A method for selective stimulation of T cells in solid tumors using oncolytic viral delivery of orthogonal IL-2

Abstract

The present disclosure provides orthogonal chimeric cytokine receptor / orthogonal cytokine pairs and compositions and methods for engineered immune cells or their precursors (e.g., engineered T cells) comprising an orthogonal chimeric cytokine receptor (e.g., an oIL2R-IL9R chimeric receptor) and a chimeric antigen receptor (CAR) or T cell receptor (TCR). The present disclosure further provides oncolytic adenoviral vectors comprising a nucleic acid sequence encoding an orthogonal cytokine (e.g., oIL2), and methods of using the engineered cells and vectors to treat cancer in a subject in need thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 158,671, filed March 9, 2021, which is incorporated by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with United States Government support under Contract U54CA244711 awarded by the National Institutes of Health. The United States Government has certain rights in the invention. [Background technology]

[0003] 2. Background of the Invention Current immunotherapy advances have been groundbreaking for the treatment of hematological malignancies, as evidenced by the FDA approval of CD19-targeted CAR-T cells for the treatment of acute lymphoblastic leukemia and diffuse large B-cell lymphoma. However, the biggest burden yet to be addressed for cancer treatment is solid tumors. CAR-T cells have been ineffective in the fight against solid tumors due to several challenges, including lack of tumor-specific antigens, overcoming the barrier of therapeutic resistance, tumor heterogeneity, poor expansion and persistence, and the extrinsic functional impairment and physical barriers to T cell infiltration posed by the dense immunosuppressive tumor microenvironment (TME). One major limitation is the poor in vivo expansion and persistence of adoptively transferred T cells, which necessitates lymphodepleting conditioning chemotherapy, a toxic regimen that limits patient eligibility. Even T cells that expand and persist end up terminally differentiated and functionally impaired. T cells with stem cell-like phenotypes can overcome these limitations and exhibit superior anti-tumor activity in mouse models and humans, but therapeutic manipulations to select or expand stem cell-like T cells are limited to the cell manufacturing stage and cannot be performed in vivo. There is a need in the art for novel cell-based therapies that overcome these barriers and challenges. The present invention addresses this need. Summary of the Invention

[0004] In some aspects, the present invention provides a system for selective activation of a receptor in a cell, comprising: (a) Below: (i) an orthogonal chimeric cytokine receptor, and (ii) at least one chimeric antigen receptor (CAR); and a modified immune cell comprising: (b) an oncolytic adenoviral vector comprising a nucleic acid sequence encoding an orthogonal IL2 cytokine; Including, The system provides that the orthogonal chimeric cytokine receptor comprises the extracellular domain of an orthogonal IL2 receptor (oIL2R) and the intracellular signaling domain of a cytokine receptor that is not an IL2R.

[0005] In some embodiments, said extracellular domain of oIL2R is the extracellular domain of the orthogonal IL2 receptor beta (oIL2Rb).

[0006] In some embodiments, the intracellular signaling domain of said orthogonal chimeric cytokine receptor comprises an IL9R intracellular signaling domain.

[0007] In some embodiments, said IL9R intracellular signaling domain is an IL9R-alpha (IL9Ra) intracellular signaling domain.

[0008] In some embodiments, the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain.

[0009] In some embodiments, the antigen-binding domain is selected from the group consisting of a full-length antibody or antigen-binding fragment thereof, a Fab, a single-chain variable fragment (scFv), or a single domain antibody.

[0010] In some embodiments, the antigen binding domain targets a tumor antigen.

[0011] In some embodiments, the tumor antigen is selected from the group consisting of CD19, CD20, HER2, NY-ESO-1, MUC1, CD123, FLT3, B7-H3, CD33, IL1RAP, CLL1 (CLEC12A) PSA, CEA, VEGF, VEGF-R2, CD22, ROR1, mesothelin, c-Met, glycolipid F77, FAP, EGFRvIII, MAGE A3, 5T4, WT1, KG2D ligand, folate receptor (FRa), and Wnt1 antigen.

[0012] In some embodiments, the antigen binding domain is an scFv.

[0013] In some embodiments, the antigen binding domain is an anti-mesothelin scFv.

[0014] In some embodiments, the intracellular domain of the CAR comprises a costimulatory domain of a protein selected from the group consisting of the TNFR superfamily of proteins, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or a variant thereof, or an intracellular domain from a killer immunoglobulin-like receptor (KIR).

[0015] In some embodiments, the intracellular domain of the CAR comprises an intracellular signaling domain of a protein selected from the group consisting of human CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor bearing an immunoreceptor tyrosine-based activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof.

[0016] In some embodiments, (a) the orthogonal chimeric cytokine receptor comprises the extracellular domain of oIL2Rb and the intracellular signaling domain of IL9Ra, and (b) the CAR comprises an anti-mesothelin antigen binding domain.

[0017] In some aspects, the present invention provides a method of treating cancer in a subject, comprising: (a) to the subject; (i) an orthogonal chimeric cytokine receptor, and (ii) at least one CAR administering an effective amount of modified immune cells or precursors thereof (a population of modified immune cells); (b) administering to the subject an oncolytic adenoviral vector comprising a nucleic acid sequence encoding an orthogonal IL2 cytokine. Including, The orthogonal chimeric cytokine receptor comprises the extracellular domain of an orthogonal IL2 receptor (oIL2R) and the intracellular signaling domain of a cytokine receptor that is not an IL2R.

[0018] In some embodiments, said extracellular domain of oIL2R is the extracellular domain of the orthogonal IL2 receptor beta (oIL2Rb).

[0019] In some embodiments, the intracellular signaling domain of said orthogonal chimeric cytokine receptor comprises an IL9R intracellular signaling domain.

[0020] In some embodiments, said IL9R intracellular signaling domain is an IL9R-alpha (IL9Ra) intracellular signaling domain.

[0021] In some embodiments, the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain.

[0022] In some embodiments, the antigen-binding domain is selected from the group consisting of a full-length antibody or antigen-binding fragment thereof, a Fab, a single-chain variable fragment (scFv), or a single domain antibody.

[0023] In some embodiments, the antigen binding domain targets a tumor antigen.

[0024] In some embodiments, the tumor antigen is selected from the group consisting of CD19, CD20, HER2, NY-ESO-1, MUC1, CD123, FLT3, B7-H3, CD33, IL1RAP, CLL1 (CLEC12A) PSA, CEA, VEGF, VEGF-R2, CD22, ROR1, mesothelin, c-Met, glycolipid F77, FAP, EGFRvIII, MAGE A3, 5T4, WT1, KG2D ligand, folate receptor (FRa), and Wnt1 antigen.

[0025] In some embodiments, the antigen binding domain is an scFv.

[0026] In some embodiments, the antigen binding domain is an anti-mesothelin scFv.

[0027] In some embodiments, the intracellular domain of the CAR comprises a costimulatory domain of a protein selected from the group consisting of the TNFR superfamily, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or a variant thereof, or an intracellular domain from a killer immunoglobulin-like receptor (KIR).

[0028] In some embodiments, the intracellular domain of the CAR comprises an intracellular signaling domain of a protein selected from the group consisting of human CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor bearing an immunoreceptor tyrosine-based activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof.

[0029] In some embodiments, (a) the orthogonal chimeric cytokine receptor comprises the extracellular domain of oIL2Rb and the intracellular signaling domain of IL9Ra, and (b) the CAR comprises an anti-mesothelin antigen binding domain.

[0030] In some embodiments, the population of modified immune cells exhibit stem cell memory (Tscm) characteristics with improved trafficking and effector function, thereby treating cancer.

[0031] In some embodiments, said step of administering a vector comprises intratumoral injection.

[0032] In some embodiments, the cancer is selected from the group consisting of pancreatic cancer and melanoma.

[0033] In some embodiments, the pancreatic cancer is pancreatic ductal adenocarcinoma.

[0034] In some aspects, the present invention provides a system for selective activation of a receptor in a cell, comprising: (a) Below: (i) an orthogonal chimeric cytokine receptor, and (ii) at least one T cell receptor (TCR); and a modified immune cell engineered to express (b) an oncolytic adenoviral vector comprising a nucleic acid sequence encoding an orthogonal IL2 cytokine; Including, The system provides that the orthogonal chimeric cytokine receptor comprises the extracellular domain of an orthogonal IL2 receptor (oIL2R) and the intracellular signaling domain of a cytokine receptor that is not an IL2R.

[0035] In some embodiments, said extracellular domain of oIL2R is the extracellular domain of the orthogonal IL2 receptor beta (oIL2Rb).

[0036] In some embodiments, the intracellular signaling domain of said orthogonal chimeric cytokine receptor comprises an IL9R intracellular signaling domain.

[0037] In some embodiments, said IL9R intracellular signaling domain is an IL9R-alpha (IL9Ra) intracellular signaling domain.

[0038] In some embodiments, the TCR targets a tumor antigen.

[0039] In some embodiments, the TCR targets gp100 melanoma antigen or NYESO1.

[0040] In some embodiments, the TCR is a pmel-1 TCR or a NYESO1-specific TCR.

[0041] In some embodiments, (a) the orthogonal chimeric cytokine receptor comprises the extracellular domain of oIL2Rb and the intracellular signaling domain of IL9Ra, and (b) the TCR is a pmel-1 TCR or a NYESO1-specific TCR.

[0042] In some aspects, the present invention provides a method of treating cancer in a subject, comprising: (a) to the subject; (i) an orthogonal chimeric cytokine receptor, and (ii) at least one T cell receptor (TCR); administering an effective amount of a modified immune cell or a precursor thereof (a population of modified immune cells) modified to express (b) administering to the subject an oncolytic adenoviral vector comprising a nucleic acid sequence encoding an orthogonal IL2 cytokine. Including, The orthogonal chimeric cytokine receptor comprises the extracellular domain of an orthogonal IL2 receptor (oIL2R) and the intracellular signaling domain of a cytokine receptor that is not an IL2R.

[0043] In some embodiments, said extracellular domain of oIL2R is the extracellular domain of the orthogonal IL2 receptor beta (oIL2Rb).

[0044] In some embodiments, the intracellular signaling domain of said orthogonal chimeric cytokine receptor comprises an IL9R intracellular signaling domain.

[0045] In some embodiments, said IL9R intracellular signaling domain is an IL9R-alpha (IL9Ra) intracellular signaling domain.

[0046] In some embodiments, the TCR targets a tumor antigen.

[0047] In some embodiments, the TCR targets gp100 melanoma antigen or NYESO1.

[0048] In some embodiments, the TCR is a pmel-1 TCR or a NYESO1-specific TCR.

[0049] In some embodiments, (a) the orthogonal chimeric cytokine receptor comprises the extracellular domain of oIL2Rb and the intracellular signaling domain of IL9Ra, and (b) the TCR is a pmel-1 TCR or a NYESO1-specific TCR.

[0050] In some embodiments, the population of modified immune cells exhibit stem cell memory (Tscm) characteristics with improved trafficking and effector function, thereby treating cancer.

[0051] In some embodiments, said step of administering a vector comprises intratumoral injection.

[0052] In some embodiments, the cancer is selected from the group consisting of pancreatic cancer and melanoma.

[0053] In some embodiments, the pancreatic cancer is pancreatic ductal adenocarcinoma. [Brief description of the drawings]

[0054] The above and other features and advantages of the present invention will be better understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings.

[0055] [Figure 1-1]Figure 1A-G illustrate the finding that chimeric orthogonal IL-2 receptors unmask IL-9R signaling properties in T cells. (Figure 1A) Schematic of wild-type IL2Rβ receptor complex, orthogonal IL2β receptor complex, or γc-family orthogonal IL2Rβ chimeric receptor complex. All orthogonal and orthogonal chimeric receptor-containing vectors also contain an IRES-YFP element. (Figure 1B) Representative histograms and quantification of pSTAT signaling in ortho chimeric receptor-expressing (YFP+) or untransduced (UTD) T cells stimulated with MSA-IL2 (100 nM) (open color) or MSA-oIL2 (5 μM) (solid color) for 20 min. Data are presented as mean fluorescence ± SEM. n=3. (Fig. 1C) Dose-response curves of pSTAT signaling in o2R-transduced T cells (red) or o9R-transduced T cells (blue) stimulated with MSA-oIL2, MSA-IL2, or IL9 for 20 min. Data are presented as mean fluorescence ± SEM. YFP+ gate. n=3. (Fig. 1D) Surface marker levels of CD62L in chimeric receptor-expressing T cells cultured for 2 days with MSA-IL2 (100 nM) or MSA-oIL2 (5 μM). (Fig. 1E) Surface marker levels of Fas in chimeric receptor-expressing T cells cultured for 2 days with MSA-IL2 (100 nM) or MSA-oIL2 (5 μM). (Fig. 1F) Surface marker levels of Sca1 in chimeric receptor-expressing T cells cultured for 2 days with MSA-IL2 (100 nM) or MSA-oIL2 (5 μM). In Fig. 1D-F, data are presented as mean fluorescence ± SEM. n=3. ns, not significant; ***, p<0.001; ****, p<0.0001 (ANOVA). (Figure 1G) Dose-response curves for o2R or o9R cells that underwent at least one division after 4 days of culture in the presence of MSA-oIL2 or MSA-IL2. Data are expressed as the percentage of cells that divided, calculated in Flowjo; YFP+ gate; n=3 biological replicates. [Figure 1-2] See description of Figure 1-1. [Figure 1-3] See description of Figure 1-1. [Figure 1-4] See description of Figure 1-1. [Diagram 2] FIG. 1 illustrates dose response curves for pSTAT signaling in orthoIL2Rβ ICD chimeric receptor expressing T cells stimulated with MSA-orthoIL2 for 20 min. Data are presented as mean fluorescence ± SEM. n=3. [Figure 3-1] Figures 3A-D show data on mIL9R expression and pSTAT signaling. (Figure 3A) Expression of IL9R in mock- or mIL9R-transduced T cells from C57BL6 transgenic mice. (Figure 3B) Expression of IL9R in mock- or mIL9R-transduced T cells from pmel1 TCR transgenic mice. (Figure 3C) Representative histograms (top) and quantification (bottom) of pSTAT signaling in mIL9R-expressing (YFP+) C57BL6 T cells stimulated with MSA-IL2 (100 nM) (open color) or IL9 (1 μM) (solid color) for 20 min. Data are presented as mean fluorescence ± SEM. n=3. (Figure 3D) Dose-response curves of pSTAT signaling in IL9R- or mock-transduced pmel T cells stimulated with MSA-IL2 or IL9. [Figure 3-2] See description of Figure 3-1. [Figure 4] Figures 4A-C show data on pSTAT signaling in o9R-expressing C57BL / 6 T cells treated for 20 min with a 10-fold dose titration of MSA-IL2 (starting at 100 nM) in the absence (open) or presence (solid) of MSA-oIL2 [5 μM]. Data are presented as mean fluorescence ± SEM. n=3. YFP(+) gate. (Figure 4A) Data on pSTAT5 signaling. (Figure 4B) Data on pSTAT3 signaling. (Figure 4C) Data on pSTAT1 signaling. [Figure 5A]5A-B are in vitro proliferation data. (FIG. 5A) Dose titration in vitro proliferation curves of orthoIL2Rb ICD chimeric receptor expressing T cells cultured for 4 days in the presence of MSA-IL2 (open symbols) or MSA-orthoIL2 (solid symbols). Data represent live CD8+YFP(+) cell total counts normalized to the respective maximum growth of the various chimeric receptor expressing cells cultured in the presence of MSA-IL2. n=2±SEM. (FIG. 5B) In vitro proliferation of o2R (red) or o9R (blue) transduced CTV-labeled T cells cultured for 4 days in the presence of MSA-oIL2 (solid curves) or MSA-IL2 (hollow curves). YFP(+) gate; one of three representative plots is shown. [Figure 5B] See legend to Figure 5A. [Figure 6] Figures 6A-B illustrate signaling and proliferation of o2R pmel and o9R pmel T cells. (Figure 6A) STAT1, STAT3, and STAT5 phosphorylation in o2R pmel and o9R pmel T cells after 30 min of stimulation with MSA-oIL2 (5 μM). MFI and range of biological duplicates gated on YFP+ cells are shown. *, adjusted p<0.05; **, adjusted p<0.005 (unpaired t-test, multiple comparisons correction using Holm-Sidak method). (Figure 6B) Seven replicate experiments measuring in vitro proliferation (measured by fold growth) of o2R pmel or o9R pmel T cells at 48 h cultured with MSA-oIL2 are shown (each data point represents the mean ± SD, n=3). *, p<0.05 (ratio-paired t-test). [Figure 7-1]Figures 7A-J illustrate the findings that oIL9R signaling alters the phenotype and function of pmel T cells, endowing them with antitumor efficacy even in the absence of lymphodepletion. (Figure 7A) Schematic of the experiment. C57BL / 6 mice implanted with B16-F10 melanoma were lymphodepleted (or not) prior to ACT with 5x106 pmel or control C57BL / 6 T cells, followed by treatment with MSA-IL2 or MSA-oIL2 (2.5x104 U / day, i.p.) for 5 days. (Figure 7B) Peripheral blood quantification of adoptively transferred T cells 7 days after ACT (mean ± SD, n = 5-6 mice / group). **, p < 0.01 (unpaired t-test). (FIG. 7C) Tumor growth in mice treated with o2R pmel ACT and MSA-IL2 or MSA-oIL2 (mean ± SEM, n=5-6 mice / group). **, p<0.01 (ANOVA). Data representative of 3 independent experiments. (FIG. 7D) Tumor growth in mice treated with o9R pmel ACT and MSA-IL2 or MSA-oIL2 (mean ± SEM, n=5-6 mice / group). **, p<0.01 (ANOVA). Data representative of 3 independent experiments. (FIG. 7E) Quantification of tumor-infiltrating o2R or o9R pmel T cells (CD8+Thy1.1+T cells / gram) 5 days after ACT in mice treated with MSA-oIL2. **, p<0.01 (unpaired t-test). (Fig. 7F) In vitro growth of nRFP+B16-F10 tumor cells co-cultured (effector:target ratio 2:1) with o2R or o9R pmel T cells pretreated with MSA-IL2 (50 nM) or MSA-oIL2 (5 μM) measured with an Incucyte live cell imaging system (mean ± SD, n=3). *, p<0.05; **, p<0.01 (ANOVA). (Fig. 7G) opt-SNE clustering of o2R and o9R pmel T cells (n=3 / group) treated in vitro for 48 h with MSA-oIL2 (5 μM) (left) with individual plots for each group illustrating only differentially abundant clusters (middle) and volcano plot of differentially abundant clusters annotated with prominent features (right).(Fig. 7H) Heatmap of genes differentially expressed between o2R pmel and o9R pmel T cells treated with MSA-oIL2 (5 μM). Groups treated with MSA-IL2 (50 nM) are also shown. (Fig. 7I) Heatmap of manually curated genes differentially expressed between o9R pmel and o2R pmel T cells treated with MSA-oIL2 (5 μM) that are related to T cell stemness and dysfunction (left) and T cell activation and effector function (right). (Fig. 7J) Normalized enrichment plot of transcription factor gene sets comparing o9R pmel and o2R pmel T cells treated with MSA-oIL2 (left). Statistically significant enrichments (adjusted p-values ​​< 0.05) are shown in red. Jun to Fos expression ratio in o9R pmel T cells and o2R pmel T cells treated with MSA-oIL2 (right panel). **, p<0.01 (unpaired t-test). [Figure 7-2] See description of Figure 7-1. [Figure 7-3] See description of Figure 7-1. [Figure 7-4] See description of Figure 7-1. [Figure 7-5] See description of Figure 7-1. [Figure 8]8A-8C illustrate the antitumor efficacy of o2R pmel T cells and o9R pmel T cells under lymphocyte-depleted and lymphocyte-eutrophic conditions. (FIG. 8A) Survival of B16-F10 tumor-bearing mice treated with o2R pmel T cells and mIL-2 or oIL-2. (FIG. 8B) Survival of B16-F10 tumor-bearing mice treated with o9R pmel T cells and mIL-2 or oIL-2. In FIG. 8A-8B, BL / 6 T cells treated with mIL-2 were used as off-target T cell control. Non-transduced pmel T cells + mIL-2 in lymphodepleted or non-lymphodepleted tumor-bearing mice served as control. (FIG. 8C) Efficacy of o9R pmel T cells in lymphodepleted hosts. Tumor growth of B16-F10 tumors in lymphodepleted C57BL / 6 mice treated with o9R pmel T cells and mIL-2, oIL-2, or no IL2 (mean ± SEM, left panel). [Figure 9A]Figures 9A-D illustrate the trafficking, phenotype, and function of o9R pmel T cells. (Figure 9A) opt-SNE clustering of CD45+ tumor-infiltrating leukocytes 7 days after adoptive transfer of o2R pmel T cells (left) and o9R pmel T cells (middle) (n=4 mice / group). Volcano plot of differentially abundant clusters in tumors from mice treated with o9R pmel T cells and mice treated with o2R pmel T cells (right). (Figure 9B) opt-SNE clustering of subsets of tumor-infiltrating o9R and o2R pmel T cells in non-lymphocyte-depleted hosts treated with oIL-2 (n=4 mice / group, left) with individual plots for each treatment group illustrating only differentially abundant clusters (middle), and Volcano plot of differentially abundant clusters annotated with prominent features (right). (FIG. 9C) Tumor infiltration of CD3+CD8+ and CD8+PD1+ T cells by multiplex IHC (red=CD3, orange=CD8, yellow=PD1, green=CD4, turquoise=FOXP3) in non-lymphocyte depleted hosts treated with either o2R and oIL-2 (left panel) or o9R pmel T cells and oIL-2 (right panel). Quantitation is shown below. (FIG. 9D) IFNγ secretion by oIL2R and oIL9R pmel T cells co-cultured with B16-F10 melanoma for 24 hours in vitro. T cells were pretreated with oIL-2 (5 μM) for 48 hours in vitro prior to co-culture. *, p<0.05, unpaired t-test. [Figure 9B] See legend to Figure 9A. [Figure 9C] See legend to Figure 9A. [Figure 9D] See legend to Figure 9A. [Figure 10]Illustrating the finding that o9R and wild-type IL-9R signaling drive the Tscm phenotype. Surface expression of CD62L, CD95 (Fas), and CD44 as a percentage of CD8+Thy1.1+ sorted o9R pmel T cells, o2R pmel T cells, or pmel T cells transduced with wild-type IL-9R (pmel-IL9R) ​​and treated with mIL-2 (0.05 μM), oIL-2 (5 μM), or IL-9 (0.05 μM) for 48 h in vitro. ns, not significant; ****, p<0.0001, unpaired t-test. [Figure 11] Unsupervised transcriptome analysis (RNA-seq) of sorted pmel-o2R (n=3) and pmel-o9R (n=3) T cells after 48 h of in vitro exposure to oIL-2 or mIL-2 is illustrated. Principal component analysis separates samples by treatment group (PC1 vs. PC2, left panel). When aligned by inter-sample distance in a heatmap, samples clustered by treatment group (right panel), with oIL-2-treated o9R pmel T cells being the most distinct of the four groups. [Figure 12] Figure 1 illustrates the frequency of CD62L+o2R or o9R pmel T cells (Thy1.1+YFP+) from tumor-draining lymph nodes (DLN), spleen or tumors in mice treated with MSA-oIL2. Tissues were collected 1 or 5 days after adoptive transfer. Each data point represents one individual mouse. n=3-5 mice per group. **, p<0.01; ***, p<0.001; ****, <0.0001; unpaired t-test. [Figure 13-1]Figures 13A-M illustrate orthogonal targeting of CAR T cells co-expressing o2R and o9R receptors. (Figure 13A) Schematic of the replication-deficient serotype 5 adenoviral vector Ad-oIL2 encoding oIL-2 under the homeostatic cytomegalovirus (CMV) promoter (top). In vitro oIL-2 expression by Ad-oIL2 in cell culture supernatants (bottom left panel; mean ± SEM, n = 3). Quantification of oIL-2 expression in tumor homogenates and serum 72 hours after intratumoral (IT) injection of 1 x 109 Ad-oIL2 viral particles or daily intraperitoneal (IP) injection of 25,000 units of MSA-oIL2 (bottom right panel; mean ± SEM, n = 5 mice / group). LITR, left terminal inverted repeat; RITR, right terminal inverted repeat. (FIG. 13B) Representative Western blot analysis of pSTAT1 / pSTAT3 / pSTAT5 expression in untransduced (UTD) T cells, CAR-o2R T cells, or CAR-o9R T cells 30 min after stimulation with MSA-IL2 (100 nM) or MSA-oIL2 (5 μM). (FIG. 13C) In vitro cell killing of mesothelin-positive PDA7940b tumor cells by CAR-o2R and CAR-o9R T cells (effector:target ratio of 2:1) preincubated with MSA-oIL2 (5 μM) for 48 h measured with the Xcelligence live cell imaging system (average normalized cell index, n=4). (FIG. 13D) Representative CD44 and CD62L surface co-expression in CAR-o2R / CAR-o9R T cells 4 days after stimulation with MSA-IL2 (100 nM) or MSA-oIL2 (5 μM). (FIG. 13E) Representative Fas expression in CAR-o2R / CAR-o9R T cells after 4 days of stimulation with MSA-IL2 (100 nM) or MSA-oIL2 (5 μM). (FIG. 13F) Luminex analysis of secreted cytokines in CAR-o2R and CAR-o9R T cell culture supernatants after 4 days of stimulation with MSA-IL2 (100 nM) or MSA-oIL2 (5 μM) (mean ± SEM, n=3 / group). ****P<0.0001 (ANOVA).(FIG. 13G) Heatmap of genes differentially expressed between o2R and o9R CAR T cells treated with MSA-oIL2 (left panel). MSA-IL2 treated groups are also shown. Heatmap of manually curated genes differentially expressed between o9R and o2R CAR T cells in MSA-oIL2 treated mice (middle and right panels) related to T cell stemness and dysfunction and T cell activation and effector function. (FIG. 13H) Schematic of syngeneic adoptive CAR T cell therapy mouse model utilizing KPC-derived PDA7940b tumors. Ad-oIL2 dose, 1×10 9 VP. CAR T cell dose, 5×10 6 cells. CTX dose, 120 mg / kg. SC, subcutaneous; CTX, cyclophosphamide; IP, intraperitoneal; IT, intratumoral; IV, intravenous. (FIG. 13I) Individual growth curves of PDA7940b tumors by indicated treatment group. (FIG. 13J) Individual growth curves of PDA7940b tumors by indicated treatment group. (FIG. 13K) Individual growth curves of PDA7940b tumors by indicated treatment group without cyclophosphamide preconditioning ("No CTX"). In FIG. 13I-K, black lines indicate deaths due to immune effector cell-associated neurotoxicity syndrome (ICANS). n=6-12 mice per group. CR, complete response. Tox, number of deaths due to neurotoxicity. Efficacy experiments were repeated twice. (FIG. 13L) Tumor volume of cured mice (from FIG. 13J) rechallenged with PDA7940b compared to age-matched naive mice (n=10) (left panel). Quantification of CD45.1+ lymphocytes in peripheral blood of rechallenged mice (right panel). *P<0.05, **P<0.01 (ANOVA). ns, not significant. (Figure 13M) Quantification of tumor-infiltrating CAR T cells on day 9 (8 days after ACT) (left panel). Frequency of IFNγ-positive tumor-infiltrating CAR T cells on day 9 (right panel). *P<0.05, ****P<0.0001 (ANOVA). [Figure 13-2] See description of Figure 13-1. [Figure 13-3] See description of Figure 13-1. [Figure 13-4] See description of Figure 13-1. [Figure 13-5] See description of Figure 13-1. [Figure 13-6] See description of Figure 13-1. [Figure 13-7] See description of Figure 13-1. [Figure 13-8] See description of Figure 13-1. [Figure 14A] Figures 14A-B illustrate co-expression of CAR and orthoIL-2Rβ in mouse T cells. (Figure 14A) Schematic of primary mouse CD3+ T cells expressing anti-mesothelin CAR and o2R (CAR-o2R) or o9R (CAR-o9R). (Figure 14B) Representative mesothelin CAR expression (top), IL-2Rβ expression (middle), and CAR / IL-2Rβ co-expression (bottom) in untransduced (UTD) T cells or in retrovirally transduced CAR-o2R and CAR-o9R T cells as determined by flow cytometry. Inset in middle panel is the mean fluorescence intensity (MFI) of IL-2Rβ in untransduced and transduced T cells. [Figure 14B-1] See legend to Figure 14A. [Figure 14B-2] See legend to Figure 14A. [Figure 14B-3] See legend to Figure 14A. [Figure 14B-4] See legend to Figure 14A. [Figure 14B-5] See legend to Figure 14A. [Figure 14B-6] See legend to Figure 14A. [Figure 15A]Figure 15A-B illustrate the expansion and phenotype of o2R and o9R CAR T cells. (Figure 15A) In vitro CAR T cell expansion. CAR-o2R and CAR-o9R cells were incubated in the presence of MSA-IL2 (100 nM) or MSA-oIL2 (5 μM). Every day, an aliquot of cells was removed from the plate, stained with Calcein AM viability dye, and counted on a Celigo image cytometer. Mean ± SEM, n = 3 replicate wells / group. (Figure 15B) CD44 and CD62L co-expression in CAR T cells. Full data for a representative flow plot shown in Figure 13D. CAR-o2R and CAR-o9R cells were incubated in the presence of MSA-IL2 (100 nM) or MSA-oIL2 (5 μM) for 4 days. Surface co-expression of CD44 and CD62L was determined by flow cytometry on live CAR+ cells. Mean ± SEM, n = 3 / group. ns, not significant. ****P < 0.0001 (ANOVA). [Figure 15B] See legend to Figure 15A. [Figure 16-1] Figures 16A-E illustrate RNA ISH and serum markers of toxicity at day 11. (Figure 16A) Images of Ad-oIL2+CAR-o2R. (Figure 16B) Images of Ad-oIL2+CAR-o9R. Figures 16A-B are representative images of brain and meningeal sections stained with fluorescent probes specific for mouse CAR (red, Cy3), mouse mesothelin (green, FITC), and counterstained with DAPI (blue). CAR-positive cells (white arrows) and mesothelin-positive meningeal cells (purple arrows) are shown in the pia arachnoid layer of the meninges. (Figure 16C) Semiquantification of stained CAR T cells in brain sections. Mean ± SEM, 2 sections / mouse, 3 mice / group. *P<0.05 (t-test with Welch's correction). (Figure 16D) Serum levels of calcium, phosphorus, potassium, and uric acid at day 11 of treatment. Mean ± SEM, n = 3 mice / group. (FIG. 16E) Serum levels of CRS-associated cytokines on day 11 of treatment. Mean ± SEM, n = 3 mice / group. [Figure 16-2] See description of Figure 16-1. [Figure 16-3] See description of Figure 16-1. [Figure 17] Figures 17A-C show data on survival and antitumor efficacy in mice. (Figure 17A) Difference in survival between mice treated with CAR-o2R+Ad-oIL2 and mice treated with CAR-o9R T cells+Ad-oIL2 with or without conditioning chemotherapy. Kaplan-Meier survival curves for treatment groups in Figures 13I-13K. **P<0.01, ****P<0.0001 (Log-rank-Mantel-Cox test). (Figure 17B) Antitumor efficacy in mice treated with Ad-oIL2 and CAR T cells (without orthogonal cytokine receptor). Established subcutaneous PDA7940b tumors were treated with Ad-oIL2 (1x109VP / tumor) on days 0 and 4 and with CAR T cells (5x106) on day 0. Mean ± SEM, n=5 mice / group. ns, not significant. (FIG. 17C) Antitumor efficacy in mice treated with Ad-Null (no transgene) and CAR-o2R T cells. Established subcutaneous PDA7940b tumors were treated with Ad-Null (1×10 9 VP / tumor) on days 0 and 4 and CAR-o2R T cells (5×10 6 ) on day 0. Mean ± SEM, n=5 mice / group. ns, not significant. [Figure 18A]Figures 18A-G illustrate the findings that human chimeric orthogonal IL-2Rβ / IL-9R promotes stemness and multi-functionality in TCR engineered T cells. (Figure 18A) pSTAT signaling in human T cells co-expressing either ho2R or ho9R with NY-ESO-1 TCR stimulated with MSA-hoIL2 or MSA-hIL2 for 20 min. Data are presented as mean fluorescence ± SEM. n=3. (Figure 18B) Magnification of ho2R / NYESO1-TCR engineered human T cells and ho9R / NYESO1-TCR engineered human T cells treated with MSA-hoIL2 (1 μM) or MSA-hIL2 (0.1 μM) for 6 days. (FIG. 18C) Immunophenotype of ho2R / NYESO1-TCR engineered human T cells and ho9R / NYESO1-TCR engineered human T cells after 6 days of culture with MSA-hoIL2 (1 μM). Representative plots of CD45RA and CD27 expression gated on YFP+ T cells (top row), and CD95 and CCR7 expression gated on the CD45RA+CD27+ population (indicated by arrows and dotted lines). Quantification of CD45RA+CD27+CD95+CCR7+ TSCM cells as a percentage of the YFP+ population is shown in the bar graph on the right. ****, p<0.0001 (unpaired t-test, n=3 / group). (FIG. 18D) Magnification of TSCM and TCM cells cultured in vitro with MSA-hoIL2 (1 μM) or MSA-hIL2 (0.1 μM), respectively, on day 2. *, p<0.05; **, p<0.01 (unpaired t-test, n=3 / group). (Figure 18E) T cells co-expressing either ho2R or ho9R with NY-ESO-1 TCR, pre-incubated with MSA-hoIL2 (1 μM) for 48 h, were co-cultured with HLA*0201+NY-ESO-1+ melanoma tumor cell line expressing nRFP (nRFP-M407) in the presence of MSA-hoIL2 at a 1:1 E:T ratio. Non-transduced T cells in the presence of MSA-hIL2 (1 nM) were used as a negative control. Tumor cells (105) were re-introduced into the co-culture every 72 h (light blue arrows), and MSA-hoIL2 (1 μM) was added to the culture 24 h before each tumor challenge.Live cell images were captured every 2 hours and percent tumor confluence was measured based on RFP expression. After the fourth tumor challenge, T cells were collected for phenotypic analysis (see FIG. 18F) and for restimulation and intracellular cytokine staining (ICS) (see FIG. 18G). (FIG. 18F) Quantification of CD45RA+CD27+ and TSCM cells as a percentage of YFP+ T cells and MFI of CD62L and CXCR3 (YFP+ gate). *, p<0.05; **, p<0.01; ***, p<0.001 (unpaired t-test, n=2 / group). (FIG. 18G) T cells after the fourth tumor challenge in FIG. 18E were restimulated for 4 hours with either αCD3 / αCD28 activating antibodies, melanoma cell lines M407 (HLA*0201+NY-ESO-1+) or M263 (HLA*0201-NY-ESO-1-). IFNγ, TNFα and IL-2 were quantified by ICS among CD8+YFP+ T cells. Donut graphs show the proportion of CD8+YFP+ T cells in each group expressing 0, 1, 2 or 3 of the three cytokines. ns, not significant; ****, p<0.0001 (2-way ANOVA, n=3 / group). [Figure 18B] See legend to Figure 18A. [Figure 18C-1] See legend to Figure 18A. [Figure 18C-2] See legend to Figure 18A. [Figure 18D] See legend to Figure 18A. [Figure 18E] See legend to Figure 18A. [Figure 18F] See legend to Figure 18A. [Figure 18G] See legend to Figure 18A. [Figure 19A]Figure 19A-D illustrates the finding that human chimeric orthogonal IL-2Rβ / IL-9R promotes multifunctional T cells with stem cell-like phenotype even after repeated antigen-specific tumor challenge. (Figure 19A) Flow plots of normal healthy donor T cells co-transduced with either ho2R and NYESO1-TCR (left) or ho9R and NYESO1-TCR (right) as detected by anti-human Vβ13.1 antibody recognizing the β-chain of NYESO1-TCR clone 1G4 and YFP (internal marker of o2R or o9R vectors). Co-transduction efficiency is shown. (Figure 19B) Gating strategy for immunophenotyping of the YFP+ population of ho2R / NYESO1-TCR and ho9R / NYESO1-TCR T cells shown in Figure 18C. (FIG. 19C) Immunophenotype of ho2R / NYESO1 and ho9R / NYESO1 TCR engineered human T cells after 2 days of culture with MSA-hoIL2 (1 μM). Bar graph quantification of TSCM cells as a percentage of the YFP+ population is shown. **, p<0.01 (unpaired t-test, n=3 / group). (FIG. 19D) T cells from FIG. 13E were collected and restimulated with either αCD3 / αCD28 dynabeads, melanoma cell lines M407 (HLA*0201+NY-ESO-1+) or M263 (HLA*0201+NY-ESO-1-) for 4 hours. IFNγ, TNFα and IL-2 were quantified by ICS among YFP+CD4 T cells (CD8-). [Figure 19B-1] See legend to Figure 19A. [Figure 19B-2] See legend to Figure 19A. [Figure 19C] See legend to Figure 19A. [Figure 19D] See legend to Figure 19A. [Figure 20-1] FIG. 20 is a table of the reagents used in the experiments described herein. [Figure 20-2] See description of Figure 20-1. [Figure 21-1]Figure 21 illustrates the creation of a mesothelin knockout cell line. Mesothelin-positive PDA7940b tumor cells were edited with CRISPR / Cas9 to knock out mesothelin and purified by FACS sorting. In real-time cell killing assays (RTCA), anti-mesothelin CAR T cells killed wild-type cells (PDA7940bMSLN+) but not KO cells (PDA7940bMSLN-). [Figure 21-2] See description of Figure 21-1. [Figure 21-3] See description of Figure 21-1. [Figure 21-4] See description of Figure 21-1. [Figure 22] Illustrates tumor antigen spread. Mice previously cured by combination treatment were rechallenged with mesothelin-positive (PDA7940bMSLN+) and mesothelin-negative (PDA7940bMSLN-) tumor cells. Tumors were allowed to grow for 24 days before tumor size was measured. Age-matched naive mice served as controls for tumor growth. [Diagram 23] Figures 23A-B are data for human orthoIL-2. Figure 23A is a graph illustrating in vitro expression of human orthoIL-2 by Ad5 / 3-D24-hoIL2. A549 cells were infected with Ad5 / 3-D24-hoIL2 or the isogenic control virus Ad5 / 3-D24, or mock infected. 96 hours after infection, 50 ul of cell culture supernatant was collected and analyzed by human IL-2 ELISA. Figure 23B is a photograph illustrating purification of the fiber-chimeric oncolytic adenovirus Ad5 / 3-D24-hoIL2 encoding human orthoIL-2 through a cesium chloride (CsCl) gradient. The red arrow indicates the band containing intact viral particles, which is collected with a needle and syringe by piercing the side of the tube. CsCl was removed by two rounds of buffer exchange. [Figure 24A-1]Figures 24A-B are data illustrating co-expression of human ortho-receptor and PSMA-retargeted CAR in human T cells. Figure 24A is flow cytometry data illustrating co-expression of PSMA28z CAR and full-length human orthoIL2Rb receptor 48 hours after infection. Figure 24B is flow cytometry data illustrating co-expression of PSMA28z CAR and chimeric orthoIL2Rb / IL9Ra switch receptor 48 hours after infection. [Figure 24A-2] See description of Figure 24A-1. [Figure 24A-3] See description of Figure 24A-1. [Figure 24B-1] See description of Figure 24A-1. [Figure 24B-2] See description of Figure 24A-1. [Figure 24B-3] See description of Figure 24A-1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0056] Detailed Description In one aspect, the present invention provides compositions and methods for engineered immune cells or their precursors (e.g., engineered T cells) comprising an orthogonal chimeric cytokine receptor (e.g., oIL2R-IL9R chimeric receptor) and at least one chimeric antigen receptor (CAR). The present disclosure further provides an oncolytic adenoviral vector comprising a nucleic acid sequence encoding an orthogonal cytokine (e.g., oIL2). Methods of using the engineered cells and vectors to treat cancer are also provided. In one aspect, the present invention provides a method of treating cancer, comprising administering to a subject having cancer (a) engineered cells comprising an orthogonal chimeric cytokine receptor and at least one CAR, and (b) an oncolytic adenoviral vector comprising a nucleic acid sequence encoding an orthogonal cytokine, wherein the engineered T cells exhibit stem cell memory (Tscm) characteristics with improved trafficking and effector function in the subject, thereby treating the cancer.

[0057] In another aspect, the present invention provides compositions and methods for engineered immune cells or precursors thereof (e.g., engineered T cells) comprising an orthogonal chimeric cytokine receptor (e.g., oIL2R-IL9R chimeric receptor) and at least one T cell receptor (TCR). The present disclosure further provides an oncolytic adenoviral vector comprising a nucleic acid sequence encoding an orthogonal cytokine (e.g., oIL2). Methods of using the engineered cells and vectors to treat cancer are also provided. In one aspect, the present invention provides a method of treating cancer, comprising administering to a subject having cancer (a) engineered cells comprising an orthogonal chimeric cytokine receptor and at least one TCR, and (b) an oncolytic adenoviral vector comprising a nucleic acid sequence encoding an orthogonal cytokine, wherein the engineered T cells exhibit stem cell memory (Tscm) characteristics with improved trafficking and effector function in the subject, thereby treating the cancer.

[0058] It is to be understood that the methods described in this disclosure are not limited to the specific methods and experimental conditions disclosed herein, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0059] Furthermore, in the experiments described herein, unless otherwise indicated, ordinary molecular and cell biological and immunological techniques are used that are within the skill of the art. Such techniques are well known to those skilled in the art and are described in detail in the literature. For example, see Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, NY (1987-2008) (including all supplements), MR Green and J. Sambrook, Molecular Cloning: A Laboratory Manual (4th edition), and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2nd edition).

[0060] A. definition Unless otherwise defined, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In the event of ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. Unless otherwise required by context, the singular includes the plural and the plural includes the singular. The use of "or" means "and / or" unless otherwise stated. The use of the term "including" and other forms, such as "includes" and "included", is not limiting.

[0061] In general, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein chemistry and nucleic acid chemistry, and hybridization described herein is well known and commonly used in the art. The methods and techniques described herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification, unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly practiced in the art, or as described herein. The nomenclature used in connection with the experimental procedures and techniques of analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein is well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and patient treatment.

[0062] In order that this disclosure may be more readily understood, selected terms are defined below.

[0063] The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0064] As used herein, "about" when referring to a measurable value, such as an amount, duration, etc., is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the stated value, as such variations are appropriate for carrying out the disclosed methods.

[0065] As used herein, "activation" refers to a state of T cells that have been stimulated sufficiently to induce detectable cell proliferation. Activation may also be accompanied by induced cytokine production and detectable effector function. The term "activated T cells" refers, inter alia, to T cells that are undergoing cell division.

[0066] As used herein, "alleviating" a disease means reducing the severity of one or more symptoms of the disease.

[0067] As used herein, the term "antigen" is defined as a molecule that elicits an immune response. This immune response may involve either the production of antibodies or the activation of specific immunologically competent cells, or both. Those skilled in the art will appreciate that any macromolecule, including virtually any protein or peptide, may function as an antigen.

[0068] Furthermore, the antigen can be derived from recombinant or genomic DNA. Thus, it will be understood by those skilled in the art that any DNA containing a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response will code for an "antigen" as the term is used herein. It will also be understood by those skilled in the art that an antigen does not necessarily have to be encoded by a full-length nucleotide sequence of a gene. It is clear that the present invention encompasses, but is not limited to, the use of partial nucleotide sequences of multiple genes, and that these nucleotide sequences are arranged in various combinations to elicit a desired immune response. It will also be understood by those skilled in the art that an antigen does not necessarily have to be encoded by a "gene" at all. It is clear that an antigen can be synthetically generated or derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0069] As used herein, the term "autologous" is intended to refer to any material derived from the same individual into which it is subsequently reintroduced.

[0070] "Costimulatory molecule" refers to the cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors.

[0071] As used herein, a "costimulatory signal" refers to a signal that, in combination with a primary signal, such as TCR / CD3 ligation, leads to T cell proliferation and / or up- or down-regulation of key molecules.

[0072] A "disease" is a condition in an animal's health in which the animal is unable to maintain homeostasis and, unless the condition is ameliorated, the animal's health will continue to deteriorate. In contrast, a "disorder" in an animal is a condition in which the animal is able to maintain homeostasis, but the animal's health is less favorable than it would be in the absence of the disorder. If left untreated, a disorder does not necessarily cause a further deterioration in the animal's health.

[0073] As used herein, the term "downregulation" refers to the reduction or elimination of gene expression of one or more genes.

[0074] "Effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, material, or composition described herein that is effective to achieve a particular biological result or provide a therapeutic or prophylactic benefit. Such results may include, but are not limited to, an amount that, when administered to a mammal, induces a detectable level of immune suppression or immune tolerance compared to an immune response detected in the absence of the composition of the present invention. Immune responses can be readily assessed by a myriad of methods recognized in the art. Those skilled in the art will appreciate that the amount of the composition administered in the present invention will vary and can be readily determined based on several factors, such as the disease or condition being treated, the age and health and physical condition of the mammal being treated, the severity of the disease, the specific compound being administered, and the like.

[0075] "Encoding" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template in biological processes for the synthesis of other polymers and macromolecules having either a given nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a given amino acid sequence and the biological properties it imparts. Thus, a gene codes for a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, i.e., the nucleotide sequence which is identical to the mRNA sequence and which is usually listed in the sequence listing, and the non-coding strand which is used as a template for transcription of the gene or cDNA, can be said to code for the protein or other product of the gene or cDNA.

[0076] As used herein, "endogenous" refers to any substance that originates or is produced within an organism, cell, tissue or system.

[0077] The term "epitope" as used herein is defined as a small chemical molecule on an antigen that can elicit an immune response and induce a B-cell response and / or a T-cell response. An antigen can have one or more epitopes. Most antigens have many epitopes; that is, they are multivalent. The size of an epitope is generally about 10 amino acids and / or sugars. Preferably, an epitope is about 4-18 amino acids, more preferably about 5-16 amino acids, even more preferably about 6-14 amino acids, more preferably about 7-12, and most preferably about 8-10 amino acids. In general, it will be understood by those skilled in the art that the overall three-dimensional structure of a molecule, rather than the specific primary sequence, is the primary criterion for antigen specificity and thus serves to distinguish one epitope from another. According to the present disclosure, a peptide used in the present invention can be an epitope.

[0078] As used herein, the term "exogenous" refers to any material that is introduced from or produced outside an organism, cell, tissue or system.

[0079] The term "expand" as used herein refers to an increase in number, such as when the number of T cells is increased. In one embodiment, the T cells expanded ex vivo are increased in number compared to the number originally present in the culture. In another embodiment, the T cells expanded ex vivo are increased in number compared to other cell types in the culture. As used herein, the term "ex vivo" refers to cells that are removed from a living organism (e.g., a human) and grown outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0080] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0081] "Expression vector" refers to a vector that contains a recombinant polynucleotide that includes an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids incorporating a recombinant polynucleotide, plasmids (e.g., naked plasmids or plasmids contained in liposomes), and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus, and adeno-associated virus).

[0082] "Identity" as used herein refers to the subunit sequence identity between two polymer molecules, particularly between two amino acid molecules, e.g., between two polypeptide molecules. If two amino acid sequences have the same residue at the same position, e.g., if a position in each of the two polypeptide molecules is occupied by arginine, then they are identical at that position. Identity, or the degree to which two amino acid sequences have the same residue at the same position in an alignment, is often expressed as a percentage. Identity between two amino acid sequences is a direct function of the number of matching or identical positions. For example, if half of the positions in two sequences (e.g., 5 positions in a polymer 10 amino acids in length) are identical, then the two sequences are 50% identical, and if 90% of the positions (e.g., 9 out of 10) are matched or identical, then the two amino acid sequences are 90% identical.

[0083] As used herein, the term "immune response" is defined as a cellular response that occurs when lymphocytes identify an antigenic molecule as foreign and induces the formation of antibodies and / or activates lymphocytes to eliminate the antigen.

[0084] The term "immunosuppressive" is used herein to refer to reducing the overall immune response.

[0085] "Isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can be present in a substantially purified form or can exist in a non-native environment, such as, for example, a host cell.

[0086] As used herein, "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells, and lentiviruses are one of the most efficient gene delivery vectors, since they can deliver a significant amount of genetic information into the DNA of host cells. HIV, SIV, and FIV are all examples of lentiviruses. Lentivirus-derived vectors provide a means to achieve significant levels of gene transfer in vivo.

[0087] The term "modified" as used herein refers to an altered state or structure of a molecule or cell of the invention. Molecules can be modified in a variety of ways, including chemically, structurally, and functionally. Cells can be modified by the introduction of a nucleic acid.

[0088] The term "modulate" as used herein means to mediate a detectable increase or decrease in the level of response in a control compared to the level of response in a control in the absence of a treatment or compound and / or compared to the level of response in an otherwise identical subject without treatment. This term encompasses perturbing and / or affecting a native signal or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

[0089] In the present invention, the following abbreviations are used for the commonly occurring nucleobases: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0090] The term "oligonucleotide" typically refers to a short polynucleotide. Where a nucleotide sequence is represented by a DNA sequence (i.e., A, T, C, G), it will be understood that this also includes RNA sequences in which "U" replaces "T" (i.e., A, U, C, G).

[0091] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerates of each other and encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA can also include introns, to the extent that a nucleotide sequence encoding a protein may contain introns in some versions.

[0092] "Parenteral" administration of the immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im) or intrasternal injection, or infusion techniques.

[0093] The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Thus, as used herein, nucleic acid and polynucleotide are interchangeable. Those skilled in the art have the general knowledge that a nucleic acid is a polynucleotide and can be hydrolyzed into monomeric "nucleotides". The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including but not limited to, recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR, and synthetic means.

[0094] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can make up a protein's or peptide's sequence. A polypeptide encompasses any peptide or protein that contains two or more amino acids joined together by peptide bonds. As used herein, the term encompasses both short chains, often referred to in the art as peptides, oligopeptides and oligomers, and the like, and longer chains, commonly referred to in the art as proteins, of which there are many types. A "polypeptide" encompasses, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, mutants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. A polypeptide encompasses natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0095] The term "specifically binds" as used herein with respect to an antibody means an antibody that recognizes a specific antigen but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind that antigen from one or more species. However, such cross-species reactivity, in and of itself, does not change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of that antigen. However, such cross-reactivity, in and of itself, does not change the classification of the antibody as specific. In some cases, the terms "specifically bind" or "specifically bind" can be used with respect to the interaction of an antibody, protein, or peptide with a second chemical species to mean that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species. For example, an antibody recognizes and binds to a specific protein structure, rather than to proteins in general. If an antibody is specific for epitope "A", then in a reaction involving labeled "A", the presence of a molecule containing epitope A (or free unlabeled A) will reduce the amount of labeled A bound to the antibody.

[0096] The term "stimulation" refers to a primary response induced by binding of a stimulatory molecule (e.g., the TCR / CD3 complex) with its cognate ligand, thereby mediating a signaling event, such as, but not limited to, signaling by the TCR / CD3 complex. Stimulation can mediate changes in the expression of certain molecules, such as downregulation of TGF-beta, and / or rearrangements of cytoskeletal structures.

[0097] "Stimulatory molecule," as that term is used herein, refers to a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell.

[0098] As used herein, a "stimulatory ligand" refers to a ligand that, when present on an antigen-presenting cell (e.g., aAPC, dendritic cell, B cell, etc.), specifically binds to a cognate binding partner (herein referred to as a "stimulatory molecule") on a T cell, thereby mediating a primary stimulatory response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, etc. Stimulatory ligands are well known in the art and include, inter alia, peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0099] The term "subject" is intended to include any living organism in which an immune response can be elicited (e.g., a mammal). As used herein, a "subject" or "patient" can be a human or non-human mammal. Non-human mammals include, for example, farm animals and pets, such as ovine, bovine, porcine, canine, feline and murine mammals, as well as simian and non-human primate mammals. Preferably, the subject is a human.

[0100] "Target site" or "target sequence" refers to a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur. In some embodiments, a target sequence refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur.

[0101] As used herein, the term "T cell receptor" or "TCR" refers to a complex of membrane proteins involved in the activation of T cells in response to the presentation of an antigen. The TCR is responsible for the recognition of antigens bound to major histocompatibility complex molecules. The TCR is composed of a heterodimer of alpha (α) and beta (β) chains, although in some cells, the TCR consists of gamma and delta (γ / δ) chains. TCRs can exist in alpha / beta and gamma / delta forms, which are structurally similar but have distinct anatomical locations and functions. Each chain is composed of two extracellular domains, a variable domain and a constant domain. In some embodiments, the TCR can be engineered on any cell that contains a TCR, such as helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and gamma delta T cells.

[0102] As used herein, the term "therapeutic" refers to treatment and / or prophylaxis. The therapeutic effect is achieved by suppression, amelioration, or eradication of the disease state.

[0103] As used herein, the terms "transfect" or "transformation" or "transduction" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected or transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0104] "Treating" a disease, as that term is used herein, means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject.

[0105] A "vector" is a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the inside of a cell. Numerous vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, etc.

[0106] "Ortholog" or "orthogonal cytokine / receptor pair" refers to an engineered protein pair that (a) exhibits significantly reduced affinity for a native cytokine or cognate receptor, and (b) has been modified by amino acid changes to specifically bind to a corresponding engineered (orthogonal) cytokine or receptor. Upon binding of the orthogonal cytokine, the orthogonal cytokine receptor activates signaling that is transmitted through native cellular components to confer biological activity that mimics the native response, but this signaling is specific to the engineered cell expressing the orthogonal receptor. Engineered orthogonal cytokine / receptor pairs are described, for example, in WO 2017 / 044464 and WO 2019 / 173773, which are incorporated herein by reference. Orthogonal cytokine / receptor pairs are used to target the activity of promiscuous cytokines to T cell subsets of interest, allowing precise control of T cell function through genetic engineering.

[0107] "Orthogonal chimeric cytokine receptor" refers to a cytokine receptor that includes an extracellular domain of an orthogonal cytokine receptor and an intracellular signaling domain of a cytokine receptor that is different from the cytokine receptor from which the orthogonal cytokine receptor is derived. Upon binding of the orthogonal cytokine, the orthogonal cytokine chimeric receptor activates signaling that is transduced through native cellular elements to confer a biological activity that mimics the native response of the receptor from which the intracellular signaling domain is derived, but that is specific to the engineered cell that expresses the orthogonal chimeric cytokine receptor.

[0108] Ranges: Throughout this disclosure, various aspects of the invention may be expressed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to specifically disclose the possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to specifically disclose the subranges 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as the individual numerical values ​​within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0109] B. Orthogonal chimeric cytokine receptor / cytokine pairs In one aspect, the present invention provides orthogonal chimeric cytokine receptor / cytokine pairs and methods of use thereof. In some embodiments, the orthogonal chimeric cytokine receptor comprises the extracellular domain of an orthogonal IL-2 receptor (oIL2R) and the intracellular signaling domain of an IL-9 receptor (IL9R). In some embodiments, the orthogonal chimeric cytokine receptor comprises the extracellular domain of an orthogonal IL-2 receptor beta (oIL2Rb) and the intracellular signaling domain of an IL-9 receptor alpha (IL9Ra). The orthogonal chimeric cytokine receptor extracellular domain (i.e., oIL2Rb) specifically binds to the corresponding engineered orthogonal cytokine (i.e., oIL2).

[0110] Upon binding to the orthogonal IL2 cytokine (oIL2) via the oIL2R extracellular domain, the orthogonal chimeric cytokine receptor activates signaling of the intracellular signaling domain (e.g., IL9R intracellular signaling domain) that is transmitted through native cellular elements to provide a biological activity that mimics the native response of the receptor from which the intracellular signaling domain is derived, but this signaling is specific to the engineered cell expressing the orthogonal chimeric cytokine receptor. The orthogonal chimeric cytokine receptor does not bind to the corresponding endogenous cytokine, including the native counterpart of the orthogonal cytokine (e.g., IL-2), while the orthogonal cytokine (e.g., oIL2) does not bind to any endogenous receptor, including the native counterpart of the orthogonal receptor from which the orthogonal extracellular domain is derived (e.g., IL-2R). In some embodiments, the affinity of the orthogonal cytokine for the orthogonal chimeric cytokine receptor is equivalent to the affinity of the native cytokine for the native receptor from which the orthogonal extracellular domain is derived.

[0111] The nucleotide and amino acid sequences of exemplary orthogonal cytokines and orthogonal chimeric cytokine receptors of the invention are listed below.

[0112] Human ortho-IL2 cDNA (SEQ ID NO:1) TIFF2024509917000001.tif51155

[0113] Human ortho-IL2 protein (SEQ ID NO:2) TIFF2024509917000002.tif17155

[0114] Human orthoIL2Rb cDNA (SEQ ID NO:3) TIFF2024509917000003.tif174155

[0115] Human orthoIL2Rb protein (SEQ ID NO:4) TIFF2024509917000004.tif58155

[0116] Human orthoIL2Rb / IL9Ra cDNA(SEQ ID NO:5) TIFF2024509917000005.tif160155

[0117] Human orthoIL2Rb / IL9Ra protein (SEQ ID NO:6) TIFF2024509917000006.tif51155

[0118] Mouse ortho-IL2 (clone 3A10) cDNA (SEQ ID NO:7) TIFF2024509917000007.tif51155

[0119] Mouse ortho-IL2 (clone 3A10) protein (SEQ ID NO:8) TIFF2024509917000008.tif17155

[0120] Mouse orthoIL2Rb (also known as o2R) cDNA (SEQ ID NO:9) TIFF2024509917000009.tif174155

[0121] Mouse orthoIL2Rb (also known as o2R) protein (SEQ ID NO:10) TIFF2024509917000010.tif58155

[0122] Mouse orthoIL2Rb (also known as o2R) extracellular domain (SEQ ID NO:11) TIFF2024509917000011.tif24155

[0123] Mouse orthoIL2Rb (also known as o2R) transmembrane domain (SEQ ID NO:12) TIFF2024509917000012.tif3128

[0124] Mouse orthoIL2Rb (also known as o2R) intracellular domain (SEQ ID NO:13) TIFF2024509917000013.tif31155

[0125] Mouse orthoIL2Rb / IL9Ra (also known as o9R) cDNA (SEQ ID NO:14) TIFF2024509917000014.tif140155

[0126] Mouse orthoIL2Rb / IL9Ra (also known as o9R) protein (SEQ ID NO:15) TIFF2024509917000015.tif44155

[0127] Mouse orthoIL2Rb / IL9Ra (also known as o9R) - orthoIL2Rb extracellular domain (SEQ ID NO:16) TIFF2024509917000016.tif24155

[0128] Mouse orthoIL2Rb / IL9Ra (also known as o9R)-transmembrane domain (SEQ ID NO:17) TIFF2024509917000017.tif3128

[0129] Mouse orthoIL2Rb / IL9Ra (also known as o9R) - IL9Ra intracellular domain with transmembrane region (SEQ ID NO:18) TIFF2024509917000018.tif24155

[0130] Mouse orthoIL2Rb / IL4R (also known as o4R) protein (SEQ ID NO:19) TIFF2024509917000019.tif85155

[0131] Mouse orthoIL2Rb / IL7R (also known as o7R) protein (SEQ ID NO:20) TIFF2024509917000020.tif51155

[0132] Mouse orthoIL2Rb / IL21R (also known as o21R) protein (SEQ ID NO:21) TIFF2024509917000021.tif58155

[0133] C. Chimeric antigen receptors (CARs) In some embodiments of the present invention, the engineered immune cell (e.g., a T cell) expressing an orthogonal chimeric cytokine receptor is a T cell engineered to co-express at least one chimeric antigen receptor (a "CAR-T" cell).

[0134] Antigen-binding domain The antigen-binding domain of a CAR is the extracellular region of the CAR for binding to a specific target antigen, including proteins, carbohydrates, and glycolipids. The antigen-binding domain can include any domain that binds to one or more antigens, including, but not limited to, a monoclonal antibody (mAb), a polyclonal antibody, a synthetic antibody, a bispecific antibody, a human antibody, a humanized antibody, a non-human antibody, a single domain antibody, a full-length antibody or any antigen-binding fragment thereof, a Fab, and a single-chain variable fragment (scFv). In some embodiments, the antigen-binding domain comprises a glycosylated antibody or a fragment thereof or a scFv thereof.

[0135] In some embodiments, the target antigen recognized by antigen binding comprises a tumor antigen. Examples of tumor antigens that can be targeted by the antigen binding domain of the CAR include one or more antigens selected from the group including, but not limited to, CD19, CD20, HER2, NY-ESO-1, MUC1, CD123, FLT3, B7-H3, CD33, IL1RAP, CLL1 (CLEC12A) PSA, CEA, VEGF, VEGF-R2, CD22, ROR1, mesothelin, c-Met, glycolipid F77, FAP, EGFRvIII, MAGE A3, 5T4, WT1, KG2D ligand, folate receptor (FRa), and Wnt1 antigen.

[0136] The term "single chain variable fragment" or "scFv" as used herein refers to a fusion protein of the variable heavy (VH) and variable light (VL) chains of immunoglobulins (e.g., mouse or human) covalently linked to form a VH::VL heterodimer. The variable heavy (VH) and variable light (VL) chains are directly linked or linked by a peptide linker that links the N-terminus of the VH to the C-terminus of the VL or the C-terminus of the VH to the N-terminus of the VL. In some embodiments, the antigen binding domain comprises an scFv with a VH-linker-VL configuration from the N-terminus to the C-terminus. In some embodiments, the antigen binding domain comprises an scFv with a VL-linker-VH configuration from the N-terminus to the C-terminus. Those skilled in the art will be able to select the appropriate configuration for use in the present invention.

[0137] The linker is usually glycine-rich for flexibility and serine- or threonine-rich for solubility. The linker can link the heavy and light chain variable regions of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6):1910-1917 (2008) and WO 2014 / 087010, the contents of which are incorporated herein by reference in their entirety. Various linker sequences are known in the art, such as (GS) n , (SG) n , (GSGGS) n , (GGGS) n and (GGGGS) n Exemplary linker sequences include, but are not limited to, glycine serine (GS) linkers, such as, for example, but not limited to, glycine serine (GS) linkers, where n represents an integer of at least 1. The amino acid sequence of the linker sequence may include, for example, TIFF2024509917000022.tif11149. Those skilled in the art will be able to select a suitable linker sequence for use in the present invention. In one embodiment, the antigen-binding domain of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the VH and the VL are separated by a linker sequence.

[0138] Despite the removal of the constant state and the introduction of the linker, the scFv protein retains the specificity of the original immunoglobulin. Single chain Fv polypeptide antibodies can be expressed from nucleic acids containing VH and VL coding sequences as described by Huston et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See also U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778, and U.S. Patent Publication Nos. 20050196754 and 20050196754. Antagonist scFvs with inhibitory activity have been described (e.g. Zhao et al., Hybridoma (Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., J Imunol 2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife et al., J Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol 1995 2 (10:31-40). Agonistic scFvs with stimulatory activity have been described (see, e.g., Peter et al., J Bioi Chem 2003 25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit Rev Immunol 1997 17(5-6):427-55; Ho et al., BioChim Biophys Acta 2003 1638(3):257-66).

[0139] As used herein, "Fab" refers to the fragment of an antibody structure that binds to an antigen but is monovalent and does not have the Fc portion. For example, digestion of an antibody with the enzyme papain produces two Fab fragments and an Fc fragment (e.g., heavy (H) chain constant region; the Fc region that does not bind to antigen).

[0140] As used herein, "F(ab')2" refers to an antibody fragment produced by pepsin digestion of a whole IgG antibody, which has two antigen-binding (ab') (bivalent) regions, each (ab') region containing two separate amino acid chains, a portion of the H chain and a light (L) chain, linked together by S-S bonds for antigen binding, with the remaining H chain portions linked together. The "F(ab')2" fragment can be split into two independent Fab' fragments.

[0141] In some embodiments, the antigen binding domain can be derived from the same species as the species that the CAR will ultimately be used in.For example, when used in humans, the antigen binding domain of the CAR can comprise a human antibody or a fragment thereof.In some embodiments, the antigen binding domain can be derived from a different species than the species that the CAR will ultimately be used in.For example, when used in humans, the antigen binding domain of the CAR can comprise a mouse antibody or a fragment thereof, or a humanized mouse antibody or a fragment thereof.

[0142] In certain embodiments, the antigen binding domain comprises a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) and a light chain variable region comprising three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3).

[0143] Transmembrane domain The CAR of the present invention can comprise a transmembrane domain that connects the antigen-binding domain of the CAR to the intracellular domain of the CAR. The transmembrane domain of the CAR is a region that can span the plasma membrane of a cell (e.g., an immune cell or its precursor). The transmembrane domain is for insertion into a cell membrane, e.g., a eukaryotic cell membrane. In some embodiments, the transmembrane domain is interposed between the antigen-binding domain and the intracellular domain of the CAR.

[0144] In some embodiments, the transmembrane domain is naturally associated with one or more of the domains in the CAR. In some embodiments, the transmembrane domain can be selected or modified by one or more amino acid substitutions to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins in order to minimize interaction with other members of the receptor complex.

[0145] The transmembrane domain can be derived from either natural or synthetic sources.When the source is natural, this domain can be derived from any membrane-bound or transmembrane protein, such as type I transmembrane protein.When the source is synthetic, the transmembrane domain can be any artificial sequence, such as an artificial hydrophobic sequence, that facilitates the insertion of CAR into cell membrane. Examples of transmembrane domains that are particularly useful in the present invention include, but are not limited to, transmembrane domains derived from (i.e., comprising at least the transmembrane region of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), ICOS, CD278, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, or transmembrane domains derived from killer immunoglobulin-like receptors (KIR).

[0146] In certain embodiments, the transmembrane domain comprises the transmembrane domain of CD8. In certain embodiments, the transmembrane domain of CD8 is the transmembrane domain of CD8α.

[0147] In some embodiments, the transmembrane domain may be synthetic, in which case it will comprise primarily hydrophobic residues such as leucine and valine. Preferably, triplets of phenylalanine, tryptophan and valine will be found at each end of the synthetic transmembrane domain.

[0148] The transmembrane domains described herein can be combined with any of the antigen binding domains described herein, with any of the intracellular domains described herein, or with any of the other domains described herein that can be included in a CAR.

[0149] In some embodiments, the transmembrane domain further comprises a hinge region. The CAR of the present invention may also comprise a hinge region. The hinge region of the CAR is a hydrophilic region located between the antigen binding domain and the transmembrane domain. In some embodiments, this domain facilitates proper protein folding of the CAR. The hinge region is an optional component of the CAR. The hinge region may comprise a domain selected from an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge sequence, or a combination thereof. Examples of hinge regions include, but are not limited to, the CD8a hinge, an artificial hinge made of a polypeptide that may be as few as three glycines (Gly), and the CH1 and CH3 domains of IgG (e.g., human IgG4).

[0150] In some embodiments, the CAR comprises a hinge region that connects the antigen binding domain to the transmembrane domain, which in turn connects to the intracellular domain. The hinge region can preferably assist the antigen binding domain in recognizing and binding to the target antigen on the target cell (see, e.g., Hudecek et al., Cancer Immunol. Res. (2015) 3(2): 125-135). In some embodiments, the hinge region is a flexible domain, thus allowing the antigen binding domain to have a structure for optimally recognizing the specific structure and density of the target antigen on a cell, such as a tumor cell (Hudecek et al., supra). The flexibility of the hinge region allows it to adopt many different conformations.

[0151] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. In some embodiments, the hinge region is a receptor-derived hinge region polypeptide (e.g., a CD8-derived hinge region). In certain embodiments, the hinge region is a CD8α hinge.

[0152] The hinge region can have a length of about 4 amino acids to about 50 amino acids, for example, about 4 aa to about 10 aa, about 10 aa to about 15 aa, about 15 aa to about 20 aa, about 20 aa to about 25 aa, about 25 aa to about 30 aa, about 30 aa to about 40 aa, or about 40 aa to about 50 aa. In some embodiments, the hinge region can have a length of more than 5 aa, more than 10 aa, more than 15 aa, more than 20 aa, more than 25 aa, more than 30 aa, more than 35 aa, more than 40 aa, more than 45 aa, more than 50 aa, more than 55 aa, or more.

[0153] A suitable hinge region can be readily selected and can be of any number of suitable lengths, for example, from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 to 15 amino acids, from 3 to 12 amino acids, such as from 4 to 10 amino acids, from 5 to 9 amino acids, from 6 to 8 amino acids, or from 7 to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids. Suitable hinge regions can have a length of more than 20 amino acids (e.g., 30, 40, 50, 60 or more amino acids).

[0154] For example, the hinge region contains a glycine polymer (G) n , glycine-serine polymers (e.g., (GS) n , (GSGGS) n and (GGGS) n where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used, with both Gly and Ser being relatively unstructured and therefore able to serve as neutral tethers between components. Glycine polymers can be used. Glycine has access to significantly more phi-psi space compared to alanine and is much less constrained than residues with longer side chains (see, e.g., Scheraga, Rev. Computational. Chem. (1992) 2:73-142). Exemplary hinge regions include, but are not limited to: It can include amino acid sequences such as TIFF2024509917000023.tif4128.

[0155] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. The amino acid sequences of immunoglobulin hinge regions are known in the art. See, for example, Tan et al., Proc. Natl. Acad. Sci. USA (1990) 87(1): 162-166 and Huck et al., Nucleic Acids Res. (1986) 14(4): 1779-1789. As a non-limiting example, an immunoglobulin hinge region can include one of the following amino acid sequences: TIFF2024509917000024.tif4128 (see, e.g., Glaser et al., J. Biol. Chem. (2005) 280:41494-41503); For example, TIFF2024509917000025.tif25144.

[0156] The hinge region can comprise the amino acid sequence of a human IgG1, IgG2, IgG3 or IgG4 hinge region. In one embodiment, the hinge region can comprise one or more amino acid substitutions and / or insertions and / or deletions compared to a wild-type (naturally occurring) hinge region. For example, His229 of the human IgG1 hinge can be replaced with Tyr such that the hinge region comprises the sequence EPKSCDKTYTCPPCP. See, for example, Yan et al., J.Biol.Chem.(2012)287:5891-5897. In one embodiment, the hinge region can comprise an amino acid sequence from human CD8, or a variant thereof.

[0157] Intracellular signaling domain The CAR of the present invention also comprises an intracellular signaling domain. The terms "intracellular signaling domain" and "intracellular domain" are used interchangeably herein. The intracellular signaling domain of a CAR is responsible for activating at least one of the effector functions of the cell (e.g., immune cell) in which the CAR is expressed. The intracellular signaling domain transmits an effector function signal, instructing the cell (e.g., immune cell) to perform its specialized function, for example, to damage and / or destroy a target cell.

[0158] Examples of intracellular domains for use in the present invention include, but are not limited to, the cytoplasmic portions of surface receptors, costimulatory molecules and any molecules that act in concert to initiate signaling in T cells, as well as any derivatives or variants of these elements, and any synthetic sequences having the same functional capabilities.

[0159] Examples of intracellular signaling domains include, but are not limited to, the ζ chain of the T cell receptor complex or any of its homologs, such as the η chain, FcsRI γ and β chains, MB1 (Iga) chain, B29 (Ig) chain, human CD3 zeta chain, CD3 polypeptides (Δ, δ and ε), syk family tyrosine kinases (Syk, ZAP70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell transduction, such as CD2, CD5 and CD28. In one embodiment, the intracellular signaling domain can be human CD3 zeta chain, FcyRIII, FcsRI, the cytoplasmic tail of the Fc receptor, cytoplasmic receptors bearing immunoreceptor tyrosine-based activation motifs (ITAMs), and combinations thereof.

[0160] In one embodiment, the intracellular signaling domain of the CAR comprises at least one signaling domain from any portion of one or more costimulatory molecules, such as CD2, CD3, CD8, CD27, CD28, ICOS, 4-1BB, PD-1, any derivative or variant thereof, any synthetic sequence thereof having the same functional capability, and any combination thereof.

[0161] Other examples of intracellular domains include, but are not limited to, TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma (common FcR gamma), FcR beta (Fc epsilon RIb), CD79a, CD79b, Fc gamma RIIa, DAP10, DAP12, T cell receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), OX9, OX40, CD30, CD40, PD-1, ICOS, KIR family proteins, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, CD83 specific binding ligand, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD1 9, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CDlib, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT Fragments or domains derived from one or more molecules or receptors including AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, other costimulatory molecules described herein, any derivative, variant or fragment thereof, any synthetic sequence of a costimulatory molecule having the same functional capability, and any combination thereof.

[0162] Further examples of intracellular domains include, but are not limited to, the intracellular signaling domains of several types of various other immune signaling receptors, such as, but not limited to, first, second and third generation T cell signaling proteins, including CD3, B7 family costimulatory and tumor necrosis factor receptor (TNFR) superfamily receptors (see, e.g., Park and Brentjens, J. Clin. Oncol. (2015) 33(6):651-653). Additionally, intracellular signaling domains can include signaling domains used by NK cells and NKT cells (see, e.g., Hermanson and Kaufman, Front. Immunol. (2015) 6:195), such as the signaling domains of NKp30 (B7-H6) (see, e.g., Zhang et al., J. Immunol. (2012) 189(5):2290-2299) and DAP12 (see, e.g., Topfer et al., J. Immunol. (2015) 194(7):3201-3212), NKG2D, NKp44, NKp46, DAP10, and CD3z.

[0163] Intracellular signaling domains suitable for use in the CAR of the present invention include any desired signaling domain that provides a characteristic detectable signal in response to activation of the CAR (i.e., activated by an antigen and a dimerization agent) (e.g., increased production of one or more cytokines by the cell; altered transcription of a target gene; altered activity of a protein; altered cell behavior, such as cell death; cell proliferation; cell differentiation; cell survival; modulation of cell signaling responses, etc.). In some embodiments, the intracellular signaling domain comprises at least one (e.g., one, two, three, four, five, six, etc.) ITAM motif, as described below. In some embodiments, the intracellular signaling domain comprises a DAP10 / CD28-type signaling chain. In some embodiments, the intracellular signaling domain is diffused in the cytoplasm, rather than being covalently attached to the membrane-bound CAR.

[0164] Intracellular signaling domains suitable for use in the CARs of the invention comprise an immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptide. In some embodiments, the ITAM motif is repeated twice in the intracellular signaling domain, with the first and second ITAM motifs separated from each other by 6-8 amino acids. In one embodiment, the intracellular signaling domain of the CAR comprises three ITAM motifs.

[0165] In some embodiments, the intracellular signaling domain comprises a signaling domain of a human immunoglobulin receptor containing an immunoreceptor tyrosine-based activation motif (ITAM), such as, but not limited to, Fc gamma RI, Fc gamma RIIA, Fc gamma RIIC, Fc gamma RIIIA, FcRL5 (see, e.g., Gillis et al., Front. Immunol. (2014) 5:254).

[0166] Suitable intracellular signaling domains can be ITAM motif-containing moieties derived from ITAM motif-containing polypeptides. For example, suitable intracellular signaling domains can be ITAM motif-containing domains from any ITAM motif-containing protein. Thus, suitable intracellular signaling domains do not need to contain the entire sequence of the whole protein from which they are derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to, DAP12, FCER1G (Fc epsilon receptor I gamma chain), CD3D (CD3 delta), CD3E (CD3 epsilon), CD3G (CD3 gamma), CD3Z (CD3 zeta) and CD79A (antigen receptor complex-associated protein alpha chain).

[0167] In one embodiment, the intracellular signaling domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX activating protein 12; KAR associated protein; TYRO protein tyrosine kinase binding protein; killer activating receptor associated protein; killer activating receptor associated protein, etc.). In one embodiment, the intracellular signaling domain is derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma chain; fc-epsilon RI-gamma; fcR gamma; fceR1 gamma; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high affinity, gamma chain, etc.). In one embodiment, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-delta; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T cell receptor T3 delta chain; T cell surface glycoprotein CD3 delta chain, etc.). In one embodiment, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T cell surface antigen T3 / Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3 epsilon, T3e, etc.). In one embodiment, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 gamma chain (also known as CD3G, T cell receptor T3 gamma chain, CD3-gamma, T3G, gamma polypeptide (TiT3 complex), etc.). In one embodiment, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T cell receptor T3 zeta chain, CD247, CD3-zeta, CD3H, CD3Q, T3Z, TCRZ, etc.). In one embodiment, the intracellular signaling domain is derived from CD79A (also known as B cell antigen receptor complex associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; ig-alpha; membrane-bound immunoglobulin-associated protein; surface IgM-associated protein, etc.).In one embodiment, the intracellular signaling domain suitable for use in the CAR of the present disclosure comprises a DAP10 / CD28 type signaling chain. In one embodiment, the intracellular signaling domain suitable for use in the CAR of the present disclosure comprises a ZAP70 polypeptide. In some embodiments, the intracellular signaling domain comprises the cytoplasmic signaling domain of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b or CD66d. In one embodiment, the intracellular signaling domain in the CAR comprises the cytoplasmic signaling domain of human CD3 zeta.

[0168] Typically the entire intracellular signaling domain can be used, but in many cases it is not necessary to use the entire chain. When a truncated portion of the intracellular signaling domain is used, the truncated portion may be used in place of the intact chain so long as it transmits an effector function signal. The intracellular signaling domain includes any truncated portion of the intracellular signaling domain sufficient to transmit an effector function signal.

[0169] The intracellular domains described herein can be combined with any of the antigen binding domains described herein, with any of the transmembrane domains described herein, or with any of the other domains described herein that can be included in a CAR.

[0170] In certain embodiments, the intracellular domain comprises a costimulatory domain of 4-1BB. In certain embodiments, the intracellular domain comprises an intracellular domain of CD3ζ or a variant thereof. In certain embodiments, the intracellular domain comprises a 4-1BB domain and a CD3ζ domain.

[0171] D. T cell receptor In some embodiments of the present invention, the engineered immune cell (e.g., T cell) expressing an orthogonal chimeric cytokine receptor is an immune cell (e.g., T cell) that has been engineered to co-express at least one T cell receptor (TCR).

[0172] In some embodiments, the TCR targets an antigen, e.g., a tumor antigen (i.e., has antigen specificity for an antigen, e.g., a tumor antigen). As used herein, phrases such as "having antigen specificity" mean that the TCR can specifically bind to and recognize an antigen or an epitope thereof.

[0173] Native TCRs are generally heterodimers. In humans, in 95% of T cells, the TCR contains an alpha (α) and a beta (β) chain (encoded by TRA and TRB, respectively), whereas in 5% of T cells, the TCR contains a gamma and delta (γ / δ) chain (encoded by TRG and TRD, respectively). The native TCR complex is an octameric assembly of type I single-pass transmembrane proteins arranged in four dimeric modules: the variable ligand-binding TCRαβ module (in the majority of T cells) (or TCRγ / δ module) and three invariant signaling modules, CD3δε, CD3γε and CD3ζ dimers. Although the TCRαβ module binds to pMHC ligands on the surface of APCs or target cells, these proteins lack intrinsic signaling capacity and therefore rely on signaling modules to transmit information by their cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs). See, e.g., Chandler et al. Int J Mol Sci (2020) 21:7424, incorporated herein by reference. Native TCRs can be cloned and modified using standard molecular biology and genetic engineering techniques known in the art.

[0174] Engineered TCR forms have also been proposed, such as TCR mimic antibodies (Chang et al., Expert Opin Biol Ther. (2016) 16(8):979-87), TCR-like CARs and TCR-CARs (Walseng et al., Sci Rep. (2017) 7:1-10; Akatsuka et al., Front Immunol. (2020) 11:257; Poorebrahim et al., Cancer Gene Ther. (2021) 28(6):581-589, and various other known techniques are known in the art. Unlike CARs, TCRs are not limited to cell surface antigens and can detect and bind to peptides presented by MHC molecules (pMHC). This feature allows the potential targets of TCRs to be broad, including tumor-specific neoepitopes. Notably, redirection of TCR-based CARs to highly tumor-specific neoepitopes can prevent the "off-tumor" toxicity commonly associated with CAR therapy.

[0175] In some embodiments, the TCR is a natural TCR. In some embodiments, the TCR is a modified TCR. In some embodiments, the TCR is an engineered TCR, such as a TCR mimic or antibody-like structure, a CAR-like TCR, or a CAR-TCR. In some embodiments, the TCR is a mouse TCR. In some embodiments, the TCR is a human TCR. In some embodiments, the TCR is a hybrid TCR that has one or more parts (e.g., constant or variable) of a human TCR and one or more parts (e.g., constant or variable) of a mouse TCR. Alternatively, the part can be a few amino acids of the human TCR, so that the almost mouse TCR is "humanized". Methods for making such hybrid TCRs are known in the art (see, for example, Cohen et al., Cancer Res., (2006) 66:8878-8886).

[0176] In some embodiments, the TCR targets gp100 melanoma antigens, e.g., human gp100 (i.e., has antigen specificity for gp100 melanoma antigens, e.g., human gp100). In some embodiments, the tumor antigen is gp100. gp100 is also known in the art as SILV, SI, SIL, ME20, PMEL17 or D12S53E. gp100 is a protein known to play an important role in the control of mammalian pigmentation (Hoashi et al., J.Biol.Chem.(2005)280:14006-14016) and is known as a cancer antigen expressed by human tumors, including melanoma and colorectal tumors (Tartaglia et al., Vaccine(2001)19(17-19):2571-5). The amino acid and nucleotide sequences of human gp100 are published in the GenBank database of the National Center for Biotechnology Information (NCBI) under GenBank Accession No. NP_008859 (amino acid sequence) and GenBank Accession No. NM_006928.3 (nucleotide sequence).

[0177] TCRs with antigen specificity for gp100 (i.e., anti-gp100 TCRs) are known in the art, such as the TCRs described in US20140219978A1. In some embodiments, the TCR is a pmel-1 TCR. The pmel-1 mouse model was developed as a system for modeling the treatment of malignant melanoma using adoptive cell therapy (ACT) (Overwijk, et al., J Exp Med. (2003), 198(4):569-80). The target antigen pmel-17 is an ortholog of the melanocyte differentiation antigen gp100, which is often overexpressed in human melanoma.

[0178] In some embodiments, the TCR targets the NY-ESO-1 antigen (i.e., has antigen specificity for the NY-ESO-1 antigen). NY-ESO-1 is a cancer testis antigen that is overexpressed in synovial sarcoma, myxoid liposarcoma, melanoma, and other tumors. In some embodiments, the TCR is NYESO1-TCR clone 1G4 (Robbins, et al., J Immunol, 2008, 180:6116-6131). In some embodiments, the TCR targets an antigen selected from MAGE-A3 / A6, MAGE-A10, AFP, PRAME, MART-1, and HPV E6.

[0179] E. Nucleic acids and expression vectors The present disclosure provides (i) nucleic acids encoding orthogonal chimeric cytokine receptors, (ii) nucleic acids encoding orthogonal cytokines, and (iii) nucleic acids encoding CARs and / or TCRs. The nucleic acids of the present disclosure can include nucleic acid sequences (i.e., polynucleotide sequences) encoding any one of the orthogonal chimeric cytokine receptors, orthogonal cytokines, CARs, and / or TCRs disclosed herein.

[0180] In certain embodiments, the nucleic acid of the present disclosure comprises a first polynucleotide sequence and a second polynucleotide sequence. The first and second polynucleotide sequences may be separated by a linker. A linker for use in the present disclosure allows multiple proteins to be encoded by the same nucleic acid sequence (e.g., a multicistronic or bicistronic sequence), which is translated as a polyprotein that is dissociated into separate protein components. In certain embodiments, the nucleic acid comprises, from 5' to 3', a first polynucleotide sequence, a linker, and a second polynucleotide sequence. In certain embodiments, the nucleic acid comprises, from 5' to 3', a second polynucleotide sequence, a linker, and a first polynucleotide sequence.

[0181] In some embodiments, the linker comprises a nucleic acid sequence encoding an internal ribosome entry site (IRES). As used herein, "internal ribosome entry site" or "IRES" refers to an element that promotes direct internal ribosome entry into the start codon, e.g., ATG, of a protein coding region, thereby resulting in cap-independent translation of the gene. A variety of internal ribosome entry sites are known to those skilled in the art, including, but not limited to, IRESs that can be obtained from viral or cellular mRNA sources, such as immunoglobulin heavy chain binding protein (BiP); vascular endothelial growth factor (VEGF); fibroblast growth factor 2; insulin-like growth factor; translation initiation factor eIF4G; yeast transcription factors TFIID and HAP4, and IRESs that can be obtained from, for example, cardioviruses, rhinoviruses, aphthoviruses, HCV, Friend murine leukemia virus (FrMLV) and Moloney murine leukemia virus (MoMLV). Those skilled in the art will be able to select the appropriate IRES for use in the present invention.

[0182] In some embodiments, the linker comprises a nucleic acid sequence that codes for a self-cleaving peptide.As used herein, "self-cleaving peptide" or "2A peptide" refers to an oligopeptide that allows multiple proteins to be encoded as a polyprotein and dissociate into its component proteins after translation.The use of the term "self-cleaving" is not intended to imply a proteolytic cleavage reaction. A variety of self-cleaving or 2A peptides are known to those of skill in the art, including, but not limited to, those found in members of the viral family Picornaviridae, such as foot and mouth disease virus (FMDV), equine rhinitis A virus (ERAV0, Thosea asigna virus (TaV) and porcine teschovirus-1 (PTV-1), and carioviruses, such as tylovirus and encephalomyocarditis virus. The 2A peptides from FMDV, ERAV, PTV-1 and TaV are referred to herein as "F2A", "E2A", "P2A" and "T2A", respectively. One of skill in the art would be able to select an appropriate self-cleaving peptide for use in the present invention.

[0183] In some embodiments, the linker further comprises a nucleic acid sequence encoding a furin cleavage site. Furin is a ubiquitously expressed protease that resides in the trans-Golgi and processes protein precursors before their secretion. Furin cleaves the COOH-terminus of its consensus recognition sequence. A variety of furin consensus recognition sequences (or "furin cleavage sites") are known to those skilled in the art, including, but not limited to, Arg-X1-Lys-Arg or Arg-X1-Arg-Arg, X2-Arg-X1-X3-Arg and Arg-X1-X1-Arg, such as Arg-Gln-Lys-Arg, where X1 is a naturally occurring amino acid, X2 is Lys or Arg, and X3 is Lys or Arg. Those skilled in the art will be able to select a suitable furin cleavage site for use in the present invention.

[0184] In some embodiments, the linker comprises a nucleic acid sequence encoding a combination of a furin cleavage site and a 2A peptide. Examples include, but are not limited to, a linker comprising a nucleic acid sequence encoding a furin cleavage site and F2A, a linker comprising a nucleic acid sequence encoding a furin cleavage site and E2A, a linker comprising a nucleic acid sequence encoding a furin cleavage site and P2A, and a linker comprising a nucleic acid sequence encoding a furin cleavage site and T2A. A person skilled in the art will be able to select an appropriate combination for use in the present invention. In such embodiments, the linker may further comprise a spacer sequence between the furin cleavage site and the 2A peptide. In some embodiments, the linker comprises a furin cleavage site 5' to the 2A peptide. In some embodiments, the linker comprises a 2A peptide 5' to the furin cleavage site. A variety of spacer sequences are known in the art, including, but not limited to, glycine serine (GS) spacers (also known as GS linkers), such as (GS)n, (SG)n, (GSGGS)n, and (GGGS)n, where n represents an integer of at least 1. Exemplary spacer sequences include, but are not limited to, TIFF2024509917000026.tif4128, etc. A person skilled in the art would be able to select an appropriate spacer sequence for use in the present invention.

[0185] In some embodiments, the nucleic acids of the disclosure may be operably linked to transcriptional control elements, such as promoters and enhancers. Suitable promoter and enhancer elements are known to those of skill in the art.

[0186] For expression in bacterial cells, suitable promoters include, but are not limited to, lacI, lacZ, T3, T7, gpt, lambda P and trc. For expression in eukaryotic cells, suitable promoters include, but are not limited to, light and / or heavy chain immunoglobulin gene promoter and enhancer elements; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoters present in retroviral long terminal repeats; mouse metallothionein-I promoter; and various tissue-specific promoters known in the art. Suitable reversible promoters are known in the art, including reversible inducible promoters. Such reversible promoters can be isolated or derived from many organisms, e.g., eukaryotes and prokaryotes. Modification of reversible promoters from a first organism for use in a second organism, e.g., a first prokaryote and a second eukaryote, a first eukaryote and a second prokaryote, is well known in the art.Such reversible promoters, and systems based on such reversible promoters but also including additional regulatory proteins, include, but are not limited to, alcohol-regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, alcohol transactivator protein responsive promoter (AlcR), etc.), tetracycline-regulated promoters (e.g., promoter systems including TetActivator, TetON, TetOFF, etc.), steroid-regulated promoters (e.g., rat glucocorticoid receptor promoter system, human estrogen receptor promoter system, retinoid promoter system, thyroid promoter system, ecdysone promoter system, mifepristone promoter system, etc.), metal-regulated promoters (e.g., metallothionein promoter system, etc.), pathogenesis-associated regulated promoters (e.g., salicylic acid-regulated promoters, ethylene-regulated promoters, benzothiadiazole-regulated promoters, etc.), temperature-regulated promoters (e.g., heat shock-inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light-regulated promoters, synthetic inducible promoters, etc.

[0187] In some embodiments, the promoter is a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or a NK-specific promoter. For example, the CD4 gene promoter can be used. See, for example, Salmon et al. Proc. Natl. Acad. Sci. USA (1993) 90: 7739 and Marodon et al. (2003) Blood 101: 3416. As another example, the CD8 gene promoter can be used. NK cell-specific expression can be achieved by using the NcrI (p46) promoter. See, for example, Eckelhart et al. Blood (2011) 117: 1565.

[0188] For expression in yeast cells, suitable promoters are constitutive promoters such as the ADH1 promoter, the PGK1 promoter, the ENO promoter, the PYK1 promoter, or controllable promoters such as the GAL1 promoter, the GAL10 promoter, the ADH2 promoter, the PHOS promoter, the CUP1 promoter, the GALT promoter, the MET25 promoter, the MET3 promoter, the CYC1 promoter, the HIS3 promoter, the ADH1 promoter, the PGK promoter, the GAPDH promoter, the ADC1 promoter, the TRP1 promoter, the URA3 promoter, the LEU2 promoter, the ENO promoter, the TP1 promoter, and the AOX1 (e.g., for use in Pichia). The selection of the appropriate vector and promoter is well within the ordinary level of skill in the art. Suitable promoters for use in prokaryotic host cells include, for example, the bacteriophage T7 RNA polymerase promoter; the trp promoter; the lac operon promoter; hybrid promoters, such as the lac / tac hybrid promoter, the tac / trc hybrid promoter, the trp / lac promoter, the T7 / lac promoter; the trc promoter; the tac promoter, and the like; the araBAD promoter; in vivo regulated promoters, such as the ssaG promoter or related promoters (see, for example, U.S. Patent Application Publication No. 20040131637), the pagC promoter (Pulkkinen and Miller, J. Bacteriol. (1991) 173(1):86-93; Alpuche-Aranda et al., Proc. Natl. Acad. Sci. USA (1992) 89(21):10079-83), the nirB promoter (Harborne et al. Mol. Micro. (1992) 6:2805-2813), and the like (see, for example, Dunstan et al. al., Infect. Immun. (1999) 67:5133-5141, McKelvie et al., Vaccine (2004) 22:3243-3255, and Chatfield et al., Biotechnol.(1992) 10:888-892); sigma 70 promoters, such as the consensus sigma 70 promoter (see, e.g., GenBank Accession Nos. AX798980, AX798961 and AX798183); stationary phase promoters, such as the dps promoter, the spv promoter, and the like; promoters from the pathogenicity island SPI-2 (see, e.g., WO 96 / 17951); the actA promoter (see, e.g., Shetron-Rama et al., Infect. Immun. (2002) 70:1087-1096); the rpsM promoter (see, e.g., Valdivia and Falkow Mol. Microbiol. (1996). 22:367); the tet promoter (see, e.g., Hillen, W. and Wissmann, A. (1989), in Saenger, W. and Heinemann, U., eds., Topics in Molecular and Structural Biology, Protein--Nucleic Acid Suitable strong promoters for use in prokaryotic cells such as Escherichia coli include, but are not limited to, Trc, Tac, T5, T7 and Plambda. Non-limiting examples of operators for use in bacterial host cells include the lactose promoter operator (the LacI repressor protein changes conformation upon contact with lactose, thereby preventing the Lad repressor protein from binding to the operator), the tryptophan promoter operator (when complexed with tryptophan, the TrpR repressor protein has a conformation that binds to the operator, and in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator), and the tac promoter operator (see, e.g., deBoer et al., Proc. Natl. Acad. Sci. USA. (1983) 80:21-25.

[0189] Another example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked to it. Other constitutive promoter sequences may also be used, such as, but not limited to, Simian Virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) or human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, EF-1 alpha promoter, and human gene promoters, such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also considered part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on the expression of the polynucleotide sequence to which it is operably linked when such expression is desired or turn off when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0190] In some embodiments, the locus or construct or transgene containing a suitable promoter is irreversibly switched by inducing an inducible system. Suitable systems for inducing irreversible switching are well known in the art, for example, Cre-lox mediated recombination can be used to induce irreversible switching (see, for example, Fuhrmann-Benzakein, et al., Proc. Natl. Acad. Sci. USA (2000) 28: e99, the disclosure of which is incorporated herein by reference). Any suitable combination of recombinases, endonucleases, and ligases known in the art can be used to create irreversibly switchable promoters. The methods, mechanisms, and requirements for performing site-specific recombination described in other sections of this specification are useful for creating irreversibly switchable promoters and are well known in the art. See, e.g., Grindley et al. Annual Review of Biochemistry (2006) 567-605 and Tropp, Molecular Biology (2012) (Jones & Bartlett Publishers, Sudbury, Massachusetts), the disclosures of which are incorporated herein by reference.

[0191] In some embodiments, the nucleic acid of the present disclosure further comprises a nucleic acid sequence encoding a CAR-inducible expression cassette. In one embodiment, the CAR-inducible expression cassette is for producing a transgenic polypeptide product that is released upon CAR signaling. See, for example, Chmielewski and Abken, Expert Opin. Biol. Ther. (2015) 15(8): 1145-1154 and Abken, Immunotherapy (2015) 7(5): 535-544. In some embodiments, the nucleic acid of the present disclosure further comprises a nucleic acid sequence encoding a cytokine operably linked to a T cell activation responsive promoter. In some embodiments, the cytokine operably linked to the T cell activation responsive promoter is present on a separate nucleic acid sequence. In one embodiment, the cytokine is IL-12.

[0192] The nucleic acids of the present disclosure may be present in an expression vector and / or a cloning vector. Expression vectors may include selectable markers, origins of replication, and other features that provide for replication and / or maintenance of the vector. Suitable expression vectors include, for example, plasmids, viral vectors, and the like. Numerous suitable vectors and promoters are known to those of skill in the art, and many are commercially available for making the present recombinant constructs. The following vectors are exemplary but should not be construed as limiting in any way: For bacteria: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA), pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). For eukaryotes: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

[0193] Expression vectors generally have convenient restriction sites located near the promoter sequence for insertion of a nucleic acid sequence encoding an exogenous protein. A selectable marker functional in the expression host may be present. Suitable expression vectors include, for example, viral vectors (e.g., vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest. Opthalmol. Vis. Sci. (1994) 35:2543-2549; Borras et al., Gene Ther. (1999) 6:515-524; Li and Davidson, Proc. Natl. Acad. Sci. USA (1995) 92:7700-7704; Sakamoto et al., H. Gene Ther. (1999) 5:1088-1097; WO 94 / 12649; WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984; and WO 95 / 00655); adeno-associated virus (see, e.g., Ali et al., al.,Hum.Gene Ther.(1998)9:81-86, Flannery et al.,Proc.Natl.Acad.Sci.USA(1997)94:6916-6921,Bennett et al.,Invest.Opthalmol.Vis.Sci.(1997)38:2857-2863,Jomary et al.,Gene Ther.(1997)4:683 690, Rolling et al.,Hum.Gene Ther.(1999)10:641-648, Ali et al.,Hum.Mol.Genet.(1996)5:591-594, Srivastava's WO 93 / 09239, Samulski et al. al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., Proc. Natl. Acad. Sci. USA (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., Proc. Natl. Acad. Sci. USA (1997) 94:10319-23; Takahashi et al., J. Virol.(1999) 73:7812-7816); retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus).

[0194] Other suitable expression vectors include, but are not limited to, lentivirus vectors, gamma retrovirus vectors, foamy virus vectors, adeno-associated virus vectors, adenovirus vectors, poxvirus vectors, herpes virus vectors, engineered hybrid virus vectors, and transposon-mediated vectors. Viral vector technology is well known in the art and described, for example, in Sambrook et al., 2012, "Molecular Cloning: A Laboratory Manual," Vols. 1-4, Cold Spring Harbor Press, NY) and other manuals on virology and molecular biology. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.

[0195] Generally, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01 / 96584, WO 01 / 29058 and U.S. Patent No. 6,326,193).

[0196] In some embodiments, an expression vector (e.g., lentiviral vector) may be used to introduce a CAR or TCR into immune cells or their precursors (e.g., T cells). Thus, an expression vector (e.g., lentiviral vector) of the present invention may comprise a nucleic acid encoding a CAR or TCR. In some embodiments, an expression vector (e.g., lentiviral vector) will comprise additional elements that aid in the functional expression of a CAR or TCR encoded therein. In some embodiments, an expression vector comprising a nucleic acid encoding a CAR or TCR further comprises a mammalian promoter. In one embodiment, the vector further comprises an elongation factor 1 alpha promoter (EF-1α promoter). Use of the EF-1α promoter may increase the efficiency of expression of a downstream transgene (e.g., a nucleic acid sequence encoding a CAR or TCR). A physiological promoter (e.g., EF-1α promoter) is less likely to induce genotoxicity upon integration and may negate the ability of a retroviral vector to transform stem cells. Other physiological promoters suitable for use in vectors (e.g., lentiviral vectors) are known to those skilled in the art and may be incorporated into the vectors of the present invention. In some embodiments, the vector (e.g., lentiviral vector) further comprises non-essential cis-acting sequences that may improve titer and gene expression. A non-limiting example of a non-essential cis-acting sequence is the central polypurine tract and central termination sequence (cPPT / CTS), which are important for efficient reverse transcription and nuclear import. Other non-essential cis-acting sequences are known to those skilled in the art and can be incorporated into the vector (e.g., lentiviral vector) of the present invention. In some embodiments, the vector further comprises a post-transcriptional regulatory element. The post-transcriptional regulatory element may improve RNA translation, improve transgene expression, and stabilize the RNA transcript. One example of a post-transcriptional regulatory element is the marmot hepatitis virus post-transcriptional regulatory element (WPRE). Thus, in some embodiments, the vector of the present invention further comprises a WPRE sequence.Various post-transcriptional regulatory elements are known to those skilled in the art and can be incorporated into the vectors (e.g., lentiviral vectors) of the present invention. The vectors of the present invention may further comprise additional elements such as a rev response element (RRE) for RNA transport, a packaging sequence, and 5' and 3' long terminal repeats (LTRs). The term "long terminal repeat" or "LTR" refers to a domain of base pairs located at the end of retroviral DNA, and includes the U3, R and U5 regions. LTRs generally provide functions necessary for retroviral gene expression (e.g., promotion, initiation and polyadenylation of gene transcripts) and viral replication. In one embodiment, the vectors (e.g., lentiviral vectors) of the present invention comprise a 3'U3 deleted LTR. Thus, the vectors (e.g., lentiviral vectors) of the present invention may comprise any combination of elements described herein to enhance the efficiency of functional expression of transgenes. For example, a vector of the invention (e.g., a lentiviral vector) can include, in addition to a nucleic acid encoding a CAR or TCR, a WPRE sequence, a cPPT sequence, an RRE sequence, a 5'LTR, a 3'U3 deleted LTR'.

[0197] The vector of the present invention can be a self-inactivating vector. As used herein, the term "self-inactivating vector" refers to a vector in which the 3'LTR enhancer promoter region (U3 region) is modified (e.g., by deletion or substitution). The self-inactivating vector can prevent viral transcription beyond the first round of viral replication. As a result, the self-inactivating vector can infect and integrate into the host genome (e.g., mammalian genome) only once, and cannot be passaged further. Therefore, the self-inactivating vector can significantly reduce the risk of generating a replicative virus.

[0198] In some embodiments, the nucleic acid of the present invention can be RNA, for example, in vitro synthesized RNA. Methods for in vitro synthesis of RNA are known to those skilled in the art, and any known method can be used to synthesize RNA comprising a sequence encoding the orthogonal chimeric cytokine receptor of the present disclosure or its corresponding orthogonal cytokine, and / or CAR or TCR. Methods for introducing RNA into host cells are known in the art. See, for example, Zhao et al. Cancer Res. (2010) 15:9053. The introduction of RNA comprising a nucleotide sequence encoding the CAR or TCR of the present disclosure into host cells can be performed in vitro, ex vivo, or in vivo. For example, host cells (e.g., NK cells, cytotoxic T lymphocytes, etc.) can be electroporated in vitro or ex vivo with RNA comprising a nucleotide sequence encoding the orthogonal chimeric cytokine receptor of the present disclosure or its corresponding orthogonal cytokine, and / or CAR or TCR.

[0199] To evaluate the expression of a polypeptide or a portion thereof, the expression vector introduced into cells may also contain a selectable marker gene or a reporter gene or both, so that expressing cells can be easily identified and selected from a population of cells that have been transfected or infected with a viral vector. In some embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection method. Both the selectable marker and the reporter gene may be flanked by appropriate control sequences to allow expression in the host cell. Useful selectable markers include, but are not limited to, antibiotic resistance genes.

[0200] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of control sequences. In general, reporter genes are genes that are not present or expressed in recipient organisms or tissues, and the expression of the polypeptides they code for is revealed by some easily detectable property, such as enzyme activity. The expression of reporter genes is evaluated at a suitable time after DNA is introduced into recipient cells. Suitable reporter genes can include, but are not limited to, genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82).

[0201] In some embodiments, the nucleic acids of the present disclosure are provided for the production, e.g., in a mammal, of (i) an orthogonal chimeric cytokine receptor, (ii) an orthogonal cytokine, and / or (iii) a CAR or TCR as described herein. In some embodiments, the nucleic acids of the present disclosure correspond to amplification of the nucleic acid.

[0202] Oncolytic adenovirus vectors Oncolytic viruses are extremely promising agents for the treatment of solid tumors, and an oncolytic herpesvirus expressing GM-CSF has been approved by the US FDA for the treatment of advanced melanoma based on its proven therapeutic benefit in clinical trials (Andtbacka RH, et al. "Talimogene laherparepvec improves durable response rate in patients with advanced Melanoma" J Clin Oncol. 2015;33(25):2780-2788). Oncolytic adenoviruses (OAds) can be programmed to specifically target, replicate in, and kill cancer cells while sparing normal cells. The release of viral progeny results in an exponential increase in the viral inoculum, which can cause direct tumor regression and provides the danger signal necessary to awaken the immune system (Lichty BD, Breitbach CJ, Stojdl DF, Bell JC. "Going viral with cancer immunotherapy." Nat Rev Cancer. 2014;14(8):559-567).Importantly, OAds can be genetically modified to selectively express therapeutic transgenes within the TME (Siurala M,et al. "Adenoviral delivery of tumor necrosis factor-α and interleukin-2 enables successful adoptive cell therapy of immunosuppressive melanoma" Mol Ther.2016;24(8):1435-1443; Nishio N,et al. "Armed oncolytic virus enhances immune functions of chimeric antigen receptor-modified T cells in solid tumors" Cancer Res.2014;74(18):5195-5205; Tanoue K,et al. "Armed oncolytic adenovirus-expressing PD-L1 mini-body enhances antitumor effects of chimeric antigen receptor T cells in solid tumors" Cancer Res.2017;77(8):2040-2051; Rosewell Shaw A,et al. "Adenovirotherapy delivering cytokine and checkpoint inhibitor Augments CAR T cells against metastatic head and neck cancer”Mol Ther.2017;25(11):2440-2451).The feasibility and safety of OAd in human patients has been demonstrated in clinical trials (Kim KH, et al. "A phase I clinical trial of Ad5 / 3-Δ24, novel serotype-chimeric, infectivity-enhanced, conditionally-replicative adenovirus (CRAd), in patients with recurrent ovarian cancer" Gynecol Oncol. 2013;130(3):518-524; Ranki T, et al. "Phase I study with ONCOS-102 for the treatment of solid tumors-an evaluation of clinical response and exploratory analyses of immune markers" J Immunother Cancer. 2016;4:17). Their ability to restore tumor immunosuppression while locally expressing therapeutic transgenes provides a rational strategy for combination with adoptive T cell transfer.

[0203] In some embodiments, the orthogonal cytokine of the present disclosure, e.g., orthogonal IL-2, is encoded by a nucleic acid sequence contained within an oncolytic adenoviral vector, such as a conditionally replicating oncolytic adenoviral vector. An example of a conditionally replicating oncolytic adenoviral vector is a serotype 5 adenoviral vector (Ad5) in which the early genes E1A and E3 are modified to allow cancer cell-specific replication and transgene expression, respectively. E1A is modified by deleting 24 base pairs of DNA in the CR2 region (also known as the D24 mutant) to obtain a virus that can selectively replicate in cancer cells carrying p16-Rb pathway mutations. The orthogonal cytokine (e.g., oIL2) transgene can be placed in the E3 region. Furthermore, the viral capsid is modified to include a chimeric 5 / 3 fiber, which allows for improved tumor cell transduction efficiency. This construct, Ad5 / 3-D24-orthoIL2, and its isogenic control, will be used in in vitro and in vivo studies to evaluate the ability of the orthogonal cytokine pair to selectively attract and stimulate lentivirally transduced orthoCAR and / or orthoTCR T cells to confer improved anti-tumor efficacy.

[0204] F. Engineered immune cells The present invention provides modified immune cells or precursors thereof (e.g., T cells) comprising (i) an orthogonal chimeric cytokine receptor and (ii) at least one CAR. Modified immune cells or precursors thereof comprising one or more nucleic acids encoding (i) an orthogonal chimeric cytokine receptor and (ii) at least one CAR are also provided. Thus, such modified cells have specificity directed by the CAR expressed therein. For example, modified cells of the present disclosure comprising a CAR have specificity for one or more antigens on a target cell (e.g., one or more tumor antigens on a cancer cell). In one aspect, the present invention provides a method of treating cancer, comprising administering to a subject having cancer (i) a modified cell comprising an orthogonal chimeric cytokine receptor and at least one CAR and (ii) an oncolytic adenoviral vector comprising a nucleic acid encoding an orthogonal cytokine, thereby treating the cancer.

[0205] The present invention further provides modified immune cells or precursors thereof (e.g., T cells) modified to express (i) an orthogonal chimeric cytokine receptor and (ii) at least one T cell receptor (TCR). Modified immune cells or precursors thereof are also provided that include one or more nucleic acids encoding (i) an orthogonal chimeric cytokine receptor and (ii) at least one TCR. Such modified cells thus have specificity directed by the TCR expressed therein. For example, modified cells of the present disclosure that include a TCR have specificity for one or more antigens on a target cell (e.g., one or more tumor antigens on a cancer cell). In one aspect, the present invention provides a method of treating cancer, comprising administering to a subject having cancer (i) a modified cell modified to express an orthogonal chimeric cytokine receptor and at least one TCR and (ii) an oncolytic adenoviral vector that includes a nucleic acid encoding an orthogonal cytokine, thereby treating the cancer.

[0206] In certain embodiments, the modified cells are modified immune cells. In certain embodiments, the modified cells are autologous cells. In certain embodiments, the modified cells are autologous cells obtained from a human subject. In certain embodiments, the modified cells are T cells.

[0207] G. Treatment Method The modified cells (e.g., T cells) described herein can be included in a composition for immunotherapy. The composition can be a pharmaceutical composition and can further include a pharma- ceutically acceptable carrier. A therapeutically effective amount of the pharmaceutical composition comprising the modified T cells can be administered.

[0208] In one aspect, the present invention includes a method for adoptive cell transfer therapy, comprising administering a modified T cell of the present invention to a subject in need thereof. In another aspect, the present invention includes a method for treating a disease or condition in a subject, comprising administering a population of modified T cells to a subject in need thereof. In one aspect, the present invention provides a method for treating cancer, comprising administering to a subject having cancer (a) modified cells comprising (i) an orthogonal chimeric cytokine receptor and (ii) at least one CAR, and (b) an oncolytic adenoviral vector comprising a nucleic acid sequence encoding an orthogonal cytokine, wherein the modified cells exhibit stem cell memory (Tscm) characteristics with improved trafficking and effector function, thereby treating the cancer. In one aspect, the present invention provides a method of treating cancer, comprising administering to a subject having cancer (a) modified cells modified to express (i) an orthogonal chimeric cytokine receptor and (ii) at least one TCR, and (b) an oncolytic adenoviral vector comprising a nucleic acid sequence encoding an orthogonal cytokine, wherein the modified cells exhibit stem cell memory (Tscm) characteristics with improved trafficking and effector function, thereby treating the cancer.

[0209] Methods of administering immune cells for adoptive cell therapy are known, and they can be used with the provided methods and compositions. For example, adoptive T cell therapy is described in U.S. Patent Application Publication No. 2003 / 0170238 to Gruenberg et al., U.S. Patent No. 4,690,915 to Rosenberg, Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85, etc. See, for example, Themeli et al. (2013) Nat Biotechnol. 31(10):928-933, Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1):84-9, Davila et al. (2013) PLoS ONE 8(4):e61338. In some embodiments, cell therapy, such as adoptive T cell therapy, is carried out by autologous transfer, in which cells are isolated and / or otherwise prepared from the subject who will receive cell therapy, or from a sample derived from such a subject.Thus, in some aspects, cells are derived from the subject, e.g., the patient, who needs treatment, and are administered to the same subject after isolation and processing.

[0210] In some embodiments, cell therapy, such as adoptive T cell therapy, is carried out by allogeneic transfer, in which cells are isolated and / or otherwise prepared from a subject other than the subject that will receive cell therapy or the subject that will ultimately receive cell therapy, such as a subject other than the first subject. In such embodiments, the cells are then administered to a different subject of the same species, such as a second subject. In some embodiments, the first subject and the second subject are genetically identical. In some embodiments, the first subject and the second subject are genetically similar. In some embodiments, the second subject expresses the same HLA class or HLA supertype as the first subject.

[0211] In some embodiments, the subject has been treated with a therapeutic agent that targets the disease or condition, e.g., tumor, prior to administering the cells or the composition containing the cells. In some aspects, the subject is resistant or non-responsive to other therapeutic agents. In some embodiments, the subject has persistent or recurrent disease after another therapeutic intervention, including, for example, chemotherapy, radiation therapy, and / or hematopoietic stem cell transplantation (HSCT), such as allogeneic HSCT. In some embodiments, the administration effectively treats the subject, even though the subject has become resistant to another treatment.

[0212] In some embodiments, the subject is responsive to the other therapeutic agent, and treatment with the therapeutic agent reduces the disease burden. In some aspects, the subject is initially responsive to the therapeutic agent, but then exhibits a recurrence of the disease or condition. In some embodiments, the subject has not relapsed. In some such embodiments, the subject is determined to be at risk of relapse, e.g., at high risk of relapse, and thus the cells are administered prophylactically, e.g., to reduce the likelihood of relapse or prevent relapse. In some aspects, the subject has not previously been treated with another therapeutic agent.

[0213] In some embodiments, the subject has persistent or recurrent disease after another therapeutic intervention, including, for example, chemotherapy, radiation therapy, and / or hematopoietic stem cell transplantation (HSCT), such as allogeneic HSCT, In some embodiments, the administration effectively treats the subject despite the subject having become refractory to another therapy.

[0214] The modified immune cells of the present invention can be administered to animals, preferably mammals, and more preferably humans, to treat cancer. In addition, the cells of the present invention can be used to treat any condition related to cancer, particularly any condition related to cell-mediated immune response against tumor cells, where it is desirable to treat or alleviate the disease. Types of cancer that can be treated by the modified cells or pharmaceutical compositions of the present invention include carcinomas, blastomas, and sarcomas, as well as certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant diseases, such as sarcomas, carcinomas, and melanomas. Other exemplary cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, thyroid cancer, and the like. The cancer can be a non-solid tumor (e.g., a hematological tumor) or a solid tumor. Adult tumors / cancers and pediatric tumors / cancers are also included. In one embodiment, the cancer is a solid tumor or a hematological tumor. In one embodiment, the cancer is a carcinoma. In one embodiment, the cancer is a sarcoma. In one embodiment, the cancer is leukemia. In one embodiment, the cancer is a solid tumor.

[0215] A solid tumor is an abnormal mass of tissue that usually does not contain cysts or fluid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (e.g., sarcoma, carcinoma, and lymphoma). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, and pheochromocytoma. These include sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors such as gliomas (e.g., brain stem glioma and mixed glioma), glioblastoma (also known as glioblastoma multiforme), astrocytoma, CNS lymphoma, germinoma, medulloblastoma, schwannoma craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastases.

[0216] Carcinomas that may be amenable to treatment by the methods disclosed herein include, but are not limited to, esophageal carcinoma, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (various tissues), bladder carcinoma including transitional cell carcinoma (malignant neoplasm of the bladder), bronchogenic carcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma such as small cell lung carcinoma and non-small cell lung carcinoma, adrenal cortical carcinoma, thyroid carcinoma, pancreatic carcinoma, breast cancer, ovarian cancer, prostate cancer, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical carcinoma, uterine carcinoma, testicular carcinoma, osteogenic carcinoma, epithelial carcinoma, and nasopharyngeal carcinoma.

[0217] Sarcomas that may be treated by the methods disclosed herein include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovium, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

[0218] In certain exemplary embodiments, the modified immune cells of the present invention are used to treat myeloma or myeloma-related conditions. Examples of myeloma or myeloma-related conditions include, but are not limited to, light chain myeloma, non-secretory myeloma, monoclonal gamopathy of undetermined significance (MGUS), plasmacytoma (e.g., solitary, multiple solitary, extramedullary plasmacytoma), amyloidosis, and multiple myeloma. In one embodiment, the method of the present disclosure is used to treat multiple myeloma. In one embodiment, the method of the present disclosure is used to treat resistant myeloma. In one embodiment, the method of the present disclosure is used to treat relapsed myeloma.

[0219] In certain exemplary embodiments, the modified immune cells of the present invention are used to treat melanoma or melanoma-related conditions. Examples of melanoma or melanoma-related conditions include, but are not limited to, superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, acral lentigo melanoma, amelanotic melanoma, or cutaneous melanoma (e.g., cutaneous melanoma, ocular melanoma, vulvar melanoma, vaginal melanoma, rectal melanoma). In one embodiment, the method of the present disclosure is used to treat cutaneous melanoma. In one embodiment, the method of the present disclosure is used to treat resistant melanoma. In one embodiment, the method of the present disclosure is used to treat recurrent melanoma.

[0220] In yet another exemplary embodiment, the modified immune cells of the present invention are used to treat sarcomas or sarcoma-related conditions. Examples of sarcomas or sarcoma-related conditions include, but are not limited to, angiosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, gastrointestinal stromal tumor, leiomyosarcoma, liposarcoma, malignant peripheral nerve sheath tumor, osteosarcoma, pleomorphic sarcoma, rhabdomyosarcoma, and synovial sarcoma. In one embodiment, the method of the present disclosure is used to treat synovial sarcoma. In one embodiment, the method of the present disclosure is used to treat liposarcoma, such as myxoid / round cell liposarcoma, differentiated / dedifferentiated liposarcoma, and pleomorphic liposarcoma. In one embodiment, the method of the present disclosure is used to treat myxoid / round cell liposarcoma. In one embodiment, the method of the present disclosure is used to treat resistant sarcomas. In one embodiment, the method of the present disclosure is used to treat recurrent sarcomas.

[0221] The cells of the invention that are administered can be autologous with respect to the subject undergoing treatment.

[0222] Administration of the cells of the invention may be carried out in any convenient manner known to those skilled in the art. The cells of the invention may be administered to a subject by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient intraarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (iv) injection, or intraperitoneally. In another example, the cells of the invention are directly injected into a subject at a site of inflammation, a site of a local disease in a subject, a lymph node, an organ, a tumor, etc.

[0223] In some embodiments, cells are administered at a desired dosage, which in some aspects includes a desired dose or a desired number of cells or cell types and / or a desired ratio of cell types.Thus, the dosage of cells is based in some embodiments on the total number of cells (or number per kg of body weight) and the desired ratio of individual populations or subtypes, such as CD4+ to CD8+ ratio.In some embodiments, the dosage of cells is based on the desired total number of cells (or number per kg of body weight) in each population or each cell type.In some embodiments, the dosage is based on a combination of such characteristics, such as the desired total number of cells, the desired ratio, and the desired total number of cells in each population.

[0224] In some embodiments, a population or subtype of cells, such as CD8 + T cells and CD4 + The T cells are administered within a tolerance or tolerance of a desired total cell dose, e.g., a desired T cell dose. In some aspects, the desired dose is a desired number of cells or a desired number of cells per unit body weight of the subject to whom the cells are administered, e.g., cells / kg. In some aspects, the desired dose is or exceeds a minimum number of cells or a minimum number of cells per unit body weight. In some aspects, the total cells administered at the desired dose include individual populations or subtypes that are in a desired output ratio (e.g., CD4 + vs. CD8 + ratio), e.g., within a certain tolerance or tolerance of such ratio.

[0225] In some embodiments, the cells are administered at or within a tolerance of the desired dose of one or more of the individual populations or subtypes of cells, e.g., the desired dose of CD4+ cells and / or the desired dose of CD8+ cells. In some aspects, the desired dose is the desired cell number of a subtype or population, or the desired number of such cells per unit body weight of the subject to whom the cells are administered, e.g., cells / kg. In some aspects, the desired dose is or exceeds the minimum cell number of a population or subtype or the minimum cell number of a population or subtype per unit body weight. Thus, in some embodiments, the dosage is based on a desired fixed dose and a desired ratio of total cells, and / or based on a desired fixed amount of one or more, e.g., each, of the individual subtypes or subpopulations. Thus, in some embodiments, the dosage is based on a desired fixed dose or minimum dose of T cells and CD4 + Cells vs. CD8 + Based on the desired ratio of cells and / or CD4 + and / or CD8 + Based on a desired fixed or minimum dose of cells.

[0226] In certain embodiments, individual populations of cells, or subtypes of cells, are administered to a subject in a range of about 1 million to about 100 billion, such as 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, or a range defined by any two of the foregoing values). 0 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells), or any value between these ranges.

[0227] In some embodiments, the total cell dose and / or the dose of individual cell subpopulations is 1×10 5 Or about 1 x 10 5 cells / kg ~ approx. 1×10 11 cells / kg, 10 4 ~10 11 Or about 10 11 cells / kilogram (kg) body weight, e.g. 10 5 ~10 6 in the range of cells / kg body weight, e.g. 1×10 5 cells / kg, 1.5×10 5 cells / kg, 2×10 5 cells / kg or 1×10 6 cells / kg body weight, or approximately 1 x 10 5 cells / kg, 1.5×10 5 cells / kg, 2×10 5 cells / kg or 1×10 6cells / kg body weight. For example, in some embodiments, the cells are 4 Or about 10 4 ~10 9 Or about 10 9 T cells / kilogram (kg) body weight, e.g., 10 5 ~10 6 T cells / kg body weight, e.g. 1 × 10 5 T cells / kg, 1.5×10 5 T cells / kg, 2×10 5 T cells / kg or 1×10 6 T cells / kg body weight, or approximately 1 × 10 5 T cells / kg, 1.5×10 5 T cells / kg, 2×10 5 T cells / kg or 1×10 6 In another exemplary embodiment, a suitable dosage range of modified cells for use in the methods of the present disclosure is about 1×10 5 cells / kg ~ approx. 1×10 6 cells / kg, approximately 1×10 6 cells / kg ~ approx. 1×10 7 cells / kg, approximately 1×10 7 cells / kg ~ approx. 1×10 8 cells / kg, approximately 1×10 8 cells / kg ~ approx. 1×10 9 cells / kg, approximately 1×10 9 cells / kg ~ approx. 1×10 10 cells / kg, approximately 1×10 10 cells / kg ~ approx. 1×10 11 In an exemplary embodiment, a suitable dosage for use in the methods of the present disclosure is about 1×10 cells / kg. 8 In an exemplary embodiment, a suitable dosage for use in the methods of the present disclosure is about 1×10 7 In another embodiment, the suitable dosage is about 1×10 7 Total cells ~ approximately 5 x 10 7 In some embodiments, the appropriate dosage is about 1×10 total cells. 8 Total cells ~ approximately 5 x 108 In some embodiments, the appropriate dosage is about 1.4×10 total cells. 7 Total cells ~ approx. 1.1 x 10 9 In an exemplary embodiment, a suitable dosage for use in the methods of the present disclosure is about 7×10 9 is the total cell number.

[0228] In some embodiments, the cells are 4 Or about 10 4 ~10 9 Or about 10 9 CD4 + and / or CD8 + cells / kilogram (kg) body weight, e.g. 10 5 ~10 6 CD4 + and / or CD8 + For example, 1 x 10 cells / kg body weight 5 CD4 + and / or CD8 + cells / kg, 1.5×10 5 CD4 + and / or CD8 + cells / kg, 2×10 5 CD4 + and / or CD8 + cells / kg, or 1 x 10 6 CD4 + and / or CD8 + in cells / kg body weight, or approximately 1 x 10 5 CD4 + and / or CD8 + cells / kg, 1.5×10 5 CD4 + and / or CD8 + cells / kg, 2×10 5 CD4 + and / or CD8 + cells / kg, or 1 x 10 6 CD4 + and / or CD8 +In some embodiments, the cells are administered at about 1×10 cells / kg body weight or within a certain margin of error thereof. 6 , about 2.5×10 6 , about 5×10 6 , about 7.5×10 6 Or about 9 x 10 6 More than and / or at least about 1 × 10 6 , about 2.5×10 6 , about 5×10 6 , about 7.5×10 6 Or about 9 x 10 6 CD4 + cells, and / or at least about 1 x 10 6 , about 2.5×10 6 , about 5×10 6 , about 7.5×10 6 Or about 9 x 10 6 CD8+ cells, and / or at least about 1 x 10 6 , about 2.5×10 6 , about 5×10 6 , about 7.5×10 6 , or about 9 × 10 6 In some embodiments, the cells are administered at about 10 T cells or within a certain margin of error thereof. 8 ~10 12 Or about 10 10 ~10 11 T cells, approximately 10 8 ~10 12 Or about 10 10 ~10 11 CD4 + cells, and / or about 10 8 ~10 12 Or about 10 10 ~10 11 CD8 + The dose is administered at or within a certain margin of error in cells.

[0229] In some embodiments, cells are administered at a desired output ratio, or within a tolerance range of a desired output ratio, of multiple cell populations or subtypes, e.g., CD4+ and CD8+ cells or subtypes. In some aspects, the desired ratio can be a specific ratio or a range of ratios. For example, in some embodiments, the desired ratio (e.g., CD4 + Cells vs. CD8 + The ratio of cells is 5:1 or about 5:1 to 5:1 or about 5:1 (i.e., greater than about 1:5 and less than about 5:1), or 1:3 or about 1:3 to 3:1 or about 3:1 (i.e., greater than about 1:3 and less than about 3:1), for example, 2:1 or about 2:1 to 1:5 or about 1:5 (i.e., greater than about 1:5 and less than about 2:1, for example, 5 :1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1: 3.5, 1:4, 1:4.5, or 1:5, or about 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9:1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5. In some aspects, the tolerance is within about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of the desired ratio, including any value between these ranges.

[0230] In some embodiments, the dose of modified cells is administered to the subject in need thereof in a single dose or multiple doses.In some embodiments, the dose of modified cells is administered in multiple doses, for example, once a week or once every 7 days, once every 2 weeks or once every 14 days, once every 3 weeks or once every 21 days, once every 4 weeks or once every 28 days.In an exemplary embodiment, a single dose of modified cells is administered to the subject in need thereof.In an exemplary embodiment, a single dose of modified cells is administered to the subject in need thereof by rapid intravenous infusion.

[0231] For the prevention or treatment of disease, the appropriate dosage may depend on the type of disease being treated, the type of cells or recombinant receptor, the severity and course of the disease, whether the cells are administered for prophylactic or therapeutic purposes, previous treatments, the subject's clinical history and response to the cells, and the discretion of the attending physician. The compositions and cells are, in some embodiments, suitably administered to the subject at one time or over a series of treatments.

[0232] In some embodiments, the cells are administered as part of a combination treatment with another therapeutic intervention, such as an antibody or engineered cell or receptor or agent, such as a cytotoxic agent or therapeutic agent, e.g., simultaneously therewith, or sequentially in any order. In some embodiments, the cells are co-administered with one or more additional therapeutic agents, or are co-administered in conjunction with another therapeutic intervention, simultaneously, or sequentially in any order. In some situations, the cells are co-administered with another treatment close enough in time to the cell population to enhance the effect of the one or more additional therapeutic agents, or vice versa. In some embodiments, the cells are administered prior to the one or more additional therapeutic agents. In some embodiments, the cells are administered after the one or more additional therapeutic agents. In some embodiments, the one or more additional agents include a cytokine, such as IL-2, e.g., to enhance persistence. In some embodiments, the method includes administration of a chemotherapeutic agent.

[0233] In certain embodiments, the engineered cells of the present invention (e.g., engineered cells comprising (i) an orthogonal chimeric cytokine receptor and (ii) at least one CAR or TCR) may be administered to a subject in combination with an immune checkpoint antibody (e.g., an anti-PD1 antibody, an anti-CTLA-4 antibody, or an anti-PDL1 antibody). For example, the engineered cells may be administered in combination with an antibody or antibody fragment that targets, for example, PD-1 (programmed death 1 protein). Examples of anti-PD-1 antibodies include, but are not limited to, pembrolizumab (KEYTRUDA®, formerly known as lambrolizumab, also known as MK-3475), and nivolumab (BMS-936558, MDX-1106, ONO-4538, OPDIVA®) or antigen-binding fragments thereof. In certain embodiments, the engineered cells may be administered in combination with an anti-PD-L1 antibody or antigen-binding fragment thereof. Examples of anti-PD-L1 antibodies include, but are not limited to, BMS-936559, MPDL3280A (TECENTRIQ®, atezolizumab) and MEDI4736 (durvalumab, Imfinzi). In certain embodiments, the modified cells may be administered in combination with an anti-CTLA-4 antibody or an antigen-binding fragment thereof. Examples of anti-CTLA-4 antibodies include, but are not limited to, ipilimumab (trade name Yervoy). Other types of immune checkpoint modulators may also be used, such as, but are not limited to, small molecules, siRNA, miRNA, and CRISPR systems. The immune checkpoint modulator may be administered before, after, or simultaneously with the modified cells comprising a CAR or TCR. In certain embodiments, combination treatments including immune checkpoint modulators may increase the therapeutic efficacy of therapies including the modified cells of the present invention.

[0234] After administration of the cells, the biological activity of the modified cell population in some embodiments is measured, for example, by any of a number of known methods. Parameters evaluated include specific binding of modified or natural T cells or other immune cells to antigens in vivo, for example, by imaging, or ex vivo, for example, by ELISA or flow cytometry. In certain embodiments, the ability of the modified cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays as described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7):689-702 (2009), and Herman et al. J. Immunological Methods, 285(1):25-40 (2004). In certain embodiments, the biological activity of the cells is measured by assaying the expression and / or secretion of one or more cytokines, for example, CD107a, IFNγ, IL-2, and TNF. In some aspects, the biological activity is measured by evaluating a clinical outcome, for example, reduction in tumor burden or tumor mass.

[0235] In certain embodiments, the subject is provided with a secondary treatment, including, but not limited to, chemotherapy, radiation therapy, surgery, and drug therapy.

[0236] In some embodiments, the subject can be administered a conditioning treatment prior to adoptive cell therapy (e.g., CAR T cell therapy). In some embodiments, the conditioning treatment comprises administering an effective amount of cyclophosphamide to the subject. In some embodiments, the conditioning treatment comprises administering an effective amount of fludarabine to the subject. In a preferred embodiment, the conditioning treatment comprises administering an effective amount of a combination of cyclophosphamide and fludarabine to the subject. The administration of a conditioning treatment prior to adoptive cell therapy (e.g., CAR T cell therapy) can increase the efficacy of adoptive cell therapy. Methods for conditioning patients for T cell therapy are described in U.S. Patent No. 9,855,298, which is incorporated herein by reference in its entirety.

[0237] In some embodiments, one of the specific dosing regimens of the present disclosure includes a lymphodepletion step prior to administration of the modified T cells. In an exemplary embodiment, the lymphodepletion step includes administration of cyclophosphamide and / or fludarabine.

[0238] In some embodiments, the lymphodepletion step comprises administering about 200 mg / m 2 / day~about 2000mg / m 2 / day (e.g. 200 mg / m 2 / day, 300mg / m 2 / day or 500 mg / m 2 / day). In an exemplary embodiment, the dose of cyclophosphamide is about 300 mg / m 2 In some embodiments, the lymphodepletion step is about 20 mg / m 2 / day~about 900mg / m 2 / day (e.g. 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day or 60 mg / m 2 / day). In an exemplary embodiment, the dose of fludarabine is about 30 mg / m 2 / day.

[0239] In some embodiments, the lymphodepletion step comprises administering about 200 mg / m 2 / day~about 2000mg / m 2 / day (e.g. 200 mg / m 2 / day, 300mg / m 2 / day or 500 mg / m 2 / day) and cyclophosphamide at a dose of approximately 20 mg / m 2 / day~about 900mg / m 2 / day (e.g. 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day or 60 mg / m 2 In an exemplary embodiment, the lymphodepletion step comprises administration of fludarabine at a dose of about 300 mg / m 2 / day dose of cyclophosphamide and approximately 30 mg / m 2 / day dose of fludarabine.

[0240] In an exemplary embodiment, the administration of cyclophosphamide is 300 mg / m for 3 days. 2 / day and fludarabine at 30 mg / m for 3 days. 2 / day.

[0241] Administration of lymphodepleting chemotherapy may be scheduled for days -6 to -4 (with a -1 day window, administration on days -7 to -5) relative to infusion of T cells (e.g., CAR-T cells, TCR-T cells, modified T cells, etc.) on day 0.

[0242] In an exemplary embodiment, for a subject with cancer, the subject is administered 300 mg / m 200 mg / ml by intravenous infusion 3 days prior to administration of the modified T cells. 2 In an exemplary embodiment, for subjects with cancer, the subject receives lymphodepleting chemotherapy including cyclophosphamide at 300 mg / m2 by intravenous infusion for three days prior to administration of the modified T cells. 2 The patient underwent lymphodepleting chemotherapy, including cyclophosphamide.

[0243] In an exemplary embodiment, for a subject with cancer, the subject receives about 20 mg / m 2 / day~about 900mg / m 2 / day (e.g. 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day or 60 mg / m 2 / day). In an exemplary embodiment, for a subject with cancer, the subject receives lymphodepleting chemotherapy including fludarabine at a dose of 30 mg / m 2 Patients will receive lymphodepleting chemotherapy containing Fludarabine at a dose of 100 mg / kg for 3 days.

[0244] In an exemplary embodiment, for a subject with cancer, the subject receives about 200 mg / m 2 / day~about 2000mg / m 2 / day (e.g. 200 mg / m 2 / day, 300mg / m 2 / day or 500 mg / m 2 / day) and cyclophosphamide at a dose of approximately 20 mg / m 2 / day~about 900mg / m 2 / day (e.g. 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day or 60 mg / m 2 / day) and fludarabine. In an exemplary embodiment, for a subject with cancer, the subject receives lymphodepleting chemotherapy comprising about 300 mg / m 2 / day dose of cyclophosphamide and 30 mg / m 2 and fludarabine at a dose of 0.01 mg / kg for 3 days.

[0245] The cells of the invention can be administered at dosages and routes, and at times, as determined in appropriate preclinical and clinical experiments and trials. The cell compositions can be administered multiple times at dosages within these ranges. Administration of the cells of the invention can be combined with other methods useful for treating the desired disease or condition, as determined by one of skill in the art.

[0246] It is known in the art that one of the adverse effects following infusion of CAR T cells is the development of immune activation known as cytokine release syndrome (CRS). CRS is immune activation resulting in elevated inflammatory cytokines. CRS is a known on-target toxicity, and its occurrence likely correlates with efficacy. Clinical and laboratory measures range from mild CRS (systemic symptoms and / or grade 2 organ toxicity) to severe CRS (sCRS; grade 3 or higher organ toxicity, aggressive clinical intervention and / or potentially life-threatening). Clinical features include high fever, malaise, fatigue, myalgia, nausea, anorexia, tachycardia / hypotension, capillary leak, cardiac dysfunction, renal impairment, liver failure and disseminated intravascular coagulation. Dramatic elevation of cytokines including interferon-gamma, granulocyte-macrophage colony-stimulating factor, IL-10 and IL-6 has been shown following infusion of CAR T cells. One of the CRS hallmarks is the elevation of cytokines including IL-6 (severe elevation), IFN-gamma, TNF-alpha (moderate), and IL-2 (mild). Elevations of clinically available inflammatory markers including ferritin and C-reactive protein (CRP) have also been observed to correlate with CRS syndrome. The presence of CRS generally correlates with the expansion of adoptively transferred cells and progressive immune activation. It has also been demonstrated that CRS severity is determined by the disease burden at the time of infusion, as patients with high tumor burden develop more severe sCRS.

[0247] Therefore, the present invention provides a suitable CRS management strategy to alleviate the physiological symptoms of uncontrolled inflammation after the diagnosis of CRS without compromising the anti-tumor efficacy of engineered cells (e.g., CAR T cells). CRS management strategies are known in the art. For example, systemic corticosteroids can be administered to rapidly reverse sCRS (e.g., grade 3 CRS) without compromising initial anti-tumor response.

[0248] In some embodiments, anti-IL-6R antibodies may be administered. One example of an anti-IL-6R antibody is the monoclonal antibody tocilizumab, also known as atlizumab (sold as Actemra or RoActemra), approved by the Food and Drug Administration. Tocilizumab is a humanized monoclonal antibody against the interleukin-6 receptor (IL-6R). Administration of tocilizumab has shown almost immediate reversal of CRS.

[0249] CRS is generally managed based on the severity of the syndrome observed, and interventions are adjusted accordingly. CRS management decisions can be based not just on laboratory values, but also on clinical signs and symptoms and response to interventions.

[0250] Mild to moderate cases are generally treated with symptom management with fluid therapy, nonsteroidal anti-inflammatory drugs (NSAIDs) and antihistamines as needed for adequate symptom relief. More severe cases include patients with any degree of hemodynamic instability, any of which is recommended for administration of tocilizumab. First-line management of CRS may be tocilizumab, in some embodiments at a labelled dose of 8 mg / kg IV over 60 minutes (not to exceed 800 mg / dose). Tocilizumab may be repeated every 8 hours. If the response to the first dose of tocilizumab is suboptimal, further doses of tocilizumab may be considered. Tocilizumab may be administered alone or in combination with corticosteroid therapy. Patients with persistent or progressive CRS symptoms, inadequate clinical improvement in 12-18 hours, or poor response to tocilizumab may be treated with high-dose corticosteroid therapy, typically hydrocortisone 100 mg IV or methylprednisolone 1-2 mg / kg. For patients with more severe hemodynamic instability or more severe respiratory symptoms, patients may be placed on high-dose corticosteroid therapy early in the course of CRS. CRS management guidance may be based on published criteria (Lee et al. (2019) Biol Blood Marrow Transplant, doi.org / 10.1016 / j.bbmt.2018.12.758, Neelapu et al. (2018) Nat Rev Clin Oncology, 15:47, Teachey et al. (2016) Cancer Discov, 6(6):664-679).

[0251] In patients treated with CAR-T therapy, features consistent with macrophage activation syndrome (MAS) or hemophagocytic lymphohistiocytosis (HLH) have been observed concomitantly with the clinical findings of CRS (Henter, 2007). MAS appears to be a response to immune activation resulting from CRS and should therefore be considered a manifestation of CRS. MAS resembles HLH, which is also a response to immune stimulation. The clinical syndrome of MAS is characterized by high-grade unremitting fever, cytopenias affecting at least two of the three lineages, and hepatosplenomegaly. This is accompanied by high serum ferritin, soluble interleukin-2 receptor, and triglycerides, as well as reduced circulating natural killer (NK) activity.

[0252] Adoptive cell therapy that provides immune cells as described herein engineered to express any of the chimeric orthogonal cytokine receptors described herein in conjunction with a CAR may result in increased on-target off-tumor toxicity. Thus, it is envisioned herein that the present adoptive cell therapy (i.e., method of treating cancer) may further include additional cell engineering strategies (e.g., on / off systems, synthetic circuits) in certain embodiments to maximize patient safety (see, e.g., Caliendo, et al., Front Bioeng Biotechnol. (2019) 7:43).

[0253] In one aspect, the invention includes a method of treating cancer in a subject in need thereof, comprising administering to the subject an engineered immune cell or progenitor cell disclosed herein. Yet another aspect of the invention includes a method of treating cancer in a subject in need thereof, comprising administering to the subject an engineered immune cell or progenitor cell made by any of the methods disclosed herein.

[0254] H. Source of immune cells In certain embodiments, a source of immune cells (e.g., T cells) is obtained from a subject for ex vivo manipulation. The source of target cells for ex vivo manipulation can also include, for example, autologous or heterologous donor blood, umbilical cord blood, or bone marrow. For example, the source of immune cells can be derived from the subject to be treated with the modified immune cells of the present invention, such as the subject's blood, the subject's umbilical cord blood, or the subject's bone marrow. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Preferably, the subject is a human.

[0255] Immune cells can be obtained from several sources, including blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, lymph, or lymphoid organs. Immune cells are cells of the immune system, such as cells of innate or adaptive immunity, such as cells of myeloid or lymphatic system, such as lymphocytes, typically T cells and / or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). In some aspects, the cells are human cells. With respect to the subject to be treated, the cells can be allogeneic and / or autologous. The cells are typically primary cells, such as those directly isolated from the subject and / or those isolated and frozen from the subject.

[0256] In certain embodiments, the immune cell is a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, a central memory T cell, or an effector memory T cell), a CD4+ T cell, a natural killer T cell (NKT cell), a regulatory T cell (Treg), a stem cell memory T cell, a lymphoid progenitor cell, a hematopoietic stem cell, a natural killer cell (NK cell), or a dendritic cell. In some embodiments, the cell is a monocyte or a granulocyte, e.g., a myeloid cell, a macrophage, a neutrophil, a dendritic cell, a mast cell, an eosinophil, and / or a basophil. In one embodiment, the target cell is an induced pluripotent stem (iPS) cell or an iPS cell-derived cell, e.g., an iPS cell generated from a subject and engineered to alter (e.g., induce mutations therein) or manipulate the expression of one or more target genes, and differentiated into, e.g., a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, a central memory T cell, or an effector memory T cell), a CD4+ T cell, a stem cell memory T cell, a lymphoid progenitor cell, or a hematopoietic stem cell.

[0257] In some embodiments, the cells include one or more subsets of T cells or other cell types, such as the total T cell population, CD4+ cells, CD8+ cells and subpopulations thereof, such as those defined by function, activation state, maturity, differentiation potential, expansion potential, recirculation potential, localization potential, and / or persistence potential, antigen specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. Subtypes and subpopulations of T cells, and / or of CD4+ T cells, and / or of CD8+ T cells include naive T (TN) cells, effector T cells (TEFF), memory T cells and subtypes thereof, such as stem cell memory T (T SCM ) cells, central memory T(T CM ) cells, effector memory T(T EM) cells or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, endogenous and inducible regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and delta / gamma T cells. In certain embodiments, any of a number of T cell lines available in the art may be used.

[0258] In some embodiments, the method includes steps of isolating immune cells from a subject and preparing, treating, culturing, and / or manipulating them. In some embodiments, the preparation of the engineered cells includes one or more culturing and / or preparation steps. The cells for manipulation as described can be isolated from a sample, e.g., a biological sample, e.g., obtained from or derived from a subject. In some embodiments, the subject from which the cells are isolated is one that has a disease or condition or is in need of cell therapy or is being performed on. The subject in some embodiments is a human in need of a particular therapeutic intervention, e.g., adoptive cell therapy, for which the cells are isolated, treated, and / or manipulated. Thus, the cells are, in some embodiments, primary cells, e.g., primary human cells. Samples include tissues, body fluids, and other samples taken directly from a subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic modification (e.g., transduction with a viral vector), washing, and / or incubation, and the like. The biological sample can be a sample obtained directly from a biological source or a sample that has been processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

[0259] In some aspects, the sample from which the cells are derived or from which the cells are isolated is blood or blood-derived sample, or is apheresis product or leukapheresis product, or is derived from apheresis product or leukapheresis product.Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMC), leukapheresis, bone marrow, thymus, tissue biopsy, tumor, leukaemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil, or other organ, and / or cells derived therefrom.In the context of cell therapy, such as adoptive cell therapy, samples include samples from autologous and allogeneic sources.

[0260] In some embodiments, the cells are derived from a cell line, such as a T cell line. In some embodiments, the cells are obtained from heterologous sources, such as mouse, rat, non-human primate and pig. In some embodiments, the isolation of cells comprises one or more preparation steps and / or non-affinity-based cell separation steps. In some examples, the cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, and lyse or remove cells that are sensitive to a particular reagent. In some examples, the cells are separated based on one or more properties, such as density, adhesion, size, sensitivity and / or resistance to a particular component.

[0261] In some examples, cells are obtained from the circulating blood of a subject, for example, by apheresis or leukapheresis. The sample contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells and / or platelets, and in some aspects cells other than red blood cells and platelets. In some embodiments, blood cells collected from a subject are washed, for example to remove the plasma fraction and place the cells in a suitable buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some aspects, the washing step is performed by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in various biocompatible buffers after washing. In certain embodiments, components of the blood cell sample are removed and the cells are resuspended directly in culture medium. In some embodiments, the method includes preparation of white blood cells from peripheral blood by density-based cell separation methods, for example, lysis of red blood cells and centrifugation through a Percoll or Ficoll gradient.

[0262] In one embodiment, immune cells obtained from the circulating blood of an individual are obtained by apheresis or leukapheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. The cells collected by apheresis can be washed to remove the plasma fraction and place the cells in a suitable buffer or medium, such as phosphate-buffered saline (PBS), or the washing solution can be calcium-free, magnesium-free, or many, if not all, divalent cations, for subsequent processing steps. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as Ca-free, Mg-free PBS. Alternatively, the undesirable components of the apheresis sample can be removed and the cells can be resuspended directly in culture medium.

[0263] In some embodiments, the isolation method comprises the separation of different cell types based on the expression or presence of one or more specific molecules in cells, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known separation method based on such markers can be used. In some embodiments, the separation is based on affinity or immunoaffinity. For example, the isolation in some aspects comprises the separation of cells and cell populations based on the expression or expression level of one or more markers, typically cell surface markers, for example by incubation with an antibody or binding partner that specifically binds to the marker, followed generally by a washing step and separation of cells that are bound to the antibody or binding partner from cells that are not bound to the antibody or binding partner.

[0264] Such separation steps can be based on positive selection, where cells bound to the reagent are retained for further use, and / or on negative selection, where cells that are not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection is particularly useful when an antibody that specifically identifies a certain cell type in a heterogeneous population is not available, and separation is best performed based on a marker expressed by cells other than the desired population. Separation does not need to result in 100% enrichment or removal of a specific cell population or cells expressing a specific marker. For example, positive selection or enrichment of a specific type of cell, such as cells expressing a marker, refers to increasing the number or percentage of such cells, but does not need to result in a complete lack of cells that do not express the marker. Similarly, negative selection, removal or depletion of a specific type of cell, such as cells expressing a marker, refers to decreasing the number or percentage of such cells, but does not need to result in a complete removal of all such cells.

[0265] In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another step, such as subsequent positive or negative selection.In some examples, cells expressing multiple markers can be simultaneously depleted in a single separation step, such as by incubating cells with multiple antibodies or binding partners, each specific to the marker targeted for negative selection.Similarly, multiple cell types can be simultaneously positively selected by incubating cells with multiple antibodies or binding partners expressed on different cell types.

[0266] In some embodiments, one or more of the T cell populations are identified as cells that are positive for one or more particular markers, e.g., surface markers (marker+) or that express high levels of one or more particular markers, e.g., surface markers (marker high ) cells, or cells that are negative for one or more markers (marker-), or cells that express relatively low levels of one or more markers (marker low) cells are enriched or depleted. For example, in some aspects, specific subpopulations of T cells, such as cells that are positive for or express high levels of one or more surface markers, such as CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+ and / or CD45RO+ T cells, are isolated by positive or negative selection techniques. In some cases, such markers are absent or expressed at relatively low levels on certain populations of T cells (e.g., non-memory cells) but present or expressed at relatively high levels on certain other populations of T cells (e.g., memory cells). In one embodiment, cells (e.g., CD8+ cells or T cells, e.g., CD3+ cells) are enriched for cells that are positive for or express high surface levels of CD45RO, CCR7, CD28, CD27, CD44, CD127 and / or CD62L (i.e., positive selection), and / or depleted for cells that are positive for or express high surface levels of CD45RA (e.g., negative selection). In some embodiments, cells are enriched or depleted for cells that are positive for or express high surface levels of CD122, CD95, CD25, CD27 and / or IL7-Ra (CD127). In some examples, CD8+ T cells are enriched for cells that are positive for CD45RO (or negative for CD45RA) and positive for CD62L. For example, CD3+,CD28+ T cells can be positively selected using CD3 / CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0267] In some embodiments, T cells are separated from PBMC samples by negative selection of markers expressed on non-T cells such as B cells, monocytes, or other white blood cells, e.g., CD14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper T cells and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into subpopulations by positive or negative selection for markers expressed or relatively highly expressed on one or more naive, memory, and / or effector T cell subpopulations. In some embodiments, CD8+ cells are further enriched or depleted of naive, central memory, effector memory, and / or central memory stem cells, e.g., by positive or negative selection based on surface antigens associated with each subpopulation. In some embodiments, enrichment of central memory T (TCM) cells results in increased potency, e.g., improved long-term survival, expansion, and / or engraftment after administration, which in some aspects is particularly robust for such subpopulations. In some embodiments, efficacy is further enhanced by combining TCM-enriched CD8+ T cells with CD4+ T cells.

[0268] In some embodiments, memory T cells are present in both CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMCs can be enriched or depleted for CD62L-CD8+ and / or CD62L+CD8+ fractions, such as by using anti-CD8 and anti-CD62L antibodies. In some embodiments, CD4+ T cell populations and CD8+ T cell subpopulations, such as subpopulations enriched for central memory (TCM) cells. In some embodiments, enrichment of central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127, and in some aspects, it is based on negative selection of cells that express or highly express CD45RA and / or granzyme B. In some aspects, the isolation of CD8+ population enriched with TCM cells is carried out by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment of cells expressing CD62L. In one aspect, the enrichment of central memory T (TCM) cells is carried out starting from the negative fraction of cells selected based on CD4 expression, which fraction is subjected to negative selection based on CD14 and CD45RA expression, and positive selection based on CD62L. Such selections are carried out in either order, in some aspects simultaneously, and in other aspects sequentially. In some aspects, the same selection step based on CD4 expression that is used to prepare CD8+ cell population or CD8+ cell subpopulation is also used to create CD4+ cell population or CD4+ cell subpopulation, so that both positive and negative fractions from CD4-based separation are retained and used in subsequent steps of the method, optionally after one or more additional positive or negative selections.

[0269] CD4+ T helper cells are sorted into naive cells, central memory cells, and effector cells by identifying cell populations with cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L- and CD45RO. In one example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as magnetic or paramagnetic beads, to allow for the separation of cells for positive and / or negative selection.

[0270] In some embodiments, the cells are incubated and / or cultured prior to or in conjunction with the genetic manipulation. The incubation step can include culturing, cultivating, stimulating, activating and / or growing. In some embodiments, the composition or cells are incubated in the presence of stimulatory conditions or stimulatory agents. Such conditions include those designed to induce proliferation, expansion, activation and / or survival of cells in the population, and / or to mimic antigen exposure, and / or to prime cells for genetic modification, for example, for the introduction of recombinant antigen receptors. The conditions can include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, agents such as nutrients, amino acids, antibiotics, ions and / or stimulatory factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate cells. In some embodiments, the stimulatory conditions or stimulatory agents include one or more agents, such as ligands, that can activate the intracellular signaling domain of the TCR complex. In some aspects, the agent activates or initiates the TCR / CD3 intracellular signaling cascade in T cells. Such agents can include, for example, antibodies bound to a solid support such as beads, for example, antibodies specific for TCR components and / or costimulatory receptors, for example, anti-CD3, anti-CD28, and / or one or more cytokines. Optionally, the expansion method can further include adding anti-CD3 and / or anti-CD28 antibodies to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulatory agent includes IL-2 and / or IL-15, for example, an IL-2 concentration of at least about 10 units / mL.

[0271] In another embodiment, T cells are isolated from peripheral blood by lysing red blood cells and depleting monocytes, such as by centrifugation through a PERCOLL™ gradient. Alternatively, T cells can be isolated from umbilical cord. In either case, specific subpopulations of T cells can be further isolated by positive or negative selection techniques.

[0272] The cord blood mononuclear cells thus isolated can be depleted of cells expressing certain antigens, including but not limited to, cells expressing CD34, CD8, CD14, CD19 and CD56. Depletion of these cells can be accomplished using isolated antibodies, antibody-containing biological samples such as ascites fluid, antibodies bound to a physical support, and antibodies bound to cells.

[0273] Enrichment of T cell populations by negative selection can be achieved using a combination of antibodies against surface markers unique to the cells being negatively selected. A preferred method is sorting and / or selection of cells by negative magnetic immunoadherence or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the cells being negatively selected. For example, negative selection can be used to enrich for CD4 + When enriching cells, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR and CD8.

[0274] When isolating a desired cell population by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can vary. In certain embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum cell-to-bead contact. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, more than 100 million cells / ml is used. In a further embodiment, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a cell concentration from 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, concentrations of 125 or 150 million cells / ml can be used. The use of higher concentrations can result in increased cell yield, cell activation, and cell expansion.

[0275] T cells can also be frozen after the washing step, which does not require a monocyte removal step. Without wishing to be bound by theory, the freezing and subsequent thawing step removes granulocytes and, to some extent, monocytes in the cell population, resulting in a more uniform product. After a washing step that removes plasma and platelets, the cells can be suspended in a freezing solution. As a non-limiting example, one method uses PBS or other suitable cell freezing medium containing 20% ​​DMSO and 8% human serum albumin, although numerous freezing solutions and freezing parameters are known in the art and would be useful in this context. The cells are then frozen to -80°C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other controlled freezing methods can also be used, with uncontrolled freezing occurring immediately at -20°C or in liquid nitrogen.

[0276] In one embodiment, the population of T cells is comprised within cells such as peripheral blood mononuclear cells, umbilical cord blood cells, purified T cell populations, and T cell lines. In another embodiment, peripheral blood mononuclear cells comprise the population of T cells. In yet another embodiment, purified T cells comprise the population of T cells.

[0277] In certain embodiments, regulatory T cells (Tregs) can be isolated from a sample. Samples include, but are not limited to, umbilical cord blood or peripheral blood. In certain embodiments, Tregs are isolated by flow cytometry sorting. Samples can be enriched for Tregs by any means known in the art prior to isolation. Isolated Tregs can be cryopreserved and / or expanded prior to use. Methods for isolating Tregs are described in U.S. Patent Nos. 7,754,482, 8,722,400 and 9,555,105 and U.S. Patent Application Publication No. 13 / 639,927, the contents of which are incorporated herein by reference in their entirety.

[0278] I. Expansion of immune cells The CAR or TCR may be expressed by any of the methods described in, for example, U.S. Patent Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,316, 7,144,575, 7,067,316, 7,144,576, 7,067,316, 7,144,577, 7,067,316, 7,144,578, 7,144,579, 7,067,316, 7,144,579 ... The cells can be activated and expanded in number using methods such as those described in US Patent Publication Nos. 18, 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, 6,867,041, and US Patent Publication No. 20060121005. For example, the T cells of the present invention can be expanded by contact with a surface that is affixed with an agent that stimulates CD3 / TCR complex-associated signals and a ligand that stimulates costimulatory molecules on the T cell surface. Specifically, the T cell population can be stimulated by contact with a surface-immobilized anti-CD3 antibody or antigen-binding fragment thereof or anti-CD2 antibody, or by contact with a protein kinase C activator (e.g., bryostatin) together with a calcium ionophore. To costimulate accessory molecules on the surface of T cells, ligands that bind to accessory molecules are used. For example, T cells can be contacted with anti-CD3 and anti-CD28 antibodies under conditions suitable for stimulating T cell proliferation. Examples of anti-CD28 antibodies include 9.3, B-T3, and XR-CD28 (Diaclone, Besançon, France), which can be used in the present invention, as well as other methods and reagents known in the art. (See, e.g., ten Berge et al., Transplant Proc. (1998) 30(8):3975-3977; Haanen et al., J. Exp. Med. (1999) 190(9):1319-1328; and Garland et al., J. Immunol. Methods (1999) 227(1-2):53-63).

[0279] Expansion of T cells by the methods disclosed herein can be about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold or more, and any and all whole or partial integers therebetween. In one embodiment, T cells are expanded in the range of about 20-fold to about 50-fold.

[0280] Following culturing, the T cells can be incubated in the culture device in cell medium for a period of time or until the cells become confluent or reach a high cell density for optimal passaging, and then the cells can be transferred to another culture device. The culture device can be any culture device commonly used for culturing cells in vitro. Preferably, the level of confluence is 70% or more before transferring the cells to another culture device. More preferably, the level of confluence is 90% or more. The period of time can be any time suitable for culturing cells in vitro. The T cell medium can be replaced at any time during the culture of the T cells. Preferably, the T cell medium is replaced about every 2-3 days. The T cells are then harvested from the culture device, after which the T cells can be used immediately or cryopreserved for storage for later use. In one embodiment, the invention includes a step of cryopreserving the expanded T cells. The cryopreserved T cells are thawed prior to introducing the nucleic acid into the T cells.

[0281] In another embodiment, the method includes isolating T cells and expanding the T cells. In another embodiment, the invention further includes cryopreserving the T cells prior to expansion. In yet another embodiment, the cryopreserved T cells are thawed for electroporation of RNA encoding the chimeric membrane protein.

[0282] Another procedure for ex vivo expansion of cells is described in U.S. Patent No. 5,199,942, which is incorporated herein by reference. Expansion as described in U.S. Patent No. 5,199,942 may be an alternative to or in addition to other expansion methods described herein. Briefly, ex vivo culture and expansion of T cells includes the addition of factors described in U.S. Patent No. 5,199,942 or other factors such as flt3-L, IL-1, IL-3 and c-kit ligand. In one embodiment, expansion of T cells includes culturing T cells with factors selected from the group consisting of flt3-L, IL-1, IL-3 and c-kit ligand.

[0283] The culturing steps described herein (after contact with an agent described herein or electroporation) can be very short, for example less than 24 hours, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours. The culturing steps described further herein (after contact with an agent described herein) can be longer, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days.

[0284] Various terms are used to describe cells in culture. Cell culture generally refers to cells taken from a living organism and grown under controlled conditions. A primary cell culture is a culture of cells, tissues, or organs taken directly from an organism before the first passage. Cells are expanded in culture when placed in a growth medium under conditions that are conducive to cell growth and / or division, resulting in a larger cell population. When cells are expanded in culture, the rate of cell proliferation is typically measured by the time required for the cells to double their number, also known as doubling time.

[0285] Each time of transfer is called a passage. When cells are transferred, they are said to be passaged. A particular cell population or cell line may be called or characterized by the number of times they have been passaged. For example, a cultured cell population that has been passaged 10 times may be called a P10 culture. The primary culture, i.e., the first culture after isolating cells from tissue, is referred to as P0. After the first transfer, the cells are described as a secondary culture (P1 or passage 1). After the second transfer, the cells become a tertiary culture (P2 or passage 2), and so on. Those skilled in the art will understand that many populations may double during the period of passaging, and therefore, the population doubling number of a culture is greater than the number of passages. The expansion of cells during the period between passaging (i.e., the number of population doublings) depends on many factors, including, but not limited to, seeding density, substrate, medium, and time between passaging.

[0286] In one embodiment, the cells may be cultured for a few hours (about 3 hours) to about 14 days, or any integer value of time units therebetween. Suitable conditions for T cell culture include a suitable medium (e.g., Minimum Essential Medium or RPMI Medium 1640 or X-vivo15 (Lonza)), which may contain factors necessary for cell growth and viability, such as serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-gamma, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF-beta, and TNF-α, or any other additive known to those of skill in the art for growing cells. Other additives for growing cells may include, but are not limited to, detergents, plasmanate, and reducing agents, such as N-acetyl-cysteine ​​and 2-mercaptoethanol. The culture medium may include RPMI1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15 and X-Vivo 20, Optimizer, which are serum-free or supplemented with an appropriate amount of serum (or plasma) or a set of hormones and / or sufficient amounts of cytokines for the growth and expansion of T cells, and are supplemented with amino acids, sodium pyruvate and vitamins. Antibiotics, such as penicillin and streptomycin, are included only in the experimental cultures and not in the cultures of cells injected into the subject. Target cells are maintained under the conditions necessary to support growth, such as an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air+5% CO2).

[0287] The medium used to culture T cells may contain an agent capable of costimulating T cells. For example, an agent capable of stimulating CD3 is an antibody to CD3, and an agent capable of stimulating CD28 is an antibody to CD28. The cells isolated by the methods disclosed herein may be expanded approximately 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 200x, 300x, 400x, 500x, 600x, 700x, 800x, 900x, 1000x, 2000x, 3000x, 4000x, 5000x, 6000x, 7000x, 8000x, 9000x, 10,000x, 100,000x, 1,000,000x, 10,000,000x, or more. In one embodiment, T cells are expanded in the range of about 20-fold to about 50-fold, or more. In one embodiment, human regulatory T cells are expanded by KT64.86 artificial antigen presenting cells (aAPCs) coated with anti-CD3 antibodies. Methods for expanding and activating T cells can be found in U.S. Patent Nos. 7,754,482, 8,722,400, and 9,555,105, the contents of which are incorporated herein by reference in their entireties.

[0288] In one embodiment, the method of expanding T cells can further include isolating the expanded T cells for further application. In another embodiment, the method of expansion can further include subsequent electroporation of the expanded T cells followed by culturing. The subsequent electroporation can include introducing a nucleic acid encoding an agent into the expanded T cell population, e.g., transducing the expanded T cells, transfecting the expanded T cells, or electroporating the expanded T cells with a nucleic acid, where the agent further stimulates the T cells. The agent can stimulate the T cells, e.g., by stimulating further expansion, effector function, or another T cell function.

[0289] J. Pharmaceutical Compositions and Formulations Also provided are populations of immune cells of the present invention, compositions containing such cells and / or enriched with such cells, for example, cells expressing orthogonal chimeric cytokine receptors or cells co-expressing orthogonal chimeric cytokine receptors and at least one CAR or TCR, which account for at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the total cell number in a certain type of composition or cell, such as T cells or CD8+ or CD4+ cells.Compositions include pharmaceutical compositions and formulations for administration, for example, for adoptive cell therapy.Therapeutic methods for administering the cells and compositions to subjects, for example, patients, are also provided.

[0290] Also provided are compositions that contain cells for administration, such as pharmaceutical compositions and formulations, such as unit-dose compositions that contain a given dose or a fraction thereof of the number of cells to be administered.Pharmaceutical compositions and formulations generally contain one or more optional pharma- ceutically acceptable carriers or excipients.In some embodiments, the composition contains at least one additional therapeutic agent.

[0291] The term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective and does not contain additional components that are unacceptably toxic to the subject to which the formulation is to be administered. "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. In some aspects, the choice of carrier depends, in part, on the particular cell and / or on the method of administration. Thus, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain a preservative. Suitable preservatives can include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, for example, in "Remington's Pharmaceutical Sciences", 16th Edition, Osol, A. Ed. (1980).Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and may include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants, such as ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polymers; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as poly(vinylpyrrolidone); amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, such as glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as polyethylene glycol (PEG).

[0292] In some aspects, a buffering agent is included in the composition. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixture is typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail, for example, in "Remington: The Science and Practice of Pharmacy" Lippincott Williams & Wilkins; 21st Edition (May 1, 2005).

[0293] The formulation may comprise an aqueous solution. The formulation or composition may also contain two or more active ingredients useful for the particular indication, disease or condition being treated with the cells, preferably those with complementary activities for the cells, where each activity does not adversely affect the other. Such active ingredients are present in combination, where appropriate, in amounts effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further comprises other pharma- ceutical active agents or drugs, such as chemotherapeutic agents, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and / or vincristine. The pharmaceutical composition, in some embodiments, contains an amount of cells sufficient to treat or prevent the disease or condition, such as a therapeutically or prophylactically effective amount. Therapeutic or prophylactic efficacy is, in some embodiments, monitored by periodic evaluation of the treated subject. The desired dosage can be delivered by a single bolus of cells, by multiple boluses of cells, or by continuous infusion of cells.

[0294] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell population is administered parenterally. As used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection. The compositions in some embodiments are provided as sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which in some aspects may be buffered to a selected pH. Liquid preparations are usually easier to prepare than gels, other viscous compositions, and solid compositions. In addition, liquid compositions offer some convenience for administration, particularly administration by injection. On the other hand, viscous compositions can be formulated within an appropriate viscosity range that provides longer contact periods for specific tissues. A liquid or viscous composition can include a carrier, which can be a solvent or dispersion medium, for example, containing water, saline, phosphate buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

[0295] Sterile injectable solutions can be prepared by incorporating the cells in a solvent, for example by mixing with a suitable carrier, diluent, or excipient, such as sterile water, physiological saline, glucose, dextrose, etc. Depending on the route of administration and the desired formulation, the composition can contain auxiliary substances, such as wetting agents, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or thickening additives, preservatives, flavoring agents, and / or coloring agents, etc. For preparing suitable preparations, in some aspects standard texts can be consulted.

[0296] Various additives can be added to improve the stability and sterility of the composition, such as antimicrobial preservatives, antioxidants, chelating agents and buffers.Prevention of microbial action can be ensured by various antibacterial and antifungal agents, such as paraben, chlorobutanol, phenol and sorbic acid.Prolonged absorption of injectable pharmaceutical forms can be achieved by using agents that delay absorption, such as aluminum monostearate and gelatin.

[0297] Formulations to be used for in vivo administration are generally sterile. Sterility is readily accomplished, for example, by filtration through sterile filtration membranes.

[0298] The contents of the articles, patents and patent applications, and all other documents and electronically available information mentioned or cited in this specification are each and in their entirety incorporated herein by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated herein by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

[0299] Although the present invention has been described with respect to specific embodiments thereof, those skilled in the art should understand that various modifications may be made and equivalents may be substituted without departing from the true spirit and scope of the present invention. Those skilled in the art will readily recognize that other suitable modifications and adaptations of the methods described herein may be made using appropriate equivalents without departing from the scope of the embodiments disclosed herein. Furthermore, many modifications may be made to adapt a particular situation, material, composition, process, process step or steps to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the appended claims. Although certain embodiments have been described in detail above, they will be more clearly understood by reference to the following examples. The following examples are illustrative only and are not intended to be limiting. EXAMPLES

[0300] Experimental Example The present invention is described below with reference to examples, which are provided for illustrative purposes only, and are not intended to limit the invention to these examples, but rather, all variations that become evident as a result of the teachings provided herein are encompassed by the present disclosure.

[0301] material and method Protein production DNA encoding mouse and human wild-type and orthogonal IL-2 and wild-type IL-9 was cloned into the insect expression vector pAcGP67-A, which contains a C-terminal 8xHIS tag for affinity purification. DNA encoding mouse serum albumin (MSA) was purchased from Integrated DNA Technologies (IDT) and cloned as an N-terminal fusion into pAcGP67-A. Insect expression DNA constructs were transfected into Trichoplusia ni (High Five) cells (Invitrogen) using the BaculoGold Baculovirus Expression System (BD Biosciences) for secretion, and clarified supernatants were purified by Ni-NTA followed by size exclusion chromatography on a Superdex-200 column and formulated in sterile phosphate-buffered saline (PBS) for injection. Endotoxin was removed using a Proteus NoEndo HC spin column kit according to the manufacturer's recommendations (VivaProducts) and endotoxin removal was confirmed using the Pierce LAL chromogenic endotoxin quantification kit (ThermoFisher). Protein was concentrated and stored at -80°C until ready for use.

[0302] Mammalian Expression Vectors The cDNA encoding mouse orthogonal IL-2Rβ, as well as geneblock cDNAs encoding the mouse intracellular domains (ICDs) of IL4R, IL7R, IL9R, and IL21R (Integrated DNA Tech) were cloned into the retroviral vector pMSCV-MCS-IRES-YFP by PCR and ITA. Similarly, human orthogonal IL2Rβ and chimeric orthogonal IL9R were cloned into the pMSCV vector.

[0303] animal Mice were maintained in an Association for the Assessment and Accreditation of Laboratory Care-approved animal facility and used under protocols approved by the Institutional Animal Care and Use Committees at the University of California, Los Angeles (UCLA), the University of Pennsylvania, and Stanford University. For experiments performed at UCLA, C57BL / 6J mice were bred and housed at the Radiation Oncology Vivarium, and pmel-1 TCR / Thy1.1 transgenic mice (pmel mice) on a C57BL / 6 background were obtained from the Jackson Laboratory. For experiments performed at the University of Pennsylvania, C57BL / 6J mice and B6 CD45.1 "Pepboy" mice were purchased from Jackson Labs.

[0304] Cell lines and cell culture The B16-F10 mouse melanoma cell line was purchased from ATCC and cultured in L-glutamine-containing RPMI 1640 (Fisher Scientific) containing 10% fetal bovine serum (FBS, Omega Scientific), penicillin (100 U / mL, Omega Scientific), streptomycin (100 μg / mL, Omega Scientific) and amphotericin B (0.25 μg / ml, Omega Scientific). The KPC-derived mouse pancreatic cancer cell line PDA7940b was maintained in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin / streptomycin. The human melanoma cell lines M407 and M263 were established from patient biopsies under UCLA IRB approval number 11-003254 and were maintained in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin / streptomycin. M407 cells were stably transduced to express nuclear RFP (nRFP) for use in live cell imaging assays (Zaretsky, et al., The New England Journal of Medicine, 2016, 375:819-829). T cells from C57BL / 6 or pmel transgenic mice were cultured in RPMI 1640 containing L-glutamine supplemented with 10% FBS (Hyclone Characterized fetal bovine serum), antibiotics, 50 μM 2-mercaptoethanol (Gibco), 1% NEAA, 1% NaPyr and HEPES. Primary human T cells were cultured in RPMI 1640 containing L-glutamine supplemented with 10% FBS (Hyclone Characterized fetal bovine serum), antibiotics, 1% NEAA, 1% NaPyr and HEPES. HEK293T cells were purchased from ATCC and maintained in DMEM supplemented with 10% FBS, 1× GlutaMax (Gibco), and penicillin and streptomycin. Cell lines were routinely authenticated and routinely tested for mycoplasma infection using a mycoplasma detection kit (Biotool).

[0305] Retrovirus production The production of retroviruses encoding orthogonal cytokine receptors has been described previously (Sockolosky et al., Science (2018) 359:1037-1042). Briefly, 3 × 10 cells were cultured per 10 cm tissue culture dish. 6 HEK293T cells were seeded and allowed to adhere overnight. Cells were transfected with pMSCV retroviral vector and pCL-Eco packaging vector at a 1.5:1 ratio using X-tremeGENE HP (Roche), Turbofect (ThermoFisher) or TransIT reagent (Thermofisher) and cultured overnight in DMEM containing 5% FBS. After 24 h, the medium was replaced with fresh DMEM containing 5% FBS and cultured for an additional 24 h. The medium was collected, clarified by centrifugation, and flash frozen in liquid nitrogen for storage at -80°C. Cells were replenished with fresh DMEM containing 5% FBS and cultured for an additional 24 h, and retroviral supernatants were collected and stored as described. To generate retroviruses for transduction of pmel T cells and human T cells, the same procedure was used with the following modifications: 18 h after transfection, the medium was replaced with 10% FBS-containing DMEM containing 20 mM HEPES and 10 mM sodium butyrate and incubated for 8 h. The medium was then replaced with 10% FBS-containing DMEM containing 20 mM HEPES and no sodium butyrate and incubated overnight. The next day, the medium was collected and filtered through a 0.45 μm filter. If not used immediately, the virus was frozen at -80 °C for later use. Retroviruses encoding chimeric antigen receptors were produced as previously described (Watanabe et al., JCI Insight (2018) 3 (7): e99573). Briefly, pMSGV vectors were transfected into Plat-E packaging cells (Cell Biolabs) using Lipofectamine 2000 (ThermoFisher). Culture medium was replaced after 24 hours and after a further 24 hours the medium was harvested, clarified by centrifugation, passed through a 0.45 μm filter and stored at -80°C.

[0306] Adenovirus construction and purification Replication-deficient E1 / E3 deleted adenoviral vectors Ad5-CMV-oIL2 (Ad-oIL2) and Ad5-CMV-Null (Ad-Null) were constructed using the AdEasy™ XL adenoviral vector system (Agilent). Mouse orthogonal IL-2 clone 3A10 cDNA with 5' KpnI and 3' HindIII restriction sites was synthesized (GenScript) and subcloned into the multiple cloning site of pShuttle-CMV (Addgene). pAdEasy-1-containing BJ5183-AD-1 E. coli cells were transformed with PmeI-linearized pShuttle-CMV-oIL2 for homologous recombination. The resulting recombinant Ad plasmids were sequence verified and expanded in XL10-Gold Ultracompetent cells before being linearized with PacI and transfected into HEK293T cells. After multiple rounds of amplification, high titer adenovirus was purified by cesium chloride (CsCl2) gradient centrifugation. CsCl2 was exchanged into A195 buffer (Evans et al., J Pharm Sci (2004) 93:2458-2475) using Amicon Ultra-15 centrifugal filter units (Millipore). Viral titers (VP / ml) were determined spectrophotometrically (Nanodrop, ThermoFisher).

[0307] Activation, retroviral transduction and sorting of primary murine T cells Viral transduction of C57BL / 6-derived mouse T cells has been described previously (Sockolosky et al., Science (2018) 359:1037-1042). Briefly, 12-well tissue culture plates were coated overnight with a 2.5 μg / ml solution of anti-mouse CD3ε (clone 145-2C11, Biolegend) in sterile PBS. Single cell suspensions were prepared from spleens and lymph nodes of 6-8 week-old C57BL / 6J mice by dissociation through a 70 μm cell strainer followed by RBC lysis in ACK lysis buffer (Gibco). Cells were resuspended in mouse T cell medium containing 100 IU / ml recombinant mouse IL-2 and activated with plate-bound anti-mouse CD3ε and soluble anti-mouse CD28 (5ug / ml, clone 37.51, BioXCell) for 24 hours. Activated mouse T cells were transduced with retroviral supernatant containing polybrene (10ug / ml) and 100IU / ml mIL-2 by spinfection at 2600 rpm, 32°C for 90 minutes. The viral supernatant was replaced with fresh mouse T cell medium containing 100IU / ml IL-2 and cultured for 24 hours. Cells were harvested from the plate by gentle pipetting and transferred to fresh T cell medium containing 100IU / ml IL-2 at 1x10 6 The cells were resuspended at 100 / ml and expanded overnight at 37°C before further downstream cell assays.

[0308] For retroviral transduction of pmel T cells, splenocytes were collected from 5- to 10-week-old pmel mice 1–3 days before transduction and activated with 50 U / mL mouse IL-2 (Peprotech) and 1 μg / mL mouse gp100 peptide (Anaspec). One day before transduction, 6-well tissue culture plates were coated with Retronectin (Takara) and kept in a 4 °C refrigerator overnight. The next day, plates were blocked with 0.5% FBS in PBS for 30 min and washed with PBS. Viral supernatant (2 mL) was added to each well and centrifuged at 2000 g for 2 h. Activated pmel T cells (3 × 10 6) were added to each well along with 50 U / mL mouse IL-2, centrifuged at 2000 g for 10 min, and then cultured at 37°C for 18–24 h. Viable transduced cells were then sorted on an Aria II cell sorter (BD Biosciences) based on YFP expression and 7-AAD exclusion, rested overnight, and then used for downstream in vitro or in vivo assays.

[0309] Retroviral transduction of murine CAR T cells has been previously described (Watanabe et al., JCI Insight (2018) 3(7):e99573). Briefly, primary donor CD45.1 murine splenocytes were enriched for CD3+ cells by magnetic bead separation (Stemcell Technologies). T cells were activated with murine CD3 / CD28 Dynabeads (ThermoFisher) in the presence of 50 U / ml recombinant human IL-2 (Peprotech) for 48 h before spinfection on retronectin-coated (Takara Bio) plates. Two days after spinfection, cells were harvested for in vitro assays or for intravenous injection.

[0310] Activation, retroviral transduction and selection of primary human T cells Primary human peripheral blood mononuclear cells (PBMCs) isolated from healthy human donor leukapheresis were thawed, rested overnight, and activated with anti-human CD3 / 28 magnetic Dynabeads (ThermoFisher) and human IL-2 (500 U / mL) for 2 days. T cells were co-transduced by spinfection in 6-well plates coated with Retronectin (Takara) and loaded with 1 mL / well of each retrovirus (encoding ho2R and NYESO1-TCR clone 1G4 or ho9R and NYESO1-TCR clone 1G4) for 48 h. Activated and transduced cells were collected and beads were removed by placing on an EasySep cell separation magnet for 2 min. Cells were stained with anti-human Vβ13.1 PE antibody (Beckman Coulter, recognizing the β-chain of NYESO1-TCR clone 1G4) and 7-AAD viability dye, followed by cell sorting based on expression of YFP, Vβ13.1, and exclusion of 7-AAD using an Aria II cell sorter (BD Biosciences).

[0311] Phosphoflo signaling assay Actively growing primary mouse or human T cells were rested for 24 h in RPMI-C lacking IL-2 before signaling assays. Cells were plated in ultra-low attachment 96-well round-bottom plates in 50 μl of warm RPMI-C medium. Cells were then stimulated by adding 50 μl of serial dilutions of recombinant cytokines for 20 min at 37°C, and the reaction was terminated by fixation with 1.5% PFA for 15 min at room temperature (RT) with agitation. Cells were washed and permeabilized with 150 μl of ice-cold 100% methanol for 60 min on ice or stored overnight at -80°C. Cells were washed with FACS buffer and then stained with Alexa647-labeled anti-STAT5 pY694 (BD Biosciences), anti-STAT3 (BD ​​Biosciences) or anti-STAT1 (Cell Signaling), diluted 1:100 in FACS buffer, and incubated for 1 h at 4°C in the dark. Cells were washed and analyzed using CytoFlex (Beckman Coulter). Data represent mean fluorescence intensity (MFI) and points were fitted to a log(agonist) vs. dose-response model using Prism 8.4 (GraphPad).

[0312] Western blot IL-2 and FBS-starved CAR T cells were stimulated with cytokines for 20 min and lysed in ice-cold RIPA buffer supplemented with protease / phosphatase inhibitor cocktail (Halt, ThermoFisher) for protein extraction. 30 μg of total protein was loaded onto an SDS-PAGE gel (NuPage Bis-Tris, ThermoFisher) and then transferred onto a PVDF membrane (Immobilon-FL, Millipore). Detection of pSTAT1 / pSTAT3 / pSTAT5 and GAPDH was performed with the respective primary antibodies followed by IRDye-labeled or HRP-linked secondary antibodies. Membranes were imaged on an Odyssey CLx (LI-COR Biosciences).

[0313] Mouse T cell proliferation assay Actively growing primary mouse T cells were rested in RPMI-C lacking IL-2 for 48 h before labeling with CellTracer Violet (CTV, ThermoFisher). Labeled cells were seeded at 50,000 T cells / well in 50 μl in 96-well round-bottom plates. Serial dilutions of MSA-oIL2 (50 μl) were added to a total volume of 100 μl and cultured at 37°C for 2 days. On day 2, freshly diluted cytokines (100 μl) were added to the cells and cultured for an additional 2 days. On day 4, CTV-labeled cell proliferation was assessed by FACS using CytoFlex. Live cell gates were based on FSC and SSC.

[0314] CAR T cell proliferation was assessed by seeding 50,000 cells / well in the presence of MSA-oIL2 or MSA-IL2 in round-bottom 96-well plates. Cells were fed with fresh medium and cytokines on day 2. Daily cell counts were obtained by staining an aliquot of cells with Calcein AM viability dye (ThermoFisher) and analyzed on a Celigo image cytometer (Nexcelom Bioscience).

[0315] Cytokine assays For the Luminex assay, transduced CAR T cells were incubated in triplicate in round-bottom 96-well plates (50,000 cells / well in 200 μl) in the presence of cytokines for 4 days, after which the supernatants were analyzed with a Th1 / Th2 / Th9 / Th17 / Th22 / Treg cytokine 17plex mouse ProcartaPlex™ panel (ThermoFisher). IFNγ was measured separately from the supernatants of co-cultures with B16-F10 pmel T cells using a Cytokine Bead Array (BD Biosciences) according to the manufacturer's instructions. oIL-2 expression from PDA7940b cells (10,000 cells / well, 96-well plates) was assessed by mouse IL-2 ELISA (Abcam) in cell culture supernatants at different time points after infection with Ad-Null or Ad-oIL2 (100VP / cell). In vivo expression was assessed by injecting PDA7940b with PBS, Ad-Null or Ad-oIL2 (1e9 VP / tumor) and harvesting 72 hours later. Tumors were dissociated by three freeze-thaw cycles and homogenates were analyzed by ELISA for mouse IL-2. Terminal blood was collected by cardiac puncture and processed to serum by centrifugation. IL-2 concentrations were normalized to total protein content.

[0316] Real-time cell killing assay PDA7940b tumor cells were seeded at 10,000 cells / well in 96-well xCELLigence E-plates (Agilent). After 24 h, transduced CAR T cells pre-incubated for 48 h in the presence of oIL-2 were added at a 2:1 ratio, and target cell index was recorded every 15 min on a real-time cell analysis (RTCA) analyzer (Agilent). T cell killing of the nuclear-localized RFP-transduced B16-F10 cell line has been previously described (Kalbasi et al., Science Translational Medicine (2020) 12:eabb0152). Briefly, B16-F10-RFP+ cells pulsed with 100 ng / mL IFNγ for 18 h were plated in triplicate at 5,000 cells per well in flat-bottom 96-well plates for IncuCyte live cell analysis (Essen Bioscience). pmel T cells (o2R or o9R, pretreated with MSA-IL2 or MSA-oIL2 for 48 hours) were added at a 2:1 effector-to-target ratio and two phase contrast and fluorescence images of each well were acquired every 2 hours using an IncuCyte live imaging system and quantified by percent confluence. Human T cell repetitive killing assays were also performed using IncuCyte live cell analysis. Human melanoma cells (nRFP-M407, 5 × 10 5 5 × 10 cells) were plated into each well of a 6-well plate. Non-transduced or co-transduced human T cells (co-transduced with either ho2R / NYESO1-TCR or ho9R / NYESO1-TCR and pre-incubated with MSA-hoIL2 for 48 h) were added in duplicate at a 1:1 effector:target ratio. Every 72 h, melanoma cells (nRFP-M407, 5 × 10 cells) were added at 1:1 effector:target ratio. 5 ) were added to each well and supplemented with orthogonal cytokine (MSA-hoIL2) 24 hours prior to each tumor rechallenge.

[0317] In vivo tumor studies For in vivo B16-F10 tumor growth experiments, early passage cell lines (<10 passages) were used. B16-F10 cells (5 × 10 5 C57BL / 6 mice were injected subcutaneously into the right flank of 5 × 10 cells / mouse. Where indicated, mice were lymphodepleted with 500 cGy total body irradiation 1 day prior to adoptive transfer of T cells. T cells were adoptively transferred approximately 7 days after tumor inoculation or when tumors became palpable. Specifically, 5 × 10 cells / mouse were injected subcutaneously into the right flank of C57BL / 6 mice. 6 Sorted T cells were resuspended in 50 μl of PBS and administered by retroorbital injection. Where indicated, mice were treated with cytokines, namely mouse serum albumin (MSA)-conjugated murine IL-2 (MSA-IL2) or MSA-orthogonal IL2 (MSA-oIL2) (25,000 units per day, intraperitoneally), for 5 consecutive days starting on the day of adoptive transfer. Tumor size (length × width) was monitored with calipers 3 times a week, and volume (length × width) was measured. 2 Peripheral blood (10 μL) was collected from the tail vein at the indicated time points for quantification of adoptively transferred pmel T cells by flow cytometry. 3 When the body weight exceeded 100 mg / kg, the mice were euthanized.

[0318] The syngeneic PDA tumor model has been described previously (Watanabe et al., JCI Insight (2018) 3(7):e99573). Briefly, established subcutaneous PDA7940b tumors were treated intratumorally on days 0 and 4 with control virus Ad-Null (1e9 VP / injection) or Ad-oIL2 (1e9 VP / injection) in 50 μl PBS. CAR T cells (5e6 live CAR-positive cells) in 200 μl PBS were administered by tail vein injection on day 1. Cyclophosphamide preconditioning was performed on day -1 by intraperitoneal injection (120 mg / kg). Tumor dimensions were measured with digital calipers and volume was calculated as follows: Volume = (Length × Width) x 100 μl. 2) / 2. Cured mice were rechallenged with PDA7940b cells by subcutaneous injection into the contralateral flank, and tumor size was recorded by caliper measurement 24 days later. Age-matched naive mice were similarly injected and served as controls for tumor growth.

[0319] Immunophenotyping by flow and mass cytometry For in vitro immunophenotyping of orthogonal cytokine receptor transduced T cells, sorted T cells were plated in triplicate with equipotent doses of MSA-oIL2 or MSA-IL2. After 48 hours, T cells were harvested and surface stained. For in vivo evaluation, peripheral blood was obtained by tail vein sampling at the indicated time points. At necropsy, splenocytes were harvested by crushing the spleen on a 70 μm cell strainer and washing with PBS. Splenocyte and peripheral blood samples were treated with ACK lysis buffer prior to antibody staining.

[0320] B16 tumors were minced and dissociated using a Mouse Tumor Dissociation Kit (Miltenyi Biotec) and a gentleMACS Octo dissociator (Miltenyi Biotec), and the cells were then resuspended in PBS and filtered through a 70 μm cell strainer to obtain a single cell suspension.

[0321] Cells were stained with antibodies in phosphate-buffered saline (PBS), 5% fetal bovine serum, and 2 mM EDTA for 30 min at 4° C. Antibodies are listed in FIG. 20. 7-AAD viability dye was used to distinguish live from dead cells. Cells were analyzed by flow cytometry using an LSRFortessa (BD Biosciences). Data were analyzed using FlowJo software (version 10, BD Biosciences).

[0322] PDA7940b tumors were excised, weighed, minced with a scalpel, and dissociated using an enzyme cocktail consisting of hyaluronidase (2.5 U / ml), DNAse (50 U / ml), and collagen types I / II / IV (75 U / ml, 35 U / ml, and 75 U / ml, respectively) in RPMI1640 supplemented with 1% penicillin / streptomycin. CD45-positive cells were isolated from single-cell tumor suspensions using CD45(TIL)MicroBeads according to the manufacturer's instructions (Miltenyi Biotec) and stored in liquid nitrogen. Quantification of tumor-infiltrating CAR T cells was performed using CountBright Beads (ThermoFisher) and normalized to tumor weight.

[0323] For mass cytometry, cells were first fixed with 1.6% PFA for 5 min at room temperature. Cells were washed with 10 mL MaxPar cell staining buffer (Fluidigm) and centrifuged at 970×g for 10 min at 4° C. Cells were then resuspended in surface antibody cocktail for 30 min at RT. Cells were washed with 5 mL PBS and resuspended in 1 mL ice-cold methanol for 15 min on ice. Cells were washed again with MaxPar cell staining buffer and stained with intracellular antibody cocktail for 30 min at RT. Finally, cells were washed with 10 mL MaxPar cell staining buffer and stained overnight at 4° C. with intercalation solution (Cell-ID™ Intercalator-Ir, Cat. No. 201192B) diluted 1:6000 in Maxpar Fixation and Permeabilization Buffer (Fluidigm Cat. No. 201067) containing 1.6% PFA. Data were acquired using a Fluidigm® Helios™ (San Francisco, CA) mass cytometer. Analyses were performed using Omiq (San Francisco, CA) based on arcsinh-scaled data gated on live singlet CD45+ leukocytes or CD8+ T cells. Cells were embedded into two-dimensional visualizations using opt-SNE and clustered using FlowSOM with elbow metaclustering using Euclidean distance. Clusters of differential abundance were determined using edgeR with a p-value significance threshold of 0.05 and log(fold change) ≥ 1. Graphs were generated using the R package ggplot.

[0324] Intracellular cytokine staining Human co-transduced T cells (either ho2R / NYESO1-TCR or ho9R / NYESO1-TCR) were harvested from repeated tumor-challenged co-cultures 72 h after the last tumor challenge and 24 h after the culture medium was supplemented with orthogonal cytokines. T cells (1 × 10 5) were cultured in 96-well plates in the presence of Brefeldin A and Monensin with anti-human CD3 / CD28 Dynabeads (ThermoFisher) or with melanoma cells at a 1:1 effector:target ratio (nRFP-M407 or M263). After 4 h, cells were surface stained for 30 min at RT, fixed, and permeabilized for intracellular cytokine staining for 30 min at RT. For intracellular cytokine staining of CD45+ TILs enriched from PDA740b tumors, cells were stimulated with Cell Activation Cocktail (with Brefeldin A) (Biolegend) for 6 h, fixed / permeabilized in Cyto-Fast™ Fixation / Permeabilization Buffer Set (Biolegend), and stained with anti-IFNγ antibody. Cells were washed and analyzed by flow cytometry using an LSRII (BD Biosciences). Data were analyzed using FlowJo software (version 10, BD Biosciences).

[0325] Multiplex immunohistochemistry Formalin-fixed paraffin-embedded tumor specimens were cut into 4-μm-thick sections onto glass slides for staining. In this study, the tyramide signal amplification (TSA)-based Opal method was used for immunofluorescence (IF) staining (Opal Polaris 7-Color Automation IHC Kit, Akoya Biosciences, Marlborough, MA, USA, Cat. No. NEL871001KT). The Opal fluorophore was used at a 1:150 dilution according to the manufacturer's recommendations. Staining performance was assessed by performing fluorescent singleplexes for each biomarker and comparing the appropriate chromogenic singleplexes. Once each target was optimized for singleplex staining, multiplex staining of slides was performed using the Opal 6 multiplex assay. Primary antibodies were applied to control mouse spleen specimens at optimized concentrations previously determined for singleplex staining of control tissues. Staining was performed using the BOND RX system (Leica Biosystems). The order of antibodies for multiplex staining was Foxp3 (opal 480), CD4 (opal 520), PD-1 (opal 570), CD8 (opal 620) and CD3 (opal 690). Staining was performed after 20 min of heat-induced antigen retrieval using Bond Epitope Retrieval Solution 2 (Leica Biosystems). Antibodies are listed in Figure 20. Antibodies were used at 1:200 dilution with 1 h incubation. All fluorescently labeled slides were counterstained with DAPI and scanned at 20x magnification with a Vectra Polaris (Akoya Biosciences) using appropriate exposure times. Data from the multispectral camera was analyzed by imaging InForm software (Akoya Biosciences) and quantification was performed using HALO image analysis software (Indica Labs).

[0326] Histopathology, clinical chemistry and RNA ISH Animals were euthanized by CO2 asphyxiation. Immediately after death, blood (n=3 per group) was collected into Microvette tubes (Sarstedt) by cardiac puncture and allowed to clot for 30 min at RT before centrifugation at 12,000g for 10 min. Serum was stored at -80°C until analysis. Cytokine levels in serum were measured with a mouse 25-plex cytokine panel (IDEXX Bioanalytics). Serum levels of Ca, P, K and uric acid were measured with a custom clinical chemistry panel (IDEXX Bioanalytics).

[0327] A complete necropsy including gross postmortem examination was performed on all animals, and the following organs / tissues were sampled and fixed in 10% neutral buffered formalin for histopathological examination: head and brain, lumbar spinal cord, dorsal skin, ears, quadriceps, sternum, salivary glands, larynx and trachea, esophagus, lungs, heart, GI tract, mesenteric ligament, pancreas, liver and gallbladder, reproductive organs, kidneys, urinary bladder, and any organs / tissues showing gross lesions. Weights of liver, right kidney, left kidney, and heart were recorded prior to fixation. Formalin-fixed tissue samples were trimmed according to RITA guidelines (1, 2, 3) and then routinely processed for paraffin embedding, sectioning, and H&E staining. Resulting slides were analyzed by a board-certified pathologist blinded to the experimental design.

[0328] The distribution of mesothelin expression and CAR T cell infiltration in the meninges was examined by multiplex fluorescent RNA in situ hybridization (RNAscope® Multiplex Fluorescence Assay, ACD Bio) with custom probes designed for the mouse retroviruses used to transduce CAR and ortho receptors into T cells. The assay was performed on formalin-fixed paraffin-embedded brain sections. Whole-slide imaging of the resulting fluorescently labeled sections was performed using an Aperio VERSA 200 slide scanner (Leica Biosystems). Finally, CAR T cells and mesothelin-positive cells were counted using the object counting tool included in the Aperio ImageScope software (Leica Biosystems).

[0329] RNA sequencing and analysis For the in vitro experiments described in Figures 7H-7J, o2R and o9R pmel T cells were stimulated with 5 μM oIL-2 or 0.05 μM oIL-2 for 48 h. RNA was extracted using the RNeasy mini kit (Qiagen). RNA sequencing (RNAseq) libraries were prepared using the KAPA mRNA stranded library preparation kit according to the manufacturer's recommendations. Libraries were pooled and sequenced (50 bp single-end reads) on an Illumina HiSeq3000 platform. Reads were aligned to the mouse reference genome (mm9 / GRCm38) using HISAT2 (v2.0.4) (Kim et al., Nature Biotechnology (2019) 37: 907-915). Gene expression was quantified using HTSeq-counts (v0.6.1) (Anders et al., Bioinformatics (2015) 31: 166-169). Differential expression analysis was performed using DESeq2 (Love et al., Genome Biol (2014) 15:550), followed by gene set enrichment analysis using the fgsea (Sergushichev et al., bioRxiv, 060012 (2016)) and msigdbr (Liberzon et al., Cell Systems (2015) 1:417-425) R packages, specifically for the TFactS annotated gene set (Essaghir et al., Nucleic Acids Res (2010) 38:e120), and visualized using the ggplot2 R package. Differentially expressed genes were filtered for adjusted p-values ​​<0.01 and log2(fold change) ≥1. Normalized gene expression (calculated using the rlog transformation of DESeq2 and scaled row-wise) was used to visualize gene expression using the pheatmap R package. Principal component analysis and sample-to-sample heat maps were generated using the R functions prcomp and dist, respectively.

[0330] For the in vitro experiments described in Figure 13G, CAR-o2R and CAR-o9R cells were stimulated with MSA-oIL2 or MSA-IL2 for 48 h and total RNA was extracted using the RNeasy mini kit (Qiagen). RNA expression was analyzed using the nCounter Mouse Immunology Panel (Nanostring Technologies). Analysis was performed as described above.

[0331] FIG. 20 is a table of the reagents used in the experiments described herein.

[0332] The results of the experiment are set forth below.

[0333] Example 1: Synthetic IL-9 receptor signaling confers a combination of stem cell memory and effector anti-tumor activity to T cells. Synthetic receptor signaling has the potential to endow adoptively transferred T cells with new functions that overcome major barriers in the treatment of solid tumors, including the need for conditioning chemotherapy. Here, we engineered chimeric receptors with the orthogonal IL-2 receptor (o2R) extracellular domain fused with the intracellular domain (ICD) of the receptor for the common gamma chain (γc) cytokines IL-4, IL-7, IL-9 and IL-21, such that the orthogonal IL-2 cytokine elicits the corresponding γc cytokine signal. Of these, T cell signaling by the chimeric orthogonal IL-2R-ECD / IL-9-ICD receptor (o9R) was identified by the simultaneous activation of STAT1, STAT3 and STAT5, and is a key regulator of stem cell memory (T scm o9R T cells exhibit characteristics of IL-9R and effector T cells. o9R T cells had superior antitumor efficacy in two refractory syngeneic murine solid tumor models of melanoma and pancreatic cancer, and were effective even in the absence of conditioning lymphodepletion. Thus, by repurposing IL-9R signaling with a chimeric orthogonal cytokine receptor, T cells acquire novel functions and provide improved antitumor activity against difficult-to-treat solid tumors.

[0334] Adoptively transferred engineered T cell therapy has shown significant antitumor activity in patients with hematopoietic malignancies but has provided limited benefit in patients with solid tumors (Rosenberg and Restifo, Science (2015) 348:62-68). One major limitation is the poor in vivo expansion and persistence of adoptively transferred T cells, necessitating lymphodepleting conditioning chemotherapy, a toxic regimen that limits patient eligibility (Goff et al., J Clin Oncol. (2016) 34:2389-2397; Dudley et al., J Clin Oncol. (2008) 26:5233-5239; Dutcher et al., Journal for ImmunoTherapy of Cancer (2014) 2:26). Even expanded and persistent T cells end up terminally differentiated and functionally impaired (Philip et al., Nature (2017) 545: 452-456; Schietinger et al., Immunity (2016) 45: 389-401). Synthetic cytokine receptor signaling can reprogram T cells with a stem cell-like phenotype, overcoming these constraints and allowing them to exhibit superior antitumor activity in mouse models and humans (Gattinoni et al., Nature Reviews Cancer (2012) 12: 671-684; Krishna et al., Science (2020) 370: 1328-1334). Until now, therapeutic manipulations to select or expand stem cell-like T cells have been limited to the cell manufacturing stage and could not be performed in vivo.

[0335] Orthogonal cytokine receptors are variants of native cytokine receptors that selectively bind variants of the native cytokine. These orthogonal cytokine-receptor pairs are mutually exclusive with native cytokine-receptor pairs. Thus, when transduced into T cells and stimulated with their respective orthogonal cytokines, orthogonal cytokine receptors provide a means to selectively and biologically engineer adoptively transferred cells in vivo. This was recently demonstrated using an orthogonal mouse IL-2 cytokine-receptor pair (oIL2 and o2R) for in vivo conditioning of adoptively transferred T cells for cancer immunotherapy (Sockolosky et al., Science (2018) 359: 1037-1042), as IL-2R signaling potently stimulates T cell survival, expansion, and enhanced antitumor function.

[0336] The orthogonal murine IL-2 receptor (o2R) consists of an IL-2Rβ chain with a modified extracellular domain that selectively binds oIL-2 (clone 3A10) but not wild-type IL-2 (Figure 1A). Similarly, oIL-2 cannot bind to the wild-type IL-2 receptor. To transmit signals, both the orthogonal IL-2 receptor and the wild-type IL-2 receptor cooperate with wild-type γc. We wanted to use the modular nature of the oIL-2 system to explore the potential functional and therapeutic utility of other members of the γc cytokine receptor family, some of which confer favorable characteristics to antitumor T cells (Dwyer et al., Frontiers in Immunology (2019) 10:263; Leonard et al., Immunity (2019) 50:832-850).

[0337] To do so, we generated chimeric orthogonal receptors containing the mouse oIL-2-binding extracellular domain (ECD) fused to the intracellular domain (ICD) of each γc cytokine family receptor by replacing the intracellular domain (ICD) of o2R with the ICD of the receptors for the γc cytokines IL-4, IL-7, IL-9, and IL-21 (Figure 1A, SEQ ID NOs: 15, 19, 20, 21). Importantly, activated primary T cells (YFP+) retrovirally transduced to express the chimeric orthogonal receptors did not lose wild-type IL-2-induced STAT5 phosphorylation (Figure 1B, left panel).

[0338] Stimulation of chimeric orthogonal receptors with mouse serum albumin-bound oIL-2 (MSA-oIL2, clone 3A10) resulted in a phospho-STAT signaling pattern (Figure 1B) consistent with known wild-type signaling by the respective γc cytokines (Leonard, et al., Immunity, 2019, 50:832-850; Demoulin, et al., Mol Cell Biol., 1996, 16:4710-4716). Signaling by o9R resulted in robust phosphorylation of STAT1, STAT3 and STAT5, a profile that was distinct from signaling by o2R, o4R, o7R and o21R (Figure 1B, Figure 2) and consistent with known signaling by the wild-type IL-9 receptor (CD129) (Demoulin, et al., J Biol. Chem., 1999, 274:25855-25861). o9R signaling was dose-dependent and specific to MSA-oIL2 (Figure 1C).

[0339] The IL-9 receptor, IL9R (also known as CD129), is naturally expressed by mast cells, memory B cells, innate lymphoid cells, and hematopoietic progenitor cells, but is a less well-studied member of the γc cytokine receptor family (Demoulin, et al., Mol Cell Biol., 1996, 16:4710-4716; Knoops et al., Growth Factors (2004) 22:207-215; Bauer, et al., J Biol Chem., (1998) 273:9255-9260; Takatsuka et al., Nature Immunology (2018) 19:1025-1034; Townsend et al., Immunity (2000) 13:573-583; Williams et al., al., Blood(1990)76:906-911, Turner et al., J Exp Med(2013)210:2951-2965). Although IL-9-producing T cell subsets have been described (Lu et al., J Clin Invest (2012) 122: 4160-4171; Lu et al., Proc Natl Acad Sci USA (2014) 111: 2265; Purwar et al., Nat Med (2012) 18: 1248-1253; Liu et al., Nat Commun. (2020) 11: 5902; Schanz, et al., J Immunother Cancer (2021) 9), the effects of IL-9R signaling on T cells are less well characterized (Elyaman et al., Proc Natl Acad Sci USA (2009) 106: 12885-12890; Nowak et al., J Exp Med (2009) 206: 1653-1660; Li et al., J Immunother Cancer (2021) 9). al.,Eur J Immunol(2011)41:2197-2206, Houssiau et al.,J Immunol(1993)150:2634-2640, Louahed et al.,J Immunol(1995)154:5061-5070, Lehrnbecher et al. al., Cytokine (1994) 6:279-284).For example, it has been reported that naive T cells are insensitive to IL-9 and T cell development is not impaired in IL-9-deficient mice, suggesting that IL-9 is not a critical natural cytokine in T cell biology (Townsend et al., Immunity (2000) 13:573-583; Houssiau et al., J Immunol (1993) 150:2634-2640; de Heusch, et al., Eur J Immunol (2020) 50:1034-1043).

[0340] In this study, activated mouse T cells did not support IL-9 signaling (Figure 1C) due to the absence of IL-9R expression (Figure 3A-B), highlighting the unorthodoxy of o9R signaling in these cells (Nowak et al., J Exp Med (2009) 206:1653-1660; Druez et al., J Immunol (1990) 145:2494-2499; Cosmi et al., Blood (2004) 103:3117-3121). To verify whether o9R signaling is a true mimic of wild-type IL-9R signaling, we overexpressed wild-type IL-9R in both mouse T cells from C57BL / 6 mice and in mouse T cells from transgenic pmel mice (Figure 3A-B). Activation of wild-type IL-9R by IL-9 resulted in a STAT1, STAT3, and STAT5 phosphorylation profile similar to that observed in response to o9R signaling (Figures 3C-D).

[0341] Since o9R cells also express endogenous wild-type IL-2Rβ, we assessed the competition between native and orthogonal signaling. Co-exposure to saturating doses of MSA-oIL2 did not affect peak pSTAT5 phosphorylation by MSA-IL2 in o9R T cells (Figure 4A). However, increasing doses of MSA-IL2 partially alleviated MSA-oIL2 induction of pSTAT1 and pSTAT3 in o9R T cells (Figure 4B-C). Nevertheless, the unique signaling of o9R T cells remained active even at saturating doses of MSA-IL2.

[0342] We next investigated the effect of signaling through various orthogonal receptors on the memory phenotype of T cells. We observed that T cells signaling through o9R (as well as o21R) were enriched in the CD62L+ population and had high expression of Fas and Sca-1, a subset of T cells known to have superior antitumor activity in models of adoptive cell therapy (ACT) and in cancer patients. SCM phenotype (Fig. 1D-1F) (Krishna et al., Science (2020) 370, 1328-1334; Gattinoni et al., J Clin Invest (2005) 115: 1616-1626; Hinrichs et al., Proc Natl Acad Sci USA (2009) 106: 17469-17474; Klebanoff et al., Proc Natl Acad Sci USA (2005) 102: 9571-9576). o9R T cells also proliferated less and underwent fewer cell divisions than o2R-expressing T cells, even at MSA-oIL2 concentrations that saturated STAT5 phosphorylation (Fig. 1G and Fig. 5A-B). The limited proliferation was orthogonal receptor specific, since o9R and o2R T cells responded equally to wild-type IL-2. Among the orthogonal receptors, only o21R (which also signals through STAT3) conferred less proliferation than o9R ( Fig. 5A ).

[0343] Unique STAT signaling, a little-known aspect of the γc cytokine receptor family, and T SCM Considering the characteristics, the impact of o9R signaling in vivo was studied using a mouse model of ACT against solid tumors. First, we used an ACT model that utilizes T cells from transgenic pmel mice expressing an endogenous TCR specific for gp100, a melanocyte antigen overexpressed in B16 melanoma (Overwijk et al., J Exp Med (2003) 198: 569-580). Given the benefits and efficacy of o2R in this model (Sockolosky et al., Science (2018) 359: 1037-1042), we modified the protocol to be more stringent and to omit lymphodepleting radiation therapy, a conditioning regimen that enhances ACT by inducing homeostatic proliferation of adoptively transferred T cells by γc cytokine signaling (Surh et al., Immunity (2008) 29: 848-862). B16 tumor-bearing C57BL / 6 mice were treated intravenously with pmel T cells retrovirally transduced with either o2R or o9R, and then treated with either MSA-IL2 or MSA-oIL2 (Figure 7A). Similar to the profile of o2R and o9R T cells from C57BL / 6 mice, MSA-oIL2 activated phosphorylation of STAT1, STAT3, and STAT5 in o9R pmel T cells, but only phosphorylated STAT5 in o2R pmel T cells (Figure 6A). o9R pmel T cells also proliferated less than o2R pmel T cells (Figure 6B).

[0344] In the absence of lymphodepletion, higher frequencies of o2R pmel and o9R pmel T cells were observed in peripheral blood 7 days after ACT in mice treated with oIL-2 compared to mice treated with MSA-IL2 (Figure 7B). Although both o2R pmel and o9R pmel T cells expanded without lymphodepletion, more consistent antitumor efficacy and extended survival were observed with o9R pmel T cells treated with MSA-oIL2 in this model (Figures 7C, 7D, 8A, 8B), achieving antitumor efficacy comparable to pmel T cells in lymphodepleted mice. Superior antitumor efficacy of o9R pmel T cells was observed in lymphodepleted mice treated with MSA-oIL2, despite their weaker proliferation signal (Figure 8C).

[0345] These results suggested that factors other than selective expansion underlie the enhanced antitumor effect of o9R pmel T cells. Stronger tumor infiltration by o9R pmel T cells compared with o2R pmel T cells was observed 5 days after ACT in mice treated with MSA-oIL2 (Figure 7E). 7 days after ACT with o2R pmel and o9R pmel T cells and MSA-oIL2, 18 tumor-infiltrating CD45+ leukocyte population clusters were identified using CyTOF. Among these, a striking feature based on differential abundance analysis was the presence of a cluster corresponding to pmel T cells, which was significantly enriched in o9R-treated mice (8.9% vs. 1.6% of tumor-infiltrating CD45+ cells; Figure 9A). Among tumor-infiltrating pmel T cells, o9R pmel T cells were enriched for clusters associated with T cell activation, including clusters co-expressing CD39, PD-1, and TBET, whereas o2R pmel T cells were enriched for clusters expressing CD73 but lacking PD-1 and CD39 expression (Figure 9B).

[0346] The superior tumor infiltration of o9R pmel T cells was supported by the infiltration of CD3+CD8+ and CD8+PD1+ T cells by multiplex immunohistochemistry (Figure 9C). In addition to improved tumor infiltration, o9R pmel T cells exhibited superior cytolytic capacity compared to their o2R counterparts (Figure 7F) and showed greater IFNγ production in response to tumor cells in vitro (Figure 9D).

[0347] We next investigated the biological program responsible for the improved invasion, effector function and in vivo potency of o9R pmel T cells. In vitro exposure to MSA-oIL2 (48 h) resulted in a thoroughly divergent phenotype by high-dimensional mass cytometry, with 8 of 14 clusters differing in abundance between o2R pmel and o9R pmel T cells (Figure 7G). Consistent with the data in Figures 1D, 1E and 1F using T cells from C57BL / 6 mice, o9R pmel T cells were characterized as stem cell memory (T SCM o2R pmel T cells acquired markers of the IL-9R phenotype (Sca-1, CD127, Fas, and CD62L) (Figure 7G, Figure S10), whereas o2R pmel T cells acquired markers of terminally differentiated effectors (CD44, EOMES, and Tbet) (Figure 7G). These effects of o9R signaling were consistent with those of wild-type IL-9R signaling. SCM This phenotype was also observed in pmel T cells transduced with wild-type IL-9R and exposed to wild-type IL-9 (Figure 10).

[0348] Compared to o2R pmel T cells, bulk populations of o9R pmel T cells exposed to MSA-oIL2 undergo striking global bidirectional transcriptome changes identified by RNA-seq of sorted pmel T cells 48 hours after exposure to MSA-oIL2 or MSA-IL2 in vitro (Figure 7H, Figure S11). These changes induced by o9R signaling are consistent with the enrichment of genes including Tcf7, Ccr7, Cd127, Sell, and Bach2. SCMThis further supports the acquisition of a phenotype (Figure 7I). However, genes classically associated with effector function (Ifng, Gzma, Prf1) and T cell activation (Pdcd1, Icos, Entpd1, Lag3, Havcr2) have traditionally been implicated in T SCM Co-enrichment of genes excluded from the phenotype was also observed (Figure 7I). This may represent the co-existence of novel hybrid phenotypes or heterogeneous subpopulations unique to o9R signaling in T cells. Induction of granzyme A by IL-9R activation has been reported to depend on the co-activation of STAT1 and STAT3, a hallmark of o9R signaling that is distinct from o2R, o4R, o7R and o21R signaling (Demoulin, et al., J Biol. Chem., 1999, 274:25855-25861).

[0349] Transcriptome analysis of transcription factor pathway enrichment revealed significant (p<0.05) enrichment of genes associated with the AP-1 transcription factor JUN, as well as the expected transcription factors STAT1 and STAT3 (Figure 7J). This was accompanied by an increased ratio of Jun to Fos expression, a hallmark of tumor-specific T cells that are resistant to tumor-induced exhaustion (Lynn et al., Nature (2019) 576: 293-300). In parallel, o9R signaling downregulated genes associated with T cell exhaustion or dysfunction. These genes include Nr4a1 and Tox, both of which mediate the dysfunction of antitumor T cells (Figure 7I) (Chen et al., Nature (2019) 567: 530-534; Seo et al., Proc Natl Acad Sci USA (2019) 116: 12410-12415; Khan et al., Nature (2019) 571: 211-218; Scott et al., Nature (2019) 571: 270-274).

[0350] To examine the in vivo activity of the o9R signaling program, CD62L expression on adoptively transferred T cells was examined. CD62L expression was higher on adoptively transferred o9R pmel T cells compared to o2R pmel T cells in the draining lymph nodes and spleens of tumor-bearing mice treated with MSA-oIL2 at 1 and 5 days after ACT (Figure 12). No difference in CD62L expression was observed in tumors where antigen-specific T cell activation would likely predominate.

[0351] To further investigate o9R signaling in the context of CAR-based ACT, we used an immunotherapy-resistant model of pancreatic cancer expressing mesothelin. To ensure high intratumoral concentrations of oIL-2 (clone 3A10) in the tumor and to evaluate the impact of o9R signaling on T cell dysfunction in the tumor microenvironment, we chose vectored delivery as the oIL-2 source for in vivo CAR T cell studies (Figure 13A). This model contrasted with pmel studies, where the effects of the o9R signaling program were mostly observed in peripheral tissues. oIL-2 production from an adenoviral vector (Ad-oIL2) was assessed in vitro in PDA7940b cells (Figure 13A, bottom left) and in vivo by intratumoral injection (Figure 13A, bottom right), confirming tumor-restricted oIL-2 expression and no detectable cytokine in peripheral blood.

[0352] We generated CAR-o2R and CAR-o9R T cells by transducing primary mouse T cells with a retrovirus encoding a second-generation anti-mouse mesothelin CAR together with the orthogonal receptor (Figure S14A-B). Stimulation of CAR-o9R T cells with MSA-oIL2 resulted in phosphorylation of STAT1 and STAT3, and to a lesser extent, STAT5, which was distinct from the STAT5 phosphorylation profile of CAR-o2R T cells treated with MSA-oIL2 (Figure S13B). Unlike CAR-o2R cells, CAR-o9R cells did not expand in response to oIL-2 (Figure S15A).

[0353] Despite the growth advantage of CAR-o2R cells, CAR-o9R cells were not able to express KPC (LSL-Kras G12D / + ;LSL-Trp53 R172H / + The results showed superior antitumor efficacy in vitro for the elimination of the mesothelin-positive pancreatic ductal adenocarcinoma (PDA) cell line PDA7940b, which was derived from spontaneous tumors arising in ( ;Pdx-1-Cre) mice (Figure 13C).

[0354] Similar to the pmel model, CAR-o9R cells were SCM Phenotype (CD44 lo CD62L hi Fas + ), whereas CAR-o2R cells retained the central memory / effector phenotype (CD44 hi CD62L hi The phenotype shifted to T Fas- (Fig. 13D to E and Fig. 15B). SCM Although CAR-o9R T cells appear to be resistant to oIL-2, they secrete higher levels of IFNγ, TNFα, IL-2, IL-9, IL-10, IL-18, IL-22, and IL-23 than CAR-o2R cells in response to oIL-2 (Figure S13F). This diverse cytokine profile mirrors the complex transcriptome signature of o9R pmel T cells, which was confirmed in CAR-o9R T cells using the nCounter mouse immunology panel (Figure S13G). In CAR-o9R cells, genes associated with effector function (Ifng, Prf1, Gzmb, Gzma) and T cell activation (Pdcd1, Icos, Entpd1) were upregulated, along with a shift towards T cell stemness (upregulation of Il7r, Sell, and Bcl6 and downregulation of Eomes, Tox1).

[0355] The syngeneic in vivo model used to evaluate the antitumor properties of CAR-o2R and CAR-o9R cells utilizes two immunocompetent mouse strains with different CD45 alleles (CD45.1 and CD45.2) to allow for differentiation of adoptively transferred (donor) T cells from endogenous (host) T cells (Figure 13H). In this model, treatment of mice bearing established subcutaneous PD7940b tumors with either Ad-oIL2 or CAR-o2R / CAR-o9R alone was ineffective in suppressing the rapidly growing tumors (Figure 13I). This experimental monotherapy showed no improvement over mock treatment (PBS injection), demonstrating the refractoriness of this tumor model.

[0356] Nonetheless, combined treatment with Ad-oIL2+CAR-o2R or Ad-oIL2+CAR-o9R resulted in robust antitumor responses that eliminated tumors in 8 of 12 mice (67% CR) and 6 of 12 mice (50% CR), respectively (Figure 13J). Early toxicity was observed in the Ad-oIL2+CAR-o9R group, as 40% of mice (5 of 12) died by day 10, but notably, fewer tumor recurrences were seen in surviving animals in the Ad-oIL2+CAR-o9R group (1 of 7 mice), compared with 4 of 12 mice in the Ad-oIL2+CAR-o2R group that succumbed to recurrence.

[0357] Toxicity of Ad-oIL2+CAR-o9R in lymphodepleted mice was characterized by clinical signs of immune effector cell-associated neurotoxicity syndrome (ICANS) (tremors, delirium, seizures). RNA in situ hybridization (RNA ISH) analysis of mice with ICANS revealed CAR T cell infiltration in the meningeal layer of the brain, where mesothelin-expressing meningeal cells were also detected (Figure 16A-B). Significantly more meningeal-infiltrating CAR T cells were observed in the Ad-oIL2+CAR-o9R group compared to the Ad-oIL2+CAR-o2R group, suggesting an association between CAR-driven on-target off-tumor activity and the observed ICANS (Figure 16C). Serum analysis did not show evidence of cytokine release syndrome (CRS) or tumor lysis syndrome (TLS) (Figure 16D-E). Based on these findings and the lack of similar toxicity in the pmel model, the observed ICANS appear to be specific to the specificity of the CAR and not inherent to o9R engineered T cells. Nonetheless, translational studies of o9R signaling in CAR T cells must consider the addition of potential on-target off-tumor toxicities, and additional engineering strategies (e.g., on / off systems, synthetic circuits) may be required to maximize patient safety.

[0358] The superiority of Ad-oIL2(3A10)+CAR-o9R over Ad-oIL2(3A10)+CAR-o2R was more evident in the absence of cyclophosphamide preconditioning, as suggested by the complete response rate of 42% (5 of 12) and 8.3% (1 of 12), respectively, and the prolonged survival time (Figure 17A), since only 1 of 12 mice with Ad-oIL2+CAR-o9R showed toxicity without preconditioning (Figure 13K). Efficacy was dependent on orthogonal receptor expression on T cells and oIL-2 expression by Ad-oIL2, since CAR T cells had no antitumor effect without o2R in combination with Ad-oIL2 (Figure 17B), and the control virus Ad-Null was unable to enhance CAR-o2R treatment (Figure 17C). When rechallenged with PDA7940b, tumor growth was significantly suppressed in animals previously cured by the combination treatment compared to naive animals (Figure 13L). MSLN- ) were created (FIG. 21) and then implanted into previously cured mice (FIG. 22). At day 24, these tumors were smaller in cured mice compared to naive mice, indicating induction of tumor antigen (=epitope) spreading. CD45.1+ donor T cells were detected in the peripheral blood of animals 24 days after rechallenge and 94 days after the first injection, suggesting that in vivo oIL-2 stimulation allows long-term persistence of CAR-o2R / CAR-o9R T cells in immunocompetent mice.

[0359] We next examined the effect of Ad-oIL2 on the intratumoral quantity and quality of tumor-infiltrating CAR-o9R or CAR-o2R T cells. In contrast to the pmel model, fewer CAR-o9R T cells were observed in the tumor 8 days after ACT than CAR-o2R T cells (Figure 13M). The contrasting tumor infiltration results were not unexpected, considering that the o9R signaling program is not active in the periphery in the Ad-oIL2 model. Among tumor-infiltrating CAR T cells, CAR-o9R cells expressed IFNγ more frequently (Figure 13M), suggesting that superior intratumoral function, rather than tumor infiltration, drives the antitumor efficacy of CAR-o9R T cells in this model.

[0360] To evaluate the translational potential of o9R signaling, we generated human orthogonal IL-2Rβ (ho2R) and human orthogonal chimeric IL-2Rβ / IL-9R (ho9R) (SEQ ID NOs: 4 and 6). Human T cells were retrovirally transduced with vectors encoding either ho2R or ho9R (each containing YFP) and a T cell receptor specific for NY-ESO-1 on HLA*0201 (NYESO1-TCR clone 1G4) (Robbins, et al., J Immunol, 2008, 180:6116-6131). NY-ESO-1 is a cancer-testis antigen that is overexpressed in synovial sarcoma, myxoid liposarcoma, melanoma, and other tumors. Transduced T cells sorted on the basis of NYESO1-TCR and YFP expression (Figure 19A) were exposed to human orthogonal IL-2 bound to MSA (MSA-hoIL2) or wild-type IL-2 (MSA-hIL2). Consistent with observations in the mouse system, human T cell signaling through ho9R activates pSTAT1, pSTAT3, and pSTAT5 signaling, whereas human T cell signaling through ho2R activates primarily pSTAT5 signaling (Figure 18A). Human T cells expressing ho2R or ho9R equally phosphorylated STAT5 after exposure to MSA-hIL2 (Figure 18A, far right panel), indicating that native IL-2-STAT5 signaling through endogenous γc is not differentially affected by orthogonal receptor expression.

[0361] Also similar to the mouse system, ho9R signaling provided a weaker proliferation signal than ho2R or wild-type IL-2 signaling (Figure 18B). Compared with ho2R / NYESO1-TCR T cells, ho9R / NYESO1-TCR T cells were significantly more potent in T cells after 2 and 6 days in culture with MSA-hoIL2. SCM The population of ho9R / NYESO1-TCR T cells (CD45RA+CD27+CCR7+CD95+) was enriched for in vivo expression (Figures 19B-19C and 18C). Furthermore, despite the reduced proliferation of ho9R / NYESO1-TCR T cells between days 2 and 6, the absolute amount of T cells (not just enrichment) was significantly increased.SCM Cells and Central Memory (T CM Expansion of CD45RA-CD27+CCR7+CD95+) cells was observed.

[0362] The difference in T cell phenotype between ho9R / NYESO1-TCR and ho2R / NYESO1-TCR T cells persisted even after four rounds of antigen-specific challenge with the HLA*0201+NY-ESO-1+ human melanoma tumor cell line nRFP-M407 in the presence of MSA-hoIL2 (Figures S18E-S18F). Compared with ho2R / NYESO1 T cells, ho9R / NYESO1 T cells exhibited a higher frequency of CD45RA+CD27+ and T SCM Repeatedly stimulated ho9R / NYESO1 T cells expressed more IFNγ, TNFα, and IL-2 and exhibited greater polyfunctionality when exposed to either activating CD3 / CD28 beads or the cognate melanoma cell line (nRFP-M407) (Figures 18G and 19D). These findings were antigen-specific, as they were not observed in response to the non-cognate melanoma cell line M263 (HLA*0201-NY-ESO-1-).

[0363] Furthermore, human orthoIL-2 was cloned into the Ad5 / 3-D24 oncolytic adenovirus vector and shown to be overexpressed in vitro. A549 cells were infected with Ad5 / 3-D24-hoIL2 or the isogenic control virus Ad5 / 3-D24, or mock infected. 50 μl of cell culture supernatant was collected 96 hours after infection and analyzed by human IL-2 ELISA (FIG. 23A). Fiber-chimeric oncolytic adenovirus Ad5 / 3-D24-hoIL2 encoding human orthoIL-2 was then purified by cesium chloride (CsCl2) gradient (FIG. 23B). 48 hours after infection, virus-infected A549 cells were harvested by scraping from 11 confluent T150 cell culture flasks, followed by three freeze-thaw cycles to release viral particles. The supernatant was clarified by two rounds of ultracentrifugation on a cesium chloride (CsCl) gradient. The red arrow indicates the band containing intact virus particles, which was collected with a needle and syringe by piercing the side of the tube. CsCl was removed by two buffer exchanges in a 15 ml ultrafiltration unit.

[0364] We next demonstrated that human orthogonal IL-2Rβ (ho2R) and human orthogonal chimeric IL-2Rβ / IL-9R (ho9R) are co-expressed with PSMA-retargeted CAR in human T cells. 1e5 primary human T cells (1:1 CD4 / CD8 ratio) were infected with lentiviral vectors encoding PSMA28z CAR and either the human orthogonal receptor, full-length orthoIL2Rb receptor (Figure 24A) or the chimeric orthoIL2Rb / IL9Ra switch receptor (Figure 24B) at varying dilutions of vector. Co-expression was assessed and demonstrated by flow cytometry 48 hours after infection.

[0365] In conclusion, the orthogonal IL-2 cytokine-receptor platform allows for signaling redirection by modularly replacing the orthogonal IL-2Rβ ICD with the ICDs of receptors for other γc cytokines. Here, we unexpectedly showed that specific in vivo stimulation of o9R signaling in TCR- and CAR-based tumor-specific T cells with the orthogonal IL-2 ligand 3A10 led to improved antitumor activity in two immunotherapy-resistant solid tumor models and retained robust activity under more stringent conditions without lymphodepletion. This benefit was mediated by co-opting γc to reroute IL-2 signaling messages through the IL-9Rα ICD, resulting in the simultaneous activation of STAT1, STAT3, and STAT5. The IL-9Rα signaling messages delivered in T cells conferred improved in vivo antitumor activity by resulting in a unique phenotype that combines beneficial functional features of stem cell memory and effector T cells.

[0366] List of Aspects The following embodiments are listed, the numbering of which should not be construed as designating any level of importance. Aspect 1 provides: 1. A system for selective activation of a receptor in a cell, comprising: (a) Below: (i) an orthogonal chimeric cytokine receptor, and (ii) at least one chimeric antigen receptor (CAR); and a modified immune cell comprising: (b) an oncolytic adenoviral vector comprising a nucleic acid sequence encoding an orthogonal IL2 cytokine; Including, The system, wherein the orthogonal chimeric cytokine receptor comprises the extracellular domain of an orthogonal IL2 receptor (oIL2R) and the intracellular signaling domain of a cytokine receptor that is not an IL2R. Aspect 2 provides: 2. The system of embodiment 1, wherein said extracellular domain of oIL2R is the extracellular domain of the orthogonal IL2 receptor beta (oIL2Rb). Aspect 3 provides: 3. The system of embodiment 1 or 2, wherein the intracellular signaling domain of the orthogonal chimeric cytokine receptor comprises an IL9R intracellular signaling domain. Aspect 4 provides the following: The system according to any one of aspects 1 to 3, wherein the IL9R intracellular signaling domain is an IL9R-alpha (IL9Ra) intracellular signaling domain. Aspect 5 provides the following:

[0023] 2. The system of any preceding embodiment, wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain. Aspect 6 provides the following: 23. The system of any one of the preceding aspects, wherein the antigen binding domain is selected from the group consisting of a full length antibody or antigen binding fragment thereof, a Fab, a single chain variable fragment (scFv), or a single domain antibody. Aspect 7 provides the following: 23. The system of any one of the preceding aspects, wherein the antigen binding domain targets a tumor antigen. Aspect 8 provides the following: 4. The system of any one of the preceding aspects, wherein the tumor antigen is selected from the group consisting of CD19, CD20, HER2, NY-ESO-1, MUC1, CD123, FLT3, B7-H3, CD33, IL1RAP, CLL1 (CLEC12A) PSA, CEA, VEGF, VEGF-R2, CD22, ROR1, mesothelin, c-Met, glycolipid F77, FAP, EGFRvIII, MAGE A3, 5T4, WT1, KG2D ligand, folate receptor (FRa), and Wnt1 antigen. Aspect 9 provides the following: 2. The system of any one of the preceding aspects, wherein the antigen binding domain is an scFv. Aspect 10 provides the following: 2. The system of any one of the preceding aspects, wherein the antigen binding domain is an anti-mesothelin scFv. Aspect 11 provides the following:

[0023] 3. The system of any one of the preceding embodiments, wherein the intracellular domain of said CAR comprises a costimulatory domain of a protein selected from the group consisting of the TNFR superfamily of proteins, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or a variant thereof, or an intracellular domain derived from a killer immunoglobulin-like receptor (KIR). Aspect 12 provides the following:

[0023] The system of any one of the preceding embodiments, wherein the intracellular domain of said CAR comprises an intracellular signaling domain of a protein selected from the group consisting of human CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor bearing an immunoreceptor tyrosine-based activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof. Aspect 13 provides the following: (a) the orthogonal chimeric cytokine receptor comprises the extracellular domain of oIL2Rb and the intracellular signaling domain of IL9Ra; (b) the CAR comprises an anti-mesothelin antigen-binding domain. 2. The system of any one of the preceding aspects. Aspect 14 provides the following: 1. A method of treating cancer in a subject, comprising: (a) to the subject; (i) an orthogonal chimeric cytokine receptor, and (ii) at least one CAR administering an effective amount of modified immune cells or precursors thereof (a population of modified immune cells); (b) administering to the subject an oncolytic adenoviral vector comprising a nucleic acid sequence encoding an orthogonal IL2 cytokine. Including, The method, wherein the orthogonal chimeric cytokine receptor comprises the extracellular domain of an orthogonal IL2 receptor (oIL2R) and the intracellular signaling domain of a cytokine receptor that is not an IL2R. Aspect 15 provides the following: The method of embodiment 14, wherein said extracellular domain of oIL2R is the extracellular domain of the orthogonal IL2 receptor beta (oIL2Rb). Aspect 16 provides the following: 16. The method of embodiment 14 or 15, wherein the intracellular signaling domain of the orthogonal chimeric cytokine receptor comprises an IL9R intracellular signaling domain. Aspect 17 provides the following: 4. The method of any one of the preceding aspects, wherein said IL9R intracellular signaling domain is an IL9R-alpha (IL9Ra) intracellular signaling domain. Aspect 18 provides the following:

[0023] 2. The method of any one of the preceding embodiments, wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain. Aspect 19 provides the following: 4. The method of any one of the preceding aspects, wherein the antigen-binding domain is selected from the group consisting of a full length antibody or antigen-binding fragment thereof, a Fab, a single chain variable fragment (scFv), or a single domain antibody. Aspect 20 provides the following: 23. The method of any one of the preceding aspects, wherein the antigen binding domain targets a tumor antigen. Aspect 21 provides the following: 4. The method of any one of the preceding aspects, wherein said tumor antigen is selected from the group consisting of CD19, CD20, HER2, NY-ESO-1, MUC1, CD123, FLT3, B7-H3, CD33, IL1RAP, CLL1 (CLEC12A) PSA, CEA, VEGF, VEGF-R2, CD22, ROR1, mesothelin, c-Met, glycolipid F77, FAP, EGFRvIII, MAGE A3, 5T4, WT1, KG2D ligand, folate receptor (FRa), and Wnt1 antigen. Aspect 22 provides the following: 2. The method of any one of the preceding aspects, wherein the antigen binding domain is an scFv. Aspect 23 provides the following: 2. The method of any one of the preceding aspects, wherein the antigen binding domain is an anti-mesothelin scFv. Aspect 24 provides the following:

[0023] 3. The method of any one of the preceding embodiments, wherein the intracellular domain of said CAR comprises a costimulatory domain of a protein selected from the group consisting of the TNFR superfamily, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or a variant thereof, or an intracellular domain from a killer immunoglobulin-like receptor (KIR). Aspect 25 provides the following:

[0023] The method of any one of the preceding embodiments, wherein the intracellular domain of said CAR comprises an intracellular signaling domain of a protein selected from the group consisting of human CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor bearing an immunoreceptor tyrosine-based activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof. Aspect 26 provides the following: (a) the orthogonal chimeric cytokine receptor comprises the extracellular domain of oIL2Rb and the intracellular signaling domain of IL9Ra; (b) the CAR comprises an anti-mesothelin antigen-binding domain. 2. The method of any one of the preceding aspects. Aspect 27 provides the following: The method of any one of the preceding embodiments, wherein said population of modified immune cells exhibit stem cell memory (Tscm) characteristics with improved trafficking and effector function, thereby treating cancer. Aspect 28 provides the following: 23. The method of any one of the preceding aspects, wherein said step of administering the vector comprises intratumoral injection. Aspect 29 provides the following: 4. The method of any one of the preceding aspects, wherein said cancer is selected from the group consisting of pancreatic cancer and melanoma. Aspect 30 provides the following: The method of embodiment 29, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma. Aspect 31 provides the following: 1. A system for selective activation of a receptor in a cell, comprising: (a) Below: (i) an orthogonal chimeric cytokine receptor, and (ii) at least one T cell receptor (TCR); and a modified immune cell engineered to express (b) an oncolytic adenoviral vector comprising a nucleic acid sequence encoding an orthogonal IL2 cytokine; Including, The system, wherein the orthogonal chimeric cytokine receptor comprises the extracellular domain of an orthogonal IL2 receptor (oIL2R) and the intracellular signaling domain of a cytokine receptor that is not an IL2R. Aspect 32 provides the following: The system of embodiment 31, wherein said extracellular domain of oIL2R is the extracellular domain of the orthogonal IL2 receptor beta (oIL2Rb). Aspect 33 provides the following: 33. The system of embodiment 31 or 32, wherein the intracellular signaling domain of the orthogonal chimeric cytokine receptor comprises an IL9R intracellular signaling domain. Aspect 34 provides the following: 34. The system according to any one of aspects 31 to 33, wherein the IL9R intracellular signaling domain is an IL9R-alpha (IL9Ra) intracellular signaling domain. Embodiment 35 provides the following: The system of any one of aspects 31 to 34, wherein the TCR targets a tumor antigen. Aspect 36 provides the following: The system of any one of aspects 31-35, wherein the TCR targets gp100 melanoma antigen or NYESO1. Aspect 37 provides the following: The system according to any one of aspects 31 to 36, wherein the TCR is a pmel-1 TCR or a NYESO1-specific TCR. Embodiment 38 provides the following: (a) the orthogonal chimeric cytokine receptor comprises the extracellular domain of oIL2Rb and the intracellular signaling domain of IL9Ra; (b) the TCR is a pmel-1 TCR or a NYESO1-specific TCR; A system according to any one of aspects 31 to 37. Aspect 39 provides the following: 1. A method of treating cancer in a subject, comprising: (a) to the subject; (i) an orthogonal chimeric cytokine receptor, and (ii) at least one T cell receptor (TCR); administering an effective amount of a modified immune cell or a precursor thereof (a population of modified immune cells) modified to express (b) administering to the subject an oncolytic adenoviral vector comprising a nucleic acid sequence encoding an orthogonal IL2 cytokine. Including, The method, wherein the orthogonal chimeric cytokine receptor comprises the extracellular domain of an orthogonal IL2 receptor (oIL2R) and the intracellular signaling domain of a cytokine receptor that is not an IL2R. Aspect 40 provides the following: The method of embodiment 39, wherein said extracellular domain of oIL2R is the extracellular domain of the orthogonal IL2 receptor beta (oIL2Rb). Aspect 41 provides the following: 41. The method of embodiment 39 or 40, wherein the intracellular signaling domain of said orthogonal chimeric cytokine receptor comprises an IL9R intracellular signaling domain. Aspect 42 provides the following: 42. The method of any one of aspects 39 to 41, wherein the IL9R intracellular signaling domain is an IL9R-alpha (IL9Ra) intracellular signaling domain. Aspect 43 provides the following: 43. The method of any one of aspects 39 to 42, wherein the TCR targets a tumor antigen. Aspect 44 provides the following: The method of any one of aspects 39 to 43, wherein the TCR targets gp100 melanoma antigen or NYESO1. Aspect 45 provides the following: The method of any one of aspects 39 to 44, wherein the TCR is a pmel-1 TCR or a NYESO1-specific TCR. Aspect 46 provides the following: (a) the orthogonal chimeric cytokine receptor comprises the extracellular domain of oIL2Rb and the intracellular signaling domain of IL9Ra; (b) the TCR is a pmel-1 TCR or a NYESO1-specific TCR; The method of any one of aspects 39 to 45. Aspect 47 provides the following: The method of any one of embodiments 39-46, wherein said population of modified immune cells exhibits stem cell memory (Tscm) characteristics with improved trafficking and effector function, thereby treating cancer. Aspect 48 provides the following: 48. The method of any one of aspects 39-47, wherein said step of administering the vector comprises intratumoral injection. Aspect 49 provides the following: The method of any one of aspects 39 to 48, wherein said cancer is selected from the group consisting of pancreatic cancer and melanoma. Embodiment 50 provides the following: The method of embodiment 49, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma.

[0367] Other Aspects The disclosures of all patents, patent applications and publications mentioned herein are hereby incorporated by reference in their entirety. Although the present invention has been disclosed with reference to specific embodiments, it is apparent that others skilled in the art may devise other embodiments and modifications of the present invention without departing from the true spirit and scope of the present invention. It is intended that the appended claims be construed to include all such embodiments and equivalent modifications.

Claims

1. 1. A system for selective activation of a receptor in a cell, comprising: (a) Below: (i) an orthogonal chimeric cytokine receptor, and (ii) at least one chimeric antigen receptor (CAR); and a modified immune cell comprising: (b) an oncolytic adenoviral vector comprising a nucleic acid sequence encoding an orthogonal IL2 cytokine; Including, the orthogonal chimeric cytokine receptor comprises the extracellular domain of an orthogonal IL2 receptor (oIL2R) and an intracellular signaling domain of a cytokine receptor that is not an IL2R; and wherein the CAR further comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain, and optionally the antigen-binding domain targets a tumor antigen. system.

2. The system of claim 1, wherein the extracellular domain of oIL2R is the extracellular domain of orthogonal IL2 receptor beta (oIL2Rb).

3. The system of claim 1 or 2, wherein the intracellular signaling domain of the orthogonal chimeric cytokine receptor comprises an IL9R intracellular signaling domain.

4. The system of claim 3, wherein the IL9R intracellular signaling domain is an IL9R-alpha (IL9Ra) intracellular signaling domain.

5. The system of any one of claims 1 to 4, wherein the antigen-binding domain is selected from the group consisting of a full-length antibody or an antigen-binding fragment thereof, a Fab, a single-chain variable fragment (scFv), or a single domain antibody.

6. The system of any one of claims 1 to 5, wherein the antigen binding domain targets a tumor antigen.

7. The system of any one of claims 1 to 6, wherein the tumor antigen is selected from the group consisting of CD19, CD20, HER2, NY-ESO-1, MUC1, CD123, FLT3, B7-H3, CD33, IL1RAP, CLL1 (CLEC12A) PSA, CEA, VEGF, VEGF-R2, CD22, ROR1, mesothelin, c-Met, glycolipid F77, FAP, EGFRvIII, MAGE A3, 5T4, WT1, KG2D ligand, folate receptor (FRa), and Wnt1 antigen.

8. The system according to any one of claims 1 to 7, wherein the antigen-binding domain is an scFv.

9. The system according to any one of claims 1 to 8, wherein the antigen-binding domain is an anti-mesothelin scFv.

10. The system of any one of claims 1 to 9, wherein the intracellular domain of the CAR comprises a costimulatory domain of a protein selected from the group consisting of proteins of the TNFR superfamily, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or a mutant thereof, or an intracellular domain from a killer immunoglobulin-like receptor (KIR).

11. The system of any one of claims 1-10, wherein the intracellular domain of the CAR comprises an intracellular signaling domain of a protein selected from the group consisting of human CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor bearing an immunoreceptor tyrosine-based activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof.

12. (a) the orthogonal chimeric cytokine receptor comprises the extracellular domain of oIL2Rb and the intracellular signaling domain of IL9Ra; (b) the CAR comprises an anti-mesothelin antigen-binding domain. A system according to any one of claims 1 to 11.

13. A pharmaceutical composition for treating cancer in a subject in need thereof, comprising: The pharmaceutical composition comprises: (i) an orthogonal chimeric cytokine receptor, and (ii) At least one CAR an effective amount of modified immune cells or precursor cells thereof (a population of modified immune cells), comprising the pharmaceutical composition is used in combination with an oncolytic adenoviral vector comprising a nucleic acid sequence encoding an orthogonal IL2 cytokine; the orthogonal chimeric cytokine receptor comprises the extracellular domain of an orthogonal IL2 receptor (oIL2R) and the intracellular signaling domain of a cytokine receptor that is not an IL2R; and the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain, and optionally the antigen-binding domain targets a tumor antigen; Pharmaceutical compositions.

14. The pharmaceutical composition of claim 13, wherein the extracellular domain of oIL2R is the extracellular domain of the orthogonal IL2 receptor beta (oIL2Rb).

15. The pharmaceutical composition of claim 13 or 14, wherein the intracellular signaling domain of the orthogonal chimeric cytokine receptor comprises an IL9R intracellular signaling domain.

16. 16. The pharmaceutical composition of claim 15, wherein the IL9R intracellular signaling domain is an IL9R-alpha (IL9Ra) intracellular signaling domain.

17. 17. The pharmaceutical composition of any one of claims 13 to 16, wherein the antigen-binding domain is selected from the group consisting of a full-length antibody or an antigen-binding fragment thereof, a Fab, a single-chain variable fragment (scFv), or a single domain antibody.

18. The pharmaceutical composition of any one of claims 13 to 17, wherein the antigen binding domain targets a tumor antigen.

19. 19. The pharmaceutical composition of any one of claims 13 to 18, wherein the tumor antigen is selected from the group consisting of CD19, CD20, HER2, NY-ESO-1, MUC1, CD123, FLT3, B7-H3, CD33, IL1RAP, CLL1 (CLEC12A) PSA, CEA, VEGF, VEGF-R2, CD22, ROR1, mesothelin, c-Met, glycolipid F77, FAP, EGFRvIII, MAGE A3, 5T4, WT1, KG2D ligand, folate receptor (FRa), and Wnt1 antigens.

20. The pharmaceutical composition of any one of claims 13 to 19, wherein the antigen-binding domain is an scFv.

21. The pharmaceutical composition of any one of claims 13 to 20, wherein the antigen-binding domain is an anti-mesothelin scFv.

22. 22. The pharmaceutical composition of any one of claims 13-21, wherein the intracellular domain of the CAR comprises a costimulatory domain of a protein selected from the group consisting of the TNFR superfamily, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or a variant thereof, or an intracellular domain from a killer immunoglobulin-like receptor (KIR).

23. 23. The pharmaceutical composition of any one of claims 13-22, wherein the intracellular domain of said CAR comprises an intracellular signaling domain of a protein selected from the group consisting of human CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor bearing an immunoreceptor tyrosine-based activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof.

24. (a) the orthogonal chimeric cytokine receptor comprises the extracellular domain of oIL2Rb and the intracellular signaling domain of IL9Ra; (b) the CAR comprises an anti-mesothelin antigen-binding domain.

24. The pharmaceutical composition of any one of claims 13 to 23.

25. The pharmaceutical composition of any one of claims 13 to 24, wherein the population of modified immune cells exhibits stem cell memory (Tscm) characteristics with improved trafficking and effector functions, thereby treating cancer.

26. A pharmaceutical composition described in any one of claims 13 to 25, wherein the oncolytic adenoviral vector is administered by intratumoral injection.

27. The pharmaceutical composition of any one of claims 13 to 26, wherein the cancer is selected from the group consisting of pancreatic cancer and melanoma.

28. 28. The pharmaceutical composition of claim 27, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma.

29. 1. A system for selective activation of a receptor in a cell, comprising: (a) Below: (i) an orthogonal chimeric cytokine receptor, and (ii) at least one T cell receptor (TCR); and a modified immune cell comprising: (b) an oncolytic adenoviral vector comprising a nucleic acid sequence encoding an orthogonal IL2 cytokine; Including, The system, wherein the orthogonal chimeric cytokine receptor comprises the extracellular domain of an orthogonal IL2 receptor (oIL2R) and the intracellular signaling domain of a cytokine receptor that is not an IL2R.

30. The system of claim 29, wherein the extracellular domain of oIL2R is the extracellular domain of the orthogonal IL2 receptor beta (oIL2Rb).

31. The system of claim 29 or 30, wherein the intracellular signaling domain of the orthogonal chimeric cytokine receptor comprises an IL9R intracellular signaling domain.

32. The system of claim 31 , wherein the IL9R intracellular signaling domain is an IL9R-alpha (IL9Ra) intracellular signaling domain.

33. The system of any one of claims 29 to 32, wherein the TCR targets a tumor antigen.

34. The system of any one of claims 29 to 33, wherein the TCR targets gp100 melanoma antigen or NYESO1.

35. The system of any one of claims 29 to 34, wherein the TCR is a pmel-1 TCR or a NYESO1-specific TCR.

36. (a) the orthogonal chimeric cytokine receptor comprises the extracellular domain of oIL2Rb and the intracellular signaling domain of IL9Ra; (b) the TCR is a pmel-1 TCR or a NYESO1-specific TCR; The system of any one of claims 29 to 35.

37. A pharmaceutical composition for treating cancer in a subject in need thereof, comprising: The pharmaceutical composition comprises: (i) an orthogonal chimeric cytokine receptor, and (ii) at least one T cell receptor (TCR); an effective amount of modified immune cells or precursor cells thereof (a population of modified immune cells), comprising the pharmaceutical composition is used in combination with an oncolytic adenoviral vector comprising a nucleic acid sequence encoding an orthogonal IL2 cytokine; The orthogonal chimeric cytokine receptor comprises the extracellular domain of an orthogonal IL2 receptor (oIL2R) and the intracellular signaling domain of a cytokine receptor that is not an IL2R. Pharmaceutical compositions.

38. 38. The pharmaceutical composition of claim 37, wherein the extracellular domain of oIL2R is the extracellular domain of the orthogonal IL2 receptor beta (oIL2Rb).

39. The pharmaceutical composition of claim 37 or 38, wherein the intracellular signaling domain of the orthogonal chimeric cytokine receptor comprises an IL9R intracellular signaling domain.

40. 40. The pharmaceutical composition of claim 39, wherein the IL9R intracellular signaling domain is an IL9R-alpha (IL9Ra) intracellular signaling domain.

41. The pharmaceutical composition of any one of claims 37 to 40, wherein the TCR targets a tumor antigen.

42. The pharmaceutical composition of any one of claims 37 to 41, wherein the TCR targets gp100 melanoma antigen or NYESO1.

43. The pharmaceutical composition of any one of claims 37 to 42, wherein the TCR is a pmel-1 TCR or a NYESO1-specific TCR.

44. (a) the orthogonal chimeric cytokine receptor comprises the extracellular domain of oIL2Rb and the intracellular signaling domain of IL9Ra; (b) the TCR is a pmel-1 TCR or a NYESO1-specific TCR; 44. The pharmaceutical composition of any one of claims 37 to 43.

45. The pharmaceutical composition of any one of claims 37-44, wherein said population of modified immune cells exhibits stem cell memory (Tscm) characteristics with improved trafficking and effector function, thereby treating cancer.

46. A pharmaceutical composition described in any one of claims 37 to 45, wherein the oncolytic adenoviral vector is administered by intratumoral injection.

47. The pharmaceutical composition of any one of claims 37 to 46, wherein the cancer is selected from the group consisting of pancreatic cancer and melanoma.

48. 48. The pharmaceutical composition of claim 47, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma.

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