Chimeric antigen receptor-modified regulatory T cells for treating cancer

Abstract

The present disclosure provides a CAR regulatory T cell. Further provided herein is a method for treating cancer, such as a solid cancer, comprising administering an effective amount of a CAR regulatory T cell. TIFF2025515324000006.tif82170

Description

[Technical field]

[0001] Claiming priority This application claims the benefit of priority to U.S. Provisional Application No. 63 / 336,137, filed April 28, 2022, the entire contents of which are incorporated herein by reference.

[0002] 1. Field The present disclosure relates generally to the field of molecular biology. More specifically, the present disclosure relates to regulatory T cells having chimeric antigen receptors and methods of use thereof. [Background technology]

[0003] 2. Related Technologies The adaptive immune system has evolved to specifically recognize and destroy a virtually limitless variety of pathogens while remaining unresponsive to self-tissues, a state known as immune tolerance. Regulatory T cells (T reg ) plays a central role in the induction and maintenance of tolerance (1). reg Manipulation of antigen-specific T cells may reverse tolerance to treat autoimmunity and organ transplant rejection. Preclinical studies have demonstrated that antigen-specific T cells reg It has been shown that T cells can reverse autoimmune diabetes in mice (6). reg Negligible abundance of and T during long-term expansion reg The instability of T reg This has hindered the implementation of T-cell-based adoptive cell therapy. reg However, the antigens recognized by H. pylori remain largely unknown, hindering progress in the field.

[0004] Chimeric antigen receptor (CAR) technology has greatly advanced the generation of antigen-specific T cells for cancer therapy. CARs are synthetic receptors that contain an extracellular antigen-binding domain and an intracellular signaling domain, allowing T cell activation by a selected antigen. CAR T cell therapy, approved by the FDA to treat B cell malignancies, has revolutionized cancer treatment by producing higher remission rates than any previously approved drug (7). CARs can be engineered to redirect Tregs to specific target antigens. For example, for type 1 diabetes, an autoimmune disorder in which insulin-producing β cells in the pancreas are destroyed by autoreactive T cells, CARs can be used to directly target Tregs to inflamed islets.

[0005] Importantly, T reg are also emerging targets in cancer immunotherapy. reg T cells migrate from the peripheral blood to the tumor microenvironment, where they accumulate, and are one of the barriers to cancer immunotherapy (2, 5). Notably, cytotoxicity is mediated by T cells to suppress immune responses. reg For example, both granzyme B and perforin are involved in optimal T cell proliferation and motility in tumors by directly eliminating either antigen-presenting cells (APCs) or CD8+ T cells and NK cells. reg has been shown to be required for inhibition ( 8 – 10 ).

[0006] Conventional T cells cannot enter solid tumors or function once in the tumor microenvironment, but T reg CAR T cells migrate to and proliferate in solid tumors (2, 5), and thus CAR T cells that directly recognize solid tumor cells may be useful. reg Generating tumor-targeting CAR T cells can greatly improve engineered immune cell therapy for cancer. reg There is an unmet need to determine the mechanisms behind the inflammatory and cytotoxic properties of . Summary of the Invention

[0007] overview In certain aspects, the present disclosure provides a method for administering to a subject an effective amount of chimeric antigen receptor (CAR) regulatory T cells (T reg The present invention provides a method of treating cancer in a subject, comprising administering to the subject a

[0008] In some aspects, the CAR comprises a CD28-CD3ζ intracellular domain. In certain aspects, the CAR binds to a tumor-associated antigen. For example, the tumor-associated antigen includes, but is not limited to, CD19, CD20, CD22, B-cell maturation antigen (BCMA), carcinoembryonic antigen (CEA), alpha fetoprotein, CA-125, MUC-1, epithelial tumor antigen, melanoma-associated antigen (MAGE), mutant p53-derived peptide-HLA, mutant ras-derived peptide-HLA, HER2 / Neu, ERBB2, folate-binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123 (IL3RA), CD319, CD23, CD30, CD56, c-Met, mesothelin, GD3 , HERV-K, IL-11Rα, κ chain, λ chain, CSPG4, ERBB2, EGFR, EGFRvIII, VEGFR2, TNFRSF17, SDC1, FAP, CD44, MS4A1, EPCAM, CA9, CD174, TNFRSF8, CD33, CD38, EPHA2, CD248 , CD274, CD276, CD5, NCAM1, CD70, ERBB2, KDR, L1CAM, ULBP1, ULBP2, IL1RAP, GPC3, IL13RA2, ROR1, CEACAM5, MET, FOLH1, CSPG4, CD133, GPNMB, or PSCA.

[0009] In certain situations, T reg is a human T reg In some aspects, human T reg CD4 + CD25 高 CD127 低 It was isolated from human peripheral blood by cell sorting.

[0010] In some aspects, the cancer is acute lymphoblastic leukemia (ALL), B-cell leukemia, myeloid leukemia, or epithelial lung cancer. In certain aspects, the cancer is oral cavity cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine cancer, or hematopoietic cancer, glioma, sarcoma, carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, kidney cancer, biliary tract cancer, pheochromocytoma, pancreatic islet cell carcinoma, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteogenic sarcoma tumor, multiple neuroendocrine neoplasia type I and type II, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer.

[0011] In some aspects, the T cells and / or at least one additional therapeutic agent are administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, topically, or by direct injection or perfusion. reg is delivered intravenously or subcutaneously.

[0012] In certain circumstances, CAR T reg The CAR T expresses IFN-γ, TNF-α, perforin, and / or granzyme B. In some aspects, the CAR T reg The CAR T expresses the pro-inflammatory cytokines IFN-γ, IL-3, CXCL9, CXCL11, IL-2, IL-9, IL-17A, CSF3, CCL3, TNFα, and / or IL-6. reg The CAR T expresses the cytolytic proteins granzyme A, granzyme B, perforin 1 (PRF1), NKG7, and / or granzyme H. In certain aspects, the CAR T reg In a specific aspect, CAR T reg are expressed in the presence of FOXP3, CD25, BATF, ICOS, GITR, and / or demethylated T regExpresses differentially demethylated regions (TSDRs).

[0013] In a further aspect, CAR T reg is conjugated to a cytotoxic agent. In some aspects, the cytotoxic agent is a chemotherapeutic agent, IL-2, IL-15, soluble TRAIL, perforin, or granzyme B.

[0014] In some aspects, the method further comprises administering to the subject at least a second anti-cancer therapy. In certain aspects, the second anti-cancer therapy is surgery, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy. In some aspects, the CAR T reg is administered multiple times.

[0015] Further embodiments include T cells engineered to express a CAR construct. regIn some aspects, the CAR construct comprises a tumor-associated antigen antibody or a fragment thereof selected from the group consisting of F(ab')2, Fab', Fab, Fv, and scFv. In certain aspects, the CAR binds to a tumor-associated antigen. For example, the tumor-associated antigen may be, but is not limited to, CD19, CD20, CD22, B-cell maturation antigen (BCMA), carcinoembryonic antigen (CEA), alpha fetoprotein, CA-125, MUC-1, epithelial tumor antigen, melanoma-associated antigen (MAGE), mutant p53-derived peptide-HLA, mutant ras-derived peptide-HLA, HER2 / Neu, ERBB2, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123 (IL3RA), CD319, CD23, CD30, CD56, c-Met, mesothelin, GD3 , HERV-K, IL-11Rα, kappa chain, lambda chain, CSPG4, ERBB2, EGFR, EGFRvIII, VEGFR2, TNFRSF17, SDC1, FAP, CD44, MS4A1, EPCAM, CA9, CD174, TNFRSF8, CD33, CD38, EPHA2, CD248, CD274, CD276, CD5, NCAM1, CD70, ERBB2, KDR, L1CAM, ULBP1, ULBP2, IL1RAP, GPC3, IL13RA2, ROR1, CEACAM5, MET, FOLH1, CSPG4, CD133, GPNMB, or PSCA. reg is autologous. In some cases, T reg In some aspects, T reg In certain aspects, T expresses the pro-inflammatory cytokines IFN-γ, IL-3, CXCL9, CXCL11, IL-2, IL-9, IL-17A, CSF3, CCL3, TNFα, and / or IL-6. reg T express the cytolytic proteins granzyme A, granzyme B, perforin 1 (PRF1), NKG7, and / or granzyme H. In some aspects, T reg secretes IL-10. In some aspects, T regare expressed in the presence of FOXP3, CD25, BATF, ICOS, GITR, and / or demethylated T reg In a specific aspect, the composition expresses a CD8 + In a further aspect, the antibody is essentially free of T cells. reg is conjugated to a cytotoxic agent. In some aspects, the cytotoxic agent is a chemotherapeutic agent.

[0016] In yet another embodiment, the T of this embodiment and aspects thereof reg and a pharmaceutical carrier. reg Also provided herein is a composition comprising:

[0017] Another aspect is a method for the preparation of ... medicament for the treatment of a disease comprising: (a) reg (b) isolating Tregs; (c) introducing a CAR expression construct into the Tregs; reg (d) expanding T reg The method comprises the step of stimulating the CAR T cell with an artificial presentation cell (APC). reg The present invention provides an in vitro method for producing

[0018] In some aspects, the CAR expression construct is a lentiviral vector or a retroviral vector. In certain aspects, the introducing step comprises administering to the subject a CAR expression construct comprising a lentiviral vector or a retroviral vector. regThe method includes contacting the APC with a lentiviral particle comprising a CAR construct. In certain aspects, the at least one cytokine is IL-2. In some aspects, the APC is a gamma-irradiated APC. In certain aspects, the APC is a CD19-K562 cell. In some aspects, the CAR expression construct is a CD19-specific construct. For example, CARs can be any of a variety of antigens, including, but not limited to, CD19, CD20, CD22, B cell maturation antigen (BCMA), carcinoembryonic antigen (CEA), alpha fetoprotein, CA-125, MUC-1, epithelial tumor antigen, melanoma associated antigen (MAGE), mutant p53-derived peptide-HLA, mutant ras-derived peptide-HLA, HER2 / Neu, ERBB2, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123 (IL3RA), CD319, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV In some embodiments, the construct may be a IL-11Rα, κ chain, λ chain, CSPG4, ERBB2, EGFR, EGFRvIII, VEGFR2, TNFRSF17, SDC1, FAP, CD44, MS4A1, EPCAM, CA9, CD174, TNFRSF8, CD33, CD38, EPHA2, CD248, CD274, CD276, CD5, NCAM1, CD70, ERBB2, KDR, L1CAM, ULBP1, ULBP2, IL1RAP, GPC3, IL13RA2, ROR1, CEACAM5, MET, FOLH1, CSPG4, CD133, GPNMB, or PSCA construct.

[0019] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. For example, a compound synthesized by one method can be used in the preparation of a final compound by a different method.

[0020] Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating specific embodiments of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0021] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0022] [Figure 1] Mechanisms of immune suppression by regulatory T cells (Tregs). Tregs use different mechanisms to carry out their immune suppression functions, including: 1) Tregs secrete the anti-inflammatory cytokine IL-10 and induce a tolerogenic state in antigen-presenting cells (APCs). Tolerogenic APCs then interact with CD4+ T effector (Teff) cells and inhibit them from secreting pro-inflammatory cytokines. Furthermore, tolerogenic APCs induce the differentiation of naive T cells into Tregs. 2) IL-2, secreted by Teff cells and essential for their activation and proliferation, is taken up by Treg cells via CD25. Tregs also induce the expression of PD-L1 in APCs, which then induces apoptosis in activated PD-1+ Teff cells via PD-1 / PD-L1 signaling. 3) Tregs induce apoptosis of APCs, e.g., B cells and dendritic cells (DCs), via perforin / granzyme-mediated cytotoxicity. 4) Tregs use the ectoenzymes CD39 and CD73 to convert extracellular ATP to adenosine (ADO), a potent immunosuppressant. ADO binds to and inhibits its receptor in Teff cells.

[0023] [Diagram 2]T cell activation via T cell receptor (TCR) and CD28, and T cell activation via chimeric antigen receptor (CAR). T cells require two signals to be fully activated. Signal 1 is provided by the binding of the T cell receptor (TCR) to its cognate antigen, which is presented on the surface of target cells, e.g., APCs, by major histocompatibility complex (MHC) molecules, also known as human leukocyte antigens (HLA) in humans. Notably, the TCR does not have a signaling motif by itself, but binds to CD3 molecules, namely, CD3δ, CD3ε, CD3γ, and CD3ζ, whose intracellular domains undergo phosphorylation and initiate a signaling cascade. Signal 2, also known as costimulation, is provided by the binding of CD28 on the T cell to CD80 or CD86 on the APC. Chimeric antigen receptors (CARs) are artificial receptors that combine an extracellular antigen-binding domain, typically a single-chain fragment variable (scFv) domain, with an intracellular signaling domain, typically the tandem CD28-CD3ζ. Thus, CARs combine antigen recognition, T cell signaling 1, and T cell signaling 2 in a single molecule.

[0024] [Diagram 3]Figures 3A-3G: Chimeric antigen receptor signaling confers both regulatory and effector cell properties to human Tregs. (Figure 3A) Apoptosis of CD19+NALM6 leukemia cells induced by CD19 CAR Tregs in vitro. Annexin V labels apoptotic cells and DAPI labels dead cells. (Figure 3B) CD19 CAR Treg single cell protein expression of pro-inflammatory cytokines and cytolytic molecules in vitro. (Figure 3C) CD19 CAR Tregs control the growth of luciferase-labeled NALM6 when co-injected in vivo in NSG mice. (Figure 3D) CD19 CAR Tregs suppress the proliferation of CD19 CAR Teff cells in vitro. (Figure 3E) CD19 CAR Tregs retain expression of FOXP3 and HELIOS upon activation by CD19+ tumor cells. (Figure 3F) CD19 CAR Tregs are cytotoxic to GFP-labeled CD19-A549 lung cancer cells in vitro. Green, GFP (CD19-A549 cells); Blue, DAPI (nuclei); Violet, PI (dead cells). (Figure 3G) CD19 CAR Tregs control the growth of luciferase-labeled CD19-A549 when co-injected in vivo in NSG mice.

[0025] [Figure 4]Probing CAR-induced gene expression programs in human Tregs using RNA sequencing. Human CD4+CD25+CD127- Treg and CD4+CD25-CD127+ Teff cells were purified from human peripheral blood, activated, and transduced with CD19CAR-2A-GFP lentivirus. The modified cells were then sorted for purity based on GFP expression and co-incubated with irradiated K562 (non-activated), OKT3-loaded CD64-CD80-K562 (TCR / CD28 activated), or CD19-K562 (CAR activated) at a 1:1 ratio. One day later, CD4+ cells were magnetically purified and used to generate bulk RNA sequencing libraries. Libraries were sequenced and data were analyzed using the Seurat pipeline. Of note, this CD19CAR construct features a CD28-CD3ζ signaling domain, and OKT3 is an agonistic anti-CD3 antibody, while CD80 is the natural agonistic ligand for CD28. Therefore, T cell signal 1 (TCR / CD3) and T cell signal 2 (CD28) were compared between delivery via CAR and endogenous TCR / CD28.

[0026] [Diagram 5]Global gene expression analysis of CAR and TCR / CD28 activated Treg and Teff cells using RNA sequencing. Left: Gene expression heatmap for the 1000 most differentially expressed genes across all 6 conditions (NoAct Treg, CAR Treg, TCR Treg, NoAct Treg, CAR Treg, TCR Treg) measured by RNA-seq. Note that CAR Treg and CAR Teff cluster close to each other (activation mode), while NoAct Treg and TCR Treg and NoAct Teff and TCR Teff cluster together (cell type), indicating that CAR activation imparts the Teff cell gene program to Treg. Right: Gene expression heatmap for the 200 most differentially expressed genes between CAR Treg and TCR Treg. Notable genes upregulated in CAR Tregs compared to TCR Tregs include the cytokine / chemokine genes IL5, CCL4, CSF2, and the transcriptional regulator genes TBX21, IRF8, and ZBED2. NoAct: non-activated.

[0027] [Figure 6] Gene expression differences between CAR-activated and TCR / CD28-activated Tregs. Expression levels of representative genes involved in Treg identity, suppressive function, and cytotoxicity, as well as inflammatory cytokine and chemokine genes. CAR Tregs have similar levels of Treg identity and suppressive function genes as TCR Tregs (except for IL-10 and EBI3, subunits of IL-35, which are higher in CAR Tregs), and also have higher levels of cytotoxicity (GZMB, GZMH, NKG7) and inflammatory cytokine and chemokine genes (e.g., IFNG, IL3, TNF).

[0028] [Figure 7]CAR Tregs upregulate inflammatory genes and associated pathways. Left: Top 20 most upregulated genes between CAR Tregs and NoAct Tregs and between CAR Tregs and TCR Tregs in RNA-seq. Upregulated genes in both pairwise comparisons include IL3, CXCL11, and IFNG. Right: Gene set enrichment analysis (GSEA) of CAR vs. TCR / CD28 activation in Tregs. Top pathways upregulated in CAR Tregs included TNF, IL6, IFNG, and inflammation. FC: fold change; pval: p-value; FDR: false discovery rate.

[0029] [Figure 8] CAR Teff cells upregulate inflammatory genes and related pathways. Left: Top 20 most upregulated genes between CAR Teff and NoAct Teff and between CAR Teff and TCR Teff in RNA-seq. Upregulated genes in both pairwise comparisons include IL2 and CSF2, but not IFNG. Between CAR Teff and NoAct Teff, IFNG is one of the top 20 genes. Right: Gene Set Enrichment Analysis (GSEA) of CAR vs TCR / CD28 activation in Teff cells. Top pathways upregulated in CAR Teff cells included TNF, IL6, and inflammation, but not the IFNG pathway. Other pathways upregulated include apoptosis and p53 pathways. FC: fold change; pval: p-value; FDR: false discovery rate.

[0030] [Figure 9]Gene expression changes upon CAR and TCR / CD28-mediated Treg activation at the whole transcriptome level using RNA-seq. Volcano plots of gene expression for pairwise comparisons between CAR Tregs and NoAct Tregs, TCR Tregs and NoAct Tregs, and CAR Tregs and TCR Tregs. Color code is as follows: red means upregulated, green means downregulated, and black means not statistically significant. FC: fold change.

[0031] [Figure 10] Gene expression changes upon CAR and TCR / CD28-mediated Teff cell activation at the whole transcriptome level using RNA-seq. Volcano plots of gene expression for pairwise comparisons between CAR Teff and NoAct Teff, TCR Teff and NoAct Teff, and CAR Teff and TCR Teff. Color code is as follows: red means upregulated, green means downregulated, and black means not statistically significant. FC: fold change.

[0032] [Figure 11]Probing CAR-induced gene expression programs in human Tregs using single-cell RNA sequencing. Human CD4+CD25+CD127- Treg and CD4+CD25-CD127+ Teff cells were purified from human peripheral blood, activated, and transduced with CD19CAR-2A-GFP lentivirus. The modified cells were then sorted for purity based on GFP expression and co-incubated with irradiated K562 (non-activated), OKT3-loaded CD64-CD80-K562 (TCR / CD28 activated), or CD19-K562 (CAR activated) at a 1:1 ratio. One day later, CD4+ cells were magnetically purified and used to generate 10x Genomics single-cell RNA sequencing libraries. The libraries were sequenced and the data were analyzed using the Seurat pipeline. Of note, this CD19CAR construct features a CD28-CD3ζ signaling domain, and OKT3 is an agonistic anti-CD3 antibody, while CD80 is the natural agonistic ligand for CD28. Therefore, T cell signal 1 (TCR / CD3) and T cell signal 2 (CD28) were compared between delivery via CAR and endogenous TCR / CD28.

[0033] [Figure 12]Single-cell RNA sequencing of activated CD4+ T cells. In total, 153,636 human CD4+ T cells were single-cell sequenced from three independent donors (79,386 Treg and 74,250 Teff cells). Single-cell sequencing data were aligned and processed using the Seurat pipeline. Data were filtered for low mitochondrial gene content and sufficient nUMI (number of unique molecular identifiers), selected for CD4+ T cells, and clustered. A gene expression cluster corresponding to contamination with K562 target cells emerged. This cluster was excluded and the remaining clean data was re-clustered. Left: UMAP (Uniform Manifold Approximation and Projection) graphic with single cells colored by gene expression Seurat clusters (14 clusters) at resolution 0.7. Right: UMAP graphic with single cells colored per condition-CAR Treg, TCR Treg, NoAct Treg, CAR Teff, TCR Teff, NoAct Teff (6 conditions). NoAct: non-activated.

[0034] [Figure 13] Magnitude of CAR-mediated and TCR / CD28-mediated CD4+ T cell activation using single cell RNA sequencing. Activation signatures were computed by combining the top 20 genes expressed in activated CD4+ T cells (CAR and TCR) compared to resting CD4+ T cells (NoAct). Left: Violin plot of activation signature expression scores for CAR CD4+ T cells, TCR CD4+ T cells, and NoAct CD4+ T cells. Right: Top 20 genes upregulated in activated cells (Treg, Teff, total CD4+ T cells) and non-activated cells (Treg, Teff, CD4+ T cells).

[0035] [Figure 14]Magnitude of activation of single cell gene expression CD4+ T cell clusters using single cell RNA sequencing. Activation signatures were computed by combining the top 20 genes expressed in activated CD4+ T cells (CAR and TCR) compared to resting CD4+ T cells (NoAct). Left: Violin plot of activation signature expression scores for single cell clusters in total CD4+ T cells. Right: Percentage of cells per condition (CAR Treg, TCR Treg, NoAct Treg, CAR Teff, TCR Teff, NoAct Teff) in single cell gene expression clusters of CD4+ T cells.

[0036] [Figure 15] Single-cell RNA sequencing of activated Tregs. Tregs were clustered based on single-cell gene expression. The left-most panel is a UMAP plot colored by activation modality (NoAct, CAR, TCR). The middle panel is a UMAP plot colored by single-cell gene expression cluster (0-12). The right-most panel is a stacked bar graph with the percentage of cells per condition in the single-cell gene expression clusters. Clusters 1, 2, 6, and 10 are enriched in NoAct Tregs. Clusters 7 and 9 are enriched in CAR Tregs. Clusters 3, 4, and 11 are enriched in TCR Tregs.

[0037] [Figure 16] Magnitude of CAR- and TCR / CD28-mediated Treg activation using single cell RNA sequencing. Activation signatures were computed by combining the top 20 genes expressed in activated Tregs (CAR and TCR) compared to resting Tregs (NoAct). Left: Violin plot of activation signature expression scores for CAR Tregs, TCR Tregs, and NoAct Tregs. Right: Top 20 genes upregulated in activated (Tregs, Teff, total CD4+ T cells) and non-activated cells (Tregs, Teff, CD4+ T cells).

[0038] [Figure 17] Magnitude of activation of single cell gene expression Treg clusters using single cell RNA sequencing. Activation signatures were computed by combining the top 20 genes expressed in activated Tregs (CAR and TCR) compared to resting Tregs (NoAct). Left: Violin plot of activation signature expression scores versus single cell clusters among total Tregs. Right: Percentage of cells per condition (CAR Treg, TCR Treg, NoAct Treg) in Treg single cell gene expression clusters.

[0039] [Figure 18] Single-cell RNA sequencing of activated Teff cells. Teff cells were clustered based on single-cell gene expression. The left-most panel is a UMAP plot colored by activation modality (NoAct, CAR, TCR). The middle panel is a UMAP plot colored by single-cell gene expression cluster (0-14). The right-most panel is a stacked bar graph with the percentage of cells per condition in the single-cell gene expression clusters. Clusters 4, 5, 6, and 7 are enriched in NoAct Teff cells. Clusters 0, 1, and 10 are enriched in CAR Teff cells. Clusters 2, 3, 8, and 9 are enriched in TCR Teff cells.

[0040] [Figure 19]Magnitude of CAR-mediated and TCR / CD28-mediated Teff cell activation using single cell RNA sequencing. Activation signatures were computed by combining the top 20 genes expressed in activated Teff cells (CAR and TCR) compared to resting Teff cells (NoAct). Left: Violin plot of activation signature expression scores for CAR Teff, TCR Teff, and NoAct Teff. Right: Top 20 genes upregulated in activated cells (Treg, Teff, total CD4+ T cells) and non-activated cells (Treg, Teff, CD4+ T cells).

[0041] [Figure 20] Magnitude of activation of single cell gene expression Teff cell clusters using single cell RNA sequencing. Activation signatures were computed by combining the top 20 genes expressed in activated Teff cells (CAR and TCR) compared to resting Teff cells (NoAct). Left: Violin plot of activation signature expression scores for single cell clusters in total Teff cells. Right: Percentage of cells per condition (CAR Treg, TCR Treg, NoAct Treg, CAR Teff, TCR Teff, NoAct Teff) in Teff cell single cell gene expression clusters.

[0042] [Figure 21] Cytotoxicity gene expression in Tregs activated via CAR or TCR / CD28 at the single cell level. Levels of PRF1, GZMA, GZMB, FASLG, TNFRSF10A, TNF gene expression in UMAP plots of Tregs (NoAct, CAR, TCR).

[0043] [Figure 22]Expression of cytotoxic genes in Tregs activated via CAR or TCR / CD28 at the single cell gene expression cluster level. Cluster 6 (enriched in NoAct Tregs) has the highest GZMA and GZMK levels, cluster 9 (enriched in CAR Tregs) has the highest GMZB, FASLG, GZMH, and NKG7 levels, and cluster 12 (even distribution of NoAct, CAR, and TCR Tregs) has the highest TNF, serpinB9, PRF1, TNFRSF10A, and LAMP1 levels.

[0044] [Diagram 23] CAR Treg cytotoxicity is perforin-dependent. Left: PRF1 CRISPR KO CD19 CAR Tregs showed reduced cytotoxicity against NALM6 cells in vitro. Left: PRF1 CRISPR KO efficiency (59.3%) measured by genomic insertion / deletion frequency in CAR Tregs assessed by TIDE (Tracking of Indels by Decomposition) analysis.

[0045] [Figure 24] Cytokine and chemokine gene expression in Tregs activated via CAR or TCR / CD28 at the single cell level. Levels of gene expression for IFNG, IL5, CCL3, IL3, CSF2, CSF3, IL17A, and IL2 in the UMAP plot of Tregs (NoAct, CAR, TCR). Note that inflammatory cytokine and chemokine gene expression is primarily restricted to the CAR Treg region of the UMAP plot.

[0046] [Diagram 25] Expression of cytokine and chemokine genes in Tregs activated via CAR or TCR / CD28 at the single cell gene expression cluster level. Cluster 9 (enriched in CAR Tregs) has the highest levels of most inflammatory cytokine and chemokine genes.

[0047] [Figure 26] IFNG is expressed in both CAR and TCR Teff cells, but at the single-cell level only in CAR Tregs.

[0048] [Figure 27]Cytokine secretion by Treg and Teff cells activated via CAR or TCR / CD28. Using multiplex ELISA, we detected secretion of 48 different cytokines by Treg and Teff (NoAct, CAR, TCR). Higher cytokines in CAR Tregs than TCR Tregs and NoAct Tregs (CAR exacerbated upregulation): sCD40L, FGF-2, fractalkine, G-CSF, GM-CSF, GROa, IFN-a2, IFNg, IL-3, IL-4, IL-6, IL-9, IL-12p40, IL-12p70, IL-13, IL-17A, IL-18, MCP-1, MCP-3, MIG / CXCL9, MIP1a, PDGF-AA, TNFb. Cytokines that are higher in CAR Tregs than NoAct Tregs but not in TCR Tregs (CAR-specific upregulation): sCD40L, G-CSF, GM-CSF, GROa, IFN-a2, IFNg, IL-3, IL-4, IL-6, IL-12p40, IL-13, MIP1a. Cytokines that are lower in CAR Tregs than TCR Tregs: MIP1b, VEGF-A. Cytokines that are higher in CAR Teff than TCR Teff: IP-10, IL-17A, TNFb. Cytokines that are higher in CAR Teff than NoAct Teff but not in TCR Teff: None. Cytokines that are lower in CAR Teff than NoAct Teff but not in TCR Tregs: None. Cytokines lower in CAR Teff than TCR Teff: sCD40L, GROa, IFNa2, IL-6, IL-10, IL-13, IL-15, MCP1, PDGF-AA, PDGF-AB / BB, VEGF-A. K562 indicates NoAct, 19K562 indicates CAR activation, and OKT3K562 indicates TCR / CD28 activation. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0049] [Figure 28]IFNG production by Treg and Teff cells activated via CAR or TCR / CD28. Intracellular staining for Treg transcription factor FOXP3 and cytokines in cells activated overnight and then treated with cellular protein transport inhibitors monensin and brefeldin A, followed by analysis by flow cytometry. Note the production of IFNG by FOXP3+CAR Tregs. PMA / Iono, i.e., phorbol myristate acetate (PMA) and ionomycin treatment, is a positive control for cytokine production.

[0050] [Figure 29] IL-2 production by Treg and Teff cells activated via CAR or TCR / CD28. Intracellular staining for Treg transcription factor FOXP3 and cytokines in cells activated overnight and then treated with the cellular protein transport inhibitors monensin and brefeldin A, followed by analysis by flow cytometry. PMA / Iono, i.e., phorbol myristate acetate (PMA) and ionomycin treatment, is a positive control for cytokine production.

[0051] [Diagram 30] IL-3 production by Treg and Teff cells activated via CAR or TCR / CD28. Intracellular staining for Treg transcription factor FOXP3 and cytokines in cells activated overnight and then treated with cellular protein transport inhibitors monensin and brefeldin A, followed by analysis by flow cytometry. PMA / Iono, i.e., phorbol myristate acetate (PMA) and ionomycin treatment, is a positive control for cytokine production.

[0052] [Diagram 31]IL-5 production by Treg and Teff cells activated via CAR or TCR / CD28. Intracellular staining for Treg transcription factor FOXP3 and cytokines in cells activated overnight and then treated with cellular protein transport inhibitors monensin and brefeldin A, followed by analysis by flow cytometry. PMA / Iono, i.e., phorbol myristate acetate (PMA) and ionomycin treatment, is a positive control for cytokine production.

[0053] [Diagram 32] Production of IL-17A by Treg and Teff cells activated via CAR or TCR / CD28. Intracellular staining for Treg transcription factor FOXP3 and cytokines in cells activated overnight and then treated with the cellular protein transport inhibitors monensin and brefeldin A, followed by analysis by flow cytometry. PMA / Iono, i.e., phorbol myristate acetate (PMA) and ionomycin treatment, is a positive control for cytokine production.

[0054] [Diagram 33] IFNG is produced by both CAR Teff and TCR Teff cells, but only by CAR Tregs, as measured by intracellular protein staining. Note that CAR Tregs and TCR Tregs had identical FOXP3 positivity, but CAR Tregs were IFNG positive, whereas TCR Tregs were not. PMA, phorbol myristate acetate (PMA) and ionomycin treatment are positive controls for cytokine production. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, not significant.

[0055] [Diagram 34]Expression of transcription factor genes in Tregs activated via CAR or TCR / CD28 at the single cell gene expression cluster level. Left: transcription factors previously implicated in polarization and maintenance of different CD4+ helper T cell types (Th1, Th2, Th17). Right: heatmap of expression of transcription factor genes across Treg single cell gene expression gene clusters. Note that HLX is the only transcription factor gene overexpressed in cluster 9 (enriched in CAR Tregs) compared to all other clusters.

[0056] [Diagram 35] Expression of TBX21, HLX, and IFNG genes in Treg and Teff cells activated via CAR or TCR / CD28 measured by RNA sequencing.

[0057] [Diagram 36] Co-expression analysis of IFNG with TBX21, HLX, IRF8, and ZEB2 in Tregs at the single cell level. IFNG is expressed in 8.02% of analyzed Tregs at the single cell level. In the UMAP plot, the expression of IFNG is marked in red and the expression of the transcription factor (TBX21, HLX, IRF8, or ZEB2) is marked in blue. ZEB2 correlates best with IFNG among the four tested transcription factor genes.

[0058] [Figure 37] Correlation of IFNG expression in Tregs via CAR or TCR / CD28 with expression of all other genes at the single cell level. 15 genes that were most correlated with IFNG expression in Tregs (CAR-blue, TCR-red, NoAct-black) with R values ​​(top) and regression curves (green).

[0059] [Figure 38]Figures 38A-38C: Chimeric antigen receptor signaling confers both regulatory and effector cell properties to mouse Tregs (Figure 38A). Mouse CD19 CAR CD4+CD25+ Tregs are cytotoxic to mouse CD19+A20 lymphoma cells in vitro to a greater extent than mouse CD19 CAR CD4+CD25-Tconv (conventional T cells) (Figure 38B). Mouse CD19 CAR CD4+CD25+ Tregs suppress proliferation of CD4+CD25-Tconv cells in vitro (Figure 38C). Mouse CD19 CAR CD4+CD25+ Tregs express the Treg lineage transcription factor Foxp3 as assessed by intracellular protein staining.

[0060] [Figure 39] CAR Tregs as a new therapy for solid tumors. The tumor microenvironment (TME) of solid tumors has immunosuppressive properties and prevents the infiltration of conventional T cells. Infiltrating T cells are exhausted. Cell types abundant in the TME include cancer-associated fibroblasts (CAFs), tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs). By engineering Tregs to recognize antigens on the surface of cancer cells using chimeric antigen receptors (CARs), these CAR Tregs are believed to infiltrate solid tumors and kill tumor cells via the perforin / granzyme pathway, IFN-γ and TNF-α secretion, and other pathways that are either directly induced by CAR activation or added using synthetic gene circuits. IFN-γ and other cytokines and chemokines made by CAR Tregs can destabilize tumor-resident Tregs and recruit effector immune cells. Overall, CAR Tregs may be a new therapy for solid tumors that directly targets tumor cells and shifts the balance in the TME from anti-inflammatory to pro-inflammatory. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] Description of exemplary embodiments Regulatory T cells (T reg Tregs are a subset of T cells dedicated to suppressing immune responses. Tregs are essential for maintaining self-tolerance and immune homeostasis, as dramatically exemplified by the severe multiorgan autoimmunity observed in human patients and mice with genetic defects in FOXP3, the master transcription factor for the Treg lineage (1). Furthermore, T reg CD19 CAR T cell therapy has achieved great success in liquid tumors, with five CD19 CAR T cell therapies for leukemia currently approved by the FDA. The same cannot be said for solid tumors: CAR T cells are either unable to enter the tumor microenvironment or are exhausted once there (4). Furthermore, thymus-derived T cells have been shown to be unable to enter the tumor microenvironment, and are unable to enter the tumor microenvironment, resulting in a lack of immune cell proliferation and proliferation. reg They migrate to solid tumors and persist in large numbers ( 2 , 5 ).

[0062] Solid tumors have proven extremely refractory to immune-based therapies, especially those involving T cells. This is in part because solid tumors create a tumor microenvironment that is difficult for T cells to invade and is replete with inhibitory immune cells that lead to T cell suppression and exhaustion. regIt was found that Tregs migrate to tumors and proliferate in their hostile microenvironment (hypoxia, glucose deprivation, and lactate overload). This study found that in preclinical humanized mouse experiments, when Tregs are redirected using chimeric antigen receptors (CARs) specific to tumor cells, such engineered CAR Tregs kill antigen-expressing tumor cells in vitro and control their growth in vivo. This idea could greatly help solid tumor patients by creating immune cells that can successfully infiltrate solid tumors, destroy tumor cells, and bring about remission in difficult-to-treat cancers. Therefore, this study focused on converting Tregs from solid tumor protector cells to antitumor effector cells.

[0063] In this study, we transduced an anti-CD19 CAR carrying a tandem CD28-CD3ζ intracellular domain into human Tregs. Upon in vitro co-incubation with CD19+ tumor cells, CAR T reg showed high FOXP3 expression and demethylated T reg They upregulated activation markers, proliferated, secreted IL-10, and suppressed T cell proliferation while maintaining the specific differentially demethylated region (TSDR). However, single-cell cytokine analysis revealed that CAR-mediated activation of Tregs also led to high production of the inflammatory and cytolytic molecules IFN-γ, TNF-α, perforin, and granzyme B. Remarkably, CAR T reg CD19 in NSG mice + The CAR T cells suppressed tumor cell growth. This phenomenon was observed across three tumor cell types (B-cell leukemia, myeloid leukemia, and epithelial carcinoma) and two delivery routes (intravenous and subcutaneous). reg Annexin V staining and quantification of tumor cells co-incubated with CD19 CAR Tregs confirmed that CD19+ tumor cells of different origins were able to kill CD19+ tumor cells. In cancer therapy, CAR signaling, originally designed to induce the secretion of inflammatory cytokines and killing of target cells by CAR T cells, has been shown to be a potent inhibitor of human T regIt was hypothesized that regulatory T cells could exacerbate the cytotoxicity of CAR-induced T cells, transforming them into a novel solid tumor therapy. Surprisingly, regulatory T cells, found as suppressor T cells that are abundant in solid tumors and impede antitumor responses and are a poor prognostic indicator, can be used to eradicate solid tumors. Thus, in certain embodiments, the present disclosure provides a novel method for the eradication of CAR-induced T cells. reg Kind Code: A1 Abstract: Methods for treating cancer through mediated cytotoxicity are provided.

[0064] Although some conventional T cells can recognize tumor cells, they are either unable to enter the solid tumor microenvironment or are exhausted once there. This is due to the combination of harsh conditions (low oxygen, low glucose, high lactate) and suppressive cells (myeloid-derived suppressor cells, regulatory T cells, tumor cells themselves expressing PD-L1, and exhaustion-inducing molecules). In contrast, T reg The cells migrate to solid tumors due to their chemokine receptor expression patterns and proliferate there due to their ability to take up, for example, lactate and use it as a fuel. reg Tregs are HLA class II restricted, as HLA class II is expressed only on professional antigen-presenting cells, and therefore Tregs usually express only HLA class I, and perhaps even at low levels (HLA downregulation is a classic tumor escape strategy). reg However, T cells specific to cancer cells do not even directly recognize tumor cells. reg Using CAR, T reg can bind directly to tumor cells with high affinity and kill the tumor cells it recognizes. In a further aspect, T reg can be operated.

[0065] I. Definition As used herein, "essentially free" with respect to a particular component is used herein to mean that none of the particular components are intentionally incorporated into the composition and / or are present only as contaminants or in trace amounts.Therefore, the total amount of the particular component resulting from any unintentional contamination of the composition is much less than 0.05%, preferably less than 0.01%.Most preferred is a composition in which the amount of the particular component cannot be detected by standard analytical methods.

[0066] As used herein, "a" or "an" can mean one or more. As used in the claims, the words "a" or "an" when used in conjunction with the word "comprising" can mean one or more than one.

[0067] Use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only or the alternatives are not mutually exclusive, however, the present disclosure supports a definition that refers to alternatives only and "and / or." As used herein, "another" can mean at least a second or more.

[0068] The term "about" generally means within the standard deviation of the stated value as determined using standard analytical techniques to measure the stated value. The term may also be used to refer to plus or minus 5% of the stated value.

[0069] The phrase "effective amount" or "therapeutically effective" refers to a dosage of a drug or agent that is sufficient to produce a desired result, which may be a subjective or objective improvement in the recipient of the dosage, i.e., increased lung growth, increased lung repair, reduced tissue edema, increased DNA repair, reduced apoptosis, reduced tumor size, slowed growth rate of cancer cells, reduced metastasis, or any combination of the above.

[0070] As used herein, the term "antibody" refers to immunoglobulins, derivatives thereof that maintain specific binding ability, and proteins that have a binding domain that is homologous or largely homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources or may be partially or fully synthetically produced. Antibodies may be monoclonal or polyclonal. Antibodies may be members of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. Antibodies may be bispecific antibodies. In exemplary embodiments, antibodies used with the methods and compositions described herein are derivatives of the IgG class. The term antibody also refers to antigen-binding antibody fragments. Examples of such antibody fragments include, but are not limited to, Fab, Faby, F(aby)2, scFv, Fv, dsFv diabody, and Fd fragments. Antibody fragments may be produced by any means. For example, antibody fragments can be enzymatically or chemically produced by fragmentation of an intact antibody, can be recombinantly produced from a gene encoding a partial antibody sequence, or can be wholly or partially synthetically produced. An antibody fragment can optionally be a single chain antibody fragment. Alternatively, the fragment can contain multiple chains linked together, for example, by disulfide bonds. The fragment can also optionally be a multimolecular complex. A functional antibody fragment will typically contain at least about 10 amino acids, and more typically will contain at least about 200 amino acids.

[0071] "Subject" and "patient" refer to either humans or non-humans, e.g., primates, mammals, and vertebrates. In certain aspects, the subject is a human.

[0072] As used herein, the terms "treat", "treatment", "treating" or "amelioration", when used in relation to a disease, disorder, or medical condition, refer to therapeutic treatment for the condition, the purpose being to reverse, alleviate, ameliorate, inhibit, slow, or stop the progression or severity of the symptoms or condition. The term "treat" includes reducing or alleviating at least one adverse effect or symptom of the condition. A treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, a treatment is "effective" if the progression of the condition is reduced or stopped. That is, "treatment" includes not only the improvement of symptoms or markers, but also the cessation or at least slowing of the progression or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desirable clinical outcomes include, but are not limited to, the alleviation of one or more symptoms, a reduction in the extent of the defect, a stabilized (i.e., not worsening) state of the tumor or malignant lesion, a delay or slowing of tumor growth and / or metastasis, and an extension of life span, compared to that expected in the absence of treatment.

[0073] The term "T cell" refers to a T lymphocyte as defined in the art and is intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells are CD4 + T cells, CD8 + T cells, CD4 + CD8 + T cells, or CD4 - CD8 - The T cells may also be helper T cells, such as T helper 1 (TH1) or T helper 2 (TH2) cells, or TH17 cells, and cytotoxic T cells, regulatory T cells, natural killer T cells, naive T cells, memory T cells, or gamma delta T cells (Wilson et al., 2009; Wynn, 2005; Ladi et al., 2006). T cells that differ from each other in at least one marker, such as CD4, are referred to herein as "subsets" of T cells.

[0074] "CD4 + "T cells" refers to a subset of T cells that express CD4 on their surface and are involved in cellular immune responses. It is characterized by a secretory profile following stimulation that may include secretion of cytokines, e.g., IFN-γ, TNF-α, IL-2, IL-4, and IL-10. "CD4" is a 55 kD glycoprotein that was originally defined as a differentiation antigen on T lymphocytes, but is also found on other cells, including monocytes / macrophages. The CD4 antigen is a member of the immunoglobulin supergene family and participates as an associative recognition element in MHC (major histocompatibility complex) class II-restricted immune responses. On T lymphocytes, it defines helper / inducer subsets.

[0075] "CD8 + "T cells" refers to a subset of T cells that express CD8 on their surface, are MHC class I restricted, and function as cytotoxic T cells. The "CD8" molecule is a differentiation antigen found on thymocytes and cytotoxic and suppressor T lymphocytes. The CD8 antigen is a member of the immunoglobulin supergene family and is the association recognition element in major histocompatibility complex class I restricted interactions.

[0076] "Regulatory T cells" refers to a subset of T cells that act to suppress immune responses, thereby maintaining homeostasis and self-tolerance. Self-tolerance refers to a state of immune unresponsiveness to self antigens, which is important for avoiding the development of autoimmune diseases.

[0077] As used herein, the term "chimeric antigen receptor (CAR)" can refer to, for example, an artificial T cell receptor, a chimeric T cell receptor, or a chimeric immune receptor, and can encompass engineered receptors that transfer artificial specificity to specific immune effector cells. CARs can be used to confer the specificity of a monoclonal antibody to T cells, thereby making it possible to generate large numbers of specific T cells, for example, for use in adoptive cell therapy. In specific embodiments, for example, CARs direct the specificity of cells to tumor-associated antigens. In some embodiments, CARs include an intracellular activation domain, a transmembrane domain, and an extracellular domain that includes a tumor-associated antigen binding region. In certain aspects, CARs include a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to a CD3ζ transmembrane domain and an endodomain. The specificity of other CAR designs can be derived from the receptor's ligand (e.g., peptide) or a pattern recognition receptor, such as dectin. In certain cases, the spacing of the antigen recognition domain can be altered to reduce activation-induced cell death. In certain cases, the CAR contains domains for additional costimulatory signaling, such as CD3ζ, FcR, CD27, CD28, CD137, DAP10, and / or OX40. In some cases, molecules can be co-expressed with the CAR, including costimulatory molecules, reporter genes for imaging (e.g., positron emission tomography), gene products that conditionally eliminate T cells upon addition of a prodrug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors.

[0078] The term "antigen-presenting cell (APC)" refers to a class of cells that can present one or more antigens in the form of peptide-MHC complexes that can be recognized by certain effector cells of the immune system, thereby inducing an effective cellular immune response against the antigen or antigens presented. APCs can be intact whole cells, such as macrophages, B cells, endothelial cells, activated T cells, and dendritic cells; or purified MHC class I molecules complexed with other molecules, naturally occurring or synthetic, such as β2-microglobulin. Although many types of cells may be capable of presenting antigens on their cell surface for T cell recognition, only dendritic cells have the ability to present antigens in amounts effective to activate naive T cells for cytotoxic T lymphocyte (CTL) responses.

[0079] II.CAR modified T cells In certain aspects, the present disclosure provides a T cell line engineered to express a CAR vector. reg Provide. CAR T reg can be used to treat a disease or disorder, for example, a solid tumor or a blood cancer.

[0080] Certain aspects of the present disclosure include reg To obtain a starting population of T reg and engineered T cells as immunotherapies to target cancer cells. reg In particular, T reg expresses CAR.

[0081] In some embodiments, T regThe starting population of cells is derived from blood, bone marrow, lymph, or lymphoid organs. In some aspects, the cells are human cells. The cells are typically primary cells, e.g., isolated directly from the subject and / or isolated and frozen from the subject. In relation to the subject to be treated, the cells can be allogeneic and / or autologous. In some embodiments, the method includes isolating cells from a subject, preparing, treating, culturing, and / or manipulating them as described herein, and reintroducing them into the same patient before or after cryopreservation.

[0082] In some embodiments, one or more of the T cell populations are enriched or depleted in cells that are positive for a specific marker, such as a surface marker, or that are negative for a specific marker. In some cases, such a marker is absent or expressed at relatively low levels in certain T cell populations (e.g., regulatory T cells) but present or expressed at relatively high levels in certain other T cell populations (e.g., regulatory T cells).

[0083] In some embodiments, regulatory T cells are isolated from PBMC samples by negative selection of markers expressed on non-T cells, e.g., B cells, monocytes, or other white blood cells, e.g., CD14. In some aspects, CD4+CD25+CD127- Tregs are purified from human peripheral blood.

[0084] In some embodiments, the T cells are autologous T cells. In this method, a tumor sample is obtained from a patient and a single cell suspension is obtained. The single cell suspension can be obtained in any suitable manner, for example, mechanically (e.g., disaggregating the tumor using a gentleMACS™ Dissociator, Miltenyi Biotec, Auburn, Calif.) or enzymatically (e.g., collagenase or DNase). The single cell suspension of the tumor enzymatic digest is cultured in interleukin-2 (IL-2). The cells are cultured at confluence (e.g., about 2×10 6 cells / mL) for, for example, about 5 to about 21 days, preferably about 10 to about 14 days.6 For example, the cells can be cultured for 5 days, 5.5 days, or 5.8 days to 21 days, 21.5 days, or 21.8 days, e.g., from 10 days, 10.5 days, or 10.8 days to 14 days, 14.5 days, or 14.8 days.

[0085] The cultured T cells can be pooled and rapidly expanded. Rapid expansion results in an increase in the number of antigen-specific T cells of at least about 50-fold (e.g., 50, 60, 70, 80, 90, or 100-fold or more) over a period of about 10 to about 14 days. More preferably, rapid expansion results in an increase of at least about 200-fold (e.g., 200, 300, 400, 500, 600, 700, 800, 900, or more) over a period of about 10 to about 14 days. Expansion can be accomplished by any of several methods known in the art. For example, T cells can be rapidly expanded using nonspecific T-cell receptor stimulation in the presence of feeder lymphocytes and either interleukin-2 (IL-2) or interleukin-15 (IL-15). Non-specific T-cell receptor stimulation can include approximately 30 ng / ml OKT3, a mouse monoclonal anti-CD3 antibody (available from Ortho-McNeil®, Raritan, NJ). Alternatively, T cells can be rapidly expanded by stimulating peripheral blood mononuclear cells (PBMCs) in vitro with one or more antigens of the cancer (including antigenic portions thereof, e.g., epitopes, or cells), e.g., human leukocyte antigen A2 (HLA-A2) binding peptides, optionally expressed from a vector, in the presence of a T cell growth factor, e.g., 300 IU / ml IL-2. In vitro induced T cells are rapidly expanded by restimulation with the same antigens of the cancer pulsed on HLA-A2 expressing antigen presenting cells. Alternatively, T cells can be rapidly expanded by restimulation with, e.g., irradiated autologous lymphocytes or with irradiated HLA-A2 + T cells can be restimulated with allogeneic lymphocytes and IL-2.

[0086] Autologous T cells can be modified to express T cell growth factors that promote the growth and activation of autologous T cells. Suitable T cell growth factors include, for example, interleukin (IL)-2, IL-7, IL-15, and IL-12. Suitable methods of modification are known in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3 rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. In certain aspects, the modified autologous T cells express high levels of T cell growth factors. T cell growth factor coding sequences, such as those for IL-12, are readily available in the art, as are promoters whose operably linked to the T cell growth factor coding sequences promote high levels of expression.

[0087] One of skill in the art would be well equipped to construct vectors by standard recombinant techniques (see, e.g., Sambrook et al., 2001 and Ausubel et al., 1996, both of which are incorporated herein by reference) for expression of the antigen receptors of the present disclosure. Vectors include, but are not limited to, plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs), such as retroviral vectors (e.g., derived from Moloney murine leukemia virus vectors (MoMLV), MSCV, SFFV, MPSV, SNV, etc.), lentiviral vectors (e.g., derived from HIV-1, HIV-2, SIV, BIV, FIV, etc.), adenovirus (Ad) vectors, including replication competent, replication defective, and gutless forms, adeno-associated virus (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papilloma virus vectors, Epstein-Barr virus vectors, herpes virus vectors, vaccinia virus vectors, Harvey murine sarcoma virus vectors, mouse mammary tumor virus vectors, Rous sarcoma virus vectors, parvovirus vectors, poliovirus vectors, vesicular stomatitis virus vectors, Maraba virus vectors, and the like. virus vectors, as well as group B adenovirus enadenotucirev vectors.

[0088] In some embodiments, the CAR comprises an extracellular antigen recognition domain that specifically binds to an antigen. In some embodiments, the antigen is a protein expressed on the surface of a cell. In some embodiments, the CAR is a TCR-like CAR, and the antigen is a processed peptide antigen, such as a peptide antigen of an intracellular protein, that is recognized on the cell surface in the context of a major histocompatibility complex (MHC) molecule, like a TCR.

[0089] Exemplary antigen receptors, including CARs and recombinant TCRs, and methods for engineering and introducing the receptors into cells are described in, for example, International Patent Application Publication Nos. WO200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061, U.S. Patent Application Publication Nos. US2002131960, US2013287748, US201301493, and the like. 37, U.S. Patent Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European Patent Application No. EP 2537416, and / or those described in Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75. In some aspects, the engineered antigen receptor includes CARs described in U.S. Patent No. 7,446,190 and those described in International Patent Application Publication No. WO / 2014055668A1.

[0090] In some embodiments, the CAR comprises a) an intracellular signaling domain, b) a transmembrane domain, and c) an extracellular domain comprising an antigen-binding region.

[0091] In some embodiments, the engineered antigen receptor comprises a CAR, e.g., an activating or stimulatory CAR, a costimulatory CAR (see WO2014 / 055668), and / or an inhibitory CAR (iCAR, see Fedorov et al., 2013). CARs generally comprise an extracellular antigen (or ligand) binding domain linked to one or more intracellular signaling components, in some aspects via a linker and / or a transmembrane domain. Such molecules typically mimic or mimic the signaling through a natural antigen receptor, the signaling through such receptor in combination with a costimulatory receptor, and / or the signaling through a costimulatory receptor alone.

[0092] Certain aspects of the present disclosure relate to the use of nucleic acids, including nucleic acids encoding antigen-specific CAR polypeptides, including humanized CARs (hCARs) that contain an intracellular signaling domain, a transmembrane domain, and an extracellular domain that contains one or more signaling motifs, to reduce immunogenicity. In certain aspects, the CAR can recognize an epitope that contains a space shared between one or more antigens. In certain aspects, the binding region can include the complementarity determining region of a monoclonal antibody, the variable region of a monoclonal antibody, and / or an antigen-binding fragment thereof. In another aspect, the specificity is derived from a peptide (e.g., a cytokine) that binds to a receptor.

[0093] It is contemplated that the human CAR nucleic acid may be a human gene used to enhance cellular immunotherapy for human patients. In a specific embodiment, the present invention includes a full-length CAR cDNA or coding region. The antigen-binding region or domain is the V of a single chain variable fragment (scFv) derived from a particular human monoclonal antibody, such as those described in U.S. Patent No. 7,109,304, which is incorporated herein by reference. H and V LThe fragments may comprise fragments of the chains of a human antigen-specific antibody. The fragments may also be any number of different antigen-binding domains of a human antigen-specific antibody. In a more specific embodiment, the fragments are antigen-specific scFvs encoded by sequences that are optimized for human codon usage for expression in human cells.

[0094] The arrangement may be multimeric, for example, a diabody or multimer. Multimers are most likely formed by cross-pairing of the variable portions of the light and heavy chains into a diabody. The hinge portion of the construct may have multiple options, ranging from being completely deleted, to maintaining the first cysteine, to substituting proline instead of serine, to being truncated to the first cysteine. The Fc portion may be deleted. Any protein that is stable and / or dimerizes may serve this purpose. Only one of the Fc domains may be used, for example, either the CH2 or CH3 domain of a human immunoglobulin. The hinge, CH2, and CH3 regions of a human immunoglobulin that have been modified to improve dimerization may also be used. Only the hinge portion of an immunoglobulin may also be used. A portion of CD8α may also be used.

[0095] In some embodiments, the CAR nucleic acid comprises a sequence encoding other costimulatory receptors, such as transmembrane domains and modified CD28 intracellular signaling domains. Other costimulatory receptors include, but are not limited to, one or more of CD28, CD27, OX-40 (CD134), DAP10, and 4-1BB (CD137). In addition to the primary signal initiated by CD3ζ, the additional signal provided by the human costimulatory receptor inserted into the human CAR is important for the complete activation of NK cells and can help improve the in vivo persistence and therapeutic success of adoptive immunotherapy.

[0096] In some embodiments, CARs are constructed with specificity for a particular antigen (or marker or ligand), e.g., an antigen expressed in a particular cell type targeted by adoptive therapy, e.g., a cancer marker, and / or an antigen intended to induce an attenuated response, e.g., an antigen expressed in a normal or non-diseased cell type. Thus, CARs typically comprise, in their extracellular portion, one or more antigen-binding molecules, e.g., one or more antigen-binding fragments, domains or portions, or one or more antibody variable domains, and / or antibody molecules. In some embodiments, CARs comprise one or more antigen-binding portions of an antibody molecule, e.g., a single-chain antibody fragment (scFv) derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb).

[0097] In certain embodiments of the chimeric antigen receptor, the antigen-specific portion of the receptor (sometimes referred to as the extracellular domain containing the antigen-binding region) comprises a tumor-associated antigen or a pathogen-specific antigen-binding domain. Antigens include carbohydrate antigens recognized by pattern recognition receptors, such as Dectin-1. The tumor-associated antigen can be of any type, so long as it is expressed on the cell surface of the tumor cell. Exemplary embodiments of tumor-associated antigens include CD19, CD20, carcinoembryonic antigen, alpha-fetoprotein, CA-125, MUC-1, CD56, EGFR, c-Met, AKT, Her2, Her3, epithelial tumor antigen, melanoma-associated antigen, mutant p53, mutant ras, and the like.

[0098] The sequence of the open reading frame encoding the chimeric receptor can be obtained from genomic DNA sources, cDNA sources, or can be synthesized (e.g., by PCR), or a combination thereof. Depending on the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof, since introns are known to stabilize mRNA. It may also be more advantageous to use endogenous or exogenous non-coding regions to stabilize mRNA.

[0099] It is contemplated that chimeric constructs can be introduced into immune cells as naked DNA or in suitable vectors.Methods for using naked DNA to stably transfect cells by electroporation are known in the art.See, for example, U.S. Patent No. 6,410,319.Naked DNA generally refers to the DNA that codes for chimeric receptors contained in plasmid expression vectors in suitable orientation for expression.

[0100] Alternatively, the chimeric construct can be introduced into immune cells using viral vectors (e.g., retroviral vectors, adenoviral vectors, adeno-associated viral vectors, or lentiviral vectors).The vectors suitable for use according to the method of the present disclosure are non-replicative in immune cells.Many virus-based vectors are known, in which the copy number of the virus maintained in cells is low enough to maintain cell viability, such as HIV, SV40, EBV, HSV, or BPV-based vectors.

[0101] In some aspects, the antigen-specific binding or recognition component is linked to one or more transmembrane and intracellular signaling domains.In some embodiments, the CAR comprises a transmembrane domain fused to the extracellular domain of the CAR.In one embodiment, the transmembrane domain that is naturally bound to one of the domains in the CAR is used.In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid such domain binding to the transmembrane domain of the same or different surface membrane protein, and to minimize interaction with other members of the receptor complex.

[0102] The transmembrane domain of some embodiments is derived from either natural or synthetic sources. When the source is natural, the domain of some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e., including at least the transmembrane region of) the α, β, or ζ chain of the T cell receptor, CD28, CD3ζ, CD3ε, CD3γ, CD3δ, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D, and DAP molecules. Alternatively, the transmembrane domain of some embodiments is synthetic. In some aspects, the synthetic transmembrane domain mainly comprises hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of the synthetic transmembrane domain.

[0103] In certain embodiments, the platform technology disclosed herein for genetically modifying immune cells, e.g., NK cells, includes (i) non-viral gene transfer using an electroporation device (e.g., nucleofector), (ii) CARs that signal through an endodomain (e.g., CD28 / CD3-ζ, CD137 / CD3-ζ, or other combinations), (iii) CARs with variable length extracellular domains connecting the antigen recognition domain to the cell surface, and in some cases, (iv) CARs + These include artificial antigen presenting cells (aAPCs) derived from K562 that can potently and numerically expand immune cells ( Singh et al., 2008 ; Singh et al., 2011 ).

[0104] Some of the target proteins of the antigen targeted by the present CAR are expressed in the context of the disease, condition, or cell type targeted via the CAR. Among the diseases and conditions are proliferative, neoplastic, and malignant diseases and disorders, such as cancers and tumors, such as blood cancers, immune system cancers, such as lymphomas, leukemias, and / or myelomas, such as B, T, and myeloid leukemias, lymphomas, and multiple myelomas. In some embodiments, the antigen is selectively expressed or overexpressed on disease or condition cells, such as tumor or pathogenic cells, compared to normal or non-target cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or on engineered cells. Any suitable antigen can be used in the present methods. Exemplary antigens include, but are not limited to, antigenic molecules derived from infectious agents, auto / self antigens, tumor / cancer-associated antigens, and tumor neoantigens.

[0105] The terms "tumor-associated antigen," "tumor antigen," and "cancer cell antigen" are used interchangeably herein. In each case, the term refers to a protein, glycoprotein, or carbohydrate that is specifically or preferentially expressed by cancer cells.

[0106] Tumor-associated antigens can be of any kind, as long as they are expressed on the cell surface of tumor cells. Tumor-associated antigens can be derived from prostate, breast, colorectal, lung, pancreatic, renal, mesothelioma, ovarian, sarcoma or melanoma cancer. Exemplary tumor-associated antigens or tumor cell-derived antigens include MAGE1, 3, and MAGE4 (or other MAGE antigens, such as those disclosed in International Patent Publication No. WO99 / 40188); PRAME; BAGE; RAGE, Lage (also known as NY ESO 1); SAGE; and HAGE or GAGE. These non-limiting examples of tumor antigens are expressed in a wide range of tumor types, such as melanoma, lung cancer, sarcoma, and bladder carcinoma. See, for example, U.S. Patent No. 6,544,518. Tumor-associated antigens for prostate cancer include, for example, prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), prostatic acid phosphate, NKX3.1, and six-transmembrane epithelial antigen of the prostate (STEAP).

[0107] Exemplary embodiments of tumor-associated antigens include, but are not limited to, CD19, CD20, carcinoembryonic antigen, alphafetoprotein, CA-125, MUC-1, CD56, EGFR, c-Met, AKT, Her2, Her3, epithelial tumor antigen, melanoma-associated antigen, mutant p53, mutant ras, etc. In certain aspects, antigens include NY-ESO, EGFRvIII, Muc-1, Her2, CA-125, WT-1, Mage-A3, Mage-A4, Mage-A10, TRAIL / DR4, and CEA. In certain aspects, antigens for two or more antigen receptors include, but are not limited to, CD19, EBNA, WT1, CD123, NY-ESO, EGFRvIII, MUC1, HER2, CA-125, WT1, Mage-A3, Mage-A4, Mage-A10, TRAIL / DR4, and / or CEA. Sequences for these antigens are known in the art and include, for example, CD19 (Accession No. NG_007275.1), EBNA (Accession No. NG_002392.2), WT1 (Accession No. NG_009272.1), CD123 (Accession No. NC_000023.11), NY-ESO (Accession No. NC_000023.11), EGFRvIII (Accession No. NG_007726.3), MUC1 (Accession No. NG_029383.1), HER2 ...EGFRvIII (Accession No. NG_007726.3), MUC1 (Accession No. NG_029383.1), HER2 (Accession No. NG_009272.1), CD123 (Accession No. NC_000023.11), EGFRvIII (Access The following genes were identified in the 1446 cells: CA-125 (accession number NG_007503.1), CA-125 (accession number NG_055257.1), WT1 (accession number NG_009272.1), Mage-A3 (accession number NG_013244.1), Mage-A4 (accession number NG_013245.1), Mage-A10 (accession number NC_000023.11), TRAIL / DR4 (accession number NC_000003.12), and / or CEA (accession number NC_000019.10).

[0108] Other tumor-associated antigens include Plu-1, HASH-1, HasH-2, Cripto, and Criptin. Additionally, tumor antigens can be self-peptide hormones, such as full-length gonadotropin hormone releasing hormone (GnRH), short 10 amino acid long peptides, which are useful in the treatment of many cancers.

[0109] Tumor antigens include tumor antigens derived from cancers characterized by the expression of tumor-associated antigens, such as HER-2 / neu expression.Tumor-associated antigens of interest include lineage-specific tumor antigens, such as melanocyte melanoma lineage antigen MART-1 / Melan-A, gp100, gp75, mda-7, tyrosinase, and tyrosinase-related proteins.Exemplary tumor-associated antigens include, but are not limited to, p53, Ras, c-Myc, cytoplasmic serine / threonine kinases (e.g., A-Raf, B-Raf, and C-Raf, cyclin-dependent kinases), MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MART-1, BAGE, DAM-6, -10, GAGE-1, -2, -8, GAGE-3, -4, -5, -6, -7B, N ... A88-A, MART-1, MC1R, Gp100, PSA, PSM, tyrosinase, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, phosphoinositide 3 kinase (PI3K), TRK receptor, PRAME, P15, RU1, RU2, SART-1, SART-3, Wilms tumor antigen (WT1), AFP, -catenin / m, caspase-8 / m, CEA, CDK-4 / m, E LF2M, GnT-V, G250, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP, annexin II, CDC27 / m, TPI / mbcr-abl, BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6 / AML, LDLR / FUT, Pml / RAR, tumor-associated calcium signal transducer 1 (TACS TD1) TACSTD2, receptor tyrosine kinases (e.g., epidermal growth factor receptor (EGFR) (especially EGFRvIII), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR)), cytoplasmic tyrosine kinases (e.g., src family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducer and activator of transcription STAT3, STATS, and STATE, hypoxia-inducible factors (e.g.,HIF-1 and HIF-2), nuclear factor kappa B (NF-B), Notch receptors (e.g., Notch 1-4), c-Met, mammalian target of rapamycin (mTOR), WNT, extracellular signal-regulated kinase (ERK) and its regulatory subunits, PMSA, PR-3, MDM2, mesothelin, renal cell carcinoma-5T4, SM22-α, carbonic anhydrase I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL / AML1, GD2, proteinase 3, hTERT, sarcoma translocation breakpoints, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, GD3, fucosyl-GM1, mesothelian, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKA P-4, SSX2, XAGE1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos-related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNC1, LRRN1, as well as tumor antigens derived from or including any of the idiotypes.

[0110] Antigens can include epitope regions or epitope peptides derived from genes mutated or differentially transcribed in tumor cells compared to normal cells, such as telomerase enzyme, survivin, mesothelin, mutant ras, bcr / abl rearrangements, Her2 / neu, mutant or wild-type p53, cytochrome P450 1B1, and aberrantly expressed intronic sequences, such as N-acetylglucosaminyltransferase-V; clonal rearrangements of immunoglobulin genes that generate unique idiotypes in myelomas and B-cell lymphomas; tumor antigens that contain epitope regions or epitope peptides derived from oncoviral processes, such as human papillomavirus proteins E6 and E7; Epstein-Barr virus protein LMP2; and non-mutated oncofetal proteins with tumor-selective expression, such as carcinoembryonic antigen and alpha-fetoprotein.

[0111] A. Antigen-presenting cells APCs, including macrophages, B lymphocytes, and dendritic cells, are distinguished by the expression of specific MHC molecules. APCs internalize antigens and re-express some of the antigens along with MHC molecules on the outer cell membrane. The MHC is a large genetic complex with multiple loci. The MHC loci code for two major classes of MHC membrane molecules, termed class I and class II MHC. T helper lymphocytes generally recognize antigens associated with MHC class II molecules, while cytotoxic T lymphocytes recognize antigens associated with MHC class I molecules. In humans, the MHC is referred to as the HLA complex, and in mice, the H-2 complex.

[0112] In some cases, aAPCs are useful in preparing therapeutic compositions and cell therapy products of this embodiment. For general guidance regarding the preparation and use of antigen-presenting systems, see, e.g., U.S. Patent Nos. 6,225,042, 6,355,479, 6,362,001, and 6,790,662.

[0113] The aAPC system may include at least one exogenous auxiliary molecule. Any suitable number and combination of auxiliary molecules may be used. The auxiliary molecules may be selected from auxiliary molecules such as costimulatory molecules and adhesion molecules. Exemplary costimulatory molecules include CD86, CD64 (FcγRI), 41BB ligand, and IL-21. Adhesion molecules include carbohydrate-binding glycoproteins such as selectins, transmembrane-binding glycoproteins such as integrins, calcium-dependent proteins such as cadherins, and single-pass transmembrane immunoglobulin (Ig) superfamily proteins such as intercellular adhesion molecules (ICAMs) that promote cell-cell or cell-matrix contact. Exemplary adhesion molecules include LFA-3 and ICAMs, such as ICAM-1. Techniques, methods, and reagents useful for the selection, cloning, preparation, and expression of exemplary auxiliary molecules, including costimulatory molecules and adhesion molecules, are exemplified, for example, in U.S. Pat. Nos. 6,225,042, 6,355,479, and 6,362,001.

[0114] B. Formulation and Administration This disclosure relates to CAR T reg Such compositions comprise a prophylactically or therapeutically effective amount of T. regand pharma- ceutically acceptable carriers. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency for use in animals, more particularly in humans, or listed in the United States Pharmacopeia or other generally recognized pharmacopeia. The term "carrier" refers to a diluent, excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, such as those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.

[0115] The compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. Oral formulations may contain standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical agents are described in "Remington's Pharmaceutical Sciences." Such compositions will contain a prophylactically or therapeutically effective amount of the antibody or fragment thereof, preferably in purified form, together with a suitable amount of carrier to provide the form for proper administration to the patient. The formulation should suit the mode of administration, which may be oral, intravenous, intra-arterial, intrabuccal, intranasal, aerosol, bronchial inhalation, or delivered by mechanical ventilation.

[0116] In general, the components of the composition of the present disclosure are supplied either separately or mixed together in unit dosage form, for example as lyophilized powder or water-free concentrate in a sealed container such as an ampoule or sachet indicating the amount of active agent.When the composition is administered by injection, it can be prepared using an injection bottle containing sterile water or saline of pharmaceutical grade.When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed prior to administration.

[0117] The compositions of the present disclosure can be formulated as neutral or salt forms.Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0118] C. Hyperproliferative disorders Hyperproliferative disease can be associated with any disease that causes cells to start replicating uncontrollably, but a typical example is cancer.One of the key elements of cancer is that the normal apoptosis cycle of cells is interrupted, therefore, agents that interrupt cell growth are important as therapeutic agents for treating these diseases.

[0119] Cancer cells that can be treated with the compounds of the present disclosure include, but are not limited to, cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, pancreas, testis, tongue, cervix, or uterus. Additionally, the cancer may be of the following histological types, among others, but is not limited to: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenomatous intrapolypoid adenocarcinoma; adenocarcinoma, familial polyposis coli; solid tumors. ;Carcinoid tumor, malignant;Bronchioloalveolar adenocarcinoma;Papillary adenocarcinoma;Chromophobe carcinoma;Eosinophilic carcinoma;Eosinophilic adenocarcinoma;Basophilic carcinoma;Clear cell adenocarcinoma;Granular cell carcinoma;Follicular adenocarcinoma;Papillary-follicular adenocarcinoma;Nonencapsulated sclerosing carcinoma;Adrenal cortical carcinoma;Endometrioid carcinoma;Skin adnexal carcinoma;Apocrine gland carcinoma;Sebaceous gland carcinoma;Ear wax adenocarcinoma;Mucoepithelial carcinoma;Cystadenocarcinoma;Papillary cystadenocarcinoma;Papillary serous cystadenocarcinoma;Mucinous cystadenocarcinoma;Mucinous adenocarcinoma;Signet ring cell carcinoma;Invasive ductal carcinoma;Medullary carcinoma;Lobular carcinoma;Inflammatory carcinoma ;Paget's disease, breast;Acinic cell carcinoma;Adenosquamous carcinoma;Adenocarcinoma with squamous metaplasia;Thymoma, malignant;Ovarian stromal tumor, malignant;Theca cell tumor, malignant;Granulosa cell tumor, malignant;Androblastoma, malignant;Sertoli cell carcinoma;Leydig cell tumor, malignant;Lipid cell tumor, malignant;Paraganoneuroma, malignant;Extramammary paraganglioma, malignant;Pheochromocytoma;Hemangiosarcoma;Malignant melanoma;Amelanotic melanoma;Superficial spreading melanoma;Malignant melanoma in giant pigmented nevus;Epithelioid cell melanoma;Blue nevus, malignant; Sarcoma;Fibrosarcoma;Fibrous histiocytoma, malignant;Myxosarcoma;Liposarcoma;Leiomyosarcoma;Rhabdomyosarcoma;Embryonal rhabdomyosarcoma;Alveolar rhabdomyosarcoma;Stromatous sarcoma;Mixed tumor, malignant;Mullerian mixed tumor;Nephroblastoma;Hepatoblastoma;Carcinosarcoma;Mesenchymoma, malignant;Brenner tumor, malignant;Phyllodes tumor, malignant;Synovial sarcoma;Mesothelioma, malignant;Dysgerminoma;Embryonal carcinoma;Teratoma, malignant;Ovarian goiter, malignant;Choriocoma;Mesonephroma, malignant;Angiosarcoma;Hemangioendothelioma, malignant;Kaposi's sarcoma;Hemangiopericytoma, malignant;Lymphangiosarcoma;Osteosarcoma;Parosteal osteosarcoma;Chondrosarcoma;Chondrosarcoma, malignant;Mesenchymal chondrosarcoma;Giant cell tumor of bone;Ewing's sarcoma;Odontogenic tumor, malignant;Ameloblastic odontosarcoma;Ameloblastoma, malignant;Ameloblastic fibrosarcoma;Pinealoma, malignant;Chordoma;Glioma, malignant;Ependymoma;Astrocytoma;Protoplasmic astrocytoma;Fibrous astrocytoma;Astroblastoma;Glioblastoma;Oligodendroglioma;Oligodendroglioma;Primitive neuroectodermal tumor;Cerebellar sarcoma;Ganglioblastoma;Neuroblastoma;Retinoblastoma;Olfactory nerve tumor;Meningioma, malignant;Neurofibrosarcoma;Schwannoma, malignant;Granular cell tumor, malignant; Malignant lymphoma; Hodgkin's disease; Hodgkin's; Paragranuloma; Malignant lymphoma, small lymphocytic; Malignant lymphoma, large cell, diffuse; Malignant lymphoma, follicular; Mycosis fungoides; Other specific non-Hodgkin's lymphoma; Malignant histiocytosis; Multiple myeloma; Mast cell sarcoma; Immunoproliferative small intestinal disease; Leukemia; Lymphoid leukemia; Plasma cell leukemia; Erythroleukemia; Lymphocytic leukemia; Myeloid leukemia; Basophilic leukemia; Eosinophilic leukemia; Monocytic leukemia; Mast cell leukemia; Megakaryoblastic leukemia; Myeloid sarcoma; and Hairy cell leukemia. In certain aspects, the tumor may include osteosarcoma, angiosarcoma, rhabdomyosarcoma, leiomyosarcoma, Ewing's sarcoma, glioblastoma, neuroblastoma, or leukemia. ;

[0120] D. Treatment Method In particular, compositions are disclosed herein that can be used in treating cancer in subjects (e.g., human subjects). The compositions are preferably administered to mammals (e.g., rodents, humans, non-human primates, canines, bovines, ovines, equines, felines, etc.) in effective amounts, i.e., amounts that can produce the desired results in the treated subjects (e.g., cause apoptosis of cancerous cells or kill bacterial cells). The toxicity and therapeutic efficacy of the compositions utilized in the disclosed methods can be determined by standard pharmaceutical procedures. As is well known in the medical and veterinary fields, the dosage for any one animal depends on many factors, including the subject's size, body surface area, weight, age, the specific composition administered, the time and route of administration, overall health, clinical symptoms of infection or cancer, and other drugs administered at the same time. The compositions described herein are typically administered at dosages that inhibit bacterial cell growth or proliferation, inhibit biofilm growth, or induce cancerous cell death (e.g., induce apoptosis of cancer cells) as assayed by identifying hematological parameters (complete blood count (CBC)) or reduced cancer cell growth or proliferation.

[0121] In some embodiments, the present disclosure provides an effective amount of the T reg In one embodiment, the medical disease or disorder is treated by the transfer of an immune cell population that elicits an immune response. In certain embodiments of the present disclosure, the cancer is treated by the transfer of an immune cell population that elicits an immune response. reg The present invention provides a method for treating or delaying the progression of cancer in an individual, comprising administering an effective amount of antigen-specific cell therapy to the individual.The method can be applied to immune disorders, solid cancers, blood cancers, and viral infection treatment.

[0122] The tumors that this treatment method is useful for include any malignant cell type, such as those found in solid tumors or blood tumors.Exemplary solid tumors can include, but are not limited to, tumors of organs selected from the group consisting of pancreas, colon, appendix, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast.Exemplary blood tumors include bone marrow tumors, T or B cell malignancies, leukemia, lymphoma, blastoma, myeloma, etc. Further cancers that can be treated using the methods provided herein include, but are not limited to, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), cancer of the peritoneum, gastric or gastric cancer (including gastrointestinal cancer and gastrointestinal stromal tumors), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

[0123] The cancer may be of the following histological types, among others, but is not limited to: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenomatous intrapolypoid adenocarcinoma; adenocarcinoma, familial polyposis coli; solid tumor; carcinoid tumor, malignant; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe ;Esinophilic carcinoma;Esinophilic adenocarcinoma;Basophilic carcinoma;Clear cell adenocarcinoma;Granular cell carcinoma;Follicular adenocarcinoma;Papillary-follicular adenocarcinoma;Nonencapsulated sclerosing carcinoma;Adrenal cortical carcinoma;Endometrioid carcinoma;Skin adnexal carcinoma;Apocrine adenocarcinoma;Sebaceous gland carcinoma;Earwax gland carcinoma;Mucous epidermoid carcinoma;Cystadenocarcinoma;Papillary cystadenocarcinoma;Papillary serous cystadenocarcinoma;Mucinous cystadenocarcinoma;Mucinous adenocarcinoma;Signet ring cell carcinoma;Invasive ductal carcinoma;Medullary carcinoma;Lobular carcinoma;Inflammatory carcinoma;Paget's disease of the breast;Acinic cell carcinoma;Adenosquamous carcinoma;Adenocarcinoma with squamous metaplasia;Thymoma, malignant;Ovarian stromal tumor, malignant;Theca cell tumor, malignant ;Granulosa cell tumor, malignant;Androblastoma, malignant;Sertoli cell carcinoma;Leydig cell tumor, malignant;Lipid cell tumor, malignant;Paraganeuroma, malignant;Extramammary paraganglioma, malignant;Pheochromocytoma;Hemangiocytoma;Malignant melanoma;Amelanotic melanoma;Superficial spreading melanoma;Malignant leptomeningeal melanoma;Acral leptomeningeal melanoma;Nodular melanoma;Malignant melanoma in giant pigmented nevus;Epithelioid cell melanoma;Blue nevus, malignant;Sarcoma;Fibrosarcoma;Fibrous histiocytoma, malignant;Myxosarcoma;Liposarcoma;Leiomyosarcoma;Rhabdomyosarcoma;Embryonal rhabdomyosarcoma;Alveolar rhabdomyosarcoma;Stromatous sarcoma;Mixed tumor, malignant; Mixed Müllerian tumor;Nephroblastoma;Hepatoblastoma;Carcinosarcoma;Mesenchymoma, malignant;Brenner tumor, malignant;Phyllodes tumor, malignant;Synovial sarcoma;Mesothelioma, malignant;Dysgerminoma;Embryonal carcinoma;Teratoma, malignant;Ovarian goiter, malignant;Choriocarcinoma;Mesonephroma, malignant;Hemangiosarcoma;Hemangioendothelioma, malignant;Kaposi's sarcoma;Hemangiopericytoma, malignant;Lymphangiosarcoma;Osteosarcoma;Parocytic osteosarcoma;Chondrosarcoma;Chondrosarcoma, malignant;Mesenchymal chondrosarcoma;Giant cell tumor of bone;Ewing's sarcoma;Odontogenic tumor, malignant;Ameloblastoma, malignant;Ameloblastic fibrosarcoma;Pinealoma, malignant;Chordoma;Glioma, malignant;Ependymoma;Astrocytoma;Protoplasmic astrocytoma;Fibrous astrocytoma;Astroblastoma;Glioblastoma;Oligodendroglioma;Oligodendroglioma;Primitive neuroectodermal tumor;Cerebellar sarcoma;Ganglioblastoma;Neuroblastoma;Retinoblastoma;Olfactory nerve tumor;Meningioma, malignant;Neurofibrosarcoma;Schwannoma, malignant;Granular cell tumor, malignant;Malignant lymphoma;Hodgkin's disease;Hodgkin;Paraguloma;Malignant lymphoma, small lymphocytic;Malignant lymphoma, large cell, diffuse;Malignant lymphoma, follicular;Mycosis fungoides;Other specified non-Hodgkin lymphoma;B-cell lymphoma;Low-grade / follicular non-Hodgkin lymphoma (NHL);Small lymphocytic (SL) NHL;Intermediate-grade / follicular NHL;Intermediate-grade diffuse NHL;High-grade High-grade immunoblastic NHL;High-grade lymphoblastic NHL;High-grade small noncleaved cell NHL;Giant mass disease NHL;Mantle cell lymphoma;AIDS-related lymphoma;Waldenstrom's macroglobulinemia;Malignant histiocytosis;Multiple myeloma;Mast cell sarcoma;Immunoproliferative small intestinal disease;Leukemia;Lymphoid leukemia;Plasma cell leukemia;Erythroleukemia;Lymphoblastic cell leukemia;Myeloid leukemia;Basophilic leukemia;Eosinophilic leukemia;Monocytic leukemia;Mast cell leukemia;Megakaryoblastic leukemia;Myeloid sarcoma;Hairy cell leukemia;Chronic lymphocytic leukemia (CLL);Acute lymphocytic leukemia (ALL);Acute myeloid leukemia (AML);And chronic myeloblastic leukemia. ;

[0124] To determine the suitability of the cells provided herein for therapeutic use, the cells can first be tested in an appropriate animal model. At one level, the ability of the cells to survive and maintain phenotype in vivo is evaluated. The cells provided herein are administered to immunodeficient animals (e.g., NOG mice, or animals rendered immunodeficient chemically or by irradiation) at a site that can undergo further observation, for example, under the kidney capsule, in the spleen, in the liver lobule, or in the bone marrow. After a period of days to weeks or more, the tissue is harvested and evaluated for whether the starting cell type, such as red blood cells, is still present. This can be done by providing the administered cells with a detectable label (e.g., green fluorescent protein or beta-galactosidase); or by measuring a constitutive marker specific for the administered human cells. When the cells provided herein are tested in rodent models, the presence and phenotype of the administered cells can be evaluated by immunohistochemistry or ELISA using human-specific antibodies, or by RT-PCR analysis using primers and hybridization conditions that cause specific amplification for human polynucleotide sequences. Suitable markers for assessing gene expression at the mRNA or protein level are provided elsewhere in this disclosure.

[0125] T provided by the method of the present disclosure reg can be tested in various animal models for its ability to treat hematological disorders and injuries. For example, the sickle cell anemia mouse model or the T / B cell deficient Rag-2 knockout mouse can be particularly useful animal models for testing the myeloid and lymphoid cells disclosed herein.

[0126] T provided in certain aspects of the present disclosure exhibits desirable functional properties or efficacy in animal models. regmay also be suitable for direct administration to a human subject in need thereof. The cells may be administered at any site with adequate access to the circulation for hemostasis purposes. Hematopoietic cells or their precursors may also be delivered to the site of injury or disease.

[0127] T provided in certain aspects of the present disclosure reg can be used for therapy of any subject in need thereof. Human conditions suitable for such therapy include various anemias and hemoglobinopathies, as well as diseases characterized by a decrease in the number of hematopoietic cells (e.g., myelodysplastic syndromes, myelofibrosis, neutropenia, agranulocytosis, Glanzmann's thrombasthenia, thrombocytopenia, and acquired immune deficiency syndrome, etc.). For human therapy, the dose is generally about 10 9 ~10 12 cells, typically about 5 x 10 9 ~5×10 10 The individual cells will be adjusted for the weight of the subject, the nature and severity of the disease, and the replicative capacity of the cells being administered. Ultimate responsibility for determining the mode of treatment and appropriate dosage rests with the supervising clinician.

[0128] A therapeutically effective dose of T reg can be administered by several routes, including parenteral administration, for example, intravenous, intraperitoneal, intramuscular, intrasternal, or intraarticular injection or infusion.

[0129] T for Use in Adoptive Cell Therapy reg A therapeutically effective amount of is an amount that achieves a desired effect in the treated subject. For example, it is an amount necessary to inhibit the progression of or cause the regression of an autoimmune or alloimmune disease, or that can reduce symptoms caused by an autoimmune disease, such as pain and inflammation. reg This may be the amount required to reduce symptoms associated with inflammation, such as pain, edema, and hyperthermia, and this may also be the amount required to reduce or prevent rejection of a transplanted organ.

[0130] T reg The population may be administered a treatment regimen consistent with the disease, for example, a single or several doses over one or several days to ameliorate the condition, or regular doses over an extended period to inhibit disease progression and prevent disease recurrence. The precise dose to be employed in the formulation will also depend on the route of administration and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. reg The therapeutically effective amount of will depend on the subject being treated, the severity and type of the disease, and the mode of administration. In some embodiments, the dose that can be used in the treatment of a human subject is at least 3.8×10 4 , at least 3.8 × 10 5 , at least 3.8 × 10 6 , at least 3.8 × 10 7 , at least 3.8 × 10 8 , at least 3.8 × 10 9 , or at least 3.8 × 10 10 immune cells / m 2 In certain embodiments, the dose used in the treatment of a human subject ranges from about 3.8×10 9 ~Approx. 3.8×10 10 immune cells / m 2 In a further embodiment, the therapeutically effective amount of immune cells ranges from about 5×10 6 Cells / kg body weight ~ approx. 7.5 x 10 8 Up to 2 x 10 cells / kg body weight, for example, 7 ~ approx. 5 x 10 cells 8 Up to 5 x 10 cells / kg body weight or approximately 7 ~ approx. 2 x 10 cells 8 The amount of immune cells can vary up to 100 cells / kg body weight.The exact amount of immune cells can be easily determined by those skilled in the art based on the age, weight, sex and physiological condition of the subject.Effective amount can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0131] T regcan be administered in combination with one or more other therapeutic agents for the treatment of immune-mediated disorders. Combination therapy can include, but is not limited to, one or more antibacterial agents (e.g., antibiotics, antivirals, and antifungals), antitumor agents (e.g., fluorouracil, methotrexate, paclitaxel, fludarabine, etoposide, doxorubicin, or vincristine), immunodepleting agents (e.g., fludarabine, etoposide, doxorubicin, or vincristine), immunosuppressants (e.g., azathioprine or glucocorticoids, such as dexamethasone or prednisone), anti-inflammatory agents (e.g., glucocorticoids, such as hydrocortisone, dexamethasone, or prednisone, or nonsteroidal anti-inflammatory agents, such as acetylsalicylic acid, ibuprofen, or naproxen sodium), cytokines (e.g., interleukin-10 or transforming growth factor-β), hormones (e.g., estrogen), or vaccines. In addition, immunosuppressive or immunotolerogenic agents can be administered, including, but not limited to, calcineurin inhibitors (e.g., cyclosporine and tacrolimus); mTOR inhibitors (e.g., rapamycin); mycophenolate mofetil, antibodies (e.g., recognizing CD3, CD4, CD40, CD154, CD45, IVIG, or B cells); chemotherapeutic agents (e.g., methotrexate, treosulfan, busulfan); irradiation; or chemokines, interleukins, or inhibitors thereof (e.g., BAFF, IL-2, anti-IL-2R, IL-4, JAK kinase inhibitors). Such additional pharmaceutical agents can be administered before, during, or after the administration of immune cells, depending on the desired effect. This administration of cells and agents may be by the same route or different routes, and may be at either the same or different sites.

[0132] The therapeutic method (including preventive treatment) of the present disclosure generally comprises administering a therapeutically effective amount of the composition described herein to a subject in need thereof, including a mammal, particularly a human. Such treatment would be suitably administered to a subject, particularly a human, who suffers from, has, is susceptible to, or is at risk of a disease, disorder, or symptoms thereof. The determination of such a subject as "at risk" can be made by any objective or subjective determination, such as by diagnostic testing or the subject's or health care provider's opinion (e.g., genetic testing, enzyme or protein markers, markers (as defined herein), family history, etc.).

[0133] In one embodiment, the present disclosure provides a method for monitoring the progress of treatment. The method includes determining the level of changes in hematological parameters and / or cancer stem cell (CSC) analysis using cell surface proteins as diagnostic markers (e.g., including but not limited to CD34, CD38, CD90, and CD117) or diagnostic measurements (e.g., screening, assays) in subjects suffering from or susceptible to cancer (e.g., leukemia)-related disorders or symptoms thereof, and administering a therapeutic amount of a composition described herein. The level of the marker determined in this manner can be compared to known marker levels in either healthy normal controls or other affected patients to ascertain the disease state of the subject. In a preferred embodiment, a second marker level is determined for the subject at a later time point than the determination of the first level, and the two levels are compared to monitor the progress of the disease or the efficacy of the therapy. In certain preferred embodiments, the subject's pre-treatment marker level is determined before treatment begins according to the methods described herein; the pre-treatment marker level can then be compared to the subject's marker level after treatment begins to determine the efficacy of the treatment.

[0134] E. Further Therapy In certain embodiments, the compositions and methods of the present embodiments are directed to T reg Includes.

[0135] In certain embodiments, the compositions and methods of the present embodiments include T reg The additional therapy may be radiation therapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the above. The additional therapy may be in the form of adjuvant or neoadjuvant therapy.

[0136] The methods and compositions, including combination therapies, enhance the therapeutic or protective effects and / or enhance the therapeutic effects of another anti-cancer or anti-hyperproliferative therapy. The therapeutic and prophylactic methods and compositions can be provided in a combined amount effective to achieve a desired effect, e.g., killing of cancer cells and / or inhibiting hyperproliferation of cells. The methods can include contacting cells with both the antibody or antibody fragment and the second therapy. The tissue, tumor, or cells can be contacted with one or more compositions or pharmacological formulations that include one or more of the agents (i.e., antibody or antibody fragment or anti-cancer agent), or by contacting the tissue, tumor, and / or cells with two or more separate compositions or formulations, where one composition provides 1) an antibody or antibody fragment, 2) an anti-cancer agent, or 3) both an antibody or antibody fragment and an anti-cancer agent. It is also contemplated that such combination therapies can be used in conjunction with chemotherapy, radiation therapy, surgery, or immunotherapy.

[0137] As applied to a cell, the terms "contacting" and "exposing" are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to or placed in direct juxtaposition with a target cell. For example, to achieve cell death, both agents are delivered to the cell in a combined amount effective to kill the cell or prevent the cell from dividing.

[0138] The inhibitory antibody can be administered before, during, after, or in various combinations with the anti-cancer treatment. Administration may be at intervals ranging from the same time to minutes, days, or weeks. In embodiments where the antibody or antibody fragment is provided to the patient separately from the anti-cancer drug, one will generally ensure that there is no effective time between the time of each delivery so that the two compounds can still exert their advantageously combined effect on the patient. In such cases, it is contemplated that the antibody therapy and the anti-cancer therapy can be provided to the patient within about 12-24 or 72 hours of each other, more particularly within about 6-12 hours of each other. In some situations, it may be desirable to significantly extend the duration of treatment, where several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) pass between the respective administrations.

[0139] In certain embodiments, a course of treatment is expected to last for 1-90 days or more (such ranges inclusive). It is contemplated that one agent may be given on any day from day 1 to day 90 (such ranges inclusive) or any combination thereof, and another agent may be given on any day from day 1 to day 90 (such ranges inclusive) or any combination thereof. Within a day (24 hour period), one or more doses of the agent may be given to the patient. It is further contemplated that after a course of treatment, there will be a period during which no anti-cancer treatment is administered. This period may last for 1-7 days and / or 1-5 weeks and / or 1-12 months or more (such ranges inclusive), depending on the patient's condition, e.g., the patient's prognosis, strength, health, etc. It is anticipated that the treatment cycle will be repeated as necessary.

[0140] In some embodiments, the additional therapy is administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional therapy is administration of a side effect limiting agent (e.g., an agent intended to reduce the occurrence and / or severity of side effects of treatment, such as an anti-nausea agent). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is a therapy that targets the PBK / AKT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent. The additional therapy can be one or more of the chemotherapeutic agents known in the art.

[0141] Various combinations can be used. In the following example, T reg The composition is "A" and the additional anti-cancer therapy is "B": TIFF2025515324000002.tif18128

[0142] The administration of any compound or therapy of the present embodiments to a patient will follow general protocols for the administration of such compounds, taking into account the toxicity, if any, of the agent. Thus, in some embodiments, there is a step of monitoring for toxicity that may result from the combination therapy.

[0143] 1.Chemotherapy A wide variety of chemotherapeutic agents can be used according to this embodiment. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to imply a compound or composition administered in the treatment of cancer. These agents or drugs are classified according to their mode of activity in cells, for example, whether or at what stage they affect the cell cycle. Alternatively, agents can be characterized based on their ability to directly crosslink and intercalate into DNA or to induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

[0144] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocalcistatin; Lumycin (including synthetic analogs, KW-2189 and CB1-TM1); erytherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobuenbiquine, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma II and calicheamicin omega II); dynemicins, such as dynemicin A; bisphosphonates, such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, authrarnycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo- L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolinodo-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycin, peplomycin, porfiromycin, piriromycin, uromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamipyridine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal agents such as mitotane and trilostane; folic acid supplements such as frolinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elformithine acetate; epothilones;etoglucide;gallium nitrate;hydroxyurea;lentinan;lonidainine;maytansinoids, e.g. maytansine and ansamitocin;mitoguazone;mitoxantrone;mopidanmol;nitraerine;pentostatin;phenamet;pirarubicin;losoxantrone;podophyllic acid;2-ethylhydrazide;procarbazine;PSK polysaccharide complex;razoxane;rhizoxin;schizophyllan;spirogermanium;tenuazonic acid;triazicon;2,2',2"-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A) A), Roridin A and Anguidine); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosine; Arabinoside ("Ara-C"); Cyclophosphamide; Taxoids, such as paclitaxel and docetaxel-gemcitabine; 6-Thioguanine; Mercaptopurine; Platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; Vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine; Vinorelbine; Novan torone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl protein transferase inhibitors, transplatinum, as well as pharmaceutically acceptable salts, acids, or derivatives of any of the above;

[0145] 2. Radiation therapy Other agents that cause DNA damage and are widely used include gamma radiation, commonly known as X-rays, and / or directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents are also contemplated, such as microwaves, proton beam irradiation (U.S. Patents Nos. 5,760,395 and 4,870,287), and UV irradiation. All of these agents most likely affect a wide range of damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-rays range in radiation dosage from daily doses of 50-200 roentgens for extended periods (3-4 weeks) to single doses of 2000-6000 roentgens. Dosage ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by the neoplastic cells.

[0146] 3. Immunotherapy Those skilled in the art will understand that additional immunotherapy can be used in combination or in conjunction with the method of this embodiment. In cancer treatment, immunotherapy generally relies on the use of immune effector cells and molecules that target and destroy cancer cells. Rituximab (RITUXAN®) is such an example. The immune effector can be, for example, an antibody specific for some marker on the surface of tumor cells. The antibody can act alone as an effector of therapy, or it can recruit other cells to actually affect the death of the cells. The antibody can also be conjugated to a drug or toxin (chemotherapeutic agent, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and can act as a targeting agent. Alternatively, the effector can be a lymphocyte that carries a surface molecule that interacts with tumor cell targets either directly or indirectly. Various effector cells include cytotoxic T cells and NK cells.

[0147] Antibody-drug conjugates have emerged as a breakthrough approach for the development of cancer therapeutics. Cancer is one of the leading causes of death worldwide. Antibody-drug conjugates (ADCs) contain a monoclonal antibody (MAb) covalently attached to a cell-killing drug. This approach combines the high specificity of the MAb for its antigen target with a highly potent cytotoxic drug, resulting in an "armed" MAb that delivers the payload (drug) to tumor cells where the levels of the antigen are high (Carter et al., 2008; Teicher 2014; Leal et al., 2014). Targeted delivery of the drug also minimizes its exposure, reduces toxicity, and improves the therapeutic index in normal tissues. The FDA approval of two ADC drugs, ADCETRIS® (brentuximab vedotin) in 2011 and KADCYLA® (trastuzumab emtansine or T-DM1) in 2013, validates this approach. Currently, there are more than 30 ADC drug candidates in various stages of clinical trials for cancer treatment (Leal et al., 2014). As antibody engineering and linker-payload optimization become increasingly mature, the discovery and development of new ADCs is increasingly dependent on the identification and validation of new targets suitable for this approach (Teicher 2009) and the generation of targeted MAbs. Two criteria for ADC targets are upregulated / high levels of expression in tumor cells and strong internalization.

[0148] In one aspect of immunotherapy, tumor cells must have some marker that is suitable for targeting, i.e., not present on the majority of other cells. Many tumor markers exist, and any of these may be suitable for targeting in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. An alternative aspect of immunotherapy is to combine anti-cancer effects with immune stimulating effects. There are also immune stimulating molecules, including: cytokines, such as IL-2, IL-4, IL-12, GM-CSF, γ-IFN, chemokines, such as MIP-1, MCP-1, IL-8, and growth factors, such as FLT3 ligand.

[0149] Examples of immunotherapies currently under investigation or in use include immune adjuvants, such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Patents. Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapies, such as interferon α, β, λ, and γ, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapies, such as TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Patent Nos. 5,830,880 and 5,846,945); and monoclonal antibodies, such as anti-CD20, anti-ganglioside GM2, and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Patent No. 5,824,311). It is contemplated that one or more anti-cancer therapies can be used in conjunction with the antibody therapies described herein.

[0150] In some embodiments, the immunotherapy can be an immune checkpoint inhibitor. Immune checkpoints are molecules of the immune system that either turn up (e.g., costimulatory molecules) or turn down signals. Inhibitory checkpoint molecules that can be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and V-domain Ig suppressor of T cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0151] Immune checkpoint inhibitors may be drugs such as small molecules, recombinant forms of ligands or receptors, or antibodies, such as human antibodies (e.g., International Patent Publication WO2015016718; Pardoll, Nat Rev Cancer, 12(4): 252-64, 2012; both of which are incorporated herein by reference). Known inhibitors of immune checkpoint proteins or their analogs may be used, and in particular chimeric, humanized, or human forms of antibodies may be used. As will be appreciated by those skilled in the art, alternative and / or equivalent names may be used for certain antibodies referred to in this disclosure. Such alternative and / or equivalent names are interchangeable in the context of the present invention. For example, lambrolizumab is also known by the alternative and equivalent names MK-3475 and pembrolizumab.

[0152] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In a specific aspect, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In a specific aspect, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In a specific aspect, the PDL2 binding partner is PD-1. The antagonist can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. US8735553, US8354509, and US8008449, all of which are incorporated herein by reference. Other PD-1 axis antagonists for use in the methods provided herein are known in the art and are described, for example, in U.S. Patent Application Nos. US20140294898, US2014022021, and US20110008369, all of which are incorporated herein by reference.

[0153] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin that includes an extracellular portion or a PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in WO 2006 / 121168. Pembrolizumab, also known as MK-3475, Merck 3475, Lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in WO2009 / 114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342.

[0154] Another immune checkpoint that can be targeted by the methods provided herein is cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has GenBank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when bound to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is similar to the T cell costimulatory protein, CD28, and both molecules bind to CD80 and CD86, also called B7-1 and B7-2, respectively, on antigen-presenting cells. CTLA4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA4 is also found in regulatory T cells and may be important for their function. T cell activation through the T cell receptor and CD28 increases the expression of CTLA-4, an inhibitory receptor for the B7 molecule.

[0155] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0156] Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art-recognized anti-CTLA-4 antibodies can be used. For example, anti-CTLA-4 antibodies disclosed in US8,119,129, WO01 / 14424, WO98 / 42752; WO00 / 37504 (CP675,206, also known as tremelimumab; formerly ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et al. (1998) Proc Natl Acad Sci USA 95(17): 10067-10071; Camacho et al. (2004) J Clin Oncology 22(145): Abstract No. 2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res 58:5301-5304 can be used in the methods disclosed herein. The teachings of each of the foregoing publications are incorporated herein by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 can also be used. For example, humanized CTLA-4 antibodies are described in International Patent Application Nos. WO2001014424, WO2000037504, and U.S. Patent No. US8017114; all of which are incorporated herein by reference.

[0157] An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof (see, e.g., WOO1 / 14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab, and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding to and / or binds to the same epitope on CTLA-4 as the aforementioned antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the aforementioned antibodies (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab).

[0158] Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors, such as those described in U.S. Patent Nos. US5844905, US5885796, and International Patent Application Nos. WO1995001994 and WO1998042752, all of which are incorporated herein by reference; and immunoadhesions, such as those described in U.S. Patent No. US8329867, which is incorporated herein by reference.

[0159] 4.Surgery Approximately 60% of people with cancer will undergo some type of surgery, including preventive surgery, diagnostic surgery, or staging surgery, curative surgery, and palliative surgery. Curative surgery includes resection, in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed, and can be used in conjunction with other therapies, such as the present treatment, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapy. Tumor resection refers to the physical removal of at least a part of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgery (Mohs surgery).

[0160] Upon removal of part or all of the cancerous cells, tissues, or tumors, a cavity may be formed in the body.Treatment may be achieved by perfusion, direct injection, or local application of the area with additional anti-cancer therapy.Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.These treatments may also be of various dosages.

[0161] 5. Other agents It is contemplated that other agents can be used in combination with certain aspects of this embodiment to improve the therapeutic efficacy of treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that enhance the sensitivity of hyperproliferative cells to apoptosis inducers, or other biological agents. It is believed that increasing intercellular signaling by increasing the number of GAP junctions enhances the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cytostatic or differentiation agents can be used in combination with certain aspects of this embodiment to improve the anti-hyperproliferative efficacy of treatment. It is contemplated that inhibitors of cell adhesion will improve the efficacy of this embodiment. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that enhance the sensitivity of hyperproliferative cells to apoptosis, such as antibody c225, can be used in combination with certain aspects of this embodiment to improve the efficacy of treatment.

[0162] III. Kit In various aspects of this embodiment, kits are contemplated that include the therapeutic agent and / or other therapeutic and delivery agents. In some embodiments, this embodiment contemplates a kit for preparing and / or administering the T cell composition of this embodiment. The kit can include one or more sealed vials that include any of the pharmaceutical compositions of this embodiment. The kit can include, for example, T cells and reagents for preparing, formulating, and / or administering the components of this embodiment or performing one or more steps of the method of the invention. In some embodiments, the kit can also include a suitable container that will not react with the components of the kit, e.g., an eppendorf tube, an assay plate, a syringe, a bottle, or a tube. The container can be made of a material that can be sterilized, e.g., plastic or glass.

[0163] The kit may further include an instruction sheet outlining the procedural steps of the methods described herein and which would substantially follow the same procedures as described herein or known to one of skill in the art. The instruction information may be in a computer readable medium that includes machine readable instructions that, when executed using a computer, display a real or virtual procedure for the delivery of a pharma- ceutical effective amount of a therapeutic agent. EXAMPLES

[0164] IV. Working Examples The following examples are included to demonstrate preferred aspects of the present disclosure. Those skilled in the art will recognize that the techniques disclosed in the following examples represent techniques that the inventors have discovered to function well in the practice of the present disclosure, and therefore may be considered to constitute preferred modes for its practice. However, those skilled in the art should recognize in light of the present disclosure that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present disclosure.

[0165] Example 1 -CD19-CAR T reg Generation and characterization of Purify lentiviral particles containing the CAR construct from human peripheral blood T reg by transducing anti-CD19 CD28-CD3ζ CAR T reg The resulting CD19 CAR T reg showed high FOXP3 expression (Figure 3E) and demethylated T reg Surprisingly, CAR T cells maintained their specific TSDR and were activated and proliferated in response to irradiated CD19-expressing K562 cells (a myeloid leukemia cell line with no HLA or CD80 / 86 expression), but not in parental K562 cells. reg also efficiently killed CD19-expressing tumor cells in vitro, including NALM6 (B cell leukemia cell line, Figure 3A), CD19-K562, and CD19-A549 (epithelial lung cancer cell lines, Figure 3F), as assessed by Annexin V and DAPI staining and microscopy. This killing activity was consistent with that of differentially labeled WT NALM6 and CD19 KO NALM6 and CAR T reg Surprisingly, single-cell cytokine analysis demonstrated that T reg It was revealed that CAR-mediated activation of CD19-A549 resulted in a cytokine production profile almost identical to that of CAR T effector (Teff) cells, i.e., high production of the inflammatory and cytolytic molecules IFN-γ, TNFα, perforin, and granzyme B (Figure 3B). Moreover, as seen in Figure 3C for NALM6 and in Figure 3G for CD19-A549 (results similar to CD19-K562), CAR T reg suppressed the growth of CD19+ tumor cells in NSG mice when co-injected with tumor cells. reg In vitro, CAR T eff CAR-dependent inhibition of cell proliferation (Figure 3D), thereby allowing these modified T reg are shown to retain T cell phenotypic and functional properties in addition to their de novo tumor cytotoxic properties.

[0166] Peripheral blood mononuclear cells (PBMCs) were isolated using a Ficoll gradient, CD4+ T cells were magnetically enriched, and CD4+CD25highCD127low Treg and CD4+CD25lowCD127high Teff cells were sorted using fluorescence-assisted cell sorting (FACS). After sorting, cells were plated and activated using anti-CD3 / 28 beads and recombinant human IL-2. Two days later, cells were transduced with anti-CD19 CD28-CD3ζ CAR-containing lentiviral particles and expanded for 1 week in the presence of IL-2. CAR Treg and CAR Teff cells were then incubated with either irradiated target cells, parental K562 cells (unstimulated), CD64-CD80-K562 cells treated with anti-CD3 antibody (TCR / CD28 stimulated), or CD19-K562 cells (CD28-CD3ζ CAR stimulated). CD64 is a high affinity Fc receptor; CD64-expressing K562 cells were previously shown to maintain surface expression of anti-CD3 after preincubation with anti-CD3 antibody (16). After 1 day of in vitro co-incubation, CAR Treg and CAR Teff cells were enriched using a human CD4+ magnetic positive selection kit and processed for either bulk RNA-seq libraries (Figures 4-10, 35) or 10x Genomics single cell RNA-seq (Figures 11-22, 24-26, 34, 36, 37).

[0167] For cytokine secretion analysis (Figure 27), CAR Treg and CAR Teff were co-incubated with the above irradiated target cell lines and after 48 hours, the supernatants were collected and sent to EveTech Technologies for 48-plex cytokine quantification. For cytokine production coupled with FOXP3 protein expression analysis at the single cell level (Figures 28-33), CAR Treg and CAR Teff were co-incubated with the above irradiated target cell lines overnight, followed by incubation with Brefeldin A, intracellular staining and analysis by flow cytometry. For Figure 39, splenocytes of C57BL / 6 mice were isolated and CD4+CD25+Treg and CD4+CD25-Tconv cells were magnetically purified from splenocytes and activated with anti-mouse CD3 / CD28 beads and recombinant human IL-2 (2,000 IU / ml IL-2 for Treg and 100 IU / ml IL-2 for Tconv). After 2 days, the beads were removed from mouse Tregs and Tconvs and transduced with a retrovirus encoding an anti-mouse CD19 mCD28-CD3ζ CAR. After 1 week of in vitro expansion in the presence of recombinant human IL-2, the cells were used for the assay. For Figure 39A, mouse CD19 CAR Tregs or mouse CD19 CAR Tconvs or untransduced (UT) Tconv cells were incubated with A20 mouse lymphoma cells at various ratios and lactate dehydrogenase (LDH) release was measured after 2 days as a measure of cell death. For Figure 39B, mouse CD19 CAR Tregs were co-incubated with mouse Tconv cells labeled with CELLTRACE™ Violet (CTV) and after 3 days, the dilution of CTV was measured by flow cytometry to assess the inhibition of Tconv cell proliferation. For Figure 39C, mouse CD19 CAR Tregs were intracellularly stained with anti-mouse Foxp3 APC and analyzed using flow cytometry.

[0168] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of this disclosure.Although the compositions and methods of the present invention are described in terms of preferred embodiments, it will be apparent to those skilled in the art that modifications can be made to the methods and steps or steps of the methods described herein without departing from the concept, spirit and scope of the invention.More specifically, it will be apparent that certain agents that are chemically and physiologically related can be substituted for the agents described herein while the same or similar results are achieved.All such similar substitutions and modifications that are apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

[0169] References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF2025515324000003.tif182161TIFF2025515324000004.tif210161TIFF2025515324000005.tif210161

Claims

1. A composition for treating cancer in a subject, wherein the composition contains an effective amount of chimeric antigen receptor (CAR) regulatory T cells (T reg A composition comprising, wherein the CAR T reg expresses the pro-inflammatory cytokines IL-3, CXCL9, CXCL11, IL-2, IL-9, IL-17A, CSF3, CCL3, and / or IL-6.

2. The composition according to claim 1, wherein the CAR comprises a CD28-CD3ζ intracellular domain.

3. The composition according to claim 1 or 2, wherein the CAR binds to a tumor-associated antigen.

4. The aforementioned tumor-associated antigens include TRP-1, EpCAM, CD19, CD20, CD22, B-cell maturation antigen (BCMA), carcinoembryonic antigen (CEA), alpha-fetoprotein, CA-125, MUC-1, epithelial tumor antigen, melanoma-associated antigen (MAGE), mutant p53-derived peptide-HLA, mutant ras-derived peptide-HLA, HER2 / Neu, ERBB2, folate-binding protein, HIV-1 coat glycoprotein gp120, HIV-1 coat glycoprotein gp41, GD2, CD123 (IL3RA), CD319, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, The composition according to claim 3, selected from the group consisting of IL-11Rα, κ chain, λ chain, CSPG4, ERBB2, EGFR, EGFRvIII, VEGFR2, TNFRSF17, SDC1, FAP, CD44, MS4A1, EPCAM, CA9, CD174, TNFRSF8, CD33, CD38, EPHA2, CD248, CD274, CD276, CD5, NCAM1, CD70, ERBB2, KDR, L1CAM, ULBP1, ULBP2, IL1RAP, GPC3, IL13RA2, ROR1, CEACAM5, MET, FOLH1, CSPG4, CD133, GPNMB, and PSCA.

5. Said T reg Human T reg The composition according to claim 1.

6. Prehit reg However, CD4 + CD25 高 CD127 低 The composition according to claim 5, which is isolated from human peripheral blood by selecting cells.

7. The composition according to claim 1, wherein the cancer is melanoma, breast cancer, pancreatic cancer, or lung cancer.

8. The composition according to claim 1, wherein the cancer is oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine cancer, or hematopoietic cancer, glioma, sarcoma, carcinoma, lymphoma, melanoma, fibroma, meningioma, brain tumor, oropharyngeal cancer, nasopharyngeal cancer, kidney cancer, biliary tract cancer, pheochromocytoma, islet cell carcinoma, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteogenic sarcoma, multiple neuroendocrine tumors type I and II, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, glioblastoma, neuroblastoma, or skin cancer.

9. The composition according to claim 1, wherein the T cells and / or at least one further therapeutic agent are administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, locally, or by direct injection or perfusion.

10. Said CAR T reg The composition according to claim 1, wherein it expresses IFN-γ, TNF-α, perforin, granzyme A, NKG7, granzyme H, and / or granzyme B.

11. The CAR T reg The composition according to claim 1, wherein the CAR T expresses granzyme A, granzyme B, perforin 1 (PRF1), NKG7, and / or granzyme H, which are cytolytic proteins.

12. Said CAR T reg The composition according to claim 1, wherein the organism secretes IL-10.

13. Said CAR T reg However, FOXP3, CD25, BATF, ICOS, GITR, HELIOS, CD40L, and / or demethylated T reg The composition according to claim 1, which expresses a specific demethylation region (TSDR).

14. Said CAR T reg The composition according to claim 1, wherein the composition is conjugated with a cytotoxic agent.

15. The composition according to claim 14, wherein the cytotoxic agent is a chemotherapeutic agent, IL-2, IL-15, IL-18, soluble TRAIL, perforin, granzyme A, or granzyme B.

16. The composition according to claim 1, further comprising the step of administering at least a second anti-cancer therapy to the subject.

17. The composition according to claim 16, wherein the second anti-cancer therapy is surgery, chemotherapy, radiotherapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy.

18. T cells engineered to express CAR constructs reg A composition containing the following:

19. CAR is TRP-1, EpCAM, CD19, CD20, CD22, B cell maturation antigen (BCMA), fetal carcinoma antigen (CEA), alpha-fetoprotein, CA-125, MUC-1, epithelial tumor antigen, melanoma-associated antigen (MAGE), mutant p53-derived peptide-HLA, mutant ras-derived peptide-HLA, HER2 / Neu, ERBB2, folate-binding protein, HIV-1 coat glycoprotein gp120, HIV-1 coat glycoprotein gp41, GD2, CD123 (IL3RA), CD319, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11Rα, κ The composition according to claim 18, which binds to a tumor-associated antigen selected from the group consisting of a chain, lambda chain, CSPG4, ERBB2, EGFR, EGFRvIII, VEGFR2, TNFRSF17, SDC1, FAP, CD44, MS4A1, EPCAM, CA9, CD174, TNFRSF8, CD33, CD38, EPHA2, CD248, CD274, CD276, CD5, NCAM1, CD70, ERBB2, KDR, L1CAM, ULBP1, ULBP2, IL1RAP, GPC3, IL13RA2, ROR1, CEACAM5, MET, FOLH1, CSPG4, CD133, GPNMB, and PSCA.

20. Said T reg but, It expresses the pro-inflammatory cytokines IFN-γ, IL-3, CXCL9, CXCL11, IL-2, IL-9, IL-17A, CSF3, CCL3, TNFα, and / or IL-6; It expresses the cell-lytic proteins granzyme A, granzyme B, perforin 1 (PRF1), NKG7, and / or granzyme H; Secretes IL-10; and / or FOXP3, CD25, BATF, ICOS, GITR, and / or demethylated T-reg-specific demethylation regions (TSDRs) express The composition according to claim 18.

21. CD8 + The composition according to claim 18, which essentially does not contain T cells.

22. Said T reg The composition according to claim 18, wherein the composition is conjugated with a cytotoxic agent.

23. The composition according to claim 22, wherein the cytotoxic agent is a chemotherapeutic agent, IL-2, IL-15, soluble TRAIL, perforin, granzyme A, or granzyme B.

24. T according to claim 18 reg A pharmaceutical composition comprising a pharmaceutical carrier.

25. CAR T reg An in vitro method for generating, (a) T from peripheral blood reg The process of isolating; (b) The T reg A step of introducing a CAR expression construct; (c) The T reg A step of expanding and growing a substance in the presence of at least one cytokine; and (d) The T reg The process of stimulating antigen-presenting cells (APCs) or beads. Methods that include...

26. The CAR expression constructs include TRP-1, EpCAM, CD19, CD20, CD22, B cell maturation antigen (BCMA), fetal carcinoma antigen (CEA), alpha-fetoprotein, CA-125, MUC-1, epithelial tumor antigen, melanoma-associated antigen (MAGE), mutant p53-derived peptide-HLA, mutant ras-derived peptide-HLA, HER2 / Neu, ERBB2, folate-binding protein, HIV-1 coat glycoprotein gp120, HIV-1 coat glycoprotein gp41, GD2, CD123 (IL3RA), CD319, CD23, CD30, CD56, c-Met, meso The method according to claim 25, selected from the group consisting of therine, GD3, HERV-K, IL-11Rα, κ chain, λ chain, CSPG4, ERBB2, EGFR, EGFRvIII, VEGFR2, TNFRSF17, SDC1, FAP, CD44, MS4A1, EPCAM, CA9, CD174, TNFRSF8, CD33, CD38, EPHA2, CD248, CD274, CD276, CD5, NCAM1, CD70, ERBB2, KDR, L1CAM, ULBP1, ULBP2, IL1RAP, GPC3, IL13RA2, ROR1, CEACAM5, MET, FOLH1, CSPG4, CD133, GPNMB, and PSCA CAR expression construct.

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