Tumor therapy compositions and methods

Abstract

The present disclosure relates generally to tumor therapy compositions and related treatment methods and, in particular, immune effector cells that include exogenous polynucleotides encode components of a therapeutic expression system. One aspect of the present disclosure can include a tumor therapy system. The tumor therapy system can include a caged inducer and an immune effector cell. The immune effector cell can include a first exogenous polynucleotide that is operably linked to a single viral vector, wherein the first exogenous polynucleotide encodes polypeptide components of a chemical induced proximity (CIP) complex and a therapeutic polypeptide operably linked to a regulatory region inducible by an uncaged, activated form of the caged inducer. The immune effector cell can further include a second exogenous polynucleotide that encodes a chimeric antigen receptor that specifically binds to an antigen.

Description

TUMOR THERAPY COMPOSITIONS AND METHODSRELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial Nos. 63 / 674,425 (filed July 23, 2024) and 63 / 684,489 (filed August 19, 2024), the entireties of which are hereby incorporated by reference for all purposes.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created July 22, 2025, is named CWR- 033680_WO_ORD_SL.xml and is 15,850 bytes in size.TECHNICAL FIELD

[0003] The present disclosure relates generally to tumor therapy compositions and related treatment methods and, in particular, to engineered immune effector cells that include exogenous polynucleotides encoding components of a therapeutic expression system.BACKGROUND

[0004] The potential of chimeric antigen receptor (CAR)-based immunotherapy as a promising therapeutic approach is often hindered by the presence of highly immunosuppressive tumor microenvironments (TME). Combination therapies with either co-administration or built-in expression of additional TME-modulating therapeutic molecules to potentiate the functions of CAR-T cells can cause systemic toxicities due to the lack of control over the delivery of biologies. Thus, although CAR-redirected T-cell therapies have been successful in treating hematological malignancies, their application against solid tumors has been proven challenging.SUMMARY

[0005] The present disclosure relates generally to tumor therapy compositions and related treatment methods and, in particular, to immune effector cells that include exogenous polynucleotides encoding components of a therapeutic expression system.

[0006] One aspect of the present disclosure can include a tumor therapy system. The tumor therapy system can comprise a caged inducer and an immune effector cell. The immune effector cell can comprise a first exogenous polynucleotide that is operably linked to a single viral vector, wherein the first exogenous polynucleotide encodes polypeptide components of a chemical induced proximity (CIP) complex and a therapeutic polypeptide operably linked to a regulatory region inducible by an uncaged, activated form of the caged inducer. The immune effector cell can further comprise a second exogenous polynucleotide that encodes a chimeric antigen receptor that specifically binds to an antigen.

[0007] Another aspect of the present disclosure can include an isolated immune effector cell. The immune effector cell can comprise a first exogenous polynucleotide that is operably linked to a single viral vector. The first exogenous polynucleotide can encode polypeptide components of a chemical induced proximity (CIP) complex and a therapeutic polypeptide operably linked to a regulatory region inducible by an uncaged, activated form of the caged inducer. The immune effector cell can also comprise a second exogenous polynucleotide that encodes a chimeric antigen receptor that specifically binds to an antigen.

[0008] Another aspect of the present disclosure can include a method for treating a solid tumor in a subject. The method can include the steps of: administering, to the subject, at least one immune effector cell; administering, to the subject, aninactive caged inducer, the inactive caged inducer being activatable to an active, uncaged inducer by at least one signal; allowing the inactive caged inducer to be converted to the active, uncaged inducer; and allowing the active inducer to enter the at least one immune effector cell and induce the CIP complex to express the therapeutic polypeptide and the chimeric antigen receptor. The at least one administered immune effector cell can comprise a first exogenous polynucleotide that is operably linked to a single viral vector. The first exogenous polynucleotide can encode polypeptide components of a chemical induced proximity (CIP) complex and a therapeutic polypeptide operably linked to a regulatory region inducible by an uncaged, activated form of the caged inducer. The immune effector cell can also comprise a second exogenous polynucleotide that encodes a chimeric antigen receptor that specifically binds to an antigen.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The foregoing and other features of the present disclosure will become apparent to those skilled in the art to which the present disclosure relates upon reading the following description with reference to the accompanying drawings, in which:

[0010] Fig. 1 is a schematic illustration showing the design of a CAR+T cell platform according to one aspect of the present disclosure. The programmable in situ production of therapeutic outputs is designed to be triggered by cancer- associated signal to enhance the efficacy and persistence of CAR-expressing T cells;

[0011] Figs. 2A-D show ABA-induced production of sTRAIL in engineered HEK-293T cells. (Fig. 2A) Cartoon illustrates sTRAIL-induced apoptosis in DeathReceptor-expressing cancer cells. (Fig. 2B) ABA-inducible therapeutic geneexpression constructs. (Fig. 2C) Induction of sTRAIL in HEK-293T cells in response to ABA (1 pM). Concentration of secreted TRAIL in the cell culture supernatant was measured by ELISA. (Fig. 2D) Percentage of MDA-MB-231 cell apoptosis after being treated with DMSO, recombinant human TRAIL (rhTRAIL), or media containing sTRAIL for 24 h. Target cells were co-stained with Annexin V and Propidium Iodide to exclude necrotic dead cells. Data are calculated from 2 biological replicates and presented as mean ± S.E.M. ****p < 0.0001 , ***p < 0.001 , ns, p > 0.05, two-tailed student t-test;

[0012] Figs. 3A-B show generation and validation of recombinant lentiviral vector encoding inducible gene cassette. (Fig. 3A) Lentiviral vector of abscisic acid (ABA)- inducible gene expression system. (Fig. 3B) ABA-induced EGFP expression in transfected HEK-293T cells. Cells were treated with 0.5% DMSO or 1 pM ABA, and the induced EGFP expression was observed under a fluorescence microscope after 24 h. The scale bar is 200 pm. Mean fluorescence intensity (MFI) was calculated from 3 random areas in a well using microscope-integrated cellular analysis tool. Error bars are SD (n=3);

[0013] Figs. 4A-C show ABA-induced expression of pro-inflammatory cytokine IL- 12 in Jurkat T cells. (Fig. 4A) Different configurations of lentiviral vectors tested.The best construct (3) is outlined in red and used in the following studies. (Fig. 4B) Jurkat T cells were then transduced with viruses encoding different inducible IL-12 (ilL-12) vectors and treated with DMSO or ABA at indicated concentrations for 24 h. IL-12 released in the cell culture supernatant was quantified by ELISA. Data are shown as mean ± S.E.M from 3 technical triplicates. ****p < 0.0001 , ***p < 0.001 , n.s., p > 0.05, two-tailed student t-test. (Fig. 4C) Dosage response of inducible IL-12 in engineered Jurkat T cells with varying ABA concentrations;

[0014] Figs. 5A-B show ABA-induced expression of pro-inflammatory cytokine IL- 12 in CAR+ilL-12 Jurkat T cells. (Fig. 5A) Lentiviral vectors encoding HER2-CAR and ABA-inducible IL-12 expression cassette. (Fig. 5B) The comparison of IL-12 expression in Jurkat T cells transduced with a single virus encoding ilL-12 only or two viruses encoding both ilL-2 and CAR (CAR+ilL-12) genes. Cells were treated with DMSO or ABA (1 pM) and IL-12 secretion was quantified by ELISA at 24 h posttreatment. Data are shown as mean ± S.E.M (n=3). ****p < 0.0001 , two-tailed student t-test;

[0015] Figs. 6A-C show show ABA-induced expression of immune checkpoint inhibitor Pembrolizumab (oc-PD1 mAb) in CAR+iPembro Jurkat T cells. (Fig. 6A) Lentiviral constructs for HER2-CAR and ABA-inducible Pembrolizumab. (Fig. 6B) CAR+T cells were co-cultured with MDA-MB-231 cancer cells at 5:1 E:T ratio in the presence of 500 nM ABA or PBS for 24 hours. T cells were stained for Pembro using A647-conjugated a-HA antibody. The shift of fluorescence signal towards the right indicated the binding of the secreted Pembro to the PD-1 ligand. (Fig. 6C) The levels of free PD-1 were detected by staining T cells with commercial FITC- conjugated a-PD1 antibody (clone EH12.2H7) and analyzed by flow cytometry. Data are mean ± S.E.M from 2 biological replicates (n=2). ***p < 0.001 , two-tailed student t-test;

[0016] Figs. 7A-C show ABA-inducible expression of immune checkpoint blockage a-CTLA4 scFv in CAR-expressing Jurkat T cells. (Fig. 7A) Lentiviral constructs for HER2-CAR and ABA-inducible a-CTLA4 scFv. (Fig. 7B) The secreted «-CTLA4 scFv in cell culture supernatant was detected using flow cytometry-based binding assay and HEK-293T target cells overexpressing the CTLA4 ligand. The scFv-ligand binding was followed by staining target cells with A647-conjugated a-Myc antibody for detection of the bound scFv to target cells. (Fig. 7C) Flow cytometry data showing the induced expression and secretion of a-CTLA4 scFv upon addition of ABA or PBS and selective binding of the scFv to CTLA-4+cells. The shown histogram is a representative of 3 biological replicates;

[0017] Figs. 8A-C show synthesis and characterization of ABA prodrug (ABA-Gal) for p-galactosidase (P-gal). (Fig. 8A) Synthesis and the activation of ABA-Gal prodrug. (Fig. 8B) The stability of ABA-Gal and its generation of ABA by p-gal analyzed using HPLC. 200 pM of prodrug was incubated with p-gal at the indicated concentration in PBS buffer (pH 7.4) at 37SC for different time points. (Fig. 8C) HEK- 293T EGFP reporter cells responded to p-gal when treated with ABA-Gal leading to EGFP expression. Reporter cells were treated with 20 pM ABA, or 20 pM prodrug with and without 10 pM p-gal. Treated cells were incubated for 24 h and EGFP expression was observed under a fluorescence microscope;

[0018] Figs. 9A-C show TME-gated expression and secretion of customized biologies in engineered Jurkat T cells. (Fig. 9A) The secretion of IL-12 by CAR+i I L12 T cells at 15 h post-drug treatment, as detected by ELISA. Data are shown as mean ± S.E.M from 3 technical triplicates. ****p < 0.0001 , n.s, p > 0.05, two-tailed student t-test. (Fig. 9B) Competitive binding assay showing % PD1 -positive T cells in a 24h co-culture of CAR+iPembro T cells and HER2-expressing cancer cell line MDA-MB- 231 . Data are mean ± S.E.M from 2 biological replicates (n=2). *p < 0.05, n.s, p > 0.05, one-way ANOVA with Dunnett multiple comparisons test. (Fig. 9C) The secretion of a-CTLA4 scFv by CAR+iCTLA4 scFv and the binding of scFv to CTLA4- expressing HEK-293T target cells, followed by staining target cells with A647- conjugated anti-Myc antibody and detected by flow cytometry. The histograms shown are representative of 2 biological replicates;

[0019] Figs. 10A-D show validation of HER2-CAR Jurkat T cells. (Fig. 10A) Lentiviral vector encoding for HER2-CAR. (Fig. 10B) Expression and localization of CAR on the surface of Jurkat T cells at Day 4 post-transduction via flow cytometry. (Fig. 10C) Detection of HER2 surface expression on different target cell lines.Target cells were stained with Phycoerythrin (PE) conjugated a-HER2 antibody; light gray histograms denote unstained cells (control). (Fig. 10D) Non-transduced control or HER2-CAR T cells were co-cultured with different target cell lines at an Effector:Target (E:T) ratio of 3:1 for 24h. Activated T cells were stained for CD69 expression with Allophycocyanin (APC)-conjugated a-CD69 antibody and analyzed by flow cytometry. ELISA assay was also performed to quantify IL-2 cytokine secretion into the cell culture media. Data represents mean ± SD from 2 biological replicates;

[0020] Figs. 11 A-C show surface expression of HER2-CAR in co-transduced Jurkat cells. Jurkat T cells were co-transduced to express HER2-CAR and the corresponding ABA-inducible therapeutic gene circuits including (Fig. 11 A) IL-12, (Fig. 11 B) Pembrolizumab, and (Fig. 11 C) a-CTLA4 scFv. At day 4 posttransduction, HER2-CAR expression was detected by surface staining of transduced cells with anti-Myc tag antibody and analyzed by flow cytometry;

[0021] Figs. 12A-G show engineering of a multi-layer control TME-iCAR-T cell platform. (Fig. 12A) TME-iCAR-T cells design incorporates a Boolean “AND” logic gate that requires combinatorial inputs ( / .e., tumor antigen plus TME signal plus inducer prodrugs) for precise spatiotemporal restricted T cell activation and responses. (Fig. 12B) Scheme of lentiviral CAR constructs targeting the HER2 antigen. (Fig. 12C) Expression of CARs in human primary T cells via lentiviral transduction as detected on day 8 post-transduction by flow cytometry usingantibodies against the Myc and Flag tags on the N terminal of each receptor subunit. (Fig. 12D) NTD, conventional CAR or split CAR-T cells were co-cultured with HER2- expressing BT-474 breast cancer cells (E:T = 3:1 ). Various concentrations of ABA were added to the co-culture for 22h, followed by flow cytometry. ABA and HER2- dependent T cell activation was quantified by measuring the surface expression of CD69. Gray histogram denotes non-transduced T cells stimulated with HER2+target cells, red histogram denotes conventional CAR-T cells stimulated with HER2+target cells, and gradient blue histograms indicate the split CAR-T cells dual-stimulated with HER2+target cells and ABA. (Figs. 12E-F) Antigen-specific secretion of IL-2 and IFN-ywas quantified by ELISA after an overnight co-culture (22 h) (n = 3 human donors, mean ± SEM). (Fig. 12G) Antigen-specific cytotoxicity of BT-474 cells by CAR-T cells was measured by WST-1 assay (n = 2 human donors, mean ± SEM));

[0022] Figs. 13A-D show CAR-T cell production process and characterization. (Fig. 13A) Schematic workflow of CAR-T cell production in human primary T cells. One day after activation, T cells are transduced with the lentiviral vectors described above; sequential transduction of the two-component split CAR was performed on day 1 and 2. (Fig. 13B) Ex vivo T cell expansion was measured during the production process. (Fig. 13C) Left: Expression of anti-PSMA conventional CAR and split CAR were determined by surface staining of Myc-tagged p1 and Flag-tagged p2. Right: Comparison of receptor expression levels for conventional CAR and split CAR for various donors (n = 3, grand mean is shown). (Fig. 13D) On day 14, prior to cryopreservation or mouse injection, T-cell differentiation phenotypes were analyzed based on relative markers expression (CD62L and CD45RO), and the summary data from 1 representative donor are shown on the right. Tscn: T naive, Tern: Central Memory T, Tern: Effector Memory T, Teff: Terminally Differentiated Effector T;

[0023] Figs. 14A-C show the gating strategy used for flow cytometry analysis. (Fig. 14A) Gating strategy for measuring antigen expression. (Fig. 14B) Gating strategy for quantifying expression of CD69 marker following antigen stimulation. (Fig. 14C) Gating strategy to analyze tumor cell’s antigen expression and CD3-T cell percentages in tumor tissues from in vivo experiment;

[0024] Figs. 15A-G show engineering and optimization of the split CAR in Jurkat T-cells. (Fig. 15A) Scheme of the first split CAR design. Conventional CAR was constructed as a control. (Fig. 15B) Surface expression of each receptor subunit in human Jurkat T cells transduced with either a single virus carrying an individual vector or two viruses carrying the full split CARs on Day 4 post-transduction. The expression of p1 and p2 was detected by flow cytometry using anti-Myc tag and antiFlag tag antibodies, respectively; NTD is non-transduced control T cells labeled with corresponding antibodies (n = 3 or 4 independent experiments, mean ± SEM). (Fig. 15C) Flow cytometry measurement of HER2 expression on CHO cells (HER2 negative) and BT-474 breast cancer cells (HER positive) (light grey: isotype control; dark grey: anti-human HER2 antibody). (Fig. 15D) Expression of T-cell surface marker CD69 24 h after initiation of co-culture with either BT-474 or CHO cells (E:T = 3:1 ) and 50 pM ABA or 0.5% DMSO were added in corresponding wells. 24 h postincubation, T cells were harvested and stained for expression of CD69 cell surface marker using anti-hCD69 antibody, followed by flow cytometry analysis. Jurkat T cells alone stimulated with PMA / lonomycin were used as control and were set at 100%. (Fig. 15E) Quantification of IL-2 secretion from the co-culture supernatant after 24 h via ELISA assay (n = 2 technical replicates, mean ± SEM). (Fig. 15F) Optimization of the split CAR structures. Three split pair variations (split_O, 1 and 2) with CD8H removed on either the signaling part (p2) or both chains of the split CARs.(Fig. 15G) Comparison of IL-2 secretion from 3 engineered split pair variations. Transduced Jurkat T cells were incubated with BT-474 cells (E:T = 3:1 ) and IL-2 secretion in the culture media was quantified after 24 h by ELISA (n = 2 technical replicates, mean ± SEM). The split_1 exhibited strong cytokine secretion in the presence of ABA and HER2+tumors with minimal background in the absence of the small molecule and was identified as the optimal pair for further experiments;

[0025] Figs. 16A-B show ABA-dosage dependence of T cell activation in Jurkat T cells. Jurkat T cells were transduced to express either conv. CAR, split CAR (split_1 ), or each component alone, and the T cells were stimulated with HER2 positive-BT-474 cancer cells at 3:1 E:T ratio. Various concentrations of ABA were added to the co-culture for 24 h. (Fig. 16A) Cell culture supernatant was harvested, and the production of IL-2 was quantified via ELISA. (Fig. 16B) T cells in the overnight co-culture were harvested, washed, and stained with anti-hCD69 antibody for detection of early activation marker CD69 by flow cytometry. The split CAR-T cells showed a dosage response to ABA to induce IL-2 secretion and CD69 expression. Neither the T cells engineered with each part alone nor NTD cells expressed the activation markers (n = 3 technical replicates, mean ± SEM).PMA / lonomycin-stimulated Jurkat T cells were used as positive control and were set at 100%;

[0026] Figs. 17A-D show split CAR-T cells exhibit target-specific activation and cytotoxicity against different HER2-expressing cell lines. (Fig. 17A) T cells were isolated, engineered to express split CAR or conventional CAR, expanded, and cocultured with Calcein AM-labeled HER2-expressing BT-474 tumor cells (E:T= 3:1 ). Varying concentrations of ABA were added to indicated wells. After overnight incubation, dual-gated T-cell activation was visually inspected under 10x automatedmicroscope. Shown are target cells imaged under EGFP channel, T cells viewed under the bright field channel and overlay of both channels. Black arrows indicate cell clumps as a result of tumor-mediated effector cell activation. Representative images of CAR-T cell-tumor co-culture at 22 h (Scale bars = 200 pm). (Fig. 17B) Flow cytometric detection of HER2 expression on tumor and normal cell lines. Cells were stained with anti-human HER2 (black histogram) or isotype control (grey histogram). (Fig. 17C) Cytotoxicity of CAR-T cells after stimulation by HER2 cells was measured by WST-1 assay. NTD, conv. CAR, or split CAR-T cells with and without ABA were cocultured with the indicated tumor cell lines (E:T = 3:1) for 24 h, percentage of target cell survival was determined by WST-1 assay (n = 3 human donors, mean ± SEM). (Fig. 17D) Cytokine secretion was measured (ELISA) in culture supernatants under each condition in Fig. 17C (n = 3 human donors, mean ± SEM);

[0027] Figs. 18A-E show split CAR-T cells are modular and can be applied to target different antigen. (Fig. 18A) Scheme of lentiviral CAR constructs engineered to target the PSMA antigen. (Fig. 18B) The NTD, conventional PSMA-CAR-T cells, or split PSMA-CAR-T cells with or without ABA were co-cultured with PSMA- expressing PC3-pip or PSMA-negative PC3-flu prostate cancer cells for 22h (E:T = 2:1 ), followed by flow cytometry analyses. Expression of cell surface marker CD69 indicated dose-dependent ABA-mediated CAR-T cell activation. Gray histogram denotes non-transduced T cells stimulated with PSMA+target cells, red histogram denotes conventional CAR-T cells stimulated with PSMA+ target cells, and gradient blue histograms indicate the split CAR-T cells dual-stimulated with PSMA+target cells and ABA. (Figs. 18C-D) Quantification of IL-2 (n = 3 human donors, mean ± SEM) and IFN-y (n = 2 human donors, mean ± SEM) in the co-culture media byELISA after overnight incubation. (Fig. 18E) In vitro cytotoxicity assay of NTD, conventional CAR or split CAR-T cells against PSMA-positive PC3-pip cells or PSMA-negative PC3-flu cells determined by WST-1 assay after 22-h co-culture (n = 4 human donors, mean ± SEM);

[0028] Figs. 19A-D show split CAR-T cells can be regulated by ABA both in vitro and in vivo. (Fig. 19A) Scheme of in vitro assay to determine the temporal control of the split CAR-T cells through stimulation and withdrawal cycles. (Fig. 19B) Drug- modulated T-cell activation and cytotoxicity of the split CAR upon ABA addition, being washed out and re-introduced after 2 days (n = 3 human donors, mean ± SEM). P values were calculated by one-way ANOVA with Dunnett’s multiple comparisons test. (Fig. 19C) Timeline of in vivo experiment to determine CAR-T cell antitumor activity. NSG mice were s.c. inoculated with PC3-pip tumor cells for 7 days before receiving NTD, conv. or split CAR-T cells, followed by daily injection of vehicle (2%DMSO in PBS) or ABA at a dose of 20 mg / kg for 14 days. (Fig. 19D) Tumor volume at different time points as measured by caliper. Lines are mean tumor volume ± SEM;

[0029] Figs. 20A-B show split CAR-T cells antitumor effect can be modulated by ABA. (Fig. 20A) Drug-modulated cytotoxicity (n = 3 technical replicates from 1 donor, mean ± SEM) and IFN-X secretion (n = 2 human donors, mean ± SEM) of the anti-HER2-split CAR-T cells upon ABA addition, being washed out and re-introduced after 2 days. Rvalues were calculated by one-way ANOVA with Dunnett’s multiple comparisons test. (Fig. 20B) PSMA expression on residual tumors analyzed on day 32 as described in Fig. 19D, showing antigen loss in conventional CAR-treated mice, but not the split CAR-treated one;

[0030] Figs. 21 A-B show the residual effects of prior antigen stimulation on CAR- T and the off-kinetic of drug removal. (Fig. 21 A) Anti-PSMA split CAR-T cells were preincubated with PC3-pip target tumor cells (E:T = 2:1 ) and ABA for 16 h. ABA was then washed out and CAR-T cells were rested without target tumor cells for 12 to 48 h before tumor cells and ABA was re-introduced. (Fig. 21 B) Anti-PSMA split CAR-T cells were preincubated with ABA for 16 hours in the absence of target tumor cells. After which ABA was removed and the T cells were allowed to rest from 12 to 48 h before tumor cells and ABA was introduced. The drug-modulated cytotoxicity after CAR-T cells rested for different time periods after ABA removal were quantified upon encountering target tumor cells and re-addition of ABA (n = 3 technical replicates from 1 donor, mean ± SEM);

[0031] Fig. 22 shows the effects of ABA injection periods on tumor antigen preservation. NSG mice were daily injected with the NTD, conventional or split CAR- T cells in the presence of vehicle (2%DMSO in PBS) or ABA at a dose of 20 mg / kg for 30 days (n = 2-5 mice / group), compared to the 14-day ABA injection timeline (as the experiments described in Fig. 19D). PSMA expression on residual tumors analyzed at the endpoint showing antigen sustain in the split CAR-treated mice regardless of the duration of drug treatment. Mice were sacrificed and the tumor cells were analyzed by flow cytometry using anti-PSMA antibody;

[0032] Figs. 23A-C show development of oxygen-sensitive ABA prodrugs. (Fig. 23A) Synthetic scheme and activation of hypoxia-activated ABA prodrugs. (Fig. 23B) Stability of ABA prodrugs and the regeneration of ABA were analyzed using HPLC. (Fig. 23C) EGFP expression in HEK293T-EGFP reporter cells treated with ABA, prodrugs 1 or 2 with or without activating signals for 24 h were observed under a fluorescence microscope. Scale bars = 200 gm;

[0033] Fig. 24 shows cell viability when treated with hypoxia-activated ABA- prodrugs. Toxicity was evaluated using the MTT assay in Hela cells. Viability% was calculated by comparing to DMSO treatment (n = 3 biological replicates, mean ±SEM);

[0034] Figs. 25A-E show TME-gated inducible T cell activation and cytotoxicity of split CAR-T cells. (Figs. 25A-B) Cytotoxicity, and secretion of IFN-y by split CAR-T cells after an overnight co-culture with (Fig. 25A) PSMA-expressing PC3-pip and (Fig. 25B) HER2-expressing BT-474 target cells. ABA, prodrugs 1 or 2 were added in the presence or absence of hypoxia-mimicked conditions for 22 h. Cytotoxicity was determined by WST-1 assay and cytokine secretion was quantified (ELISA) from the co-culture supernatant. Cytotoxicity: PSMA-CAR, n = 4; HER2-CAR, n = 3; IFN-y: PSMA-CAR, n = 2; HER2-CAR, n = 3. Mean ± SEM is shown for all graphs. (Fig. 25C) Timeline of in vivo experiments to test the activation of ABA prodrug and the antitumor activity of TME-iCAR-T cells. NSG mice were inoculated with 3x106PSMA+PC3-pip cells for a week before receiving 2x106split CAR-T cells. Vehicle (65%DMSO in PBS), ABA, or prodrug 1 were i.p injected daily at a dose of 20 mg / kg for 14 days. After drug injections were stopped, the tumor growth was monitored until day 28 post-tumor inoculation. (Fig. 25D) Tumor growth curve at different time points was presented, n = 5 mice per group, mean ± SEM is shown, two-tailed Student’s t-test. (Fig. 25E) Residual tumor tissues were harvested at the endpoint and percentage of tumor-infiltrated T cell was measured by flow cytometry using anti-hCD3 antibodies. The graph displayed individual values and mean ± SEM (veh / prodrug, n = 5 mice; ABA, n = 4 mice), two-tailed Student’s t-test;

[0035] Figs. 26A-D show in vivo activity of split CAR-T cells in the presence of prodrug 1 . (Fig. 26A) Immunohistological staining of PSMA+PC3-pip tumor tissuesat the time of T cell injection to confirm the degree of tumor hypoxia. Scale bars = 100 pm. (Fig. 26B) NSG mice were daily injected with the split CAR in the presence of vehicle (65%DMSO in PBS), ABA, or prodrug 1 at a dose of 20 mg / kg and the body weight was measured at indicated dates for 14 days. No toxicity was observed as accessed by body weights as well as behavioral and physical observations (n = 5 mice per group, mean ± SEM). (Fig. 26C) PSMA expression on residual tumors analyzed at the endpoint as described in Fig. 26B. Mice were sacrificed and the tumor cells were analyzed by flow cytometry using anti-PSMA antibody. (Fig. 26D) The percentage of accumulated T cells in the tumor tissue was also analyzed by flow cytometry using anti-hCD3 antibody. The higher number of T cells correlates with the degree of antigen loss in tumor cells as seen in [split CAR+ABA]-treated mouse no. a1 and a4; and

[0036] Figs. 27A-B are schematic illustrations showing a linear map (Fig. 27A) and circular map (Fig. 27B) of a bidirectional N103 lentiviral transfer vector constructed in accordance with the present disclosure.DETAILED DESCRIPTION

[0037] Definitions

[0038] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the present disclosure pertains.

[0039] Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps. In particular, in methods stated as comprising one or more steps or operations it is specifically contemplated that eachstep comprises what is listed (unless that step includes a limiting term such as “consisting of”), meaning that each step is not intended to exclude, for example, other additives, components, integers or steps that are not listed in the step.

[0040] In the context of the present disclosure, the term “about”, when expressed as from “about” one particular value and / or “about” another particular value, also specifically contemplated and disclosed is the range from the one particular value and / or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another, specifically contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise. Finally, it should be understood that all of the individual values and subranges of values contained within an explicitly disclosed range are also specifically contemplated and should be considered disclosed unless the context specifically indicates otherwise. The foregoing applies regardless of whether in particular cases some or all of these aspects are explicitly disclosed.

[0041] Optionally, in some aspects, when values or characteristics are approximated by use of the antecedents “about,” “substantially,” or “generally,” it is contemplated that values within up to 15%, up to 10%, up to 5%, or up to 1 % (above or below) of the particularly stated value or characteristic can be included within the scope of those aspects.

[0042] As used herein, the terms “first,” “second,” etc. should not limit the elements being described by these terms. These terms are only used to distinguishone element from another. Thus, a “first” element discussed below could also be termed a “second” element without departing from the teachings of the present disclosure. The sequence of operations (or acts / steps) is not limited to the order presented in the claims or figures unless specifically indicated otherwise.

[0043] As used herein, the terms “optionally” and “optional” can mean that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present and instances where it does not occur or is not present.

[0044] As used herein, the terms “subject” and “patient” can be used interchangeably and refer to a vertebrate, such as a mammal (e.g., a human). Mammals can include, but are not limited to, humans, dogs, cats, horses, cows, and pigs.

[0045] As used herein, the term “coding region” can refer to a nucleotide sequence that encodes a polypeptide and, when placed under the control of appropriate regulatory sequences expresses the encoded polypeptide. The boundaries of a coding region are generally determined by a translation start codon at its 5' end and a translation stop codon at its 3' end.

[0046] As used herein, the term “regulatory sequence” can refer to a nucleotide sequence that regulates expression of a coding sequence to which it is operably linked. Regulatory sequences include, for example, promoters, enhancers, transcription initiation sites, translation start sites, translation stop sites, and transcription terminators.

[0047] As used herein, the term “operably linked” can refer to a juxtaposition of components (e.g., polynucleotide or polypeptide domains) such that they are in arelationship permitting them to function in their intended manner. For example, a regulatory sequence is “operably linked” to a coding region when it is joined in such a way that expression of the coding region is achieved under conditions compatible with the regulatory sequence.

[0048] As used herein, the term “immune effector cell” can refer to an immune cell that is capable of providing an immunological function involved in an immune response, e.g., in the promotion of an immune effector response. For example, the immune effector cell may be capable of providing a cytotoxic function.

[0049] As used herein, the term “isolated”, when referring to an immune effector cell, can refer to an immune effector cell removed from its original or natural environment. An “isolated immune effector cell” is a cell that is no longer present in its natural environment.

[0050] As used herein, the term “tumor antigen” can refer to a biological molecule having antigenicity, expression of which is associated with a neoplastic cell. The tumor antigens targeted in the present disclosure can include a tumor-specific antigen (an antigen which is present only in tumor cells and is not found in other normal cells), and a tumor-associated antigen (an antigen which is also present in other organs and tissues or heterogeneous and allogeneic normal cells, or an antigen which is expressed on the way of development and differentiation).

[0051] As used herein, the terms “tumor cell” and “cancer cell” can be used interchangeably and refer to a malignant cell. A tumor cell can occur in and can be obtained from a solid tumor such as a sarcoma, carcinoma, melanoma, lymphoma or glioma.

[0052] As used herein, the term “solid tumor” can refer to an abnormal mass of tissue comprising neoplastic cells in a subject. Solid tumors may be benign ormalignant. Solid tumors that can be treated using the methods and compositions of the present disclosure are characterized by neovascularization. The tumor vasculature (also referred to as microvasculature) is characterized by rapid proliferation of the endothelial cells, poor wall structure, increased permeability to plasma proteins, and a limited ability to increase blood flow in response to demand. The tumor vasculature allows the tumor cells of the tumor mass to acquire a growth advantage compared to the normal cells. Solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas (epithelial tumors), melanomas, and glioblastomas.

[0053] As used herein, the term “exogenous”, when used with reference to a polynucleotide of the present disclosure, can refer to a heterologous nucleic acid sequence which is not naturally expressed within a cell (e.g., a tumor cell) or which expression in the cell is desired.

[0054] As used herein, the term “isolated”, when used with reference to a polypeptide, can refer to a polypeptide or a peptide fragment which either has no naturally-occurring counterpart (e.g., a peptidomimetic), or has been separated or purified from components which naturally accompany it, e.g., in biological fluids or tissues.

[0055] As used herein, the term “isolated”, when used with reference to a polynucleotide, can refer to a nucleotide sequence that is not immediately contiguous with nucleotide sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived. The term can include, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as aseparate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment), independent of other sequences. It also includes a recombinant DNA that is part of a hybrid nucleic acid encoding an additional polypeptide or peptide sequence.

[0056] As used herein, the term “polynucleotide” can refer to a single or double stranded nucleic acid sequence which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and / or a composite polynucleotide sequence (e.g., a combination of the above.

[0057] As used herein, the term “polypeptide” can refer to a polymer made up of two or more amino acids linked together by peptide bonds.

[0058] As used herein, the term “treatment” can refer to clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. For example, a subject is successfully “treated” if one or more symptoms associated with cancer are mitigated or eliminated, including, but are not limited to, reducing the proliferation of (or destroying) cancerous cells, decreasing symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, and / or prolonging survival of individuals. In some instances, “treating” a disease such as cancer refers to delaying progression of the disease, i.e., deferring, hindering, slowing, retarding, stabilizing, and / or postponing development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and / or individual being treated. Asis evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a late stage cancer, such as development of metastasis, may be delayed.

[0059] As used herein, the term “therapeutically effective amount” can refer to the amount of an agent (e.g., engineered immune effector cells of the present disclosure) determined to produce any therapeutic response in a subject (e.g., prolong the survivability of the subject, and / or inhibit overt clinical symptoms. Treatments that are therapeutically effective within the meaning of the term as used herein can include treatments that improve a subject’s quality of life even if they do not improve the disease outcome per se. Such therapeutically effective amounts are readily ascertained by one of ordinary skill in the art. Thus, to “treat” can mean to deliver such an amount. Thus, treating can prevent or ameliorate any pathological symptoms of a disease or disorder disclosed herein.

[0060] As used herein, the term “immunotherapy” can refer to treatment of a patient for the purpose of increasing immunity by modulating an immune response (e.g., stimulating or suppressing an immune response) to a disease or disorder, such as cancer. In some instances, immunotherapy can be used as a preventive measure or as a pretherapy (e.g., for a patient with a family history of prostate cancer at age 60, such immunotherapy can be initiated at age 55). In one example, the term can include prevention, elimination, or reduction of metastasis in a patient. In another example, the term can include prevention, elimination, or reduction of disease (e.g., cancer) recurrence in a patient.

[0061] As used herein, the terms “adoptive cell therapy” or “ACT” can refer to the administration of one or more engineered immune effector cells of the present disclosure to a subject with disease or disorder, such as cancer.

[0062] As used herein, the term “sequence identity” can refer to the extent to which two optimally aligned polynucleotide or polypeptide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids. “Identity” can be readily calculated by known methods including, but not limited to, those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, N.J. (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991 ).

[0063] As used herein, the term “percent sequence identity” or “percent identity” can refer to the percentage of identical amino acids in an amino acid sequence as compared to a reference polypeptide. The phrase “substantially identical,” or “substantial identity” in the context of two polypeptide or protein sequences, can refer to two or more sequences or subsequences that have at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a known sequence comparison algorithm or by visual inspection.

[0064] As used herein, the term “anti-tumor effect” can refer to a biological effect observed when treating solid tumors which manifests in a variety of ways, including, for example, by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, oramelioration of various physiological symptoms associated with the cancerous condition. An “anti-tumor effect” can also be manifested by the ability of the engineered immune effector cells and expression systems of the present disclosure in prevention of the occurrence of tumor in the first place.

[0065] As used herein, the term “encoding” can refer to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides ( / .e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0066] As used herein, the term “lentivirus” can refer to viruses in a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells. In addition, they can deliver a significant amount of genetic information into the DNA of the host cell, so lentivirus-derived vectors are one of the most efficient gene delivery vectors available. HIV, SIV, and FIV are all examples of lentiviruses.

[0067] As used herein, the terms “modified” and “engineered” can be used interchangeably and refer to a changed state or structure of a molecule or cell of the present disclosure. Molecules may be modified in many ways, including chemically,structurally, and functionally. Cells may be modified through the introduction of nucleic acids.

[0068] As used herein, the term “minimal promoter” can refer to a TATA-box promoter element upstream of the inducible gene and sequence can be located between the -35 to +35 region with respect to transcription start site (Smale ST (2001 ) Core promoters: active contributors to combinatorial gene regulation. Genes Dev 15: 2503-2508). Eukaryotic promoters of protein-coding genes have one or more of three conserved sequences in this region ( / .e., the TATA-box, initiator region, and downstream promoter element). A minimal promoter enables low basal leakiness in the absence of specific transcriptional activators and high expression when transcription activators are bound upstream of minimal promoter at their specific DNA binding sites. In some embodiments the minimal promoter is derived from the pGL4.23[luc2 / minP]; Promega and validated in (Smole, A., Lainscek, D., Bezeljak, U., Horvat, S. & Jerala, R. A Synthetic Mammalian Therapeutic Gene Circuit for Sensing and Suppressing Inflammation. Mol. Ther. 25, 102-119 (2017)). Alternative minimal promoters can be used, such as minimal TATA box promoter, minimal CMV promoter or minimal IL-2 promoter.

[0069] Overview

[0070] The present disclosure describes effective and safe cancer immunotherapy for solid tumors that involve integrating a cancer-inducible prodrug strategy with cell therapy and immune checkpoint blockade. The therapy, which has an anti-tumor effect, involves engineered immune effector cells (e.g., CAR Plus T cells) that can be selectively triggered by the tumor microenvironment to provide locally boosted immune effector cell activity and persistence as well as to produce anticancer agents in solid tumor environments. Advantageously, the presentdisclosure includes a new therapeutic strategy that allows targeted enhancement of immune effector cell (e.g., T cell) activity and persistence along with simultaneously production of extra anticancer agents only in solid tumor sites, which offers superior immunotherapy for solid tumors.

[0071] The present disclosure describes a strategy for targeted stimulation of immune effector cell activity (e.g., T cell activity) and inducing more effective therapeutic effects only within solid tumor environments. The strategy involves a prodrug concept and a chemically-induced proximity (CIP) technology that can be applied to enable small molecule-induced gene expression (Fig. 1 ). The CIP technology uses an inducer (e.g., abscisic acid, ABA) to trigger binding between unique inducer-binding adaptor proteins (e.g., PYL and ABI in the case of ABA) that can be genetically fused to any two proteins of interest (e.g., a DNA binding protein domain and a transcriptional activation protein domain for inducing gene expression). The ABA-induced interaction of engineered fusion proteins can in turn lead to tailored biological outputs (e.g., gene activation). The inducer (e.g., ABA) can be modified into an inactive caged or prodrug version that becomes activatable by a user-chosen signal such as, for example, a secondary messenger, a metal ion, light, a molecule, or other endogenous signal in local cellular environments. Using a prodrug responsive to a unique signal, presented only in a solid tumor microenvironment, and combined with the CIP technology, tailored therapeutic outputs can be specifically and locally produced for selected solid tumors. When integrating prodrug / CIP technology with CAR immune effector cell engineering, CAR immune effector cells (e.g., CAR Plus T cells) can be created that express desired therapeutic proteins (e.g., PD-L1 mAb, IL-12, or TRAIL) and achieve targeted tumor killing in solid tumor sites with limited collateral damage to healthy tissues.

[0072] Several proteases are overexpressed in solid tumor microenvironments, which degrade extracellular matrixes and facilitate cancer metastasis. For example, elevated levels of cathepsin B, MMP-2, MMP-9, uPA, plasmin, caspase 3, and others have been associated with solid tumors (e.g., melanoma) and their invasiveness. Many cancer prodrugs have been developed to be activatable by these proteases. These prodrugs typically have optimized protease-specific peptide substrates conjugated to cytotoxic small molecules that mask the activity of these molecules until these peptide “caging” groups are cleaved off in the presence of overexpressed proteases in cancer environments. Prodrugs for chemically-induced proximity (CIP) inducers (e.g., ABA) can be designed to become active only inside solid tumor local environments. Locally-produced ABA, for example, can readily induce the association of corresponding ABI and PYL fusion proteins within seconds and induced desired biological effects even if any possible diffusion of ABA occurs that may dilute ABA to become under the active concentration.

[0073] The technology described herein may employ CIP inducers other than ABA. The ABA CIP system has been proven to be suitable for the proposed strategy and ABA has been evaluated by EPA for the safety in human consumption.However, prodrugs may be designed under the technology platform described herein based on other CIP inducers such as, for example, rapamycin, gibberellic acid (GA), FK506, auxin, -galactosidase, or any synthesized molecule that can induce the association of two proteins. DNA plasmids can be rapidly modified for the use of these alternative CIP inducers. When rapamycin, for example, is generated from corresponding prodrugs by proteases, it can not only induce the therapeutic effects from the immune effector cells of the present disclosure (e.g., CAR Plus T cells), butitself can also act as an anticancer drug to kill cancer in local solid tumor environments.

[0074] CIP-mediated signal-triggered gene expression technology can be incorporated into immune effector cells (e.g., T cells) to generate engineered immune effector cells (e.g., CAR Plus T cells) that produce and secrete an immune checkpoint inhibitor {e.g., PD-L1 mAb) and / or other therapeutic proteins such as, for example, IL-12 (which enhances the activation of cytotoxic T cells and NK cells) and / or TRAIL (which induces apoptosis of cancer cells expressing death receptors). The production of these therapeutic proteins occurs only when engineered immune effector cells (e.g., CAR Plus T cells) located within solid tumor microenvironments are activated by local tumor-specific cues (e.g., overexpressed proteases, hypoxia, etc.). Such a “double targeting” strategy, i.e., recognizing both tumor surface antigens (through CARs) and tumor microenvironment reactivity properties (through ABA prodrug activation) provides unparalleled therapeutic specificity. Locally generated PD-L1 mAb and IL-12 can enhance the antitumor activity and / or persistence of, e.g., CAR Plus T cells in the solid tumor environment without causing collateral damage systemically. Combined with TRAIL production (or any other antitumor therapeutic agents), which selectively induces the apoptosis of cancer cells, these enhanced immune effector cells (e.g., T cells) can offer additional therapeutic benefits over current immunotherapy approaches.

[0075] In view of the foregoing, and as described in the Example below, the inventors applied the signal-triggered CIP strategy in the tumor context, where an ABA-prodrug bearing a galactose moiety activatable by / / -galactosidase was developed and tested. The inventors demonstrated that this signal-induced CIP technology can be used to engineer therapeutic T cells capable of sensing a cancer-related signal to selectively deliver desired therapeutic payloads. The outputs can be any genetically encodable therapeutics (including anti-cancer agents, secreted stimulatory cytokines, and immune checkpoint inhibitors) that can boost the antitumor activity of CAR-T cells and improve CAR-T cells’ persistence within the tumor milieu. The unparalleled programmability of this tumor microenvironment (TME)-controlled synthetic gene regulatory circuit has broad applications against different cancers whose microenvironment-associated signals are exclusively elevated.

[0076] To overcome the suppressive TME and address off-tumor toxicity problems encountered in current CAR-T therapies, the inventors have developed and tested a new strategy to empower CAR-T cells to modulate the immune response program, independent of the antigen recognition by CAR. The inventors coupled CAR with a parallel customized T cell response program built upon an “AND” logic gate design using integrated small molecule-induced protein dimerization and inducer caging strategies (to allow TME signal sensing and generating therapeutic responses). Using an ABA caging strategy that involves the chemical modification of ABA with different sensing units activatable by custom signals (e.g., H2O2, Fe2+’ or light) to drive different cellular responses, the inventors used this platform with CAR to engineer an advanced class of CAR-T cells (CAR+T cell) with enhanced therapeutic efficacy through tailored TME-inducible immunomodulatory gene circuits. The goal was to generate “armored” CAR-T cells capable of the local delivery of biologies to overcome immunosuppressive TME, which, however, is not dependent on CAR activation but on specific TME signals.

[0077] To implement this strategy, a single lentiviral vector was constructed by combining an ABA-responsive split transcriptional activator and an inducible geneexpression unit, which was then incorporated into T cells. As discussed below, after entering the tumor milieu, unique TME-enriched signals activated caged ABA and consequently induced gene expression through the ABA-triggered dimerization and activation of the split transcriptional activator. Combining the split transcriptional activator and the inducible gene expression unit into a single viral vector has several advantages. First, T cells need to gain all CAR components in the same cell to have full CAR function. The co-transduction efficiency (the chance of cells getting all components) is reduced when the number of vectors increases. Therefore, using fewer viral vectors is a significant advantage. A further advantage is that, for clinical applications, the fewer components (vectors) needed to go through clinical trials, the faster and less complicated it makes make the regulatory approval process and, therefore, a reduced time to treatment for patients in need of tumor therapy.

[0078] Based at least in part on the foregoing discoveries and the Examples below, provided herein are engineered immune effector cells that include exogenous polynucleotides encoding components of a therapeutic expression system that enables the engineered immune effector cells to sense a local environmental signal to activate production of customized therapeutic gene products and boost, e.g., engineered CAR-T efficacy and persistence.

[0079] Systems and Compositions

[0080] One aspect of the present disclosure can include a tumor therapy system. The tumor therapy system can comprise a caged inducer and an immune effector cell (e.g., an engineered immune effector cell). The immune effector cell can comprise a first exogenous polynucleotide that is operably linked to a single viral vector. The first exogenous polynucleotide can encode polypeptide components of a chemical induced proximity (CIP) complex and a therapeutic polypeptide operablylinked to a regulatory region inducible by an uncaged, activated form of the caged inducer. The immune effector cell can further comprise a second exogenous polynucleotide that encodes a chimeric antigen receptor that specifically binds to an antigen.

[0081] In one embodiment, the caged inducer can be an inactive prodrug of an inducer that is activatable by at least one signal to form the uncaged, activated form of the caged inducer. The inducer of the caged inducer can comprise at least one sensing moiety covalently attached thereto, wherein the sensing moiety is configured to react with the at least one signal to generate the uncaged inducer. In one example, the inducer can comprise abscisic acid (ABA), gibberellic acid (GA), rapamycin, or p-galactosidase. In another example, the sensing moiety can comprise a nitroimidazole group, a nitrobenzyl group, or a galactose moiety. In a further example, a caged inducer can comprise ABA conjugated with a nitroimidazole or nitrobenzyl group. In yet another example, a caged inducer can comprise ABA conjugated with galactose.

[0082] However, prodrugs may be designed under the technology platform described herein based on other inducers such as, for example, FK506, auxin, or any synthesized molecule that can induce the association of two proteins. DNA plasmids made in this study can be rapidly modified for the use of these alternative inducers. When rapamycin is generated from corresponding prodrugs by proteases, for example, it can not only induce the therapeutic effects from the engineered immune effector cells but itself can also act as an anticancer drug to kill cancer in local solid tumor environments.

[0083] In another embodiment, the at least one signal that activates the caged inducer and causes the caged inducer to obtain the uncaged, activated form cancomprise a physiological signal associated with a tumor microenvironment (TME) including, but not limited to, a signal molecule selected from the group consisting of NADPH, hydrogen ions, oxygen, hydrogen peroxide and iron (Fe2+). In another example, the signal can comprise light (e.g., non-visible light, such as UV light). In yet another example, the signal can comprise a change in temperature (e.g., an increase or decrease in temperature relative to an average body temperature, e.g., the average body temperature of a mammal, from about 34 to about 40 degrees Celsius, and generally accepted to be about 37 degrees Celsius for humans).

[0084] In another embodiment, the immune effector cell is a T cell, e.g., an autologous T cell or an allogeneic T cell.

[0085] In another example, the immune effector cell is a natural killer (NK) cell, e.g., an autologous NK cell or an allogeneic NK cell.

[0086] In another embodiment, the viral vector can comprise a retroviral DNA vector, such as a lentiviral vector. One example of a lentiviral vector is described by Gordon etal., Nature. 2020 Jul; 583(7816):459-468. Another example is Plasmid #141395, commercially available from Addgene, Inc. (Watertown, MA). The lentiviral vector can be designed to include cloning / cut sites that allow simple and standard operations to insert any coding region encoding a protein, polypeptide, or peptide fragment that may be desired for a specified application. In one example, the lentiviral vector is a bidirectional N103 lentiviral transfer vector. In another example, the bidirectional N103 lentiviral transfer vector is configured as shown in Figs. 27A-B.

[0087] In another embodiment, the regulatory region binds components of a CIP expression system. In the presence of the uncaged inducer in the immune effector cell, for example, assembly of the CIP expression system and expression of the therapeutic polypeptide is induced.

[0088] In one example, the first exogenous polynucleotide can comprise a single ABA-inducible cassette including an ABA-dimerizable split transcriptional activator (e.g., VP16AD-PYR*-T2A-GAL4DBD-ABI) and an ABA-inducible therapeutic protein construct made by inserting a therapeutic gene (or genes) under control of a minimal gene promoter.

[0089] In another embodiment, the polypeptide encoded by the first exogenous polynucleotide can comprise, 5' to 3', the following components, each of which is operably linked to one another: at least one upstream activating sequence (UAS); the regulatory region; the therapeutic polypeptide; a promoter selected from elongation factor 1 alpha (EF1 a) or phosphoglycerate kinase (PGK); a transcriptional activation domain; a PYR mutant domain; a T2A domain; a GAL4 DNA binding domain; and an ABI domain.

[0090] In one example, the at least one UAS can comprise a 5x UAS, the regulatory region can comprise a minimal promoter, such as CMV minimal promoter, the transcriptional activation domain can comprise VP16AD, and the PYR mutant domain can comprise one of PYR E141 L, PYR F61 L / A160C or PYR F61 L / E141 L / A160V.

[0091] In another example, the polypeptide encoded by the first exogenous polynucleotide can be configured as shown in Fig. 4A (construct (3)).

[0092] The first exogenous polynucleotide described herein can be engineered to express a variety of therapeutic polypeptides including, but not limited to, an immune checkpoint inhibitor, a cytokine, a cancer-specific therapeutic protein, a protein or polypeptide that activates T cells, a protein or polypeptide that enhances T cell persistence, and / or a protein or polypeptide that has anti-tumor activity. Exemplary immune checkpoint inhibitors include a monoclonal antibody or fragment thereof(e.g., an scFv) that specifically binds to PD-1 , PD-L1 , CTLA4, 4-1 BB (CD137), and / or 0X40. Exemplary cytokines include IFNa, IFN , INFy, IL-2, IL-7, IL-15, IL-18, and / or IL-21 . Cancer-specific therapeutic proteins can include thrombospondin 1 (TSP1 ), PEX, a magainin, a cecropin, a defensin, pleurocidin, and / or a Bax-derived pore-forming peptide.

[0093] In another embodiment, the chimeric antigen receptor can comprise a split chimeric antigen receptor (CAR) that specifically binds to an antigen, such as a tumor antigen.

[0094] Non-limiting examples of tumor antigens to which a split CAR can specifically bind are discussed below.

[0095] In another embodiment, the second exogenous polynucleotide can comprise a first polynucleotide subunit encoding an antigen recognition domain and a first dimerizable adaptor protein and a second polynucleotide subunit encoding an immune effector cell signaling domain and a second dimerizable adaptor protein. In the presence of the at least one signal and the caged inducer, the first and second adaptor proteins can be induced to dimerize and generate a functional split CAR.

[0096] In another embodiment, the first polynucleotide subunit can encode a hinge domain, a transmembrane domain, an intracellular co-stimulatory domain, and an optional epitope tag domain (e.g., a myc tag having a sequence with substantial identity to SEQ ID NO: 3). In one example, the first adaptor protein can comprise, 5' to 3', the following components, each of which is operably linked to one another: an a-PSMA scFV domain, a hinge domain and a transmembrane domain (e.g., human CD8a hinge and transmembrane domains having a sequence with substantial identity to SEQ ID NO: 6), a 4-1 BB domain (e.g., having a sequence with substantialidentity to SEQ ID NO: 7), and a mutant PYR domain. In another example, the first polynucleotide subunit can be configured as shown in Fig. 15F (“p1 ” of “split_1 ”).

[0097] In another embodiment, the second polynucleotide can encode a DNAX- activating protein (DNAP) domain (e.g., with a sequence having substantial identity to SEQ ID NO: 5), a transmembrane domain, an immunoreceptor tyrosine-based activation domain, an intracellular co-stimulatory domain, an optional hinge domain, and an optional epitope tag domain (e.g., a flag tag having a sequence with substantial identity to SEQ ID NO: 4). In one example, the second adaptor protein can comprise, 5' to 3', the following components, each of which is operably linked to one another: a DNAP 10 domain, a transmembrane domain (e.g., with the CD8a hinge segment removed), a 4-1 BB domain, an ABI domain, and a CD3£ domain (e.g., with a sequence having substantial identity to SEQ ID NO: 8). In another example, the second polynucleotide subunit can be configured as shown in Fig. 15F (“p2” of “split_1 ”).

[0098] In another embodiment, the first and second polynucleotide subunits can be operably linked to the same or different expression vector(s), e.g., viral vectors.

[0099] Another aspect of the present disclosure can include an isolated, engineered immune effector cell according to the methods and compositions provided herein. For example, an isolated, engineered immune effector cell can comprise a first exogenous polynucleotide that is operably linked to a single viral vector and which encodes polypeptide components of a chemical induced proximity (CIP) complex, and a therapeutic polypeptide operably linked to a regulatory region inducible by an uncaged, activated form of the caged inducer. The isolated, engineered immune effector cell can further comprise a second exogenouspolynucleotide that encodes a chimeric antigen receptor that specifically binds to an antigen.

[0100] Yet another aspect of the present disclosure can include an expression system, e.g., for expressing one or more therapeutic polypeptides in an immune effector cell. The expression system can comprise a first exogenous polynucleotide operably linked to a single viral vector and a second exogenous polynucleotide. The first exogenous polynucleotide can encode polypeptide components of a chemical induced proximity (CIP) complex and a therapeutic polypeptide operably linked to a regulatory region inducible by an uncaged, activated form of the caged inducer. The second exogenous polynucleotide can encode a CAR that specifically binds to an antigen.

[0101] Methods

[0102] Another aspect of the present disclosure can include a method for treating a solid tumor in a subject. In one example, the method can include an immunotherapy, such as adoptive cell transfer therapy.

[0103] One step of the method can include administering, to the subject, at least one immune effector cell as described herein, e.g., comprising a first exogenous polynucleotide that is operably linked to a single viral vector, wherein the first exogenous polynucleotide encodes polypeptide components of a chemical induced proximity (CIP) complex and a therapeutic polypeptide operably linked to a regulatory region inducible by an uncaged, activated form of the caged inducer, and wherein the immune effector cell further includes a second exogenous polynucleotide that encodes a chimeric antigen receptor that specifically binds to an antigen {e.g., a tumor antigen).

[0104] In one example, immune effector cells (e.g., T-cells) are first obtained from a subject having a solid tumor and then subjected to the processes described herein to obtain the engineered immune effector cells, whereafter the engineered immune effector cells are then administered back to the subject having a solid tumor.

[0105] In one embodiment, a scFv unit comprising the chimeric antigen receptor can be tailored to the particular solid tumor being treated. Thus, for example, the scFv unit of the chimeric antigen receptor can include an scFv that specifically binds to a melanoma antigen (e.g., GD2 or CD20). Exemplary alternative cancer- associated antigens, with exemplary targeted tumor listed in parentheses, include a- folate receptor (e.g., ovarian cancer and epithelial cancers), CAIX (e.g., renal cell carcinoma), CD19 (e.g'., B cell malignancies, ALL, CLL, lymphoma), CD22 (e.g., B cell malignancies), CD23 (e.g., CLL), CD 24 (e. '., pancreatic adenocarcinoma), CD30 (e.g., lymphomas and Hodgkin lymphoma), CD33 (e.g'., AML), CD38 (e.g., Non-Hodgkin lymphoma), CD44v7 / s (e.g., cervical carcinoma), CEA (e.g., colorectal cancer), EGFRvlll (e.g., glioblastoma), EGP-2 (e.g., multiple malignancies), EGP-40 (e.g., colorectal cancer), EphA2 (e.g., glioblastoma), erb-B2 (e.g., breast cancer, prostate cancer, colon cancer, and other tumors), erb-B 2,3,4 (e.g., breast cancer), FBP (e.g., ovarian cancer), fetal acetylcholine e receptor (e.g., rhabdomyosarcoma), GD2 (e.g., neuroblastoma, melanoma, Ewing sarcoma), GDS (e.g., melanoma), Her-2 (e.g., medulloblastoma, pancreatic adenocarcinoma, glioblastoma, osteosarcoma, ovarian cancer, breast cancer), HMW-MAA (e.g., melanoma), IL-1 1 Ra (e.g., osteosarcoma), IL-13R-a2 (e.g., glioma, glioblastoma, medulloblastoma), KDR (e.g., tumor neovasculature), K-light chain (e.g., B cell malignancies), Lewis Y (e.g., various carcinomas, epithelial-derived tumors), L1 -cell adhesion molecule (e.g., neuroblastoma), MAGE-A1 (e.g., melanoma), mesothelin (e.g., mesothelioma),MUC-1 (e.g., breast cancer, ovarian cancer), MUC16 (e.g., ovarian cancer), NKG2D ligands (myeloma, ovarian cancer, other tumors), NY-ESO-1 (157-165) (e.g., multiple myelomas, oncofetal antigen (h5T4) (e.g., various tumors), PSCA (e.g., prostate carcinoma), PSMA (e.g., prostate cancer, tumor vasculature), ROR1 (e.g., B-CLL, mantle cell lymphoma), TAG-72 (e.g., adenocarcinoma), and / or VEGF-R2 (tumor vasculature).

[0106] In one example, a scFv unit comprising the chimeric antigen receptor can comprise a-HER2 scFv (e.g., having a sequence with substantial identity to SEQ ID NO: 9), a-PSMA scFv (e.g., having a sequence with substantial identity to SEQ ID NO: 10) or a-CTLA-4 scFv (e.g., having a sequence with substantial identity to SEQ ID NO: 14).

[0107] In one embodiment, the immune effector cells can be administered to the subject via an effective route. An effective route is one that can provide for delivery of the cells to a desired compartment, system, or location in the patient. For example, an effective route is one through which the cells can be administered to provide, at a desired site of action, an amount of the cells sufficient to effectuate a beneficial or desired clinical result. Non-limiting examples of effective routes include intravenous, intraperitoneal, intracranial, intrathecal, oral, nasal, vaginal, anal, and intramuscular routes. In one example, the immune effector cells are delivered (e.g., via injection) directly into or adjacent a solid tumor in the subject.

[0108] In one embodiment, a therapeutically effective amount of engineered immune effector cells can be administered to the subject.

[0109] Engineered immune effector cells of the present disclosure can be formulated as a pharmaceutical composition.

[0110] In some instances, the purity of the cells for administration to a patient is about 100% (substantially homogeneous). In other instances, it is 95% to 100%. In further instances, it is 85% to 95%. If the administered cells are part of an admixture with other cells, the percentage can be about 10%-15%, 15%-20%, 20%-25%, 25%- 30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%, 60%-70%, 70%-80%, 80%-90%, or 90%-95%.

[0111] The choice of formulation for administering the cells for a given application will depend on a variety of factors. Prominent among these will be the species of subject, the nature of the disease or condition being treated, its state and distribution in the subject, the nature of other therapies and agents that are being administered (if any), the optimum route for administration, survivability via the route, the dosing regimen, and other factors that will be apparent to those skilled in the art . For instance, the choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form.

[0112] Final formulations of the aqueous suspension of cells / medium typically involves adjusting the ionic strength of the suspension to isotonicity (e.g., about 0.1 to 0.2) and to physiological pH (e.g., about pH 6.8 to 7.5). The final formulation can also contain a fluid lubricant.

[0113] In some embodiments, the cells can be formulated in a unit dosage injectable form, such as a solution, suspension, or emulsion. Pharmaceutical formulations suitable for injection of the cells can include sterile aqueous solutions and dispersions. Carriers for injectable formulations can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof.

[0114] The skilled artisan can readily determine the amount of cells and optional additives, vehicles, and / or carrier in compositions to be administered in methods of the present disclsoure. Typically, any additives (in addition to the cells) can be present in an amount of 0.001 to 50 wt % in solution, such as in phosphate buffered saline. The active ingredient can be present in the order of micrograms to milligrams, such as about 0.0001 to about 5 wt %, preferably about 0.0001 to about1 wt %, most preferably about 0.0001 to about 0.05 wt % or about 0.001 to about 20 wt %, preferably about 0.01 to about 10 wt %, and most preferably about 0.05 to about 5 wt %.

[0115] In some instances, the cells can be encapsulated for administration, particularly where encapsulation enhances the effectiveness of the therapy, or provides advantages in handling and / or shelf life. The cells, for example, can be encapsulated by membranes, as well as capsules, prior to administration. It is contemplated that any of the many methods of cell encapsulation known in the art may be employed.

[0116] In some instances, the cells can be incorporated into a polymer, such as a biopolymer or synthetic polymer. Examples of biopolymers include, but are not limited to, fibronectin, fibrin, fibrinogen, thrombin, collagen, and proteoglycans.Other factors, such as the cytokines discussed above, can also be incorporated into the polymer. In other instances, the cells may be incorporated in the interstices of a three-dimensional gel. A large polymer or gel, typically, will be surgically implanted. A polymer or gel that can be formulated in small enough particles or fibers can be administered by other common, more convenient, non-surgical routes.

[0117] Doses for humans or other mammals can be determined without undue experimentation by the skilled artisan, from this disclosure, the documents citedherein, and the knowledge in the art. The dose of the cells appropriate to be used in accordance with various aspects of the present disclosure will depend on numerous factors. The parameters that will determine optimal doses to be administered for primary and adjunctive therapy generally will include some or all of the following: the disease or condition being treated and its stage; the species of the subject, their health, gender, age, weight, and metabolic rate; the patient’s co-morbidities (if any); other therapies being administered; and expected potential complications from the patient’s history or genotype. The parameters may also include: whether the cells are syngeneic, autologous, allogeneic, or xenogeneic; the site and / or distribution that must be targeted for the cells to be effective; and such characteristics of the site such as accessibility to cells and / or engraftment of cells. Additional parameters include co-administration with other factors (such as growth factors and cytokines). The optimal dose in a given situation also will take into consideration the way in which the cells are formulated, the way they are administered, and the degree to which the cells will be localized at the target site(s) following administration.

[0118] In some instances, the cells may be administered in an initial dose, and thereafter maintained by further administration. The cells may be administered by one method initially, and thereafter administered by the same method or one or more different methods. The level of cells can be maintained by the ongoing administration thereof to the patient. The cells can be administered in many frequencies over a wide range of times. Generally, lengths of treatment will be proportional to the length of the disease process, the effectiveness of the therapies being applied, and the condition and response of the patient being treated.

[0119] In another embodiment, an inactive caged inducer or prodrug can be administered to the subject prior to, contemporaneous with, or subsequent to administration of the engineered immune effector cells.

[0120] In another aspect, the inactive, caged inducer can be allowed to be converted to the active inducer, whereafter the active inducer can be allowed to enter the administered immune effector cells and induce the CIP complex to express the therapeutic polypeptide and the chimeric antigen receptor.

[0121] It will be appreciated that the immune effector cells and the method described above can involve therapeutic constructs designed to target a variety of solid tumors such as, for example, a melanoma, a colon cancer tumor, a prostate cancer tumor, a breast cancer tumor, a lung cancer tumor, a skin cancer tumor, a liver cancer tumor, a bone cancer tumor, an ovarian cancer tumor, a pancreatic cancer tumor, a kidney cancer tumor, a brain cancer tumor, or a head and neck cancer tumor. In some cases, the immune effector cells and methods described herein also can involve constructs designed to target liquid tumors such as, for example, lymphoma.

[0122] It will also be appreciated that the immune effector cells of the present disclosure may be used for diagnostic or drug discovery purposes, e.g., to investigate the response of engineered immune effector cells to CIP inducer prodrugs when co-cultured with solid tumor cells.

[0123] Kits

[0124] Any of the compositions and systems described herein may be comprised in a kit. In some instances, immune effector cells of the present disclosure (e.g., engineered immune effector cells, e.g., CAR-T cells) can be provided in the kit, which also may include reagents suitable for expanding the cells, such as media,APCs, growth factors, antibodies (e.g., for sorting or characterizing the cells) and / or plasmids encoding certain cell components, such as transposase.

[0125] In a non-limiting example, an expression system of the present disclosure, one or more reagents to generate the expression system, cells (e.g., allogeneic or autologous immune effector cells) for transfection of the expression system, and / or one or more instruments to obtain allogeneic or autologous cells for transfection of the expression system (such an instrument may be a syringe, pipette, forceps, and / or any such medically approved apparatus).

[0126] In some embodiments, a kit can include, in addition to the foregoing components, or optionally, one or a combination of caged inducers or prodrugs.

[0127] In some aspects, the kit can comprise reagents or apparatuses for transfection of allogeneic or autologous immune effector cells.

[0128] The kits may comprise one or more suitably aliquoted compositions or systems of the present disclosure or reagents to generate compositions or systems of the present disclosure. The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits may include at least one vial, test tube, flask, bottle, syringe, or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit also will generally contain a second, third, or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. T he kits of the present invention also will typically include a means for containing the expression system of the present disclosure and any other reagent containers in close confinement for commercial sale. Such containers mayinclude injection or blow molded plastic containers into which the desired vials are retained, for example.

[0129] Kits of the present disclosure can additionally or optionally include an instructional material. An instructional material can include a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compositions, systems, and methods of the present disclosure. The instructional material of the kit can, for example, be affixed to a container which contains the compositions and / or systems (and / or components thereof) of the present disclosure or be shipped together with a container which contains the compositions and / or systems (and / or components thereof) of the present disclosure. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the composition and / or system of the present disclosure be used cooperatively by the recipient.

[0130] Exemplary Aspects

[0131] In view of the described compositions and methods and variations thereof, herein below are certain more particularly described aspects of the present disclosure. These particularly recited aspects should not, however, be interpreted to have any limiting effect on any different claims containing different or more general teachings described herein, or that the “particular” aspects are somehow limited in some way other than the inherent meanings of the language literally used therein.

[0132] Aspect 1 : A tumor therapy system, comprising: a caged inducer; and an immune effector cell, comprising: a first exogenous polynucleotide that is operably linked to a single viral vector, the first exogenous polynucleotide encoding: polypeptide components of a chemical induced proximity (CIP) complex; and atherapeutic polypeptide operably linked to a regulatory region inducible by an uncaged, activated form of the caged inducer; and a second exogenous polynucleotide that encodes a chimeric antigen receptor that specifically binds to an antigen.

[0133] Aspect 2: The system of Aspect 1 , wherein the caged inducer is an inactive prodrug of an inducer that is activatable by at least one signal to form the uncaged, activated form of the caged inducer.

[0134] Aspect 3: The system of any one of Aspects 1 -2, wherein the inducer of the caged inducer comprises at least one sensing moiety covalently attached thereto and configured to react with the at least one signal to generate the uncaged inducer.

[0135] Aspect 4: The system of any one of Aspects 1 -3, wherein the inducer is selected from the group consisting of abscisic acid (ABA), gibberellic acid (GA), rapamycin, and p-galactosidase.

[0136] Aspect 5: The system of any one of Aspects 1 -4, wherein the caged inducer comprises ABA conjugated with a nitroimidazole or nitrobenzyl group.

[0137] Aspect 6: The system of any one of Aspects 1 -5, wherein the caged inducer comprises ABA conjugated with galactose.

[0138] Aspect 7: The system of any one of Aspects 1 -6, wherein the at least one signal is a signal molecule or light.

[0139] Aspect 8: The system of any one of Aspects 1 -7, wherein the signal molecule is associated with a tumor microenvironment (TME).

[0140] Aspect 9: The system of any one of Aspects 1 -8, wherein the signal molecule is selected from the group consisting of NADPH, hydrogen ions, oxygen, hydrogen peroxide and iron (Fe2+).

[0141] Aspect 10: The system of any one of Aspects 1 -9, wherein the viral vector is a lentiviral vector.

[0142] Aspect 11 : The system of any one of Aspects 1 -10, wherein the lentiviral vector is a bidirectional N103 lentiviral transfer vector.

[0143] Aspect 12: The system of any one of Aspects 1 -11 , wherein the immune effector cell is a T-cell or a natural killer (NK) cell.

[0144] Aspect 13: The system of any one of Aspects 1 -12, wherein the antigen is a tumor antigen.

[0145] Aspect 14: The system of any one of Aspects 1 -13, wherein the regulatory region binds components of a CIP expression system.

[0146] Aspect 15: The system of any one of Aspects 1 -14, wherein the presence of the uncaged inducer in the immune effector cell induces assembly of the CIP expression system and expression of the therapeutic polypeptide.

[0147] Aspect 16: The system of any one of Aspects 1 -15, wherein the polypeptide encoded by the first exogenous polynucleotide comprises, 5' to 3', the following components, each of which is operably linked to one another: at least one upstream activating sequence (UAS); the regulatory region; the therapeutic polypeptide; a promoter selected from EF1 a or PGK; a transcriptional activation domain; a PYR mutant domain; a T2A domain; a GAL4 DNA binding domain; and an ABI domain.

[0148] Aspect 17: The system of any one of Aspects 1 -16, wherein the at least one UAS comprises a 5x UAS, the regulatory region comprises a minimal CMV promoter, the transcriptional activation domain comprises VP16AD, and the PYR mutant domain is one of PYR E141 L, PYR F61 L / A160C or PYR F61 L / E141 L / A160V.

[0149] Aspect 18: The system of any one of Aspects 1 -17, wherein the chimeric antigen receptor comprises a split chimeric antigen receptor.

[0150] Aspect 19: The system of any one of Aspects 1 -18, wherein the second exogenous polynucleotide further comprises: a first polynucleotide subunit encoding an antigen recognition domain and a first dimerizable adaptor protein; and a second polynucleotide subunit encoding an immune effector cell signaling domain and a second dimerizable adaptor protein; wherein, in the presence of at least one signal and the caged inducer, the first and second adaptor proteins are induced to dimerize and generate a functional split CAR.

[0151] Aspect 20: The system of any one of Aspects 1 -19, wherein the first polynucleotide subunit further encodes a hinge domain, a transmembrane domain, an intracellular co-stimulatory domain, and an optional epitope tag domain.

[0152] Aspect 21 : The system of any one of Aspects 1 -20, wherein the first adaptor protein comprises, 5' to 3', the following components, each of which is operably linked to one another: an a-PSMA scFV domain, a hinge domain, a transmembrane domain, a 4-1 BB domain, and a mutant PYR domain.

[0153] Aspect 22: The system of any one of Aspects 1 -21 , wherein the second polynucleotide further encodes a DNAX-activating protein (DNAP) domain, a transmembrane domain, an immunoreceptor tyrosine-based activation domain, an intracellular co-stimulatory domain, an optional hinge domain, and an optional epitope tag domain.

[0154] Aspect 23: The system of any one of Aspects 1 -22, wherein the second adaptor protein comprises, 5' to 3', the following components, each of which is operably linked to one another: a DNAP 10 domain, a transmembrane domain, a 4- 1 BB domain, an ABI domain, and a CD3 domain.

[0155] Aspect 24: The system of any one of Aspects 1 -23, wherein the first and second polynucleotide subunits are operably linked to the same or different expression vector(s).

[0156] Aspect 25: An isolated immune effector cell comprising: a first exogenous polynucleotide that is operably linked to a single viral vector and which encodes: polypeptide components of a chemical induced proximity (CIP) complex; and a therapeutic polypeptide operably linked to a regulatory region inducible by an uncaged, activated form of the caged inducer; and a second exogenous polynucleotide that encodes a chimeric antigen receptor that specifically binds to an antigen.

[0157] Aspect 26: The immune effector cell of Aspect 25, wherein the caged inducer is an inactive prodrug of an inducer that is activatable by at least one signal to form the uncaged, activated form of the caged inducer.

[0158] Aspect 27: The immune effector cell of any one of Aspects 25-26, wherein the inducer of the caged inducer comprises at least one sensing moiety covalently attached thereto and configured to react with the at least one signal to generate the uncaged inducer.

[0159] Aspect 28: The immune effector cell of any one of Aspects 25-27, wherein the inducer is selected from the group consisting of abscisic acid (ABA), gibberellic acid (GA), rapamycin, and p-galactosidase.

[0160] Aspect 29: The immune effector cell of any one of Aspects 25-28, wherein the caged inducer comprises ABA conjugated with a nitroimidazole or nitrobenzyl group.

[0161] Aspect 30: The immune effector cell of any one of Aspects 25-29, wherein the caged inducer comprises ABA conjugated with galactose.

[0162] Aspect 31 : The immune effector cell of any one of Aspects 25-30, wherein the at least one signal is a signal molecule or light.

[0163] Aspect 32: The immune effector cell of any one of Aspects 25-31 , wherein the signal molecule is associated with a tumor microenvironment (TME).

[0164] Aspect 33: The immune effector cell of any one of Aspects 25-32, wherein the signal molecule is selected from the group consisting of NADPH, hydrogen ions, oxygen, hydrogen peroxide and iron (Fe2+).

[0165] Aspect 34: The immune effector cell of any one of Aspects 25-33, wherein the viral vector is a lentiviral vector.

[0166] Aspect 35: The immune effector cell of any one of Aspects 25-34, wherein the lentiviral vector is a bidirectional N103 lentiviral transfer vector.

[0167] Aspect 36: The immune effector cell of any one of Aspects 25-35, wherein the immune effector cell is a T-cell or a natural killer (NK) cell.

[0168] Aspect 37: The immune effector cell of any one of Aspects 25-36, wherein the antigen is a tumor antigen.

[0169] Aspect 38: The immune effector cell of any one of Aspects 25-37, wherein the regulatory region binds components of a CIP expression system.

[0170] Aspect 39: The immune effector cell of any one of Aspects 25-38, wherein the presence of the uncaged inducer in the immune effector cell induces assembly of the CIP expression system and expression of the therapeutic polypeptide.

[0171] Aspect 40: The immune effector cell of any one of Aspects 25-39, wherein the polypeptide encoded by the first exogenous polynucleotide comprises, 5' to 3', the following components, each of which is operably linked to one another: at least one upstream activating sequence (UAS); the regulatory region; the therapeutic polypeptide; a promoter selected from EF1 a or PGK; a transcriptional activationdomain; a PYR mutant domain; a T2A domain; a GAL4 DNA binding domain; and anABI domain.

[0172] Aspect 41 : The immune effector cell of any one of Aspects 25-40, wherein the at least one UAS comprises a 5x UAS, the regulatory region comprises a minimal CMV promoter, the transcriptional activation domain comprises VP16AD, and the PYR mutant domain is one of PYR E141 L, PYR F61 L / A160C or PYR F61 L / E141 L / A160V.

[0173] Aspect 42: The immune effector cell of any one of Aspects 25-41 , wherein the chimeric antigen receptor comprises a split chimeric antigen receptor.

[0174] Aspect 43: The immune effector cell of any one of Aspects 25-42, wherein the second exogenous polynucleotide further comprises: a first polynucleotide subunit encoding an antigen recognition domain and a first dimerizable adaptor protein; and a second polynucleotide subunit encoding an immune effector cell signaling domain and a second dimerizable adaptor protein; wherein, in the presence of at least one signal and the caged inducer, the first and second adaptor proteins are induced to dimerize and generate a functional split CAR.

[0175] Aspect 44: The immune effector cell of any one of Aspects 25-43, wherein the first polynucleotide subunit further encodes a hinge domain, a transmembrane domain, an intracellular co-stimulatory domain, and an optional epitope tag domain.

[0176] Aspect 45: The immune effector cell of any one of Aspects 25-44, wherein the first adaptor protein comprises, 5' to 3', the following components, each of which is operably linked to one another: an a-PSMA scFV domain, a hinge domain, a transmembrane domain, a 4-1 BB domain, and a mutant PYR domain.

[0177] Aspect 46: The immune effector cell of any one of Aspects 25-45, wherein the second polynucleotide further encodes a DNAX-activating protein (DNAP)domain, a transmembrane domain, an immunoreceptor tyrosine-based activation domain, an intracellular co-stimulatory domain, an optional hinge domain, and an optional epitope tag domain.

[0178] Aspect 47: The immune effector cell of any one of Aspects 25-46, wherein the second adaptor protein comprises, 5' to 3', the following components, each of which is operably linked to one another: a DNAP 10 domain, a transmembrane domain, a 4-1 BB domain, an ABI domain, and a CD3£ domain.

[0179] Aspect 48: The immune effector cell of any one of Aspects 25-47, wherein the first and second polynucleotide subunits are operably linked to the same or different expression vector(s).

[0180] Aspect 49: A method for treating a solid tumor in a subject, the method comprising: administering, to the subject, at least one immune effector cell as recited in any one of Aspects 25-48; administering, to the subject, an inactive caged inducer, the inactive caged inducer being activatable to an active, uncaged inducer by at least one signal; allowing the inactive caged inducer to be converted to the active, uncaged inducer; and allowing the active inducer to enter the at least one immune effector cell and induce the CIP complex to express the therapeutic polypeptide and the chimeric antigen receptor.

[0181] Aspect 50: The method of Aspect 49, wherein the at least one immune effector cell is obtained from the subject.

[0182] Aspect 51 : The method of any one of Aspects 49-50, wherein the at least one signal is a signal molecule or light.

[0183] Aspect 52: The method of any one of Aspects 49-51 , wherein the signal molecule is associated with a tumor microenvironment.

[0184] Aspect 53: The method of any one of Aspects 49-52, wherein the signal molecule is selected from the group consisting of NADPH, hydrogen ions, oxygen, hydrogen peroxide and iron (Fe2+).

[0185] Aspect 54: An expression system comprising a first exogenous polynucleotide operably linked to a single viral vector and a second exogenous polynucleotide, wherein the first exogenous polynucleotide encodes: polypeptide components of a chemical induced proximity (CIP) complex; and a therapeutic polypeptide operably linked to a regulatory region inducible by an uncaged, activated form of the caged inducer; and wherein the second exogenous polynucleotide encodes a chimeric antigen receptor that specifically binds to an antigen.

[0186] Aspect 55: A method for delivering a therapeutic agent to a tumor cell comprising a solid tumor or a microenvironment associated with a solid tumor, the method comprising: administering, to the tumor cell, a composition comprising the expression system of Aspect 54; administering, to the tumor cell, an inactive caged inducer, the inactive caged inducer being activatable to an active, uncaged inducer by at least one signal; allowing the inactive caged inducer to be converted to the active, uncaged inducer; and allowing the active inducer to enter the tumor cell and induce the CIP complex to express the therapeutic polypeptide and the chimeric antigen receptor.

[0187] The following Examples are for the purpose of illustration only and are not intended to limit the scope of the claims, which are appended hereto.EXAMPLE 1

[0188] In this Example, the inventors provide a proof-of-concept, engineered T cell platform equipped with a therapeutic gene circuit that senses the local TME signal and subsequently activates the production of customized therapeutic geneproducts. The inventors demonstrated the integration of a chemically induced proximity (CIP) and associated signal sensing technologies with CAR to engineer an advanced class of CAR-T (CAR+T) cells. A / ?-ga / actos / dase-activatable prodrug was designed by conjugating a galactose moiety with a CIP inducer abscisic acid (ABA). The inventors validated that T cells engineered with the ABA-inducible genetic circuits can respond to cancer-associated ^-galactosidase to drive the production and secretion of various immunotherapeutics, including an anti-cancer agent, an immunomodulatory cytokine, and immune checkpoint inhibitors.

[0189] Materials and Methods

[0190] Synthesis of ABA-Gal

[0191] All starting reagents and solvents were directly used without any further purifications and air-sensitive reactions performed under Argon atmosphere. The progress of reactions was examined with the aid of thin layer chromatography (TLC) technique consisting of silica gel plate precoated with fluorescent indicator (254 nm) under UV light (260 nm). Purification of the compounds was carried out by column chromatography over silica gel (60-120 mesh, Sigma Aldrich) stationary phase. All1H and13C NMR spectra were recorded on a Bruker Avance lll-HD 500 NMR Spectrometer instrument, whereas high-resolution mass spectra (HRMS) were recorded on a Waters Micromass mass spectrometer with electrospray ionization probe using positive ion mode. The chemical shifts and apparent coupling constant values were reported in parts per million (ppm) and Hertz respectively. These chemical shifts were referenced from the TMS signal or deuterated residual solvents: CDCIs (7.26 ppm), CD3OD (3.31 ppm), DMSO- 6 (2.5 ppm), and D2O (4.79 ppm) for1H NMR spectra and CDCI3 (77.1 ppm), CD3OD (49.0 ppm), and DMSO-d6 (39.5 ppm) for13C NMR spectra. The1H NMR spin coupling multiplicities are listed suchas s (singlet), d (doublet), t (triplet), dd (doublet of doublets), and (q) quintet or m (multiplet and overlapping spin signal). ABA-Gal was synthesized following the reported procedures (Zaharia, L. I., et al. 2005. Journal of Labelled Compounds and Radiopharmaceuticals 48: 435-445).

[0192] Rf= 0.3 (DCM:MeOH = 9:1 ).1H NMR (500 MHz, DMSO-de): 5 7.73 (d, J=15.8, 1 H), 6.35 (s, =15.9, 1 H), 5.84 (s, 1 H), 5.76 (s, 1 H), 5.33 (d, =7.9 Hz, 1 H), 5.29 (s, 1 H), 5.07 (s, 1 H), 4.88 (s, 1 H), 4.67 (s, 1 H), 4.55 (s, 1 H), 3.68 (s, 1 H), 3.51- 3.34 (m, 5H), 2.1 1 (d, J=16.7, 1 H) 2.04 (s, 3H), 1 .8 (s, 3H), 1 .62 (s, 1 H), 0.96 (s, 3H), 0.93 (s, 3H);13C NMR (125 MHz, DMSO-d6): 5 197.7, 164.4, 163.5, 152.9, 139.6,127.4, 127.5, 1 16.9, 94.8, 78.9, 76.6, 73.7, 69.9, 68.4, 60.6, 49.7, 41 .8, 24.6, 23.6,21 .4, 19.3; HRMS (ESI): calcd for C21 H30O9 : [M + Na]+, 449.1782; found: [M + Na]+, 449.1787 (Am = +0.0005 and error = +1.1 ppm).

[0193] Chemical stability and reactivity of ABA-Gal towards fi-gai in vitro using HPLC

[0194] In-vitro stability test was done by incubating ABA-Gal in PBS buffer (pH 7.4) up to 48 hours at 37°C. HPLC data were obtained at Oh, 24h and 48h. The chromatograms were collected using a Dionex-UltiMate 3000 LC System with Acclaim 120 A, C18, 3 pm analytical (4.6 x 100 mm) column. Reactivity of ABA-Gal towards / ?-gal was performed by incubating 200 pM ABA-Gal with 150 pM ?-gal enzymes at 37°C for different time points. The reaction mixture was then quickly injected into HPLC and analyzed.

[0195] All HPLC analyses were run at RT and monitored at 260 nm. A gradient of Acetonitrile in 0.1 %TFA and water containing 0.1%TFA were used at a flow rate of 0.750 mL / min. Acetonitrile was increased from 5% to 75% in 15 min and kept constant for 3 min. Total running time is 20 min. HPLC chromatograms wereacquired using Dionex-UltiMate 3000 LC System with Acclaim 120 A, C18, 3 pm analytical (4.6 x100 mm) column.

[0196] Cellular stability of ABA-Gal and cleavage activity of / 3-gal in HEK-293T cells

[0197] Stock concentrations of ABA and ABA-prodrug were prepared in DMSO and further diluted in PBS for subsequent cellular experiments.

[0198] To test the cellular stability and reactivity of ABA-Gal, EGFP-expressing cells (HEK 293-GFP) were seeded in triplicate in 24-well plates (106cell / mL) at a final volume of 500 pL. 24 h after plating, cells were treated with either ABA, ABA- Gal, or ABA-Gal plus Agal at indicated concentrations. 24 h later, live cells were imaged with Axio Observer (Zeiss) fluorescence microscope under GFP channel (Chemistry department, University of New Mexico facility).

[0199] Cell lines and culture

[0200] Human embryonic kidney cell (HEK-293T) was obtained from Dr. Alex Huang’s lab (CWRU School of Medicine). The HEK-293T cell line was also used for lentiviral packaging and preparation. The reporter cell line EGFP-expressing human embryonic kidney (HEK-293T-EGFP) were generated and maintained in our laboratory. Human breast adenocarcinoma MDA-MB-231 was maintained in our lab. These cells were grown in Dulbecco's modified Eagle’s medium (DMEM) (Gibco) supplemented with 10%(v / v) heat-inactivated fetal bovine serum (FBS, Omega Scientific), 5%(v / v) GlutaMAX (Life Technologies) and 100 lU / ml penicillin / streptomycin (Life Technologies). Jurkat T cells, clone E6-1 were purchased from American Type Culture Collection (ATCC), and cultured in complete RPMI-1640 containing 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg / L glucose, and 1500 mg / L sodium bicarbonate, supplemented with 10%heat-inactivated fetal bovine serum (FBS) and 100 lU / ml penicillin / streptomycin. All cultures were maintained at 37°C in a humidified incubator containing 5% CO2.Cells were confirmed mycoplasma-free using MycoAlert™ mycoplasma detection kit (Lonza).

[0201] Plasmid construction

[0202] Construction of the ABA-split transcriptional activator construct sv40- VP16-PYR*-ires / T2A-Gal4DBD-ABlDi34A has been described previously (Chen, J. et al. 2023. Chemical science 14: 3377-3384). The ABA-inducible customized therapeutic constructs were made by inserting different therapeutic genes under the control of a minimal IL-2 (or minimal CMV) gene promoter and five copies of Gal4 DNA binding sites (CGGAGTACTGTCCTCCGAG) (SEQ ID NO: 11). Therapeutic genes used in this study are Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL), Interleukin-12, immune checkpoint inhibitors Pembrolizumab mAb and a-CTLA-4 scfv. Sources of the therapeutic genes are listed in Table 1 .Table 1 : Components of HER2-CAR and therapeutic protein plasmids

[0203] The conventional CAR construct targeting HER2 antigen comprising CD8a signal sequence-Myc tag-aHer2 scFv-CD8aHinge-CD8aTM-41 BB-CD3<; was designed and synthesized as gBIock gene fragments (IDT). The vector components are described in Table 1 . The genes were cloned into a 2ndgeneration N103 lentiviral transfer vector (Figs. 27A-B), under the control of an EF-1 a promoter using In-fusion cloning (Takara Bio). The expression cassettes for the ABA-split transcriptional activator (VP*2GA) and therapeutic gene are cloned into a bidirectional N103 lentiviral vector. The vector contains the VP*2GA is placed under the control of PGK or EF1 oc promoter while the response element (5xllAS) controlling the expression of customized therapeutic proteins (described previously) was inserted upstream of PGK or EF-1 a promoter, under the control of a minimal CMV promoter. The wildtypelentiviral genome is -9.2 kb, therefore all transgenes were made within the viral packing limit (3-9 kb) for efficient viral production.

[0204] All plasmid constructs were amplified using Stbl3 chemically competent E.coli strain and purified using Endo-free Qiagen Miniprep kit. DNA sequences of the final constructs were confirmed by Sanger sequencing (Genewiz). Plasmid concentration and purity (A 260 / 280 = 1 .88-1 .90) were measured by a Nanodrop spectrophotometer (Thermofisher).

[0205] Production of lentivirus

[0206] Lentivirus was produced by transient transfection into HEK-293T cells.One day before transfection, cells were plated at a density of 7 x 106cells per 150 mm diameter tissue culture plate in 30 mL DMEM, supplemented with 10%(v / v) FBS and 5%(v / v) GlutaMAX (Life Technologies), and cultured at 37°C in a 5% CO2 incubator. Cells were transfected with 2ndgeneration packaging plasmids system (gifted by Crabtree's lab) at -80% confluence. The transfection complex was prepared in two separate 1 .7 mL tubes. A mixture of 6 pg transfer plasmid containing corresponding GOI, 1 .5 pg ps.PAX2 (packaging plasmid), 4.5 pg pMD2.G (envelope plasmid) were mixed in 120 pL Opti-MEM (Gibco) (tube A) and 36 pL of polyethyleneimine (PEI) transfection reagent (Polysciences) were diluted in 444 pL of Opti-MEM (tube B). The content of tube A and tube B alone were incubated at RT for 5 min before combining into a single tube, followed by another 20-min incubation. The transfection complex was added into the cells and media was replaced with complete DMEM 6 hours post-transfection. The culture supernatant containing viral particles was harvested at 48-hour post-transfection, and filtered through a 0.45 pm syringe filter (VWR). Viral particles were concentrated by ultracentrifugation at100,000 x g at 4°C for 100 min with a Sorval LYNX T29-8x50 Rotor (Thermo Fisher)and resuspended in 500 pL of complete FtPMI medium. Stock viral supernatant was either used directly or aliquoted and stored in a -80°C freezer until ready for use.

[0207] Transduction of Jurkat T cells

[0208] For transduction of Jurkat T cells, an appropriate amount of the pooled virus was added to Jurkat T cells (106cells / ml) in the presence of 8 pg / mL polybrene (Santa Cruz Biotech) in a non-treated tissue culture 24-well plate, and the plate were centrifuged at 1000 x g at 33°C for 2 hours (spinoculation). The media was changed to complete fresh RPMI and the cells were cultured at 37°C and 5% CO2 for at least 3 days before subsequent experiments. Fresh media was added to the well frequently to maintain a cell density of 0.5-2 x 106cells / ml.

[0209] Apoptosis assay and quantification of secreted TRAIL

[0210] For sTRAIL apoptotic assays, HEK-293T cells were transiently transfected to express the new potent ABA-responsive split transcriptional activator and 5xGal4 response elements controlling the expression of sTRAIL. Cell culture supernatant containing sTRAIL was collected after 24 h treated with either ABA 1 pM or mock DMSO and incubated with TRAIL-sensitive MDA-MD-231 cancer cell lines for another 24 h. Cell viability and apoptosis were accessed using the Dead Cell Apoptosis Kit with Annexin V-FITC and PI, for flow cytometry according to the manufacturer’s protocol. Briefly, cancer cells were harvested by trypsinization at 24 h after drug treatment and were washed twice with cold PBS. Then 106cells, suspended in Annexin V binding buffer, were incubated with Propidium Iodide and FITC-Annexin V to a final concentration of 1 pg / mL, 1 :20 volume ratio, respectively, for 15 minutes at room temperature in the dark. Following which, 400 pl Annexin-Binding Buffer was added and the stained cells were immediately analyzed by flowBD Accuri™ C6 cytometer cytometry (BD Biosciences). The concentration ofsecreted TRAIL in the supernatant was determined via human TRAIL PicoKine™ELISA kit.

[0211] Signal-induced IL- 12 production and quantification

[0212] Human Jurkat T cells were transduced with either a therapeutic-encoding vector alone or with conventional CAR and treated with small molecule or small molecule plus TME signals to induce the production and secretion of IL-12 into the cell culture media. At the indicated time point, the cell culture supernatant was harvested and stored for analysis at -80 °C or analyzed directly using ELISA kit (Biolegend). One day before the assay, 96-well ELISA plate was coated with human IL-12 specific capture antibody and stored overnight at 4°C. After blocking plate for 1 hour, the ELISA was performed according to the manufacturer’s instructions.Human IL-12 standards were used to generate a standard curve of range 0 - 500 pg / mL. Absorbance was recorded at 450 nm and 570 nm on a microplate reader (Spectramax i3X, CWRU Core Facilities). Data were analyzed with GraphPad Prism software.

[0213] Signal-induced immune checkpoint antibodies production and quantification

[0214] Flow cytometry-based binding assay was used to detect secreted immune checkpoint antibodies in the cell culture supernatant. Jurkat T cells were transduced to inducibly express ICP antibodies including pembrolizumab, or a-CTLA-4 scfv.Transduced cells were treated with ABA or PBS (as control) for 24 hours to induce the production of antibodies. Cell culture supernatant containing the secreted antibody was harvested and stored at -80°C for analysis. To detect secreted a- CTLA-4 scfv, HEK-293T cells overexpressing human CTLA-4 were used as “target cells”. Target cells were incubated with culture supernatant containing secreted a-CTLA-4 scfv for 1 hour at RT, followed by staining with fluorescence-conjugated a- Myc to detect bound antibodies. Antibody-labeled target cells were then analyzed by flow cytometry to quantify the fluorescence intensity, which correlated with amount of antibody produced by CAR+T cells.

[0215] Secretion and binding of pembrolizumab (a-PD-1 ) to PD-1 receptor on T cells surface were accessed using competitive binding assay. CAR+T cells were incubated with HER2 / PDL1 -positive target at E:T= 5:1 and small molecule or prodrug plus activating signal was added to the cell culture. At 24 hours, T cells were harvested and stained with FITC-conjugated a-PD1 antibody (Biolegend, dilution 1 :100) to measure the percentage of positive PD1 -T cells, which correlated with the amount of free PD-1 receptor. Direct detection of Pembrolizumab was also performed using A647-conjugated anti-HA antibody (CST, dilution 1 :50). Data acquisition was performed with BD Accuri™ C6 Plus Flow Cytometer (BD Biosciences) and analyzed with FlowJo software (v10, TreeStar).

[0216] Flow cytometric analysis

[0217] For flow cytometric analysis, cells were washed and prepared in ice-cold FACS buffer (1 x DPBS, 0.5% FBS, 2.5 mM EDTA). Surface expression of HER2 antigen on various target cell lines was confirmed by analysis of cells stained with PE-conjugated anti-human CD340 (Biolegend, dilution 1 :100). Expression of CAR was quantified by staining transduced T cells with A488-conjugated anti-Myc antibody (CST, dilution 1 :50). Surface expression of CD69 was detected by staining activated T cells with APC-conjugated anti-human CD69 (Biolegend, dilution 1 :100). Cells were incubated with fluorophore-conjugated antibodies for 20 min at 4°C in the dark and washed twice before resuspension with FACS buffer. The percentage of positive cells was calculated by gating on the live cells (based on side and forwardscatters), then singlets (based on Area vs. Height), followed by gating on the fluorescent-labeled population. Data acquisitions were performed with BD Accuri™ C6 Plus Flow Cytometer (BD Biosciences) and analyzed with FlowJo software (v10, TreeStar).

[0218] Statistical analysis

[0219] Data are represented as mean ± standard deviation (SD) or standard error (S.E.M). Statistical significance was calculated in GraphPad Prism (GraphPad software, San Diego, CA) using two-tailed unpaired Student’s t-test for two independent groups, and ANOVA for multiple groups comparison.

[0220] Results

[0221] Engineering ABA-inducible therapeutics expression system in HEK-293T cells

[0222] To develop CAR+T cells capable of locally delivering desired therapeutic effects, we first constructed an ABA-inducible gene expression system controlling the expression of a therapeutic gene encoding secreted tumor necrosis factor- related (TNF) apoptosis-inducing ligand (sTRAIL) (Figs. 2A-B). TRAIL is a selective inducer of apoptosis in many transformed cells bearing Death Receptor and has been a promising candidate for cancer therapy. The ABA-inducible gene expression cassette with an enhanced ABA potency incorporating the PYR mutant (PYR*) has been developed in our previous work (Chen etal., 2023). We co-transfected HEK- 293T cells with the plasmids of ABA-inducible sTRAIL and the split transcriptional activator (VP16AD-PYR*-T2A-GAL4DBD-ABI) for 24 h, and then treated cells with or without ABA. At 24 h post-treatment, the secretion of sTRAIL in culture media was quantified by the enzyme-linked immunosorbent assay (ELISA). Cells that are treated with ABA secreted a high level of TRAIL as shown in Fig. 2C. To validate thebiological activity of secreted TRAIL, MDA-MD-231 cells (expressing Death Receptor) were incubated with culture media containing ABA-induced sTRAIL or commercial recombinant human TRAIL for 24 h, and the death of target cells (as a result of apoptosis) was measured by flow cytometry via Annexin V-FITC staining. Propidium Iodide (PI) staining was used to quantify necrotic cells and differentiate from the apoptotic cells. We observed an increase of apoptotic cells when cancer cells were incubated with ABA-induced sTRAIL, which showed comparable efficacy as the case when treated with commercial recombinant human TRAIL (Fig. 2D). These results confirmed the feasibility of this ABA inducible expression system to produce antitumor biologies.

[0223] We next constructed the inducible gene expression system in the lentiviral vector for more effective transgene delivery in human T cells. A single lentiviral vector was constructed by combining the split transcriptional activator and the inducible gene expression unit. The ABA-responsive split transcriptional activator was cloned into a 2ndgeneration pLV lentiviral expressing vector (N103) downstream of the PGK promoter. The 5x upstream-activating sequence (UAS) response element with a minimal CMV promoter controlling the expression of therapeutic genes were placed before the PGK promoter in the same vector. The whole transgene was flanked by the two Long Terminal Repeat (LTR) elements (Fig. 3A). To validate the lentiviral vector design of the inducible therapeutic gene circuit, we constructed the inducible EGFP reporter gene version (N103-iEGFP) to monitor the ABA-inducible EGFP expression using a fluorescence microscope. The N103- iEGFP was transiently transfected into HEK-293T cells, and the cells were treated with or without ABA. At 24 h post-transfection, we observed EGFP expression uponABA treatment (Fig. 3B), indicating the successful construction of the lentiviral vectors.

[0224] ABA-induced expression and secretion of inflammatory cytokine Interleukine- 12 in human Jurkat T cells

[0225] We next examined whether T cells could be engineered to secrete customized cytokines capable of modulating immune responses. Long-term persistence and expansion of CAR-engineered T cells are required to effectively combat cancer. To overcome tumor suppressive effects, strategies to potentiate the therapeutic activities of CAR-T cells by engineering CAR-T cells that produce nonnative cytokines upon tumor antigen engagement have been employed. Our strategy, in contrast, was to design CAR-T cells that can drive custom therapeutic responses in an antigen-independent manner through a TME-sensing approach.

[0226] As a hallmark of inflammatory cytokines, Interleukin 12 (IL-12), a heterodimeric protein composed of p35 and p40 subunits, plays an important role in bridging innate and adaptive immunity. IL-12 is mainly produced by innate immune cells such as macrophages, dendritic cells, and neutrophils, and has been known as a critical regulator of cell-mediated immunity. Various pre-clinical models have demonstrated that IL-12 modulates antitumor responses at various levels, for example, through the induction of TH1 differentiation, sustaining survival and reactivation of CD4-T cells, and enhancing cytolytic activity of NK cells and CD8 T cells. Local production of this therapeutic agent therefore will activate innate immune cells, which in turn will maximize the antitumor response in the targeted tumor lesion while avoiding substantial systemic toxicities associated with the high serum level of IL-12 observed in clinical trials.

[0227] With that in mind, we generated a lentiviral vector for inducible IL-12 expression and secretion upon ABA treatment by replacing EGFP from the N103- iEGFP with the IL-12 gene ( / .e., PGK / T2A in Fig. 4A). We explored different ways of designing the constructs to obtain the best signal-to-noise induction of the therapeutic gene expression. In one version, the 2A sequence was replaced by an internal ribosome entry site (IRES), and still driven by the PGK promoter ( / .e., PGK / IRES, Fig. 4A). The 2A-linked genes are expected to be expressed at an equal level, while the IRES-mediated bicistronic vector expressing the proteins at different levels, with a lower expression level in the IRES-driven gene. Additionally, the 2A elements are much shorter than IRES, with only 60 to 80 base pairs, which should increase the viral packaging efficiency. Another version was made by replacing PGK with the human elongation factor-1 alpha (EF1 a) promoter ( / .e., EF1a / T2A, Fig. 4A), which is commonly used in viral vector expression systems. We tested the ABA CIP-controlled inducibility in the secretion of IL-12 in human Jurkat T cells using the ELISA assay. These lentiviral vectors were transiently transfected into HEK-293T cells along with two other helper plasmids ( / .e., packaging and envelope) to generate lentiviruses, which were then used to infect Jurkat T cells. At day 4 posttransduction, transduced T cells were treated with or without ABA for 24 h and the amount of IL-12 secreted in the cell culture media was quantified by ELISA.Comparing these three designs, we observed that the PGK / IRES version failed to induce IL-12 production upon ABA treatment as compared to the PGK / T2A version under the control of the same promoter (Fig. 4B), which may be due to the differential expression of the two split transcriptional activator components in the IRES case. The EF1 o / T2A version resulted in the most optimal inducibility with minimal basal transcriptional activity, which we identified as the most ideal design tomove forward. We therefore adopted this vector configuration for constructing other therapeutic protein expression vectors. Additionally, the ABA-based CIP system has been reported to give dosage-dependent controls in biological outputs. To examine whether the amount of IL-12 expression is titratable by varying ABA concentrations, Jurkat T cells were transduced with inducible IL-12-encoding EF1 a / T2A lentivirus and treated with ABA at different concentrations ranging from 0-1000 nM for 24 h before quantifying IL-12 in the media using ELISA. We observed that there was a clear ABA-dependent dosage response with a maximum induction at around 250 nM (Fig. 4C). This system can potentially provide an important tool to fine-tune the secreted IL-12 dosage to achieve substantial efficacy without severe adverse effects.

[0228] Next, we tested the efficiency of generating CAR+T cells with both CAR and ABA-inducible therapeutic gene expression lentiviral vectors. We first constructed a 2ndgeneration CAR vector targeting human epidermal growth factor receptor 2 (HER2 / ErbB2). HER2 is a well-characterized antigen and therapeutic target expressed in a variety of tumors ranging from breast cancers, ovarian cancers, osterosacomas, and also expressed at low levels in normal tissues. The HER2-CAR lentiviral vector comprises a HER2-targeted scFv sequence derived from the humanized mAb trastuzumab (4D5-5 clone) (Liu, X. et al., Cancer research 2015 , 75, 3596-3607), a hinge and transmembrane domain of human CD8a molecule, a costimulatory 4-1 BB and an intracellular signaling CD3^ domains, driven by the EF1 a promoter (Fig. 10A). The CAR also contains a N-terminal CD8a signal peptide responsible for membrane targeting, and a Myc tag at the N-terminus for the quantification of surface expression of CAR. We first evaluated the surface expression of HER2-CAR on Jurkat T cells by transducing Jurket T cells with the HER2-CAR lentiviral vector and quantified the HER2-CAR expression through theMyc tag using flow cytometry. We confirmed the effective surface expression of HER2-CAR on Jurkat T cells on day 4 post-transduction (Fig. 10B). We also validated the function of HER2-CAR by stimulating HER2-CAR-engineered T cells with target cell lines expressing different levels of HER2 (Fig. 10C). The activation of the engineered T cells under each co-incubation condition was determined via the surface expression of activation maker-CD69 and the secretion of IL-2 cytokine (a critical and early landmark of T cells activation). We confirmed that the HER2-CAR T cells were only activated in the presence of HER2-expressing cells (Fig. 10D).

[0229] Next, the HER2-CAR and ABA-inducible IL-12 expression circuits were delivered into Jurkat T cells by two individual lentiviral vectors (Fig. 5A). The integrated provirus sequence between the two LTRs is about 3.5 kb and 6 kb for HER2-CAR and inducible IL-12 vector, respectively, which is within the viral packaging limit. Transduced Jurkat T cells either expressed the inducible IL-12 gene only (ilL-12), or the inducible IL-12 gene plus HER2-CAR (CAR+ilL-12) for 4 days and were then treated with ABA for 24 h before the cell culture supernatant was harvested and analyzed for the secretion of IL-12 by ELISA. We observed similar amounts of IL-12 production from both groups, indicating comparable and satisfactory efficiencies in the production of IL-12 even when co-transducing dual constructs in CAR+T cells (Fig. 5B). Furthermore, we examined the expression of HER2-CAR when co-transduced with the IL-12 expression circuits and observed a satisfactory transduction efficiency (Fig. 11 A).

[0230] ABA-induced expression and secretion of immune checkpoint (ICP) inhibitors in engineered Jurkat T cells

[0231] To counteract the negative effect of TME on CAR-T function, combining CAR-T and ICP blockades has been proven an efficacious treatment approachagainst solid tumors. ICP inhibitors, delivered either through the constitutive expression by CAR T cells, or systemic administration, come with several adverse effects. Toxicities associated with ICP blockades include but are not limited to the increased activation of autoreactive T cells due to systemic blocking of ICP pathways. A potential way to circumvent current systemic toxicities while enhancing the effectiveness of ICP inhibitors is to have T cells locally produce these therapeutics in tumors. Amongst the ICP inhibitors developed so far, blocking programmed cell death protein 1 (PD-1 ) expression on activated T cells using the clinically approved monoclonal antibody Pembrolizumab (a-PD1 mAb) has achieved remarkable successes in treating various solid cancers and lymphomas. PD-1 binds programmed death ligand 1 (PD-L1 ) that is expressed on many tumor cells, resulting in the suppression of T cell proliferation and cytokine production and correlating with the expression of “T-cell exhaustion” markers such as LAG-3, TIM-3, and CD160. Thus, we developed CAR+T cells capable of secreting Pembrolizumab (hereafter “Pembro”) directed against PD-1 in response to ABA induction. To test the production of Pembro, we used a cell-based flow cytometry binding assay in which the secreted Pembro binds to the target cell surface antigen PD-1 , and the bound Pembro can then be analyzed by flow cytometry. Pembro was cloned into the ABA- inducible lentiviral vector, which also contains an HA tag for detection purposes (Fig. 6A). Jurkat T cells were transduced by lentiviruses to co-express the HER2-CAR and the inducible Pembro (CAR+iPembro), and the transduced cells were coincubated with HER2-expressing MDA-MB-231 cancer cells, which triggered PD-1 expression on the activated CAR-T cells. These cells were treated with 500 nM ABA, or PBS (as a control) for 24 h, and the secretion and binding of Pembro to PD- 1 was then detected using flow cytometry by staining T cells with fluorophore-conjugated a-HA antibodies (ct-HA Alexa Flour 647). The increase of HA fluorescence signal indicated the expression and secretion of Pembro and its binding to PD-1 on the CAR+T cells only in the presence of ABA, but not PBS (17% vs. 1 %) (Fig. 6B). We also confirmed the satisfactory expression of HER2-CAR when cotransduced with the inducible Pembro expression circuits (Fig. 1 1 B). To confirm that the secreted Pembro specifically bound to PD-1 on T cells, we performed a competitive binding assay using a commercially available a-PD1 antibody (clone EH12.2H7) to measure the levels of free PD-1 on the cell surface. The commercial OC-PD1 antibody has been shown to bind to the same epitope on PD-1 as Pembro, therefore, any binding between Pembro and PD-1 will interfere with the binding of the commercial a-PD1 to PD-1 . CAR+iPembro-T cells treated with either ABA or PBS for 24 h were stained for PD-1 using FITC-conjugated a-PD1 antibody (a-PD1 FITC). The percent of PD-1 -positive cells was determined by flow cytometry. As shown in Fig. 60, the percentage of PD-1+T cells was significantly lower in the ABA- treated group, suggesting that Pembro was present and occupied PD-1 on the T cell surface, which blocked the interaction between the commercial a-PD1 antibody and PD-1 . It was also observed that CAR+T cells without co-cultured with HER2+tumor cells did not express PD-1 due to lack of tumor-mediated T cell activation.

[0232] Another ICP inhibitor of interest is a-CTLA4 antibodies that targets CTLA- 4 / B7 checkpoint pathway. CTLA4 checkpoint molecules are primarily expressed by T cells, and compete with CD28 for the binding of the B7-family costimulatory molecules. This competitive binding can block the costimulatory signal required for T cells activation, resulting in the inhibition of T cell proliferation and survival. To express iCTLA4 scFv, a Myc-tagged scFv version of a-CTLA4 monoclonal antibodies (derived from Ticilimumab) was cloned into the ABA-inducible therapeuticgene vector (Fig. 7A). Jurkat T cells transduced to express both HER2-CAR and the ICTLA4 scFv (CAR+ICTLA4 scFv) were treated with or without 500 nM ABA for 24 h to induce the expression and secretion of a-CTLA4 scFv, which was detected by a flow cytometry-based binding assay. We overexpressed CTLA4 antigen on HEK- 293T cells so that these cells can serve as “target cells” for the binding assay (Fig. 7B). Cell culture supernatant containing the secreted antibodies from CAR+T cells was incubated with surface CTLA4 antigen-positive or negative target HEK-293T cells, followed by staining the target cells with Alexa Flour 647-conjugated a-Myc to detect bound antibodies. The fluorescence intensity correlates with the amount of a- CTLA4 scFv produced by the CAR+T cells. As shown in Fig. 7C, CAR+T cells produced a high level of the a-CTLA-4 scFv when ABA was added (32.8% vs. 0.7%) as detected on CTLA4+ target cells, and the a-CTLA-4 scFv selectively bound only to antigen-expressing cells (32.8% vs. 0.2%). We also confirmed the efficient expression of HER2-CAR when co-transduced with the inducible a-CTLA4 scFv expression circuits (Fig. 11 C). Taken together, these data show that ABA can effectively trigger engineered CAR+T cells to express different immune checkpoint inhibitors.

[0233] Jurkat T cells equipped with CAR and conditional gene circuit can respond to [3-gai to drive the expression and secretion of customized therapeutics

[0234] The unique design in our strategy is to use cancer-associated signals to induce the local activation of the inducer small molecule ABA, which subsequently triggers the tumor-targeted production and release of therapeutics and immunomodulators in situ to reduce systemic toxicities and enhance the therapeutic efficacy of the engineered T cells. To achieve this goal, we designed and synthesized a caged ABA-prodrug (ABA-Gal) that is sensitive to tumor-associated fi-galactosidase ( 5-gal) as a proof-of-principle study (Fig. 8A). Deuterium-ABA-Gal was previously synthesized to study plant hormone profiling. We synthesized ABA- Gal following the reported procedures and used High Performance Liquid Chromatography (HPLC) to assess the chemical stability and reactivity of the ABA- Gal compound toward the recombinant / 3-galactosidase enzyme in vitro. 200 pM ABA-Gal in PBS was incubated at 37°C for up to 2 days, followed by HPLC analyses. We observed that the prodrug was stable in buffer during the observation period, ^-gal-induced cleavage of ABA-Gal was also confirmed by incubating 200 pM ABA-Gal with 150 pM recombinant ?-gal at 37°C. We found that the compound was quickly uncaged by / 7-gal to generate ABA within 30 minutes (Fig. 8B). Using HEK293T-EGFP reporter cell line, which can respond to ABA and express EGFP, we observed that ABA-Gal was stable within the 24-h observation period, while the addition of recombinant / 3-gal resulted in the induction of EGFP expression (Fig. 8C), indicating the release of functional ABA in the cell culture.

[0235] We next examined if this prodrug strategy can be integrated into the CAR+T cell therapeutic platform that allows engineered T cells to analyze and respond to unique TME signals and activate custom immunomodulating programs. Jurkat T cells were transduced to express HER2-CAR along with different ABA-inducible gene cassettes as described above, followed by the treatment with 500 nM ABA, or 500 nM ABA-Gal with or without 100 nM / 3-gal for indicated time periods. Using the same assays developed for testing each therapeutic protein as described above, we observed that the production and secretion of IL-12, Pembro or a-CTLA4 scFv were only detected when cells were treated with ABA or with ABA-Gal plus (3-gal, suggesting that / 3-gal reliably activated ABA-Gal to produce ABA, which in turn triggered the production of therapeutic proteins (Figs. 9A-C). These data establishedthat the TME signal can trigger engineered CAR+T cells to express different therapeutic and immunomodulatory modalities.EXAMPLE 2

[0236] In this Example, the inventors report a novel design that combines the existing chemically-induced proximity (CIP) technology with the prodrug strategy to engineer an advanced class of CAR-T cells with additional layers of control that allowed T cells to simultaneously sense multiple tumor-specific properties including tumor-associated antigen and TME signal (e.g., hypoxia), therefore the activation of T cells can be highly tumor site-restricted (Fig. 12A). The CIP platform in this study used abscisic acid (ABA) - a small water-soluble chemical inducer - to selectively trigger the heterodimerization of two plant-derived adaptor proteins ( / .e., ABI and PYL / PYR) that can be genetically fused to two proteins of interest (e.g., two inactive halves of the split CAR) to control tailored cellular output (e.g., T cells activation). In our design, ABA was caged and inactivated with unique sensing moieties that can only be removed by specific TME signals to reveal active free ABA. As a result, the TME-iCAR-T cells can only be activated by the co-presence of the chosen tumor antigen and TME-restricted signal, while remaining inactive in normal tissues that lack the right combination of the activating factors. Here, we show that the engineered T cells exhibit similar therapeutic activity as conventional CAR and that the antitumor response of the TME-iCAR-T cell is strictly dependent on the combinatorial triggering inputs including tumor antigen, small molecule, and TME signal.

[0237] Materials and Methods

[0238] Synthesis of hypoxia-activated ABA-prodrugs

[0239] The oxygen-sensitive ABA-prodrugs were synthesized using our previously reported method with slight modifications (Wright, CW et al., Chembiochem : a European journal of chemical biology, 16, 254-261 , 2015). Two nitroaromatic derivatives- nitroimidazole and nitrobenzyl were conjugated at the C- terminal to the carboxylic acid group of ABA via cleavable linkers to generate prodrug 1 and 2, respectively, as discussed below.

[0240] (+)-Abscisic acid (ABA) was purchased from Gold Biotechnology whereas other reagents and solvents were obtained from AA blocks, Sigma, and Thermos Fisher. These chemicals and solvents were directly used without any further purification. NMR and mass spectra of all synthesized compounds were recorded on 500 MHz Bruker Ascend Avance III HDTMand Electrospray Ion Trap MS: THERMO LCQ DECA at the Department of Chemistry facilities, Case Western Reserve University. The reaction progress was monitored under UV by thin-layer chromatography (TLC) plates precoated with silica gel and fluorescent indicators. The purifications of all compounds were carried out by column chromatography using silica gel (60-325 mesh) and a hexane / ethyl acetate solvent system. The chemical shift values in parts per million (ppm) were recorded downfield from the TMS signal (0 ppm) and referenced to the TMS signal or deuterated residual solvent (CDCI3): 7.26 ppm for1H and 77.2 ppm for13C NMR spectra.1H NMR spin coupling multiplicities are stated as s (singlet), d (doublet), t (triplet), dd (doublet of doublets), and m (multiplet and overlapping spin systems). The apparent coupling constant (J) values are mentioned in hertz (Hz).

[0241] Synthesis of prodrug 1 : To a stirred solution of ABA (0.220 g, 1 mmol) in acetonitrile (5 mL), CS2CO3 (0.975 g, 3 mmol) and compound 2 (0.264 mg, 1 mmol) were added and the reaction mixture was stirred at room temperature for overnight.After completion of the reaction, excess ACN was removed by vacuum and the residue was diluted in water (10 ml_). The compound was extracted with ethyl acetate (2 x 50 mL) and the combined organic phase was washed with brine solution (2 x 10 mL). The excess solvent was evaporated and dried over anhydrous Na2SC>4 and the crude compound was purified by chromatography (Hexane / Ethyl acetate = 1 / 1 ) over silica gel to afford the desired product as a light-yellow solid (0.210 g, 5%).1H NMR (500 MHz, CDCI3) 5 7.86 (d, J = 15.8 Hz, 1 H), 7.26 (s, 1 H), 6.23 (d, J = 16.0 Hz, 1 H), 5.97 (s, 1 H), 5.76 (s, 1 H), 5.2 (d, 7= 16.1 Hz, 2H), 4.06 (s, 3H), 2.49 (d, J = 16.0 Hz, 1 H), 2.30 (d, J = 17.0 Hz, 1 H), 2.07 (s, 3H), 1.96 (s, 1 H), 1.93 (s, 3H), 1.14 (s, 3H), 1.09 (s, 3H).13C NMR (126 MHz, CDCI3) 5 197.62, 164.87, 162.1 , 151.9, 146.32, 137.55, 132.4, 129.7, 127.95, 127.35, 1 16.85, 54.49, 49.83 41 .65, 34.47, 24.47, 23.17, 21 .46, 19.0. MS (ESI): calculated for C20H25N3O6 [M + Na]+426.2, found 426.2.

[0242] Synthesis of prodrug 2: To a stirred solution of ABA (0.264 g, 1 mmol) in acetonitrile (3 mL), CS2CO3 (0.975 g, 3 mmol) and 4-nitrobenzyl bromide (0.216 g, 1 mmol) were added. The reaction was stirred at room temperature overnight, after which the solvent was removed by vacuum and the residue was diluted in water (10 mL) and extracted with ethyl acetate (2 x 10 mL). The combined organic phase was washed with brine solution (2 x 10 mL) and dried over Na2SC>4. The solid was filtered off and the filtrate was concentrated under vacuum. The residue was purified by column chromatography (Hexane / Ethyl acetate = 1 / 1 ) over silica gel to give the desired product as a yellow solid (0.256 g, 64%).1H NMR (500 MHz, CDCI3) 5 8.30 - 8.20 (m, 2H), 7.91 (d, J = 16.0 Hz, 1 H), 7.56 (d, J = 8.4 Hz, 2H), 6.22 (d, J = 16.0 Hz, 1 H), 6.00 - 5.93 (m, 1 H), 5.86 (s, 1 H), 5.27 (s, 2H), 2.50 (d, J = 17.1 Hz, 1 H), 2.33 (d, J = 17.1 Hz, 1 H), 2.07 (s, 3H), 1.94 (s, 3H), 1.14 (s, 3H), 1.04 (s, 3H).13CNMR (126 MHz, CDCI3) 6 197.76, 165.41 , 162.35, 151.09, 147.77, 143.62, 137.19, 128.42, 128.03, 127.24, 123.92, 1 17.59, 79.80, 64.40, 49.85, 41 .65, 24.47, 23.18, 21 .43, 19.02. MS (ESI): calculated for C22H25NO6 [M + Na]+422.2, found 422.3.

[0243] Chemical stability and reactivity of AB A-prod rugs towards hypoxia in vitro using HPLC

[0244] In vitro stability test was done by incubating prodrugs in PBS buffer (pH 7.4) up to 48 hours at 37°C. HPLC data were obtained at Oh, 24h and 48h. The chromatograms were collected using a Dionex-UltiMate 3000 LC System with Acclaim 120 A, C18, 3 pm analytical (4.6 x 100 mm) column (Thermofisher).Reactivity of ABA-prodrugs towards NTR (Sigma Aldrich) and NADPH (Roche), was performed by incubating 100 pM prodrugs with 50pg / ml NTR and 1 mM NADPH at 37°C for different time points. The reaction mixture was then quickly injected into HPLC and analyzed.

[0245] All HPLC analyses were run at RT and monitored at 260 nm. A gradient of Acetonitrile in 0.1%TFA and water containing 0.1%TFA were used at a flow rate of 0.750 mL / min. Acetonitrile was increased from 5% to 75% in 15 min and kept constant for 3 min. Total running time is 20 min. HPLC chromatograms were acquired using Dionex-UltiMate 3000 LC System with Acclaim 120 A, C18, 3 pm analytical (4.6 x100 mm) column.

[0246] Cellular stability of ABA-prodrugs and cleavage activity of NTR in HEK- 293T cells

[0247] Stock concentrations of ABA and ABA-prodrugs were prepared in DMSO and further diluted in PBS for subsequent cellular experiments. To test the cellular stability and reactivity of ABA-prodrugs, EGFP-expressing cells (HEK 293-GFP) were seeded in triplicate in 24-well plates (106cell / mL) at a final volume of 500 pL.24 h after plating, cells were treated with either ABA, ABA-prodrugs, or ABA- prodrugs plus NTR and NADPH at indicated concentrations. Live cells were imaged with a Lion Heart FX Automatic Microscope (BioTek) at a 20x objective lens.

[0248] Cell lines and culture

[0249] HEK-293T (Human embryonic kidney cell line), 143B (human osteosarcoma cell line), CHO (Chinese Hamster Ovary), MDA-MB-231 , and MCF-7 (human breast cancer cell lines) were maintained in our laboratories. The HEK-293T cell line was used for lentiviral packaging and preparation. BT-474, MDA-MB-453 (human breast cancer cell lines) were obtained from Prof. Stefanie Avril’s lab (CWRU School of Medicine). Jurkat T cells (clone E6-1 ) and SK-OV3 (human ovarian cancer cell line, HTB-77) were purchased from American Type Culture Collection (ATCC). PC3-pip and PC3-flu (human prostate cancer cell lines) were obtained from Prof. James Basilion’s lab (CWRU School of Medicine). HEK-293T, CHO, MDA-MB-231 , MCF-7, SK-OV3 were grown in complete Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10%(v / v) heat-inactivated fetal bovine serum (FBS, Omega Scientific), 5%(v / v) GlutaMAX (Life Technologies) and 100 lU / ml penicillin / streptomycin (Life Technologies). Jurkat and other cancer cell lines were cultured in complete RPMI-1640 containing 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg / L glucose, and 1500 mg / L sodium bicarbonate, supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 100 lU / ml penicillin / streptomycin. All cultures were maintained at 37°C in a humidified incubator containing 5% CO2. Cells were confirmed mycoplasma-free using MycoAlert™ mycoplasma detection kit (Lonza).

[0250] Plasmid construction

[0251] The conventional CAR construct encoding CD8a signal sequence, a c- Myc tag for detecting membrane expression of CAR, human HER2 scFv (clone 4D5- 5) or human PSMA scFv (derived from mAb J591 ), the hinge and transmembrane domain of CD8oc, the costimulatory 41 BB, and the CD3^ cytosolic domain was designed and synthesized as gBIock gene fragments (Integrated DNA Technology). The split CAR part 1 was generated by replacing CD3 domain derived from conventional CAR with PYRF61 L / A160C(PYR*). The split CAR part 2 encoding CD8a signal sequence, a Flag tag for probing membrane expression of CAR, DAP10 endodomain, a CD8aTM and 4-1 BB costimulatory domain was purchased as gBIock gene fragment (IDT) and cloned into an existing Actin-ires-ABlDi34A plasmid to generate complete p2. The conventional, and individual split constructs were cloned into a 2ndgeneration pLV lentiviral expressing vector (gifted by Crabtree’s lab, namely N103) using the Notl and EcoRI restriction enzyme sites, with expression driven by the elongation factor-1 a (EF-1 a) promoter using In-fusion cloning (Takara Bio). Optimized lentiviral constructs used for human primary T cell experiments were made by subcloning the above CAR genes from N103 backbone into a modified pLVX lentiviral expressing backbone (purchased from Addgene) using ligation with EcoRI and Notl cut sites. All plasmid constructs were amplified using Stabl3 chemically competent E.coli strain and purified using Endo-free Qiagen Miniprep kit. DNA sequences of the final constructs were confirmed by Sanger sequencing (Genewiz). Plasmid concentration and purity (A 260 / 280 = 1 .88-1 .90) were measured by a Nanodrop spectrophotometer (Thermofisher). The amino acid sequences of the CAR constructs used in this paper are presented in Table 2.Table 2: Gene Names and Amino Acid Sequences Used in this Study

[0252] Recombinant lentiviral production

[0253] Lentivirus was produced by transient transfection into HEK-293T cells.One day before transfection, cells were plated at a density of 7 x 106cells per 150 mm diameter tissue culture plate in 30 mL DMEM, supplemented with 10%(v / v) FBS and 5%(v / v) GlutaMAX (Life Technologies), and cultured at 37°C in a 5% CO2 incubator. Cells were transfected with 2ndgeneration packaging plasmids system and media was replaced with complete DMEM 6 hours post-transfection. The culture supernatant containing viral particles was harvested at 48-hour and 72-hourpost-transfection and filtered through a 0.45 pm syringe filter (VWR). Viral particles were concentrated by ultracentrifugation at 100,000 x g at 4°C for 100 min with a Sorval LYNX T29-8x50 Rotor (Thermo Fisher) and resuspended in 500 pL of complete RPMI medium. Stock viral supernatant was either used directly or aliquoted and stored in a -80°C freezer until ready for use.

[0254] Transduction of Jurkat T cells

[0255] For transduction of Jurkat T cells, an appropriate amount of the pooled virus was added to Jurkat T cells (106cells / ml) in the presence of 8 pg / mL polybrene (Santa Cruz Biotech) in a non-treated tissue culture 24-well plate, and the plate were centrifuged at 1000 x g at 33°C for 2 hours (spinoculation). The media was changed to complete fresh RPMI and the cells were cultured at 37°C and 5% CO2 for at least 3 days before subsequent experiments. Fresh media was added to the well frequently to maintain a cell density of 0.5-2 x 106cells / ml.

[0256] Peripheral blood isolation and activation of primary T cells

[0257] Peripheral blood from anonymous healthy donors was purchased from the Case Comprehensive Cancer Center Cellular Therapy Core. Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coat using Ficoll Paque density gradient centrifugation (Cytiva Life Sciences). Human primary CD3+T cells were then purified by negative selection using MojoSort™ Human CD3 T Cell Isolation Kit (Biolegend). Purified T cells were then activated with Human T-Activator aCD3 / aCD28 Dynabeads (Gibco) at 1 :1 bead-to-cell ratio (according to manufacturer’s protocol) in human T cell culture media (TCM) comprising complete RPMI-1640 media (Thermofisher, ATCC formulated), 10% FBS, 1 % Pen / Strep (Gibco), 1 % N.E.A.A (Corning), 50 pM B-ME, and supplemented with 200 lU / ml human recombinant IL-2 (rh IL-2) (STEMCELL Technologies).

[0258] Transduction and CAR-T cell production

[0259] At day 1 post-activation, activated T cells were transduced with viral supernatant using “spinoculation” method, followed by an overnight incubation. Media was refreshed and transduced T cells were maintained in TCM supplemented with 100 lU / ml rh IL-2. To generate split CARs co-expressing cells, activated T cells were transduced sequentially first with one part and then with the other on day 2. At day 5 post T-cell stimulation, beads were removed from the culture, and T cells were rested and expanded in TCM supplemented with 10 ng / ml IL-7 and 5 ng / ml IL-15 (both from Miltenyi Biotech) until ready for use (Fig. 13A). CAR-positive T cells were not sorted and the mixture of non-transduced T cells with CAR+T cells was used in all assays. Fresh medium supplemented with cytokines was added every 2-3 days to maintain the cell density at 0.5-2x106cells / mL CAR-T cells were freshly used or cryopreserved in a solution of 50% FBS and 10% DMSO at 108cells / viaL During CAR T cell production process, the T cells were characterized based on expansion kinetics, transduction efficiency, and differentiation phenotypes (Figs. 13B-D). For all in vitro experiments, the number of CAR-positive cells was normalized to the lowest CAR expression by adding non-transduced cells, reaching the same number of CAR-positive and total T cells per group.

[0260] CAR-T cell activation analysis

[0261] Target cells were seeded at 10,000 cell / well in duplicate or triplicate in 96- well plates and allowed to adhere for 4 hours. Effector cells were washed and resuspended in TCM without exogenous cytokines. The effector cells were then combined with target cells at indicated E:T ratio. The plates were briefly spun down at 400xg in 3 min to force the effector-target interaction. After overnight culture, the T cells were harvested and washed with ice-cold FACS buffer and stained with APC-conjugated a-human CD69 for the expression of activation marker CD69. Cells were analyzed by BD Accuri™ C6 Plus flow cytometer (BD Biosciences).

[0262] In addition, the level of cytokine release in the co-culture supernatant (IL-2,I FN-y) was quantified using commercial enzyme-linked immunosorbent assay (ELISA) kit (Biolegend). 96-well ELISA plate was coated with human IL-2 or human IFN-y specific capture antibody and stored overnight at 4°C. After blocking the plate for 1 hour, the ELISA was performed according to the manufacturer’s instructions. Human IL-2 or I FN-y standards were used to generate a standard curve of range 0 - 500 pg / mL. Absorbance was recorded at 450 nm and 570 nm on a microplate reader (Spectramax i3X, CWRU Chemistry department). Data were analyzed with GraphPad Prism software.

[0263] Flow cytometric analysis

[0264] For flow cytometric analysis, cells were washed and prepared in ice-cold FACS buffer. To detect surface expression of CAR constructs, T cells engineered with CAR-expressing plasmids were labeled with A488-conjugated a-Myc tag (CST, dilution 1 :50) to detect conventional and p1 , and APC-conjugated a-Flag tag (Biolegend, dilution 1 :500) to detect expression of p2. Non-transduced T cells labeled with respective antibodies served as background control. Surface expression of HER2 and PSMA antigens on various target cell lines was confirmed by analysis of cells stained with PE-conjugated anti-human CD340 (Biolegend, dilution 1 OO) or PE-conjugated anti-human PSMA (FOLH1 ) antibodies (Biolegend, dilution 1 OO). For surface staining of CD3 on T cells, cells were pre-incubated in human Fc blocking antibody for 20 minutes prior to incubation with PE / Cyanide7 anti-human CD3 (Biolegend, dilution 1 M OO). Respective isotype control antibodies were used to set the gate (Figs. 14A-C). Cells were incubated with fluorophore-conjugated antibodies for 15-20 min at 4°C in the dark and washed twice prior to resuspension with FACS buffer. Data acquisitions were performed with BD Accuri™ C6 Plus Flow Cytometer (BD Biosciences) or CytoFLEX Flow Cytometer (Beckman Coulter) and analyzed with FlowJo software (v10, TreeStar).

[0265] In vitro cytotoxicity assay

[0266] In vitro assessment of cytolytic activity by CAR-T cells were performed using Water-soluble tetrazolium salt (WST-1 ) assay. Target cells were seeded at 10,000 cells / well in triplicate in a 96-well flat bottom plate. Effector cells were prepared and added to target cells at the indicated E:T ratio. Target cells cultured in media alone were used as a control. After an overnight co-culture (22h), T cells were gently removed from the wells, and the tumor monolayers were washed once with PBS, and then incubated with WST-1 reagent for 2 h. Absorbance was recorded at 440 nm on a microplate reader (Spectramax i3X, CWRU Chemistry department). The following formula was used to calculate the killing efficiency: Killing efficiency (%) = 100- [(Absorbance of tumor+T cells) / Absorbance of tumor+medium control) x 100].

[0267] In vivo studies

[0268] All animal experiments were performed in 6-8 weeks male or female NSG mice in accordance with regulations and protocols approved by the Institutional Animal Care and Use Committee of CWRU (#2015-0118). NSG mice were obtained either from the CWRU Athymic Animal & Xenograft Core or The Jackson Laboratory and housed in a pathogen-free BSL2 biohazard facility at the CWRU Animal Resource Center, Wolstein Research Building with unrestricted access to water and food. All animals were observed daily for signs of distress during drug injection. Mice were euthanized at the endpoint by carbon dioxide overdose.

[0269] Subcutaneous tumor models

[0270] 3 x 106PC3-pip tumor cells in 50 pL HBSS were subcutaneously injected into the left leg of the NSG mice. To confirm the degree of hypoxia of tumors at the time of CAR T cells injection, mice were given hypoxyprobe-1 (pimonidazole hydrochloride) (Hypoxyprobe, Inc.) by i.p. injection at a dose of 60 mg / kg. Two hours later, tumor tissues were obtained and fixed, followed by paraffin embedding and sectioning for immunofluorescence assay. Then 2 x 106T cells (conventional CAR, split CAR or non-transduced) resuspended in 50 pL HBSS supplemented with IL-15 / IL-7 were given to the mice by peritumoral injection, followed by i.p. injection of either vehicle or drugs at a dose of 20 mg / kg. Injection of the drugs was performed daily for a duration of 14 days or 30 days. The numbers of injected T cells were calculated based on CAR+T cells. Tumor volumes were measured with calipers twice a week and were calculated as Volume = (length x width x width) / 2.

[0271] Statistical analysis

[0272] Data are represented as mean ± SEM as stated in the figure legends.Statistical significance was calculated using two-tailed unpaired Student’s t-test or by one-way ANOVA as stated in the text. P < 0.05 was considered significant. All data analysis was performed using GraphPad Prism (version 8.4.0).

[0273] Results

[0274] Design and optimization of ABA-dependent split chimeric antigen receptors

[0275] To establish a TME-inducible CAR through ABA prodrug, we first developed an ABA-dependent CAR. We constructed the ABA-inducible receptors based on ligand inducible CIP-controlled CAR systems in which the two key structural elements of CAR: the antigen recognition domain and the T-cell signalingdomain are encoded by two separate subunits. These include a split CAR part 1 (p1 ), and a split CAR part 2 (p2), each fused to distinct dimerizing partners of the CIP inducer. To enable the TME signal sensing by integrating the ABA-based sensing technology, we incorporated the ABA-CIP system in the split CAR, with PYRF6I UAI6OC (namely PYR*) (Chen, J. etal., Chemical science, 14, 3377-3384 (2023)) and ABI inserted into p1 and p2, respectively (Fig. 15A). Specifically, ABI was cloned into p1 which contains the single chain variable fragment (scFv) targeting human epidermal growth factor receptor 2 (HER2 / ErbB2) (clone 4D5-5) (Liu, X. et al., Cancer research, 75, 3596-3607 (2015)). HER2 is a well-characterized antigen and therapeutic target expressed in a variety of tumors ranging from breast cancers, ovarian cancers, and osteosarcomas. The p1 subunit also consists of a hinge and transmembrane domain of the human CD8a molecule (CD8HTM) and a 4-1 BB intracellular co-stimulatory domain. The second subunit (p2) of the split CAR contains the ectodomain of DNAX-activating protein 10 (DAP10), a 4-1 BB costimulatory segment, followed by PYR*, and human T cell CD3£ immunoreceptor tyrosine-based activation motifs (ITAMs) that are critical for T cell signaling and activation. All split receptors contain an N-terminal CD8a signal peptide responsible for membrane targeting, and epitope tags for probing membrane localization of each split CAR subunit. A conventional non-split version of the second-generation CAR was constructed as a positive control. The CARs were cloned into 2ndgeneration lentiviral-expressing vectors, which were used to transduce Jurkat T cells. In our first split CAR design, high levels of expression (-80%) and the membrane localization for each receptor subunit were detected in Jurkat T cells on day 4 post-transduction (Fig. 15B). Moreover, co-infection of two separate viruses encoding individual parts of the split CAR also revealed satisfactory transduction efficiency (> 60%). We nexttested whether the constructed split CAR components can be reconstituted through ABA-mediated ABI-PYR* dimerization to generate a functional receptor. Jurkat T cells expressing either the conventional CAR or split CAR treated with or without ABA were analyzed for the expression of early activation marker, CD69, and the secretion of lnterleukin-2 (IL-2) cytokine (a critical and early landmark of T cell activation) after co-culturing with BT-474 target cells expressing HER2 antigen (Fig. 15C). The split CAR dual-stimulated by HER2 antigen and ABA expressed a high level of CD69 and IL-2 at a comparable level to conventional CAR (Figs. 15D-E). Neither the T-cell engineered with each part alone in the presence of HER2 antigen nor split CAR in the absence of target antigen exhibited the expression of CD69 or IL-2 secretion. This confirms that our split CAR constructs are functional and antigen-specific and that the dimerization mediated by ABA is required for maximal activation.

[0276] Without ABA treatment, however, a significant expression level of activation markers of the split CAR-T cells was also observed when incubated with HER2-expressing target cells. This indicates that these two parts spontaneously assembled into a functional receptor, which caused unwanted background activation of the engineered T cells. The spontaneous dimerization of split CAR entities could be attributed to the structural aspect of the CD8a hinge region, which normally exists on the cell surface as a disulf ide-linked homo- or heterodimer (with CD8P chain).The incorporation of CD8a hinge into the CAR structure has been shown to enhance T-cell activation signals and consequently, greater antitumor activity. However, the presence of CD8a hinge on both parts of the split CAR could result in small molecule-independent dimerization. Therefore, we sought to optimize the constructs to obtain an optimal design with minimal background signal. We generated twoadditional structural variations of the split pairs containing the CD8a transmembrane domain without the hinge segment on p2 or on both p1 and p2 (Fig. 15F). The three split CAR combinations (split_O, spl it_1 , split_2) were introduced into Jurkat T cells and stimulated with HER2+BT-474 cells in the presence or absence of ABA. The split_1 design that has CD8 hinge segment removed only on p2 showed a minimal background level of IL-2 secretion without ABA while still producing high levels of cytokine in the presence of ABA (Fig. 12G). This design was therefore selected for further characterization. More interestingly, we observed that removal of the hinge region on both chains impaired the ability of the split receptors to mediate T cell activation, presumably due to the inflexibility of the antigen-targeting domain and the lack of antigen-binding efficiency. This data also highlights the important role of the hinge region in CAR signaling and efficacy.

[0277] In vitro ABA-tunable activation and cytotoxicity of split CAR-engineered human primary T cells

[0278] Next, we evaluated whether the functions of the split CAR-T cells could be controlled by ABA in human primary T cells. The ability to fine-tune the magnitude of therapeutic activities is important for keeping toxicities in check. Previous split CARs have shown the dosage response of the engineered T cells to different dimerizing small molecules. We tested whether our split CAR exhibits the same dosage behavior towards ABA and that varying ABA concentrations can tune the degree of T cell activity. We sequentially transduced human primary T cells with two separate lentiviral vectors encoding for split CAR receptors (Fig. 12B). Here, we detected an adequate level of co-expression of both subunits on day 8 post-transduction (Fig. 12C). Split CAR, conventional CAR, or non-transduced (NTD)-T cells were cocultured with HER2-expressing BT-474 cells at 3:1 effector to target (E:T) ratio, andvarious concentrations of ABA were added. We tested T cell activation in multiple ways including the expression of CD69 and the secretion of IL-2 and interferongamma (IFN-y). After 20-h incubation, dual-stimulated split CAR-T cells with increasing ABA concentration showed dose-dependent increases in CD69 expression, IL-2, and IFN-y cytokine production with a level comparable to that of conventional CAR at ABA concentrations over 5 nM (Fig. 12D-F). Similar dosage controls in split CAR-T activation were also observed in Jurkat T cells (Figs. 16A-B). HER2-dependent activation of the conventional CAR was unaffected by ABA. Furthermore, dual-stimulated split CAR-T cells displayed ABA dose-dependent killing efficiency (Fig. 12G). This is also consistent with the formation of T cells-tumors conjugates as seen under the fluorescence microscope when tumors cells were labeled with Calcein-AM (Fig. 17A). Additionally, we comprehensively tested the antitumor activity of the split CAR-T cells against several cancerous and normal cell lines expressing HER2 antigen at different levels (Figs. 17B-D). Interestingly, we found that while the conventional CAR-T cells are reactive against all HER2- expressing cells regardless of their antigen expression levels, the split CAR-T cells only showed strong reactivity to cell lines expressing HER2 at moderate to high levels.

[0279] We next explored the modularity of the split CAR design to target other tumor antigens. We generated split CAR targeting the prostate-specific membrane antigen (PSMA) by switching the scFv on the antigen recognition subunit (Fig. 18A). PSMA is a glycosylated type-ll membrane protein that is upregulated on the surface of many malignant prostate cancers and is also present on the neovasculature of several solid tumors. We found that the anti-PSMA split CAR led to elevated production of CD69 (Fig. 18B), IL-2 (Fig. 18C), and IFN-y (Fig. 18D) in the presenceof PSMA-expressing target cells (PC3-pip) and ABA, but not of PSMA-negative PC3- flu cells, demonstrating a similar killing efficiency and ABA dose-responsiveness to the anti-HER2 split CAR (Fig. 18E).

[0280] One important need for CAR-T cell regulation is that the timing of CAR-T cell responses could be transiently delayed and resumed by the removal or addition of the small molecule. Hence, we designed experiments to evaluate the temporal control of this split CAR system (Fig. 19A). First, we stimulated split CAR-T cells with target cells and ABA, confirming the killing of target cells and secretion of IFN-y. ABA was then removed, the CAR-T cells were rested for 2 days, and additional target cells were added in fresh media with or without ABA. We observed that removal of the small molecule reduced the T cell’s killing, and then resumed killing upon ABA re-introduction (Fig. 19B and Fig. 20A). To understand the residual effects of CAR-T from prior antigen stimulation and the off-kinetics of the split CAR-T activity after ABA removal, we conducted experiments to compare the cytotoxicity of CAR-T cells with or without prior tumor antigen exposure and their re-activation after different resting periods. We observed that when CAR-T cells had prior stimulation with tumor antigens, the reversal of split CAR-T activity occurred within 48 h but was much slower than in the cases when CAR-T cells never exposed to target tumor cells, which completely reversed within 24 h (Figs. 21 A-B).

[0281] We then evaluated the antitumor efficacy of the split CAR-T cells in vivo using the PSMA-CAR as a model. PC3-pip cells were subcutaneously engrafted into the NSG mice. A week later, mice with established tumor burden received NTD, conventional CAR, or split CAR-T cells, followed by daily injection of the vehicle or ABA (20 mg / kg) for 14 days (Fig. 19C). We observed that the tumor volume in the mice injected with the split CAR-T cells plus ABA reduced slowly in the first twoweeks, eventually reaching similar tumor clearance capacity as conventional CAR T cells (Fig. 19D), although it occurred at a slower pace. Mice treated with NTD or the split CAR-T cells without ABA showed similar tumor burden. Daily injections of ABA without T cell treatment did not impair tumor growth, confirming that the observed tumor suppressive effect was caused by the ABA-dependent split CAR-T cells. Notably, analysis of the residual tumors at the end point showed that the tumor in the conventional CAR-T cell treated group relapsed and had lost the antigen expression, while the tumors treated with the split CAR-T cells still maintained the antigen regardless of the time period length of ABA injection (Fig. 19D, Fig. 20B, and Fig. 22), suggesting a correlation between relapse and antigen loss.

[0282] TME signal-triggered regulation of split CAR T cell activation and antitumor effect

[0283] Next, we sought to incorporate cancer signal-sensing prodrug strategy into the split CAR design to generate CAR-T cells that can sense the local tumor microenvironmental signals to deliver precise therapeutic functions. Targeting hypoxic tumor microenvironment has been an important strategy for cancer therapies. Hypoxia in solid tumors also induces upregulation of many enzymes including nitro-reductase (NTR), whose activities could be utilized to activate various cancer therapeutic prodrugs and represent a target that might be exploited to achieve tumor selectivity. Nitroaromatic derivatives such as the 4-nitrobenzyl, 4- nitrofuryl, and 2-nitroimidazole groups are well-characterized substrates for NTR activity in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) as an electron donor under the hypoxic environment. These common chemical moieties have been widely employed for the development of bio-reductive prodrugs, as well as fluorescence probes for imaging tumor hypoxia. In this study, wedesigned and synthesized two ABA conjugates bearing a nitroimidazole or nitrobenzyl group as hypoxia / NTR-responsive caging groups (prodrug 1 and 2, Fig. 23A). Under mimic hypoxic conditions using NTR and NADPH, the two ABA prodrugs undergo reductase-mediated reduction to form an electron-donating substituent, which subsequently releases the active ABA inducer ligand. In the presence of NTR / NADPH, we observed that both prodrugs were rapidly uncaged to generate free ABA within 60 min using HPLC analysis (Fig. 23B). We also confirmed the chemical stability of the two prodrugs in PBS buffer. Using HEK293T- EGFP reporter cell line, which can respond to ABA and express EGFP, we observed that these prodrugs were stable within the 24-h observation period, while the addition of NTR and NADPH resulted in the induction of EGFP expression, indicating the successful uncaging to release the functional ABA (Fig. 23C). We also confirmed that neither prodrug has impact on cell viability at ABA concentration up to 10 pM (Fig. 24).

[0284] We next tested the incorporation of these hypoxia-responsive prodrugs in controlling T-cell activation and antitumor activity. Primary T cells transduced with the split CAR were stimulated with tumor cells expressing the targeted antigens at the indicated E:T ratios for 22h. The cell mixture was exposed to two hypoxia- responsive prodrugs or prodrugs along with their activating signal. We observed specific activation of both HER2- and PSMA-split CAR-T cells against their respective target tumor cells when ABA or ABA-prodrugs plus NTR were added (Figs. 25A-B). Without the addition of ABA, or TME signal, no appreciable cell killing, or IFN-y production was detected, as compared to cytotoxicity and cytokine secretion level at baseline (when target cells were incubated with the split CAR-T cells only). Killing of cancer cells was not observed in the presence of TME signalalone without ligands added, indicating that the tumor cell killing is a direct result of cell-mediated cytotoxicity executed by effector T cells. The split CAR and prodrugs were then evaluated in vivo using PSMA-expressing PC3-pip cells as a tumor model. We tested prodrug 1 bearing nitroimidazole moiety due to its favorable water solubility. One week after tumor engraftment when the tumor was palpable and proved to be hypoxic (Fig. 26A), the mice received split CAR-T cells, followed by daily injection of the vehicle, ABA, or prodrug 1 (20 mg / kg) for 14 days (Fig. 25C). The progressive tumor growth was observed in the mice treated with vehicle as a control. In contrast, mice treated with either ABA or prodrug 1 exhibited significant antitumor effects (Fig. 25D), indicating that prodrug 1 was effectively uncaged in vivo and released ABA. We also confirmed that daily injections of both the ABA and ABA-prodrug were tolerable to the mice (Fig. 26B). At the experiment endpoint, we harvested the residue tumors to assess the possibility of antigen loss and quantified the number of human CD3+T cells accumulated at the tumor site. ABA- and prodrug 1 -treated mice show high percentages of T cells, while control-treated mice displayed low or undetectable amounts of T cells (Fig. 25E). Interestingly, the number of accumulated T cells was seen to be correlated to the loss of tumor antigen (Figs. 26C-D), suggesting a relationship between the treatment intensity and antigen loss.

[0285] From the above description of the present disclosure, those skilled in the art will perceive improvements, changes and modifications. Such improvements, changes, and modifications are within the skill of those in the art and are intended to be covered by the appended claims. All patents, patent applications, and publications cited herein are incorporated by reference in their entirety.

Claims

CLAIMSThe following is claimed:1 . A tumor therapy system, comprising: a caged inducer; and an immune effector cell, comprising: a first exogenous polynucleotide that is operably linked to a single viral vector, the first exogenous polynucleotide encoding: polypeptide components of a chemical induced proximity (CIP) complex; and a therapeutic polypeptide operably linked to a regulatory region inducible by an uncaged, activated form of the caged inducer; and a second exogenous polynucleotide that encodes a chimeric antigen receptor that specifically binds to an antigen.

2. The system of claim 1 , wherein the caged inducer is an inactive prodrug of an inducer that is activatable by at least one signal to form the uncaged, activated form of the caged inducer.

3. The system of claim 2, wherein the inducer of the caged inducer comprises at least one sensing moiety covalently attached thereto and configured to react with the at least one signal to generate the uncaged inducer.

4. The system of claim 3, wherein the inducer is selected from the group consisting of abscisic acid (ABA), gibberellic acid (GA), rapamycin, and - galactosidase.

5. The system of claim 4, wherein the caged inducer comprises ABA conjugated with a nitroimidazole or nitrobenzyl group.

6. The system of claim 4, wherein the caged inducer comprises ABA conjugated with galactose.

7. The system of claim 2, wherein the at least one signal is a signal molecule or light.

8. The system of claim 7, wherein the signal molecule is associated with a tumor microenvironment (TME).

9. The system of claim 8, wherein the signal molecule is selected from the group consisting of NADPH, hydrogen ions, oxygen, hydrogen peroxide and iron (Fe2+).

10. The system of claim 1 , wherein the viral vector is a lentiviral vector.11 . The system of claim 10, wherein the lentiviral vector is a bidirectionalN103 lentiviral transfer vector.

12. The system of claim 1 , wherein the immune effector cell is a T-cell or a natural killer (NK) cell.

13. The system of claim 1 , wherein the antigen is a tumor antigen.

14. The system of claim 1 , wherein the regulatory region binds components of a CIP expression system.

15. The system of claim 14, wherein the presence of the uncaged inducer in the immune effector cell induces assembly of the CIP expression system and expression of the therapeutic polypeptide.

16. The system of claim 1 , wherein the polypeptide encoded by the first exogenous polynucleotide comprises, 5' to 3', the following components, each of which is operably linked to one another: at least one upstream activating sequence (UAS); the regulatory region; the therapeutic polypeptide; a promoter selected from EF1 a or PGK; a transcriptional activation domain; a PYR mutant domain; a T2A domain; a GAL4 DNA binding domain; and an ABI domain.

17. The system of claim 16, wherein the at least one UAS comprises a 5x UAS, the regulatory region comprises a minimal CMV promoter, the transcriptional activation domain comprises VP16AD, and the PYR mutant domain is one of PYR E141 L, PYR F61 L / A160C or PYR F61 L / E141 L / A160V.

18. The system of claim 1 , wherein the chimeric antigen receptor comprises a split chimeric antigen receptor.

19. The system of claim 18, wherein the second exogenous polynucleotide further comprises: a first polynucleotide subunit encoding an antigen recognition domain and a first dimerizable adaptor protein; and a second polynucleotide subunit encoding an immune effector cell signaling domain and a second dimerizable adaptor protein; wherein, in the presence of at least one signal and the caged inducer, the first and second adaptor proteins are induced to dimerize and generate a functional split CAR.

20. The system of claim 19, wherein the first polynucleotide subunit further encodes a hinge domain, a transmembrane domain, an intracellular co-stimulatory domain, and an optional epitope tag domain.21 . The system of claim 20, wherein the first adaptor protein comprises, 5' to 3', the following components, each of which is operably linked to one another: ana-PSMA scFV domain, a hinge domain, a transmembrane domain, a 4-1 BB domain, and a mutant PYR domain.

22. The system of claim 19, wherein the second polynucleotide further encodes a DNAX-activating protein (DNAP) domain, a transmembrane domain, an immunoreceptor tyrosine-based activation domain, an intracellular co-stimulatory domain, an optional hinge domain, and an optional epitope tag domain.

23. The system of claim 22, wherein the second adaptor protein comprises, 5' to 3', the following components, each of which is operably linked to one another: a DNAP 10 domain, a transmembrane domain, a 4-1 BB domain, an ABI domain, and a CD3 domain.

24. The system of claim 19, wherein the first and second polynucleotide subunits are operably linked to the same or different expression vector(s).

25. An isolated immune effector cell comprising: a first exogenous polynucleotide that is operably linked to a single viral vector and which encodes: polypeptide components of a chemical induced proximity (CIP) complex; and a therapeutic polypeptide operably linked to a regulatory region inducible by an uncaged, activated form of the caged inducer; and a second exogenous polynucleotide that encodes a chimeric antigen receptor that specifically binds to an antigen.

26. The immune effector cell of claim 25, wherein the caged inducer is an inactive prodrug of an inducer that is activatable by at least one signal to form the uncaged, activated form of the caged inducer.

27. The immune effector cell of claim 26, wherein the inducer of the caged inducer comprises at least one sensing moiety covalently attached thereto and configured to react with the at least one signal to generate the uncaged inducer.

28. The immune effector cell of claim 27, wherein the inducer is selected from the group consisting of abscisic acid (ABA), gibberellic acid (GA), rapamycin, and p-galactosidase.

29. The immune effector cell of claim 28, wherein the caged inducer comprises ABA conjugated with a nitroimidazole or nitrobenzyl group.

30. The immune effector cell of claim 28, wherein the caged inducer comprises ABA conjugated with galactose.31 . The immune effector cell of claim 26, wherein the at least one signal is a signal molecule or light.

32. The immune effector cell of claim 31 , wherein the signal molecule is associated with a tumor microenvironment (TME).

33. The immune effector cell of claim 32, wherein the signal molecule is selected from the group consisting of NADPH, hydrogen ions, oxygen, hydrogen peroxide and iron (Fe2+).

34. The immune effector cell of claim 25, wherein the viral vector is a lentiviral vector.

35. The immune effector cell of claim 34, wherein the lentiviral vector is a bidirectional N103 lentiviral transfer vector.

36. The immune effector cell of claim 25, wherein the immune effector cell is a T-cell or a natural killer (NK) cell.

37. The immune effector cell of claim 25, wherein the antigen is a tumor antigen.

38. The immune effector cell of claim 25, wherein the regulatory region binds components of a CIP expression system.

39. The immune effector cell of claim 38, wherein the presence of the uncaged inducer in the immune effector cell induces assembly of the CIP expression system and expression of the therapeutic polypeptide.

40. The immune effector cell of claim 25, wherein the polypeptide encoded by the first exogenous polynucleotide comprises, 5' to 3', the following components, each of which is operably linked to one another: at least one upstream activating sequence (UAS); the regulatory region; the therapeutic polypeptide; a promoter selected from EF1 a or PGK; a transcriptional activation domain; a PYR mutant domain; a T2A domain; a GAL4 DNA binding domain; and an ABI domain.41 . The immune effector cell of claim 40, wherein the at least one UAS comprises a 5x UAS, the regulatory region comprises a minimal CMV promoter, the transcriptional activation domain comprises VP16AD, and the PYR mutant domain is one of PYR E141 L, PYR F61 L / A160C or PYR F61 L / E141 L / A160V.

42. The immune effector cell of claim 25, wherein the chimeric antigen receptor comprises a split chimeric antigen receptor.

43. The immune effector cell of claim 42, wherein the second exogenous polynucleotide further comprises: a first polynucleotide subunit encoding an antigen recognition domain and a first dimerizable adaptor protein; anda second polynucleotide subunit encoding an immune effector cell signaling domain and a second dimerizable adaptor protein; wherein, in the presence of at least one signal and the caged inducer, the first and second adaptor proteins are induced to dimerize and generate a functional split CAR.

44. The immune effector cell of claim 43, wherein the first polynucleotide subunit further encodes a hinge domain, a transmembrane domain, an intracellular co-stimulatory domain, and an optional epitope tag domain.

45. The immune effector cell of claim 44, wherein the first adaptor protein comprises, 5' to 3', the following components, each of which is operably linked to one another: an a-PSMA scFV domain, a hinge domain, a transmembrane domain, a 4-1 BB domain, and a mutant PYR domain.

46. The immune effector cell of claim 43, wherein the second polynucleotide further encodes a DNAX-activating protein (DNAP) domain, a transmembrane domain, an immunoreceptor tyrosine-based activation domain, an intracellular co-stimulatory domain, an optional hinge domain, and an optional epitope tag domain.

47. The immune effector cell of claim 46, wherein the second adaptor protein comprises, 5' to 3', the following components, each of which is operably linked to one another: a DNAP 10 domain, a transmembrane domain, a 4-1 BB domain, an ABI domain, and a CD3 domain.

48. The system of claim 43, wherein the first and second polynucleotide subunits are operably linked to the same or different expression vector(s).

49. A method for treating a solid tumor in a subject, the method comprising: administering, to the subject, at least one immune effector cell as recited in any one of claims 25-48; administering, to the subject, an inactive caged inducer, the inactive caged inducer being activatable to an active, uncaged inducer by at least one signal; allowing the inactive caged inducer to be converted to the active, uncaged inducer; and allowing the active inducer to enter the at least one immune effector cell and induce the CIP complex to express the therapeutic polypeptide and the chimeric antigen receptor.

50. The method of claim 49, wherein the at least one immune effector cell is obtained from the subject.51 . The method of claim 49, wherein the at least one signal is a signal molecule or light.

52. The method of claim 51 , wherein the signal molecule is associated with a tumor microenvironment53. The method of claim 52, wherein the signal molecule is selected from the group consisting of NADPH, hydrogen ions, oxygen, hydrogen peroxide and iron (Fe2+).

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