Chimera cytokine receptor

Abstract

To provide a chimera cytokine receptor.SOLUTION: A chimera cytokine receptor may contain IL-7 moored to an extracellular domain of an IL-7 receptor, and an intracellular domain of IL-21 receptor connected to the extracellular domain of the IL-7 receptor. A T-cell containing a chimera cytokine receptor can be easily activated in the absence of an exogenous cytokine and / or can be easily expanded and propagated.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO PRIORITY AND RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 62 / 802,847, filed February 8, 2019, which is expressly incorporated herein by reference in its entirety.

[0002] Electronic sequence listing This application has been filed via EFS-Web with an electronic Sequence Listing as an ASCII text file. The electronic Sequence Listing was created with the filename SCRI215WOSEQLIST.txt, was last updated on February 3, 2020, and is 5,841 bytes in size. The information set forth in the electronic Sequence Listing is incorporated herein by reference in its entirety.

[0003] Some of the embodiments of the methods and compositions provided herein relate to chimeric cytokine receptors. In some embodiments, the chimeric cytokine receptor may comprise IL-7 tethered to the extracellular domain of the IL-7 receptor and the intracellular domain of the IL-21 receptor linked to the extracellular domain of the IL-7 receptor. In some embodiments, T cells comprising the chimeric cytokine receptor can be easily activated and / or expanded in the absence of exogenous cytokines. [Background technology]

[0004] In cell-based adoptive immunotherapy, T cells isolated from a patient are modified to express synthetic proteins and then reintroduced into the patient to exert a new therapeutic function. Examples of such synthetic proteins are chimeric antigen receptors (CARs) and recombinant T cell receptors (TCRs). One example of a currently used CAR is a fusion protein consisting of an extracellular recognition domain (e.g., an antigen-binding domain), a transmembrane domain, and one or more intracellular signaling domains. Upon antigen binding to the CAR, the intracellular signaling portion of the CAR initiates an activation-associated response (e.g., release of cytolytic molecules) in immune cells, inducing tumor cell death.

[0005] Preclinical models have shown that CAR T cell therapy can be improved by supplementation with cytokines that bind to the gamma chain, soluble factors that promote T cell proliferation and survival. However, systemic administration of cytokines to patients has been shown in clinical trials to result in toxic side effects, making systemic administration of cytokines not a viable therapeutic solution. Summary of the Invention [Means for solving the problem]

[0006] Some embodiments of the methods and compositions provided herein include a polynucleotide encoding a chimeric cytokine receptor polypeptide, the chimeric cytokine receptor polypeptide comprising: IL-7 tethered to the extracellular domain of the IL-7 receptor; A transmembrane domain; and The intracellular domain of the IL-21 receptor Including, The present invention includes a polynucleotide characterized in that the extracellular domain of the IL-7 receptor is linked to the intracellular domain of the IL-21 receptor by the transmembrane domain.

[0007] Some embodiments include a first nucleic acid encoding said IL-7; a second nucleic acid encoding the tether; a third nucleic acid encoding the extracellular domain of the IL-7 receptor to which the IL-7 is linked via the tether; a fourth nucleic acid encoding the transmembrane domain; and A fifth nucleic acid encoding the intracellular domain of the IL-21 receptor. Includes.

[0008] In some embodiments, the tether is between 3 amino acids in length and 30 amino acids in length.

[0009] In some embodiments, the transmembrane domain comprises the transmembrane domain of an IL-7 receptor.

[0010] In some embodiments, the first nucleic acid comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 2. In some embodiments, the first nucleic acid comprises the nucleotide sequence of SEQ ID NO:2.

[0011] In some embodiments, the second nucleic acid comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 3. In some embodiments, the second nucleic acid comprises the nucleotide sequence of SEQ ID NO:3.

[0012] In some embodiments, the third nucleic acid comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 4. In some embodiments, the third nucleic acid comprises the nucleotide sequence of SEQ ID NO: 4.

[0013] In some embodiments, the combined sequence of the third and fourth nucleic acids comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 4. In some embodiments, the combined sequence of the third and fourth nucleic acids comprises the nucleotide sequence of SEQ ID NO: 4.

[0014] In some embodiments, the fifth nucleic acid comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 5. In some embodiments, the fifth nucleic acid comprises the nucleotide sequence of SEQ ID NO:5.

[0015] 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 thymidine kinase, thymidine kinase fused to thymidylate kinase, oxidoreductase, deoxycytidine kinase, uracil phosphoribosyltransferase, cytosine deaminase, and cytosine deaminase fused to uracil phosphoribosyltransferase. In some embodiments, the cytotoxic gene comprises thymidine kinase. In some embodiments, the thymidine kinase comprises SR39TK.

[0016] Some embodiments further comprise a ribosomal skipping sequence. In some embodiments, the ribosomal skipping sequence comprises a T2A skipping sequence.

[0017] In some embodiments, the polynucleotide encodes a transduction marker, hi some embodiments, the transduction marker comprises a truncated CD19 (CD19t).

[0018] Some embodiments of the methods and compositions provided herein include a vector comprising any of the 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, and an adenoviral vector. In some embodiments, the vector comprises a lentiviral vector.

[0019] Some embodiments of the methods and compositions provided herein include a polypeptide encoded by any of the above polynucleotides.

[0020] Some embodiments of the methods and compositions provided herein include a cell comprising any of the above polynucleotides, or a cell comprising a protein encoded by any of the above polynucleotides.

[0021] Some embodiments further comprise a polynucleotide encoding a chimeric antigen receptor (CAR), or a CAR protein.

[0022] 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 an ex vivo cell.

[0023] Some embodiments of the methods and compositions provided herein include a method of treating or alleviating cancer in a subject comprising administering to a subject in need of such treatment or alleviation of cancer any of the cells.

[0024] In some embodiments, treating or alleviating the cancer does not include co-administering a cytokine to the subject.

[0025] Some embodiments further comprise co-administering a cytokine to the subject, the amount of the cytokine being less than the amount of cytokine co-administered to a subject administered cells comprising a chimeric antigen receptor (CAR), which does not comprise the polynucleotide of any one of claims 1-23 or the protein encoded by the polynucleotide.

[0026] In some embodiments, the cytokine is selected from IL-7, IL-15, and IL-21. In some embodiments, the cytokine comprises IL-21.

[0027] In some embodiments, the cells are autologous cells to the subject.

[0028] In some embodiments, the cancer comprises a solid tumor, such as colorectal cancer, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, melanoma, renal cancer, pancreatic cancer, brain cancer, glioblastoma, neuroblastoma, medulloblastoma, sarcoma, bone cancer, liver cancer, etc.; or a non-solid tumor, such as leukemia, multiple myeloma, etc. In some embodiments, the cancer comprises a brain tumor.

[0029] In some embodiments, the subject is a mammal, hi some embodiments, the subject is a human.

[0030] Some embodiments of the methods and compositions provided herein include a method of preparing a cell population comprising a chimeric antigen receptor (CAR), comprising: (a) transducing a T cell with a polypeptide comprising any of the preceding polynucleotides and encoding a chimeric receptor; (b) transducing the T cells with another polynucleotide encoding a chimeric antigen receptor (CAR); and (c) culturing the transduced T cells under conditions sufficient to stimulate activation and expansion of the T cells. Including, The method includes a method in which the medium used for the culture contains an amount of exogenous cytokines that is less than an amount sufficient to stimulate the activation and expansion of T cells that do not contain a polynucleotide encoding the chimeric receptor.

[0031] Some embodiments of the methods and compositions provided herein include a method of preparing a cell population comprising a chimeric antigen receptor (CAR), comprising: (a) transducing a T cell with any of the preceding polynucleotides; (b) transducing the T cells with another polynucleotide encoding a chimeric antigen receptor (CAR); and (c) culturing the transduced T cells in a culture medium that does not contain exogenous cytokines under conditions that stimulate activation and expansion of the transduced T cells. The present invention includes a method comprising the steps of:

[0032] In some embodiments, step (b) is performed before step (a).

[0033] In some embodiments, steps (a) and (b) are performed simultaneously.

[0034] In some embodiments, the cytokine is selected from IL-7, IL-15, and IL-21. In some embodiments, the cytokine comprises IL-21.

[0035] 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 an ex vivo cell. [Brief description of the drawings]

[0036] [Figure 1] 1 is a schematic diagram of a chimeric cytokine receptor for IL-21 signaling in T cells (CCRIL21). The left diagram of FIG. 1 shows the design of the CCRIL21 protein, and the right diagram of FIG. 1 shows the representative functions of CCRIL21.

[0037] [Figure 2A]FIG. 1 is a schematic diagram of a lentiviral construct encoding CCRIL21.

[0038] [Figure 2B] This is the result of FACS analysis of CCRIL21-expressing CD8+ T cells purified using CD19-specific magnetic sorting after transduction.

[0039] [Diagram 3] These are the results of an analysis regarding the presence or absence of phosphorylated STAT3 (pSTAT3) or phosphorylated STAT5 (pSTAT5) in CD8+ T cells.

[0040] [Figure 4A] Graph showing the percentage of cells in cell cycle progression in T cells contacted with exogenous IL-21 or T cells containing a chimeric cytokine receptor (CCRIL21). Error bars indicate standard deviation.

[0041] [Figure 4B] FIG. 1 is a graph showing the percentage (%) of cells that underwent apoptosis when T cells were contacted with exogenous IL-21 and when T cells containing a chimeric cytokine receptor (CCRIL21) were used.

[0042] [Figure 5A] Graph showing the percentage of tumor cells lysed by T cells containing 806CAR or T cells containing 806CAR and a chimeric cytokine receptor (CCRIL21).

[0043] [Figure 5B] Results of FACS analysis for the presence or absence of Lamp-1 (LAMP-1), granzyme B (GZMB) or perforin (PRF).

[0044] [Figure 6]Graph showing the percentage of on-target and off-target tumor cells killed by T cells containing 806CAR or T cells containing 806CAR and a chimeric cytokine receptor (CCRIL2).

[0045] [Figure 7] These are the results of the analysis regarding the presence or absence of BATF or T-bet.

[0046] [Figure 8A] Graph showing the change in tumor burden (measured by luminescence) over time in mice administered T cells containing 806CAR or T cells containing 806CAR and CCRIL21.

[0047] [Figure 8B] Kaplan-Meier curves showing the time course of survival rates in tumor-bearing mice administered T cells containing 806CAR or T cells containing 806CAR and CCRIL21.

[0048] [Figure 9A] Graph of data showing that B7H3CAR T cells expressing CCRIL21 are primed and have enhanced cytotoxicity against tumor cells (Example 8).

[0049] [Figure 9B] 1 is a bar graph of data showing that increased expression of effector proteins in CCRIL21-expressing CAR T cells primes the CAR T cells and enhances their cytotoxicity (Example 9).

[0050] [Figure 10] 1 is a bar graph of data showing that CCRIL21 regulates effector functions through key transcription factors (Example 10). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] Successful adoptive T cell therapy requires that administered T cells have robust expansion and persistence, a behavior that is highly dependent on the signals they receive from the surrounding environment. Preclinical models have shown that chimeric antigen receptor (CAR) T cell therapy can be improved by supplementation with cytokines (soluble factors that promote T cell proliferation and survival) that bind to the gamma chain. However, systemic administration of such cytokines is not a viable therapeutic solution, as clinical trials have shown that systemic administration of cytokines in patients leads to toxic side effects (Jeught V. et al. (2014) Oncotarget 6:1359-81). In light of this, to reap the benefits of combining CAR T cell therapy with cytokine supplementation without causing systemic toxicity, we constructed various chimeric cytokine receptors to provide constitutive interleukin signaling endogenous to T cells. These chimeric cytokine receptors recapitulated the signaling events of specific gamma chain cytokines. Moreover, unexpectedly, CAR T cells containing specific chimeric cytokine receptors were more active.

[0052] CAR T cells expressing chimeric cytokine receptors were used in vitro to investigate the survival, proliferation, and tumor cell elimination of T cell populations through a series of tumor challenges. CAR T cells expressing chimeric cytokine receptors were found to exhibit improved proliferation and survival in the absence of exogenous cytokines. Furthermore, specific chimeric cytokine receptors conferred different effects on the induction of mitosis in T cells and T cell cytotoxicity, and these effects were confirmed by investigating the regulation of key transcription factors by specific cytokines. Furthermore, by using a glioblastoma orthotopic xenograft mouse model, CAR T cells expressing these chimeric cytokine receptors were shown to exhibit significantly improved efficacy in vivo.

[0053] Three signals are required for optimal T cell activation and expansion: T cell receptor activation, costimulation, and stimulatory cytokines. CAR T cells can provide the first two signals by the CAR, but the third signal is dependent on environmental or exogenous cytokines, which may be scarce in the tumor microenvironment. Although cytokine administration can improve the efficacy of CAR T cell therapy, systemic cytokine supplementation has been shown to cause toxicity in clinical trials. Developing a method to selectively provide this third signal to CAR T cells without or with minimal systemic toxicity would be clinically beneficial.

[0054] In some embodiments of the methods and compositions provided herein, CAR T cells are constructed that contain a chimeric receptor for interleukin 21 (CCRIL21). CCRIL21 improves the efficacy of CAR T cell therapy by providing stimulatory cytokine signals to CAR T cells. Figure 1 shows CCRIL21 on T cells. The left diagram shows the protein design, which shows a chimeric receptor protein in which the extracellular and transmembrane domains of the IL-7 receptor and the intracellular domain of the IL-21 receptor are linked to a tethered IL-7 cytokine molecule via a flexible linker. The right diagram of Figure 1 shows a representative function of CCRIL21, in which tethered IL-7 forms a complex with the endogenous gamma chain receptor, thus dimerizing the receptor and activating intracellular signaling through the intracellular domain of the IL-21 receptor.

[0055] In vitro data in primary human T cells showed that CD8+ T cells expressing CCRIL21 induced downstream signaling of IL-21 signaling, promoted proliferation and survival without the need for cytokines, and conferred enhanced cytotoxicity and effector function.In vivo data in mice orthotopically implanted with glioblastoma showed that CCRIL21-expressing CAR T cells significantly improved tumor elimination and prolonged overall survival compared to CAR T cells not expressing CCRIL21.

[0056] Definition of Terms The terms used in this disclosure have their common and ordinary meanings when read in this specification, and a person skilled in the art would understand each term used by reading the entire specification.

[0057] As used herein, "a" or "an" may mean one or more than one.

[0058] As used herein, "about" indicates that the particular numerical value includes the degree of error inherent in the method employed to determine the numerical value or experimental variation.

[0059] As used herein, a "chimeric receptor" refers to a synthetically engineered receptor that includes a ligand binding domain of an antibody or other protein sequence that binds to a molecule associated with a disease or disorder, and the ligand binding domain may include receptors linked, preferably via a spacer domain, to one or more intracellular signaling domains (e.g., costimulatory domains) derived from a T cell receptor or other receptor. Chimeric receptors may also be referred to as artificial T cell receptors, chimeric T cell receptors, chimeric immune receptors, or chimeric antigen receptors (CARs).

[0060] As used herein, a "chimeric cytokine receptor" may include a synthetically engineered receptor comprising a cytokine tethered to an extracellular domain of a cytokine receptor polypeptide, a transmembrane domain, and an intracellular domain of a cytokine receptor linked to the extracellular domain of said cytokine receptor polypeptide via said transmembrane domain. In some embodiments, the cytokine may be selected from cytokines that bind to type I cytokine receptors, such as IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-21, etc. In some embodiments, the extracellular domain of the cytokine receptor polypeptide may be derived from a type I cytokine receptor, such as IL-2 receptor, IL-4 receptor, IL-7 receptor, IL-9 receptor, IL-13 receptor, IL-15 receptor, IL-21 receptor, etc. In some embodiments, the transmembrane domain may be derived from a type I cytokine receptor, such as IL-2 receptor, IL-4 receptor, IL-7 receptor, IL-9 receptor, IL-13 receptor, IL-15 receptor, IL-21 receptor, etc. In some embodiments, the intracellular domain of the cytokine receptor polypeptide may be derived from a type I cytokine receptor, such as the IL-2 receptor, IL-4 receptor, IL-7 receptor, IL-9 receptor, IL-13 receptor, IL-15 receptor, or IL-21 receptor.

[0061] As used herein, "chimeric antigen receptor (CAR)" may refer to an artificial T cell receptor, also known as a chimeric T cell receptor, which is a genetically engineered receptor, capable of grafting any specificity onto an effector immune cell. Chimeric antigen receptors can be used to graft, for example, the specificity of a monoclonal antibody onto a T cell, and the coding sequence of the monoclonal antibody can be easily transferred, for example, by using a retroviral vector or other suitable gene delivery system. The structure of a CAR may include a single chain variable fragment (scFv) from a monoclonal antibody, which is fused to the transmembrane and intracellular domains of CD3 zeta. These molecules induce zeta signaling in response to the scFv being recognized by its target. In some embodiments, a gene delivery vector with a self-inactivating transposase system is utilized. In some embodiments, the gene delivery vector further comprises a sequence encoding at least one protein. In some embodiments, the protein is a chimeric antigen receptor. Chimeric receptors can also be referred to as artificial T cell receptors, chimeric T cell receptors, chimeric immune receptors, and / or chimeric antigen receptors (CARs). CAR is a genetically engineered receptor that can be grafted with any specificity into cells expressing immune receptors. CAR may include an antibody or antibody fragment, a spacer, a signaling domain and / or a transmembrane domain. However, CAR has been modified in various components or domains (e.g., epitope binding regions (e.g., antibody fragments, scFvs or parts thereof), spacers, transmembrane domains and / or signaling domains, etc.), which has resulted in surprising effects, and in some contexts herein, the components of CAR are described as including characteristics as independent individual components. The various components included in CAR have various variations, which, for example, enhance the binding affinity to a particular epitope.

[0062] Artificial T cell receptors, or CARs, can be used as a treatment for cancer or viral infections using a technique called adoptive cell transfer. T cells are taken from a subject and modified to express a receptor specific for a molecule presented by cancer cells, viruses, or virus-infected cells. The genetically modified T cells can recognize and kill cancer cells or virus-infected cells or promote viral clearance, and the genetically modified T cells thus produced are reintroduced into the subject. In some embodiments, the gene delivery vector may include a sequence encoding a chimeric antigen receptor. In some embodiments, a method is provided for producing recombinant multiplex T cells for adoptive T cell immunotherapy. In the broadest sense, the method may include providing a gene delivery vector according to any of the embodiments described herein, introducing the gene delivery vector into T cells, and selecting the cells containing the gene delivery vector, the selection including isolating the T cells expressing a particular phenotype under selective pressure.

[0063] To generate an effective immune response, it is desirable to costimulate T cells, which occurs when lymphocytes are activated. Costimulatory signals are antigen non-specific and are generated by interaction of T cells with costimulatory molecules expressed on the cell membrane of antigen-bearing cells. Costimulatory molecules include, but are not limited to, CD28, CD80, and CD86. In some embodiments, a method is provided for generating recombinant multiplex T cells for adoptive T cell immunotherapy. In some embodiments, the T cells are T cells bearing a chimeric antigen receptor. In some embodiments, the T cells bearing a chimeric antigen receptor are genetically modified to express a costimulatory ligand. In some embodiments, a method is provided for treating, inhibiting, or alleviating cancer or a viral infection in a subject. In the broadest sense, the method may include administering to the subject T cells according to any of the embodiments described herein. In some such embodiments, the subject is an animal, such as a livestock or pet animal, and in other embodiments, the subject is a human. In some such embodiments, the T cells bearing a chimeric antigen are genetically modified to express a costimulatory molecule. In some embodiments, the gene delivery vector comprises a sequence encoding at least one costimulatory molecule, hi some embodiments, the gene delivery vector is at least 1 kb to 20 kb.

[0064] As used herein, "genetically modifying" and "genetic recombination" may include methods of modifying organisms and cells, such as bacteria, T cells, bacterial cells, eukaryotic cells, insects, plants, and mammals, using genetic material (such as nucleic acids) modified using genetic engineering techniques. For example, nucleic acids such as DNA can be inserted into a host genome by first isolating the genetic material of interest using molecular cloning techniques, replicating it to create a DNA sequence, or synthesizing the DNA, and then inserting such resulting constructs into the host organism. Nucleases can be used to delete or "knock out" genes. Gene targeting is another technique for modifying endogenous genes by homologous recombination and can be used for gene deletion, exon removal, gene addition, or point mutation introduction.

[0065] Herein, genetic modification by transduction is described. "Transduction" refers to the method of transferring genetic material, such as DNA or RNA, into cells using vectors. Common techniques use viral vectors, electroporation, or chemical reagents that increase cell permeability. DNA can be transferred by viruses or viral vectors. Herein, methods of modifying immune cells, CD4+ T cells and / or CD8+ T cells, are described. T cells are transduced with genetic material, for example, encoding a specific protein or chimeric antigen receptor, to achieve high expression of a therapeutic gene and / or to increase the amount of chimeric antigen receptor expressed on the cell surface. T cells can be genetically modified, for example, using viruses. Viruses commonly used in gene therapy are, for example, adenoviruses, adeno-associated viruses (AAV), retroviruses, or lentiviruses.

[0066] A variety of transduction techniques have been developed that utilize recombinant infectious viral particles to deliver nucleic acids encoding chimeric antigen receptors. These transduction techniques are currently the preferred approach for transducing T lymphocytes. As described herein, viral vectors used for transduction can include viral vectors derived from Simian Virus 40, adenovirus, adeno-associated virus (AAV), lentiviral vectors, or retroviruses. Thus, there are a variety of gene transfer and expression methods, the essential function of which is to introduce and express genetic material in mammalian cells. Some of the transduction techniques mentioned above can be used to transduce hematopoietic cells or lymphocytes, including calcium phosphate transfection; protoplast fusion; electroporation; or infection with recombinant adenovirus, adeno-associated virus, retrovirus, lentivirus, or retrovirus vectors. Primary T lymphocytes have been successfully transduced by electroporation or infection with retroviruses or lentiviruses. Thus, retroviral or lentiviral vectors can be used to transfer genes into eukaryotic cells (e.g., T cells) very efficiently. Furthermore, retroviral or lentiviral integration can be controlled, and one or several copies of new genetic information can be stably integrated into each cell. Also, as described herein, cells can be transduced in situ.

[0067] As used herein, a "vector" or "construct" may include a nucleic acid used to introduce a heterologous nucleic acid into a cell, and may include various regulatory elements, thereby allowing the 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, minicircle, 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 a virion-associated protein encoded by HIV type 2 and some simian immunodeficiency virus strains. Vpx can enhance the replication of HIV-2 in humans. The use of lentiviral vectors packaged with Vpx protein for transfection can increase the infection rate of myeloid cells. In some embodiments, the lentiviral vector is packaged with Vpx protein. As used herein, "Vpr" protein may refer to Viral Protein R. Vpr is a 14 kDa protein that plays a key role in controlling the nuclear translocation of HIV-1 preintegration complexes and is required for viral replication in non-dividing cells. Non-dividing cells include, for example, macrophages. In some embodiments, the lentiviral vector can be packaged with the Vpr protein or a 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. Such accessory proteins, such as vif, Vpx, vpu, and nef, function as adapter molecules by interacting with intracellular ligands, allowing the virus to exploit the normal functions of host factors for its own purposes.HIV accessory proteins have been reported by Strebel et al. (HIV Accessory Proteins versus Host Restriction Factors, Curr Opin Virol. 2013 Dec; 3(6): 10.1016 / j.coviro.2013.08.004; which is expressly incorporated herein by reference in its entirety).

[0068] As used herein, a "promoter" may include a nucleotide sequence that induces transcription of a structural gene. In some embodiments, the promoter is located in the non-coding region at the 5' end of the gene and is located near the transcription start site of the structural gene. Elements of a promoter sequence that initiate transcription are often characterized by consensus nucleotide sequences. Elements of a promoter sequence include RNA polymerase binding sites, TATA sequences, CAAT sequences, differentiation specific elements (DSEs; McGehee et al., Mol. Endocrinol. 7:551 (1993); expressly incorporated herein by reference in its entirety), cyclic AMP response elements (CREs), serum response elements (SREs; Treisman et al., Seminars in Cancer Biol. 1:47 (1990); expressly incorporated herein by reference in its entirety), glucocorticoid response elements (GREs), and other transcription factor binding sites, such as CRE / ATF (O'Reilly et al., J. Biol. Chem. 267:19938 (1992); expressly incorporated herein by reference in its entirety), AP2 (Ye et al., J. Biol. Chem. 269:25728 (1992); expressly incorporated herein by reference in its entirety), and other transcription factor binding sites. (1994); expressly incorporated herein by reference in its entirety), SP1, cAMP response element binding protein (CREB; Loeken et al., Gene Expr. 3:253 (1993); expressly incorporated herein by reference in its entirety), and octameric factor (for reviews, see Watson et al., eds., Molecular Biology of the Gene, 4th ed. (The Benjamin / Cummings Publishing Company, Inc. 1987; expressly incorporated herein by reference in its entirety) and Lemaigre and Rousseau, Biochem. J. 303:1 (1994) (expressly incorporated herein by reference in its entirety)).In this specification, a promoter may be a constitutively active promoter, a repressible promoter, or an inducible promoter. If a promoter is an inducible promoter, the transcription rate increases in response to an inducer. In contrast, if a promoter is a constitutive promoter, the transcription rate is not regulated by an inducer. Repressible promoters are also known.

[0069] As used herein, "treat," "treating," "treated," or "treatment" can refer to both therapeutic and prophylactic treatment, depending on the context.

[0070] As used herein, the terms "alleviate", "alleviating", "alleviation" or "alleviated" in relation to a disorder may refer to the alleviation of symptoms of the disorder, the stabilization of the disease, or the prevention of the progression of the disorder. In the case of a disorder such as cancer, these terms may include the reduction in tumor size, the inhibition of the growth or proliferation of cancer cells or the reduction in the number of cancer cells, the complete or partial elimination of the tumor (e.g., complete or partial response), the stabilization of the disease, the prevention of the progression of the cancer (e.g., progression-free survival), or other effects on the cancer that are considered by the physician to be therapeutic effects.

[0071] As used herein, "administer," "administering," or "administered" may refer to a method for introducing a compound or a pharma- ceutically acceptable salt thereof, or a recombinant cell composition into a patient or subject, including, but not limited to, oral, intravenous, intramuscular, subcutaneous, or transdermal administration.

[0072] As used herein, a "subject" or "patient" may refer to any organism to which an embodiment described herein may be used or administered, for example, for experimental, diagnostic, prophylactic and / or therapeutic purposes. A subject or patient may include, for example, an animal. In some embodiments, the subject is a mouse, a rat, a rabbit, a non-human primate, or a human. In some embodiments, the subject is a cow, a sheep, a pig, a horse, a dog, a cat, a primate, or a human.

[0073] As used herein, "co-administration" may refer to the administration of two or more therapeutic agents in combination with another therapeutic agent. Each therapeutic agent may be administered sequentially or simultaneously such that they are present in the bloodstream of an organism at the same time.

[0074] Specific Polynucleotides Some of the embodiments of the methods and compositions provided herein include a polynucleotide encoding a chimeric cytokine receptor polypeptide. In some embodiments, the chimeric cytokine receptor polypeptide may comprise a cytokine (such as IL-7) tethered to a portion consisting of an extracellular domain and a transmembrane domain of a type I cytokine receptor (such as the extracellular domain and the transmembrane domain of the IL-7 receptor) and an intracellular domain of a type I cytokine receptor (such as the intracellular domain of the IL-21 receptor). In some such embodiments, the extracellular domain of the type I cytokine receptor is linked to the intracellular domain of the type I cytokine receptor by the transmembrane domain. In some embodiments, the chimeric cytokine receptor polypeptide may comprise IL-7 tethered to the extracellular domain of the IL-7 receptor; a transmembrane domain; and an intracellular domain of the IL-21 receptor, wherein the extracellular domain of the IL-7 receptor is linked to the intracellular domain of the IL-21 receptor by the transmembrane domain. An exemplary embodiment of such a chimeric cytokine receptor is shown in FIG. 2A. In some embodiments, each domain of the IL-7 receptor is derived from each domain of the IL-7 receptor alpha chain.

[0075] In some embodiments, the polynucleotide may comprise a first nucleic acid encoding IL-7. In some embodiments, the polynucleotide may comprise a second nucleic acid encoding a tethering chain. In some embodiments, the polynucleotide may comprise a third nucleic acid encoding an extracellular domain of the IL-7 receptor to which the IL-7 is linked via the tethering chain. In some embodiments, the polynucleotide may comprise a fourth nucleic acid encoding a transmembrane domain of the IL-7 receptor. In some embodiments, the polynucleotide may comprise a fifth nucleic acid encoding an intracellular domain of the IL-21 receptor.

[0076] In some embodiments, the polynucleotide may comprise a first nucleic acid encoding IL-7. In some embodiments, the IL-7 is from a mammal, such as a human. In some embodiments, the first nucleic acid comprises a nucleotide sequence having identity to the nucleotide sequence of SEQ ID NO:2. In some such embodiments, the identity is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or is 100%, or is within a range bounded by any two of the aforementioned percentages. In some such embodiments, the identity is greater than about 95%. In some such embodiments, the identity is greater than 95%.

[0077] In some embodiments, the polynucleotide may comprise a second nucleic acid encoding a tethered strand. In some embodiments, the length of the tethered strand may be 2 to 50 amino acids, 5 to 40 amino acids, 10 to 35 amino acids, 10 to 30 amino acids, or 15 to 25 amino acids. In some embodiments, the second nucleic acid comprises a nucleotide sequence having identity to the nucleotide sequence of SEQ ID NO: 3. In some such embodiments, the identity is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or is 100%, or is within a range bounded by any two of the foregoing percentages. In some such embodiments, the identity is greater than about 95%. In some such embodiments, the identity is greater than 95%.

[0078] In some embodiments, the polynucleotide may include a third nucleic acid encoding an extracellular domain of an IL-7 receptor to which the IL-7 is linked via the tether. In some embodiments, the extracellular domain of the IL-7 receptor is from a mammal, such as a human. In some embodiments, the third nucleic acid includes a nucleotide sequence having identity to the nucleotide sequence of SEQ ID NO:4. In some such embodiments, the identity is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or is 100%, or is within a range bounded by any two of the percentages. In some such embodiments, the identity is greater than about 95%. In some such embodiments, the identity is greater than 95%.

[0079] In some embodiments, the polynucleotide may include a fourth nucleic acid encoding a transmembrane domain. In some embodiments, the transmembrane domain comprises the transmembrane domain of an IL-7 receptor or the transmembrane domain of an IL-21 receptor. In some embodiments, the transmembrane domain comprises the transmembrane domain of an IL-7 receptor. In some embodiments, the transmembrane domain of an IL-7 receptor is from a mammal, such as a human. In some embodiments, the sequence of the third nucleic acid and the fourth nucleic acid together comprises a nucleotide sequence encoding the extracellular domain of an IL-7 receptor and the transmembrane domain of an IL-7 receptor and having identity to the nucleotide sequence of SEQ ID NO: 4. In some such embodiments, the identity is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or is 100%, or is within a range of any two of the percentages. In some such embodiments, the identity is greater than about 95%. In some such embodiments, this identity is greater than 95%.

[0080] In some embodiments, the polynucleotide may include a fifth nucleic acid encoding an intracellular domain of the IL-21 receptor. In some embodiments, the intracellular domain of the IL-21 receptor is from a mammal (such as a human). In some embodiments, the fifth nucleic acid includes a nucleotide sequence having identity to the nucleotide sequence of SEQ ID NO:5. In some such embodiments, the identity is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or is 100%, or is within a range bounded by any two of the percentages. In some such embodiments, the identity is greater than about 95%. In some such embodiments, the identity is greater than 95%.

[0081] In some embodiments, the polynucleotide may further comprise an inducible promoter, hi some such embodiments, transcription of the chimeric cytokine receptor can be induced in the presence of a particular agent that interacts with the inducible promoter.

[0082] In some embodiments, the polynucleotide can further code for an inducible cytotoxic gene. In some such embodiments, the transcription of the cytotoxic gene can be induced to induce the killing of cells containing the polynucleotide. Examples of cytotoxic genes include genes encoding proteins such as thymidine kinase, thymidine kinase fused to thymidylate kinase, oxidoreductase, deoxycytidine kinase, uracil phosphoribosyltransferase, cytosine deaminase, and cytosine deaminase fused to uracil phosphoribosyltransferase. In some embodiments, thymidine kinase is preferred. In some such embodiments, the thymidine kinase comprises SR39TK. An example of a nucleotide sequence comprising SR39TK is the nucleotide sequence of SEQ ID NO:7.

[0083] In some embodiments, the polynucleotide may comprise two or more sequences encoding proteins. In some of such embodiments, the polynucleotide may comprise a ribosomal skip sequence. An example of a ribosomal skip sequence is a T2A skip sequence. An example of a nucleotide sequence comprising a T2A skip sequence is the nucleotide sequence of SEQ ID NO:6.

[0084] In some embodiments, the polynucleotide may include a nucleic acid encoding a transduction marker. Such a marker is useful for identifying and obtaining cells that have been successfully transduced with the polynucleotide and have successfully expressed the marker encoded by the polynucleotide. One example of a transduction marker is the truncated CD19 (CD19t) molecule.

[0085] Some of the embodiments of the methods and compositions provided herein include polypeptides encoded by the polynucleotides provided herein.

[0086] vector Some embodiments of the methods and compositions provided herein relate to a vector comprising the polynucleotide described herein. In some embodiments, the vector is suitable for or configured to transduce cells. In some embodiments, the vector comprises a viral vector. Examples of viral vectors include lentiviral vectors, adeno-associated viral vectors, and adenoviral vectors. In some embodiments, the vector comprises a lentiviral vector.

[0087] In some embodiments, a vector may comprise a polynucleotide encoding a chimeric cytokine receptor and a polynucleotide encoding a CAR, as provided herein.

[0088] cell Some embodiments of the methods and compositions provided herein include a cell comprising a polynucleotide provided herein or a polypeptide encoded by a polynucleotide provided herein. In some embodiments, the cell further comprises 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.

[0089] Treatment method Some of the embodiments of the methods and compositions provided herein relate to a method for treating or alleviating cancer in a subject. Some of such methods include administering to a subject in need of treating or alleviating cancer a cell provided herein. 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 the CAR.

[0090] In some embodiments, the cancer may include solid tumors such as colon cancer, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, malignant melanoma, kidney cancer, pancreatic cancer, brain tumor, glioblastoma, neuroblastoma, medulloblastoma, sarcoma, bone cancer, liver cancer, etc.; or non-solid tumors such as leukemia, multiple myeloma, etc. In some embodiments, the cancer comprises a brain tumor.

[0091] Utilizing some of the chimeric cytokine receptors provided herein reduces or eliminates the need to supplement a subject's therapy with co-administration of cytokines. For example, when CAR T cells that do not comprise a chimeric cytokine receptor are administered to a subject, exogenous cytokines can be co-administered to further stimulate or activate the CAR T cells administered to the subject. As described herein, T cells that comprise a chimeric cytokine receptor provided herein can be provided in a fully stimulated and / or activated state in the absence of exogenous cytokines, or can be provided in a fully stimulated and / or activated state in the presence of exogenous cytokines at a dose that is significantly less than that of T cells that do not comprise a chimeric cytokine receptor provided herein. In some embodiments, treating or alleviating the cancer does not include co-administering a cytokine to the subject. Some embodiments further include co-administering a cytokine to the subject, the dose of which is less than the dose of the cytokine co-administered to a subject administered cells that comprise a chimeric antigen receptor (CAR) and do not comprise a chimeric cytokine receptor provided herein. In some such embodiments, the dosage of a cytokine co-administered to a subject administered cells comprising a CAR and a chimeric cytokine receptor can be less than the dosage of a cytokine co-administered to a subject administered cells comprising a CAR but not a chimeric cytokine receptor, and the difference in dosage can be more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, or more than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100%, or can be within a range bounded by any two of the aforementioned percentages.

[0092] In some embodiments, administration of CAR T cells that comprise a chimeric cytokine receptor provided herein improves tumor elimination from a subject compared to a subject administered CAR T cells that do not comprise a chimeric cytokine receptor provided herein.

[0093] In some such embodiments, administration of CAR T cells comprising a chimeric cytokine receptor provided herein reduces tumor volume in the subject compared to tumor volume in a subject administered CAR T cells that do not comprise a chimeric cytokine receptor provided herein. In some such embodiments, tumor volume in a subject administered CAR T cells comprising a chimeric cytokine receptor provided herein can be reduced compared to tumor volume in a subject administered CAR T cells that do not comprise a chimeric cytokine receptor provided herein, and the difference in tumor volume can be greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, or greater than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100%, or can be a percentage within a range bounded by any two of the percentages.

[0094] In some such embodiments, administering CAR T cells that comprise a chimeric cytokine receptor provided herein reduces tumor volume in a subject at a rate that is faster than the rate of reduction in tumor volume in a subject administered CAR T cells that do not comprise a chimeric cytokine receptor provided herein.

[0095] In some embodiments, administering CAR T cells that comprise a chimeric cytokine receptor provided herein extends the overall survival of a subject compared to the overall survival of a subject administered CAR T cells that do not comprise a chimeric cytokine receptor provided herein.

[0096] In some of the embodiments, the cytokine is selected from IL-7, IL-15, and IL-21. In some embodiments, the cytokine comprises IL-21. In some embodiments, the cells are autologous cells of the subject. In some embodiments, the subject is a mammal, such as a human.

[0097] Method for preparing cell populations Some of the embodiments of the methods and compositions provided herein relate to methods for preparing a cell population comprising a CAR. Some of such methods include: (a) transducing a T cell with a polynucleotide encoding a chimeric cytokine receptor provided herein; (b) transducing the T cells with another polynucleotide encoding a chimeric antigen receptor (CAR); and (c) culturing the transduced T cells under conditions sufficient to stimulate activation and expansion of the T cells. Including, The medium used for the culture is characterized in that it contains an amount of exogenous cytokine that is less than an amount sufficient to stimulate the activation and expansion of T cells that do not contain a chimeric cytokine receptor provided herein. In some embodiments, step (b) is performed before step (a). In some embodiments, steps (a) and (b) are performed simultaneously.

[0098] 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 less than the amount of exogenous cytokine sufficient to stimulate activation and expansion of T cells not comprising a chimeric cytokine receptor and comprising a CAR provided herein, the difference being greater than, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about 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, such as a human cell.

[0099] Some embodiments provide a method of preparing a cell population comprising a chimeric antigen receptor (CAR), comprising: (a) transducing a T cell with a polynucleotide encoding a chimeric cytokine receptor provided herein; (b) transducing the T cells with another polynucleotide encoding a chimeric antigen receptor (CAR); and (c) culturing the transduced T cells under conditions that stimulate activation and expansion of the T cells. The present invention includes a method comprising the steps of: In some embodiments, the culturing of the T cells is performed using a culture medium that does not contain exogenous cytokines. In some embodiments, step (b) is performed before step (a). In some embodiments, steps (a) and (b) are performed simultaneously. In some embodiments, the cytokine is selected from IL-7, IL-15 and 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, such as a human cell.

[0100] Kits and Systems Some embodiments of the methods and compositions provided herein relate to kits for the preparation of T cell populations. In some embodiments, the kits may include a polynucleotide or vector provided herein.

[0101] Some of the embodiments of the methods and compositions provided herein include a system for preparing a T cell population. In some embodiments, the system may include a polynucleotide or vector provided herein. In some embodiments, the system may further include a cell provided herein. EXAMPLES

[0102] Example 1 - Method Design of chimeric cytokine receptor (CCRIL21) constructs for IL21 signaling The CCRIL21 construct contains one reading frame encoding the CD19 marker protein, the T2A ribosomal skip sequence, and the CCRIL21 protein in that order (FIG. 2A). Expression is driven by the human elongation factor 1 alpha (EF1α) promoter. The CCRIL21 protein comprises an extracellular domain that includes the extracellular and transmembrane domains of endogenous IL7R, to which the cytokine IL7 is tethered via a flexible linker sequence. At the intracellular end of the transmembrane domain of IL7R is the intracellular domain of endogenous IL21R. Table 1 provides the nucleic acid sequences of the components of the chimeric cytokine receptor for IL21 signaling (CCRIL21). The sequences in Table 1 are concatenated in the order listed to provide the complete sequence of CCRIL21. Table 2 provides the nucleic acid sequences of additional components useful in certain embodiments provided herein.

[0103] Generation and culture of T cells CD8+ T cells were isolated from human peripheral blood mononuclear cells (PBMCs) by magnetic activated cell sorting and stimulated with CD3 / CD28 beads using Dynabeads (Life Technologies). Two days later, the cells were transduced with CCRIL21 and / or CAR transgenes using lentivirus. From day 10 to day 24 after stimulation, methotrexate (MTX) was added to the culture of CAR-transduced T cells to select the population expressing CAR. Also, on day 24 after stimulation, cells expressing CCRIL21 were enriched by magnetic sorting for the presence or absence of CD19 expression (Figure 2B). The selected population was subjected to the rapid expansion culture method (REP method). For this, see, for example, Wang, X. et al. J. Immunother. 35, 689-701 (2012). MTX was supplemented from day 5 to day 14 of the REP method. On day 14 after initiation of the REP procedure, purified T cell populations expressing CCRIL21 and / or CAR constructs were confirmed by flow cytometry and the T cells were cryopreserved for later assays. After thawing, the T cells were immediately subjected to a second REP procedure and assays were started 14 days later.

[0104] Apoptotic status assay Cells were stained with Annexin V and 7-AAD (BioLegend) to assess viability and apoptosis. Cells were detached from cultures, added to 96-well round-bottom plates, and washed twice with Annexin V staining buffer (BioLegend). Cells were stained with Annexin V and 7-AAD, washed twice more with Annexin V buffer, and analyzed by flow cytometry within 12 hours.

[0105] Cell cycle assay Quantitative assessment of T cell mitotic activity was performed using the Millipore Muse Cell Cycle Analysis kit, which was used to determine the cell cycle stage of individual cells by flow cytometry. Cells were gated and classified into cell cycle stages based on DNA content as determined by propidium iodide staining.

[0106] Cytotoxicity assay Target cells were labeled with chromium 51 and co-cultured with T cells at various ratios for 4 hours. The rate of target cell lysis was quantified by measuring the chromium 51 released into the supernatant (Cooper, LJN et al. Blood 101, 1637-1644 (2003)).

[0107] Intracellular cytokine staining T cells and K562 cells expressing EGFRvIII (K562-vIII) were co-cultured for 6 hours at an effector to target cell ratio of 2:1. Two hours after the start of co-culture, a transport inhibitor cocktail was added to block the release of secreted proteins from T cells. After the end of co-culture, cells were stained for surface markers, fixed and permeabilized using CytoFix / CytoPerm kit (BD), and then stained for intracellular secreted proteins.

[0108] Phosphoflow cytometry Phosphorylation status of signaling proteins was assessed by flow cytometry as described by Krutzik, PO et al., Cytometry 55A, 61-70 (2003). Concentrated PFA was added directly to cell culture wells, followed by permeabilization of cells with 70% ethanol. Staining was performed with anti-phosphorylated STAT antibody (BioLegend).

[0109] Flow cytometric cytotoxicity assay using a mixture of target cells CAR T cells were co-cultured with a mixture of K562 cells expressing CD19t (off-target) and EGFRvIII (on-target). After co-culture, populations were sorted by flow cytometry and the killing rate of each target cell population was assessed using a dye that selectively labels dead cells.

[0110] Experiments with mice The in vivo antitumor efficacy of CAR T cells expressing chimeric cytokine receptors (CCRs) was evaluated using a glioblastoma orthotopic implantation model according to an Institutional Animal Care and Use Committee (IACUC)-approved protocol. 200,000 human U87 cells expressing GFP and firefly luciferase were injected intracranially into 10-12 week-old male NSG mice. Tumor engraftment was confirmed by subcutaneous injection of D-luciferin into the mice and bioluminescence imaging detecting the luminescence of firefly luciferase. Mice were then distributed into multiple experimental groups consisting of 4-5 mice per group. After 7 days, 1 × 10 6 T cells were injected directly into the intracranial tumor injection site, and tumor development was then monitored by bioluminescence imaging for up to 90 days.

[0111] Example 2 - Recapitulation of IL-21 signaling by CCRIL21 In CD8+ T cells, exogenous IL-21 binds to the IL-21 receptor complexed with the common γ chain, and this heterodimeric receptor complex induces IL-21-associated signaling events, including phosphorylation of STAT3 and STAT5.

[0112] Primary CD8+ human T cells were cultured for 14 hours in the absence of cytokines or in the presence of IL-21 (10 ng / mL) and then analyzed for the presence of phosphorylated STAT3 and STAT5 by flow cytometry. As shown in Figure 3, CCRIL21-containing cells cultured in the absence of IL-21 exhibited substantially similar levels of phosphorylated STAT3 and STAT5 as non-CCRIL21-containing control cells cultured in the presence of IL-21. Thus, CCRIL21 was able to recapitulate IL-21 signaling events in primary CD8+ human T cells.

[0113] Example 3 - CCRIL21-mediated cytokine-independent cell cycle progression and cell survival To assess the effect of CCRIL21 on T cell proliferation and survival in vitro, CCRIL21-expressing or Mock T cells were cultured in the absence of exogenous cytokines. Cell cycle progression was examined after 2 days (Figure 4A), and the apoptotic status of cells was examined after 6 days (Figure 4B). As a control, Mock T cells were also cultured in the presence of 10 ng / mL IL-21.

[0114] As shown in Figure 4A, the percentage of cells in S phase and G2 / M phase in CCRIL21-containing cells cultured in the absence of IL-21 was substantially similar to the percentage of cells in S phase and G2 / M phase in control cells without CCRIL21 cultured in the presence of IL-21. Moreover, the combined percentage of cells in S phase and G2 / M phase in CCRIL21-containing cells cultured in the absence of IL-21 was significantly higher than the combined percentage of cells in S phase and G2 / M phase in control cells cultured in the absence of IL-21.

[0115] As shown in Figure 4B, the combined percentage of apoptotic and dead cells was essentially similar in CCRIL21-containing cells cultured in the absence of IL-21 and in non-CCRIL21-containing control cells cultured in the presence of IL-21.

[0116] Example 4 - Priming CAR T cells expressing CCRIL21 enhances their cytotoxicity The activity of T cells containing EGFRvIII-targeting CAR (806CAR) or 806CAR and CCRIL21 was measured by cytotoxicity assays. A 4-h chromium release assay demonstrated enhanced cytotoxicity of CCRIL21-expressing CAR T cells against the human glioblastoma U87 cell line (Figure 5A).

[0117] We also measured the levels of proteins involved in cytotoxicity, including Lamp-1, granzyme B, and perforin. After co-culture of each T cell with K562-EGFRvIII cells for 6 hours, we examined the presence or absence of Lamp-1 on the cell surface and the baseline expression of the cytotoxic proteins granzyme B and perforin by flow cytometry (Figure 5B). Cells expressing CCRIL21 had higher expression of the degranulation protein Lamp-1 (LAMP-1) and the cytotoxic proteins granzyme B (GZMB) and perforin (PRF) than T cells containing 806CAR alone.

[0118] Example 5 - Limited effect of CCRIL21 primed cytotoxicity on CAR target cells The cytotoxic activity of T cells containing 806CAR or T cells containing 806CAR and CCRIL21 was measured using target cells expressing the target (EGFRvIII) or cells expressing CD19t as a non-target control. Each T cell was co-cultured with a mixture of K562 cells expressing CD19t (off-target) and K562 cells expressing EGFRvIII (on-target). After co-culture, cell populations were sorted by flow cytometry and the killing rate of each target population was assessed.

[0119] As shown in Figure 6, cytotoxicity against off-target tumor cells was not enhanced by CCRIL21, indicating that the enhancement of cytotoxicity by CCRIL21 is limited to cells targeted by CAR.

[0120] Example 6 - 806 Regulation of effector function by CCRIL21 through key transcription factors in CAR T cells T cells containing 806CAR or 806CAR and CCRIL21 were cultured with EGFRvIII-expressing K562 cells at a 1:1 ratio for 24 hours. Cultures containing T cells containing only 806CAR were exposed to IL-21.

[0121] The levels of transcription factors BATF and T-bet were measured (Figure 7). BATF and T-bet are involved in the acquisition and maintenance of cytotoxic function. CCRIL21 recapitulated the effect of IL-21 on CAR T cells by upregulating transcription factors such as BATF and T-bet. This result indicated that CCRIL21-expressing CAR T cells were primed and their cytotoxicity was enhanced.

[0122] Example 7 - Expression of CCRIL21 enhances the ability of CAR T cells to eliminate tumors in vivo Human glioblastoma tumors were implanted intracranially in mice. Seven days later, T cells expressing a tumor-specific CAR (806CAR) were administered intracranially to the mice. As measured by bioluminescence imaging, mice administered 806CAR T cells expressing CCRIL21 had a higher tumor elimination rate than mice administered T cells expressing 806CAR alone (Figure 8A). Furthermore, mice administered 806CAR T cells expressing CCRIL21 showed significantly longer survival than mice administered T cells expressing 806CAR alone (Figure 8B). [Table 1] TIFF2025061188000002.tif113161 [Table 2]

[0123] Example 8 - Priming B7H3CAR T cells expressing CCRIL21 enhances their cytotoxicity against tumor cells To assess whether priming CCRIL21-expressing CAR T cells enhanced their cytotoxicity against tumor cells, CD8+ B7H3CAR T cells were co-cultured with K562 tumor cells at a ratio of 1 T cell to 2 K562 tumor cells, and tumor cell growth was monitored over a week using an IncuCyte S3 live-cell fluorescence imaging system.

[0124] As shown by the data in Figure 9A, although B7H3CAR T cells ultimately outnumbered tumor cells, CCRIL21-expressing CAR T cells were able to suppress tumor growth over the course of the study.

[0125] These data demonstrated that CCRIL21-expressing CAR T cells had sustained cytotoxicity. Furthermore, these results demonstrated that CCRIL21 enhanced the cytotoxicity of B7H3 CAR T cells, similar to 806 CAR T cells.

[0126] Example 9 - Enhanced cytotoxicity by priming CCRIL21-expressing B7H3CAR T cells with increased expression of effector proteins To assess whether the enhanced cytotoxic potential of CCRIL21-expressing CAR T cells primes the CAR T cells to enhance their cytotoxicity, effector protein expression was analyzed by intracellular flow cytometry after co-culture of CD8+ B7H3CAR T cells with K562 tumor cells.

[0127] The graph in Figure 9B shows the mean median fluorescence intensity (MFI) of three biological replicates. Error bars indicate standard deviation. Statistical analysis was performed by one-way ANOVA (ns = not significant, *p<0.05, ***p<0.005).

[0128] The data show that CCRIL21-expressing CAR T cells expressed higher levels of the cytotoxic proteins granzyme B, interferon-gamma, and perforin than B7H3CAR T cells.

[0129] These results demonstrated that, similar to 806CAR T cells, CCRIL21 enhanced the cytotoxicity of B7H3CAR T cells by promoting high expression of effector proteins.

[0130] Example 10 - Regulation of effector function by CCRIL21 through key transcription factors in B7H3CAR T cells To determine whether CCRIL21 regulates effector functions through key transcription factors, the expression of the transcription factors Tbet and BATF was assessed by intracellular flow cytometry.

[0131] The graph in Figure 10 shows the mean median fluorescence intensity (MFI) of three biological replicates. Error bars indicate standard deviation. Statistical analysis was performed by one-way ANOVA (**p<0.01, ****p<0.001). Statistically higher levels of Tbet and BATF were observed in CAR T cells expressing CCRIL21.

[0132] These data demonstrated that, similar to 806CAR T cells, CCRIL21 also globally altered gene expression in B7H3CAR T cells by regulating the expression of key transcription factors.

[0133] As used herein, the term "comprising" is synonymous with the terms "including," "containing," or "characterized by" and is open-ended and inclusive and does not exclude additional elements or steps not described herein.

[0134] The foregoing description discloses some methods and materials of the present invention. The methods and materials of the present invention may be modified, as may the manufacturing methods and equipment. Such modifications will be readily apparent to those of skill in the art in light of this disclosure or practice of the invention disclosed herein. Therefore, the present invention is not limited to the specific embodiments disclosed herein, but will encompass all modifications and other aspects that fall within the true scope and spirit of the invention.

[0135] All references cited herein, including but not limited to published patent applications, unpublished patent applications, patents, and journal articles, are incorporated herein by reference in their entirety and made a part of this specification. In the event that any reference, patent, or patent application incorporated by reference conflicts with the disclosure of this specification, the disclosure of this specification will govern and / or take precedence over such conflicting matter.

Claims

[Claim 1] 1. A polynucleotide encoding a chimeric cytokine receptor polypeptide, the chimeric cytokine receptor polypeptide comprising: IL-7 tethered to the extracellular domain of the IL-7 receptor; A transmembrane domain; and The intracellular domain of the IL-21 receptor Including, A polynucleotide, wherein the transmembrane domain links the extracellular domain of the IL-7 receptor to the intracellular domain of the IL-21 receptor.

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