proteins.

Phosphorus flow (phosphorus-flow). Phosphorylation status of signaling proteins was assessed by flow Cytometry as described in Krutzik, P.O. et al, Cytometry 55A,61-70 (2003). Concentrated PFA was added directly to the cell culture wells, and the cells were then permeabilized with 70% ethanol. Staining was performed using anti-phostat antibody (BioLegend).

Flow-based cytotoxicity assays with mixed targets. CAR T cells were co-incubated with a mixture of K562 cells expressing CD19T (off-target) and K562 cells expressing EGFRvIII (on-target). After co-incubation, the populations were sorted by flow cytometry and the killing of each target population was assessed using a dye that selectively labeled dead cells.

Mouse studies. The in vivo anti-tumor efficacy of CCR expressing CAR T cells was evaluated using the in situ glioblastoma model according to IACUC approved protocols. Male NSG mice 10-12 weeks old were injected intracranially with 200,000 human U87 cells expressing GFP and firefly luciferase. Following subcutaneous injection of D-luciferin, tumor implantation was assessed using bioluminescent imaging for firefly luciferase luminescence. The mice were then sorted into experimental groups of 4-5 mice each. After 7 days, 1X 10⁶Individual T cells were injected intracranially directly into the tumor injection site. Thereafter, tumor progression in mice was monitored by bioluminescence imaging until day 90.

Example 2CCRIL21 recurrence of IL-21 signaling events

In CD8+ T cells, exogenous IL-21 can bind to the IL-21 receptor complexed with the common gamma chain, and the heterodimeric receptor complex can induce IL-21-related signaling events, including phosphorylation of STAT3 and STAT 5.

Prior to analysis of the presence of phospho-STAT3 and phospho-STAT5 by flow cytometry, primary CD8+ human T cells were cultured for 14 hours without cytokines or with IL-21(10 ng/mL). As shown in figure 3, cells containing CCRIL21 and not cultured with IL-21 exhibited substantially similar levels of phosphorylated STAT3 and STAT5 as control cells cultured without CCRIL21 and with IL-21. Thus, CCRIL21 recapitulates the IL-21 signaling event in primary CD8+ human T cells.

Example 3CCRIL21 mediates cytokine-independent cell cycle progression and survival

To assess the effect of CCRIL21 on T cell proliferation and survival in vitro, CCRIL 21-expressing T cells and mock T cells were cultured in the absence of exogenous cytokines. Cell cycle progression was examined two days later (fig. 4A), and apoptotic status of the cells was examined six days later (fig. 4B). As a control, mock T cells were also cultured with 10ng/mL IL-21.

As shown in FIG. 4A, cells containing CCRIL21 and not cultured with IL-21 had substantially similar proportions of cells undergoing S phase and G2/M phase to control cells not containing CCRIL21 and cultured with IL-21. In addition, the total percentage of cells containing CCRIL21 and not cultured with IL-21 that underwent S and G2/M phases was significantly greater than the total percentage of control cells that underwent S and G2/M phases that were not cultured with IL-21.

As shown in FIG. 4B, the total percentage of cells undergoing apoptosis or dead cells was substantially similar for cells containing CCRIL21 and not cultured with IL-21 and control cells not containing CCRIL21 and cultured with IL-21.

Example 4 CAR T cells expressing CCRIL21 were primed to increase cytotoxicity

In a cytotoxicity assay, the activity of T cells containing an EGFRvIII-targeted CAR (806CAR) or T cells containing 806CAR and CCRIL21 was determined. CAR T cells expressing CCRIL21 showed enhanced cytotoxicity in a 4 hour chromium release assay against the human glioblastoma cell line U87 (fig. 5A).

Protein levels associated with cytotoxicity, including Lamp-1, granzyme B, and perforin, were also determined. Surface presence of Lamp-1 was assessed after 6 hours of co-incubation with K562-EGFRvIII cells, and baseline expression of cytotoxic proteins granzyme B and perforin was assessed by flow cytometry (fig. 5B). Cells expressing CCRIL21 showed higher levels of degranulation protein Lamp-1 (LAMP-1); and cytotoxic proteins such as granzyme b (gzmb) and Perforin (PRF).

Example 5 CCRIL 21-sensitized cytotoxicity was limited to CAR-targeted cells

The cytotoxic activity of the 806 CAR-containing T cells or the 806CAR and CCRIL 21-containing T cells was determined with target cells expressing target (EGFRvIII) or non-target control CD 19T. Cells were co-incubated with a mixture of K562 cells expressing CD19t (off-target) and K562 cells expressing EGFRvIII (on-target). After co-incubation, the populations were sorted by flow cytometry and the killing of each target population was assessed using flow cytometry techniques.

As shown in figure 6, CCRIL21 did not cause increased toxicity to off-target tumor cells, indicating that the increased cytotoxicity facilitated by CCRIL21 was limited to CAR-targeted cells.

Example 6 CCRIL21 regulates effector function through key transcription factors in 806CAR T cells.

T cells containing 806CAR or 806CAR and CCRIL21 were cultured with K562 cells expressing EGFRvIII at a ratio of 1:1 for 24 hours. A culture containing T cells comprising 806CAR only is contacted with IL-21.

The levels of transcription factors, BATF and T-beta were determined (FIG. 7). BATF and T-beta are involved in the acquisition and maintenance of cytotoxic functions. CCRIL21 reproduces the effects of IL-21 on CAR T cells by up-regulating transcription factors including BATF and T-beta. This suggests that CAR T cells expressing CCRIL21 were sensitized to increase cytotoxicity.

Example 7 CCRIL21 expression increases tumor clearance of CAR T cells in vivo

Mice were implanted with intracranial human glioblastoma tumors. After 7 days, mice were given intracranial T cells expressing tumor specific CARs (806 CARs). 806CAR T cells expressing CCRIL21 caused higher tumor removal as measured by bioluminescence imaging (fig. 8A). Furthermore, mice treated with 806CAR T cells expressing CCRIL21 survived significantly longer than mice treated with T cells expressing CAR only (fig. 8B).

TABLE 1

TABLE 2

Example 8 sensitization of B7H3CAR T cells expressing CCRIL21 to increase cytotoxicity to tumor cells

To assess whether CAR T cells expressing CCRIL21 were primed to increase cytotoxicity to tumor cells, CD8+ B7H3CAR T cells were co-cultured with K562 tumor cells at a ratio of 2 tumor cells to each T cell. Tumor cell growth was monitored over the course of one week using an Incucyte S3 live cell fluorescence imager.

As shown by the data in fig. 9A, B7H3CAR T cells were eventually exceeded by tumor cells, but CAR T cells expressing CCRIL21 were able to inhibit tumor growth during the study.

These data demonstrate the sustained cytotoxic ability of CAR T cells expressing CCRIL 21. Furthermore, the results indicate that CCRIL21 enhances cytotoxicity in B7H3CAR T cells as well as in 806CAR T cells.

Example 9B 7H3CAR T cells expressing CCRIL21Cellular sensitization is thereby increased by increased expression of effector proteins Cytotoxicity is added.

To assess whether CAR T cells expressing CCRIL21 were primed to increase cytotoxicity due to increased cytotoxic capacity, CD8+ B7H3CAR T cells were co-cultured with K562 tumor cells prior to intracellular flow cytometry analysis of effector protein expression.

The graph in fig. 9B shows the mean Median Fluorescence Intensity (MFI) in three biological replicates. Error bars reflect standard deviations and were analyzed by one-way ANOVA statistics (n.s.. not significant,. p <0.05,. p < 0.005).

As shown by the data, CAR T cells expressing CCRIL21 exhibited higher levels of cytotoxic proteins, granzyme B, interferon gamma, and perforin, compared to B7H3CAR T cells.

These findings demonstrate that CCRIL21 sensitizes cytotoxic capacity by supporting higher expression levels of effector proteins in B7H3CAR T cells, as found in 806CAR T cells.

Example 10 CCRIL21 regulates effector function through key transcription factors in B7H3CAR T cells.

To determine whether CCRIL21 regulates effector function through a key transcription factor, expression of the transcription factors Tbet and BATF was assessed using intracellular flow cytometry.

The graph in fig. 10 shows the mean Median Fluorescence Intensity (MFI) in three biological replicates. Error bars reflect standard deviations and were subjected to one-way ANOVA statistical analysis (. p <0.01,. p < 0.001). CAR T cells expressing CCRIL21 showed statistically higher levels of Tbet and BATF.

The data indicate that CCRIL21 affects the overall change in gene expression in B7H3CAR T cells by regulating expression of key transcription factors, as in 806CAR T cells.

As used herein, the term "comprising" is synonymous with "including", "containing" or "characterized by" and is inclusive or open-ended and does not exclude additional unrecited elements or method steps.

The above description discloses several methods and materials of the present invention. The present invention allows for modifications to methods and materials, and for variations to manufacturing methods and apparatus. Such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the invention disclosed herein. Therefore, it is not intended that the invention be limited to the particular embodiments disclosed herein, but that the invention cover all modifications and alternatives falling within the true scope and spirit of the invention.

All references cited herein (including but not limited to published and unpublished applications, patents, and literature references) are hereby incorporated by reference in their entirety and thus are part of the present disclosure. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

Sequence listing

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<212> DNA

<213> Artificial sequence

<220>

<223> synthetic; human IL7 cytokine sequence (removal of initiation codon)

<400> 2

gattgtgata ttgaaggtaa agatggcaaa caatatgaga gtgttctaat ggtcagcatc 60

gatcaattat tggacagcat gaaagaaatt ggtagcaatt gcctgaataa tgaatttaac 120

ttttttaaaa gacatatctg tgatgctaat aaggaaggta tgtttttatt ccgtgctgct 180

cgcaagttga ggcaatttct taaaatgaat agcactggtg attttgatct ccacttatta 240

aaagtttcag aaggcacaac aatactgttg aactgcactg gccaggttaa aggaagaaaa 300

ccagctgccc tgggtgaagc ccaaccaaca aagagtttgg aagaaaataa atctttaaag 360

gaacagaaaa aactgaatga cttgtgtttc ctaaagagac tattacaaga gataaaaact 420

tgttggaata aaattttgat gggcactaaa gaacac 456

<210> 3

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic; flexible linker sequence (Glycine 4 serine 1) 2

<400> 3

ggaggcggtg ggagcggagg cggtgggagc 30

<210> 4

<211> 792

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic; human IL7R extracellular and transmembrane domains

<400> 4

atgacaattc taggtacaac ttttggcatg gttttttctt tacttcaagt cgtttctgga 60

gaaagtggct atgctcaaaa tggagacttg gaagatgcag aactggatga ctactcattc 120

tcatgctata gccagttgga agtgaatgga tcgcagcact cactgacctg tgcttttgag 180

gacccagatg tcaacatcac caatctggaa tttgaaatat gtggggccct cgtggaggta 240

aagtgcctga atttcaggaa actacaagag atatatttca tcgagacaaa gaaattctta 300

ctgattggaa agagcaatat atgtgtgaag gttggagaaa agagtctaac ctgcaaaaaa 360

atagacctaa ccactatagt taaacctgag gctccttttg acctgagtgt catctatcgg 420

gaaggagcca atgactttgt ggtgacattt aatacatcac acttgcaaaa gaagtatgta 480

aaagttttaa tgcacgatgt agcttaccgc caggaaaagg atgaaaacaa atggacgcat 540

gtgaatttat ccagcacaaa gctgacactc ctgcagagaa agctccaacc ggcagcaatg 600

tatgagatta aagttcgatc catccctgat cactatttta aaggcttctg gagtgaatgg 660

agtccaagtt attacttcag aactccagag atcaataata gctcagggga gatggatcct 720

atcttactaa ccatcagcat tttgagtttt ttctctgtcg ctctgttggt catcttggcc 780

tgtgtgttat gg 792

<210> 5

<211> 855

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic; human IL21R intracellular domain

<400> 5

agcctgaaaa cacaccctct gtggcggctg tggaagaaaa tctgggccgt gccatctcct 60

gagcggttct tcatgcctct gtacaagggc tgcagcggcg acttcaagaa atgggtcgga 120

gcccctttta ccggcagctc tctggaactt ggaccttgga gccctgaggt gcccagcaca 180

ctggaagtgt acagctgtca ccctcctaga agccccgcca agagactgca gctgacagag 240

ctgcaagagc ctgccgagct ggtggaatct gatggcgtgc ccaagcctag cttctggccc 300

acagctcaga atagcggcgg ctctgcctac agcgaggaaa gagatagacc ctacggcctg 360

gtgtccatcg acaccgtgac agtgctggat gccgagggac cttgtacctg gccttgtagc 420

tgcgaggacg atggctaccc tgctctggat ctggacgctg gccttgagcc ttctccagga 480

ctggaagatc ctctgctgga cgccggaaca accgtgctgt cttgtggctg tgtgtctgcc 540

ggatctcctg gacttggagg ccctctggga agcctgctgg atagactgaa acctcctctg 600

gccgacggcg aagattgggc tggtggactt ccttggggcg gaagatctcc aggcggagtg 660

tctgagtctg aagccggttc tccactggcc ggcctggaca tggatacctt cgattctggc 720

ttcgtgggca gcgactgtag cagccctgtg gaatgcgact tcacaagccc tggcgacgag 780

ggcccaccta gaagctatct gagacagtgg gtcgtgatcc ctccacctct gtctagtcct 840

ggacctcagg cttct 855

<210> 6

<211> 63

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic; T2A

<400> 6

ggcggcggag agggcagagg aagtcttcta acatgcggtg acgtggagga gaatcccggc 60

cct 63

<210> 7

<211> 993

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic; SR39TK gene

<400> 7

atgcccacgc tactgcgggt ttatatagac ggtccccacg ggatggggaa aaccaccacc 60

acgcaactgc tggtggccct gggttcgcgc gacgatatcg tctacgtacc cgagccgatg 120

acttactggc gggtgctggg ggcttccgag acaatcgcga acatctacac cacacaacac 180

cgcctcgacc agggtgagat atcggccggg gacgcggcgg tggtaatgac aagcgcccag 240

ataacaatgg gcatgcctta tgccgtgacc gacgccgttc tggctcctca tatcgggggg 300

gaggctggga gctcacatgc cccgcccccg gccctcacca tcttcctcga ccgccatccc 360

atcgccttca tgctgtgcta cccggccgcg cggtacctta tgggcagcat gaccccccag 420

gccgtgctgg cgttcgtggc cctcatcccg ccgaccttgc ccggcaccaa catcgtgctt 480

ggggcccttc cggaggacag acacatcgac cgcctggcca aacgccagcg ccccggcgag 540

cggctggacc tggctatgct ggctgcgatt cgccgcgttt acgggctact tgccaatacg 600

gtgcggtatc tgcagtgcgg cgggtcgtgg cgggaggact ggggacagct ttcggggacg 660

gccgtgccgc cccagggtgc cgagccccag agcaacgcgg gcccacgacc ccatatcggg 720

gacacgttat ttaccctgtt tcgggccccc gagttgctgg cccccaacgg cgacctgtat 780

aacgtgtttg cctgggcctt ggacgtcttg gccaaacgcc tccgttccat gcacgtcttt 840

atcctggatt acgaccaatc gcccgccggc tgccgggacg ccctgctgca acttacctcc 900

gggatggtcc agacccacgt caccaccccc ggctccatac cgacgatatg cgacctggcg 960

cgcacgtttg cccgggagat gggggaggct aac 993