Compositions and methods of SLC7a1 and SLC7a2

Abstract

Compositions and methods for reducing toxicity in T cells, among other inventions, are provided.

Description

Attorney Docket No.: ARCI-003WO COMPOSITIONS AND METHODS OF SLC7A1 AND SLC7A2 CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of United States Provisional Application No.63 / 661,511, filed June 18, 2024, the disclosure of which is incorporated herein by reference in its entirety. INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS AN XML FILE

[0002] A Sequence Listing is provided herewith as a Sequence Listing XML, “ARCI- 003WO_SEQ_LIST.xml” created on June 17, 2025, and having a size of 35,301 bytes. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety. BACKGROUND OF THE INVENTION

[0003] A hallmark of cancer is the presence of double-stranded DNA in the cytosol, resulting from chromosomal instability, damaged mitochondria, extrachromosomal DNA replication or treatment-induced chromatin bridges1–4. The innate immune pattern recognition receptor cGAS, upon recognizing cytosolic dsDNA, is activated to produce the second messenger cGAMP. cGAMP is a potent activator of its ER transmembrane receptor STING (Stimulator of Interferon Genes), which scaffolds the recruitment and activation of kinase TBK1 and transcription factor IRF3, and ultimately stimulating type-I interferon gene expression5. Cancer cells can avoid cGAMP-induced STING activation and interferon production by exporting cGAMP using transporters such as ABCC16,7. Once released into the tumor microenvironment, cGAMP can enter bystander cells including tumor endothelial cells8, tumor-infiltrating macrophages and dendritic cells6,9, and tumor associated fibroblasts10through cell type specific transporters. cGAMP-mediated activation of STING and type-I interferons in these cell types promotes immune infiltration into the tumor microenvironment11,12, producing an intercellular signaling cascade that can turn “cold” tumors “hot”.

[0004] cGAMP mimetics and traditional small molecule STING agonists have been developed as innate immune therapies with the goal of improving the efficacy of checkpoint blockade therapies – which target the adaptive immune arm and therefore often fail to produce strong responses in immunologically “cold” tumors13–16. Intratumoral injection ofAttorney Docket No.: ARCI-003WO STING agonists in mice exerts remarkable tumor shrinkage effects in injected tumors and abscopal effects on distal tumors in a T cell-dependent manner14,17. Intriguingly, some studies have shown a loss of efficacy at higher dosages, which has been attributed to toxic effects of STING activation in T cells recruited to the tumor microenvironment18–25. Realizing the full potential of STING agonist treatment will require new strategies to overcome T cell-specific toxicity that can negate its beneficial antitumoral action.

[0005] New strategies, among other inventions, are presented herein. SUMMARY OF THE INVENTION

[0006] The present disclosure relates generally to methods for inducing toxicity in a T cell, methods for inducing toxicity in a T cell population, methods for treating a solid cancerous tumor in a subject, methods for reducing toxicity in a T cell population, methods for inhibiting cGAMP binding to SLC7A1 on a T cell, methods of identifying an agent that inhibits SLC7A1, and an agent that binds to the cGAMP binding pocket of SLC7A1.

[0007] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative embodiments and features described herein, further aspects, embodiments, objects and features of the disclosure will become fully apparent from the drawings and the detailed description and the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings.

[0009] FIG 1A shows an experimental overview. BALB / c mice were injected with 50,0004T1-Luciferase cells into the mammary fat pad. Once the tumors reached 100 mm3, the tumors were irradiated with 0, 12 or 20 Gy, and injected with nonbinding (NB) or neutralizing (Neu) STING 24 h later. The mice were euthanized the following day, and the tumors were extracted and prepared for flow cytometry.Attorney Docket No.: ARCI-003WO

[0010] FIG 1B shows CD3+cells as a percentage of live cells in irradiated tumors from NB and Neu STING groups. Significance are calculated by unpaired, two-tailed t-test. *p < 0.05.

[0011] FIG 1C shows activated primary human T cells from 2 healthy donors were incubated with increasing concentrations of cyclic dinucleotides (CDNs): 2’3’-cGAMP, 2’3’- cGSASMP, 2’3’-CDAS, and 3’3’-cGAMP for 24 h in the presence of ENPP1 inhibitor, STF- 1623. Relative viability was measured using Cell Titer Glo Assay (n = 3-4 biological replicates). Data are shown as mean + / - SD.

[0012] FIG 1D & 1E show activated human primary T cells from the same 2 donors in FIG 1C were treated 65 µM cGAMP for 2 h, with or without 20 µM DCPIB, 0.5 mM methotrexate (MTX), or 0.5 mM reduced folic acid (RFA), and signaling was assessed by Western blot (n = 3-6 biological replicates). One representative Western blot from donor 2 is shown in FIG 1F and quantification of multiple blots are shown in FIG 1G. Data are shown as the mean + / - SD.

[0013] FIG 1F shows U937 SLC19A1- / -cells were incubated with increasing concentrations of 2’3’-cGAMP, 2’3’-cGSASMP, 2’3’-CDAS, and 3’3’-cGAMP, for 24 h in the presence of ENPP1 inhibitor, STF-1623. Relative viability was measured using Cell Titer Glo Assay (n = 3-4 biological replicates). Data are shown as the mean + / - SD.

[0014] FIG 1G shows TIME cells were treated with increasing concentration of 2’3’- cGAMP, 2’3’-cGSASMP or 2’3’-CDASfor 2 h, and signaling was assessed by Western blot (n = 2 biological replicates). One representative Western blot is shown in (FIG 1P) and quantification of multiple blots is shown in FIG 1G.

[0015] FIG 1H shows structures of mammalian (1), synthetic (2-3) and bacterial (4- 6) cyclic dinucleotides.

[0016] FIG 1I shows human transcript database adopted from Schmiedel et al., 2018 for known cGAMP importers in different human T cell subsets.

[0017] FIG 1J shows activated primary human T cells from 1 healthy donor were incubated with increasing concentrations of cyclic dinucleotides (CDNs): 3’3’-CDA and 3’3’-CDG for 24 h in the presence of ENPP1 inhibitor, STF-1623. Relative viability was measured using Cell Titer Glo Assay (n = 4 biological replicates). Data are shown as mean + / - SD.Attorney Docket No.: ARCI-003WO

[0018] FIG 1K shows combined quantification of Western blots from FIG 1E. Significance are calculated by unpaired, two-tailed t-test. Data are shown as the mean + / - SD.

[0019] FIGs 1L & 1M show activated primary mouse CD4+T cells were treated 75 µM cGAMP for 3 h, with or without 20 µM DCPIB or 0.5 mM reduced folic acid (RFA), and signaling was assessed by Western blot (n = 6 biological replicates). One representative Western blot is shown in FIG 1L and quantification of multiple blots is shown in FIG 1L & 1M. Data are shown as the mean + / - SD.

[0020] FIGs 1N & 1O show activated primary mouse CD8+T cells were treated 75 µM cGAMP for 3 h, with or without 20 µM DCPIB or 0.5 mM reduced folic acid (RFA), and signaling was assessed by Western blot (n = 4 biological replicates). One representative Western blot is shown in FIG 1N and quantification of multiple blots is shown in FIG 1O. Data are shown as the mean + / - SD.

[0021] FIG 1P shows representative Western Blot from FIG 1G.

[0022] FIG 1Q shows an overview of cGAMP signaling in T cells.

[0023] FIG 1R shows activated primary human T cells from 2 healthy donors were incubated with increasing concentrations of cyclic dinucleotides (CDNs): 2’3’-cGAMP, 2’3’- cGSASMP, 2’3’-CDAS, 3’3’-cGAMP, 3’3’-CDA, and 3’3’-CDG, for 24 h in the presence of ENPP1 inhibitor, STF-1623. Relative viability was measured using Cell Titer Glo Assay (n = 3-4 biological replicates). Data are shown as mean + / - SD.

[0024] FIG 1S shows TIME cells were treated with increasing concentration of 2’3’- cGAMP, 2’3’-cGSASMP or 2’3’-CDAS for 2 h, and signaling was assessed by Western blot (n = 2 biological replicates). One representative Western blot is shown in (FIG 1P) and quantification of multiple blots is shown in (FIG 1S).

[0025] FIG 1T & 1U shows activated human primary T cells from two healthy donors were treated with 50 μM cGAMP for 2 h, and with or without 20 μM DCPIB, in serum-free media, and and signaling was assessed by Western blot (n = 6 biological replicates). One representative Western blot from donor 2 is shown in (FIG 1T) and quantification of multiple blots are shown in (FIG 1U). Data are shown as the mean + / - SD. Significance are calculated by unpaired, two-tailed t-test.

[0026] FIGs 2A & 2B show Jurkat cells were treated with 5 ng / mL PMA and 500 ng / mL ionomycin for 6 hrs.72 hours later, stimulated cells were treated with 75 µM cGAMPAttorney Docket No.: ARCI-003WO for 2 h, without or without 0.5 mM methotrexate (MTX), 1 mM sulfasalazine (SSZ) or 20 µM DCPIB, and signaling was assessed by Western blot (n = 9 biological replicates). One representative Western blot is shown in FIG 2A and quantification of multiple blots is shown in FIG 2B. Data are shown as the mean + / - SD.

[0027] FIGs 2C & 2D show Jurkat cells were treated with 5 ng / mL PMA and 500 ng / mL ionomycin for 6 hrs.72 hours later, stimulated cells were treated with increasing concentration of 2’3’-cGAMP, 2’3’-cGSASMP or 2’3’-CDASfor 2 h, and signaling was assessed by Western blot (n = 5 biological replicates). One representative Western blot is shown in FIG 2C and quantification of multiple blots is shown in FIG 2B. Data are shown as the mean + / - SD.

[0028] FIG 2E shows the CRISPR screen overview. Whole genome-targeting sgRNAs were introduced lentivirally into a Jurkat-Cas9 cell line, which was then treated with PMA and ionomycin.24 hrs later, stimulated library cells were treated daily with LD30cGAMP or untreated for 10 days. Genomic DNA was harvested, deep sequenced, and analyzed for sgRNA enrichment or depletion.

[0029] FIG 2F shows a plot of casTLE score with positive effect size. Known regulators of STING pathway and SLC7A1 are marked in red. A p<0.005 significance threshold is indicated as a dotted line.

[0030] FIGs 2G & 2H show Jurkat cells were transduced with Cas9 along with non- targeting (scramble) or SLC7A1-targeting sgRNA, were treated with 75 uM cGAMP for 2h, and signaling was assessed by Western blot (n = 9 biological replicates). Average knock-out score across independent experiments was determined by ICE (Synthego) to be 68%. One representative Western blot is shown in FIG 2G and quantification of multiple blots is shown in FIG 2H. Significance calculated by unpaired, two-tailed t-test. Data are shown as mean + / - SD. ***p < 0.001.

[0031] FIGs 2I & 2J show Jurkat cells treated with 75 µM cGAMP for 2 h, without or without 0.5 mM methotrexate (MTX), 1 mM sulfasalazine (SSZ) or 20 µM DCPIB, and signaling was assessed by Western blot (n = 9 biological replicates). One representative Western blot is shown in (FIG 2A) and quantification of multiple blots is shown in (FIG 2B). Data are shown as the mean + / - SD.

[0032] FIGs 2K & 2L show Jurkat cells treated with increasing concentration of 2’3’-cGAMP, 2’3’-cGSASMP or 2’3’-CDASfor 2 h, and signaling was assessed by WesternAttorney Docket No.: ARCI-003WO blot of pIRF3 or pSTING (n = 3 - 5 biological replicates as indicated). One representative Western blot is shown in (FIG 2C) and quantification of multiple blots is shown in (FIG 2D). Data are shown as the mean + / - SD.

[0033] FIG 2M shows human transcript database adopted from Schmiedel et al., 2018 for SLC7A1 expression in different immune cell subsets.

[0034] FIG 2N shows human proteomic database adopted from Cano-Gamez et al., 2020 for SLC7A1 expression in resting and activated T cell subsets.

[0035] FIGs 2O & 2P show Jurkat cells transduced with Cas9 along with non- targeting (scramble) or SLC7A1-targeting sgRNA, were treated with 75 uM cGAMP for 2h, and signaling was assessed by Western blot (n = 3 biological replicates). Knock-out score was determined by ICE (Synthego) to be 70%. One representative Western blot is shown in FIG 2O and quantification of multiple blots is shown in FIG 2P. Significance calculated by unpaired, two-tailed t-test. Data are shown as mean + / - SD. *p < 0.05.

[0036] FIG 2Q shows Jurkat cells were treated with 5 ng / mL PMA and 500 ng / mL ionomycin for 6 hrs.72 hours later, stimulated cells were treated with increasing concentration of 2’3’-cGAMP, 2’3’-cGSASMP or 2’3’-CDASfor 2 h, and signaling was assessed by Western blot (n = 5 biological replicates). One representative Western blot is shown in (FIG 2C) and quantification of multiple blots is shown in (FIG 2Q). Data are shown as the mean + / - SD.

[0037] FIG 2R shows Jurkat cells were treated with increasing concentration of 2’3’- cGAMP, 2’3’-cGSASMP or 2’3’-CDASfor 2 h, and signaling was assessed by Western blot of pIRF3 or pSTING (n = 3 - 5 biological replicates as indicated). One representative Western blot is shown in (FIG 2K) and quantification of multiple blots is shown in (FIG 2R). Data are shown as the mean + / - SD.

[0038] FIGs 3A & 3B show activated human primary CD3+T cells from 2 healthy donors electroporated with Cas9 protein and non-targeting (scramble) or SLC7A1-targeting sgRNA. Cells were treated with 75 µM cGAMP for 2 h three days later, and signaling was assessed by Western blot (n = 3 biological replicates). One representative Western blot from Donor 2 is shown in FIG 3A and quantification of multiple blots from 2 donors are shown in FIG 3B. Significance calculated by unpaired, two-tailed t-test. Data are shown as the mean + / - SD. *p < 0.05.Attorney Docket No.: ARCI-003WO

[0039] FIGs 3C & 3D show activated mouse CD4+and CD8+T cells electroporated with Cas9 protein and Rosa26-targeting or SLC7A1-targeting sgRNA. Cells were treated with 75 µM cGAMP for 3 h three days later, and signaling was assessed by Western blot (n = 4 - 6 biological replicates). One representative Western blot from each group of CD4+and CD8+T cells are shown in FIG 3C and quantification of multiple blots from the two groups are shown in FIG 3D. Significance calculated by unpaired, two-tailed t-test. Data are shown as the mean + / - SD. *p < 0.05, ****p<0.0001.

[0040] FIGs 3E & 3F show Rosa26- and SLC7A1-knocked out CD4+and CD8+mouse T cells from FIGs 3C & 3D electroporated with 150 nM of cGAMP for 2 h, and signaling was assessed by Western blot (n = 3 biological replicates). One representative Western blot from each group of CD4+and CD8+T cells are shown in FIG 3E and quantification of multiple blots from the two groups are shown in FIG 3F. Data are shown as the mean + / - SD.

[0041] FIG 3G shows indel frequency of donor 1 and 2 human T cells from FIGs 3A & 3B based on ICE (Synthego).

[0042] FIG 3H shows average indel frequency across independent experiments for mouse CD4+and CD8+T cells from FIGs 3C & 3D based on ICE (Synthego).

[0043] FIG 3I shows a schematic of the diazirine probe crosslinking assay. Purified FLAG-SLC7A1 proteins were incubated with a 3’3’-cGAMP probe containing a diazirine motif, and then UV irradiated at 365 nm. Crosslinked proteins were then conjugated with a R110 fluorescence tag containing an alkyne handle (R110-N3) via click chemistry. Proteins were visualized were visualized on SDS-PAGE gel and R110 fluorescence and Flag signal was assessed by Western Blot.

[0044] FIG 3J shows Purified FLAG-SLC7A1 protein was incubated with 500 μM of 3’3’-cGAMP diazirine probe, and with or without 2’3’- or 3’3’-cGAMP (lower concentration: 500 μM, higher concentration: 2mM), and irradiated at 365 nm for 20 min. Crosslinked protein signals were assayed by Western Blot. Flag blot is shown in FIG 3L and R110 fluorescence blot is shown in FIG 3J.

[0045] FIG 3K shows the synthesis schematic of 2’3’- and 3’3’ cGAMP-Diazirine probe.

[0046] FIG 3L shows Flag blot of purified FLAG-SLC7A1 from FIG 3J.Attorney Docket No.: ARCI-003WO

[0047] FIG 4A shows AlphaFold-predicted structure of human SLC7A1. The modeled cGAMP and Arginine binding sites are outlined in red.

[0048] FIG 4B shows U937 SLC19A1- / --tet-SLC7A1 (WT, single, double or triple mutant variants)-Flag cells were incubated with or without 1 µg / mL doxycycline (dox) for 24 h. Cells were then treated with 30 µM 2’3’-cGAMP for 24 h in the presence of ENPP1 inhibitor, STF-1623. Relative viability was measured using Cell Titer Glo Assay (n = 6 biological replicates). Significance calculated by unpaired, two-tailed t-test. Data are shown as the mean + / - SD. *p<0.05, ****p<0.0001.

[0049] FIG 4C U937 SLC19A1- / --tet-SLC7A1 (WT, single, double or triple mutant variants)-Flag cells were incubated with or without 1 µg / mL doxycycline (dox) for 24 h. Cells were then treated with 21 nM3H-Arginine for 1 min (n = 1-2 biological replicates, as indicated), and radioactivity was measured using a scintillation counter and normalized to protein amount.

[0050] FIG 4D shows AlphaFold-predicted structure of human SLC7A1 with cGAMP (gray sticks) docked into a pocket on the extracellular-facing surface. Residues mutated are shown as sticks; residues validated to have an effect on cGAMP transport shown in dark red. Residues 229-251 (a flexible loop with low AlphaFold prediction score) are omitted for ease of visualization.

[0051] FIGs 4E & 4F show U937 SLC19A1- / --tet-SLC7A1-Flag cells were incubated with or without 1 µg / mL doxycycline (dox) for 24 h. Cells were then treated with 75 µM cGAMP for 2 h, and signaling was assessed by Western blot (n = 7 biological replicates). One representative Western blot is shown in FIG 4E and quantification of multiple blots is shown in FIG 4F. Significance calculated by unpaired, two-tailed t-test. Data are shown as mean + / - SD. *p < 0.05.

[0052] FIG 4G shows U937 SLC19A1- / --tet-SLC7A1-Flag cells were incubated with or without 1 µg / mL doxycycline (dox) for 24 h. Cells were then treated with 21 nM3H- Arginine for 1 min (n = 3 biological replicates), and radioactivity was measured using a scintillation counter and normalized to protein amount. Data are shown as the mean + / - SD.

[0053] FIG 4H shows U937 SLC19A1- / -cells were incubated with or without 1 µg / mL doxycycline (dox) for 24 h. Cells were then treated with 21 nM3H-Arginine for 1 min (n = 2 biological replicates), and radioactivity was measured using a scintillation counter and normalized to protein amount. Data are shown as the mean + / - SD.Attorney Docket No.: ARCI-003WO

[0054] FIG 4I shows U937 SLC19A1- / --tet-SLC7A1 (WT, R59A / D404A / R130A or S354M)-Flag cells were incubated with or without 1 μg / mL doxycycline (dox) for 24 h. Cells were then treated with 20 μM 2’3’-cGAMP for 24 h in the presence of ENPP1 inhibitor, STF- 1623. Relative viability was measured using Cell Titer Glo Assay (n = 6-14 biological replicates, as indicated). Significance calculated by unpaired, two-tailed t-test. Data are shown as the mean + / - SD. **p<0.01, ****p<0.0001.

[0055] FIG 4J shows U937 SLC19A1- / --tet-SLC7A1 (WT, R59A / D404A / R130A or S354M)-Flag cells were incubated with or without 1 μg / mL doxycycline (dox) for 24 h. Cells were then treated with 21 nM3H-Arginine for 1 min (n = 2-4 biological replicates, as indicated), and radioactivity was measured using a scintillation counter and normalized to protein amount.

[0056] FIG 4K shows U937 SLC19A1- / --tet-SLC7A1 (WT or R59A / D404A / R130A)-Flag cells were incubated with or without 1 μg / mL doxycycline (dox) for 24 h. Cells were then treated with 2 μM 2’3’-cGSASMP, 4 μM 2’3’-CDASor 40 μM 3’3’- cGAMP for 24 h in the presence of ENPP1 inhibitor, STF-1623. Relative viability was measured using Cell Titer Glo Assay (n = 11-19 biological replicates, as indicated). Significance calculated by unpaired, two-tailed t-test. Data are shown as the mean + / - SD. ****p<0.0001.

[0057] FIG 4L shows U937 SLC19A1- / --tet-SLC7A1-Flag cells were incubated with or without 1 μg / mL doxycycline (dox) for 24 h. Cells were then treated with 21 nM3H- Arginine and different concentrations of 2’3’-cGAMP or L-Lysine for 1 min (n = 2 biological replicate), and radioactivity was measured using a scintillation counter and normalized to protein amount.

[0058] FIG 4M shows differential gene analysis (DESeq2) of U937 SLC19A1- / --tet- SLC7A1-Flag cells incubated with or without 1 μg / mL doxycycline (dox) for 24 h (n = 2 biological duplicate). Significantly enriched or depleted coding mRNAs and known extracellular cGAMP / STING signaling elements are annotated.

[0059] FIG 4N shows U937 SLC19A1- / --tet-SLC7A1 (WT, single or double mutant)-Flag cells were incubated with or without 1 μg / mL doxycycline (dox) for 24 h. Cells were then treated with 20 μM 2’3’-cGAMP for 24 h in the presence of ENPP1 inhibitor, STF- 1623. Relative viability was measured using Cell Titer Glo Assay (n = 6 biological replicates,Attorney Docket No.: ARCI-003WO as indicated). Significance calculated by unpaired, two-tailed t-test. Data are shown as the mean + / - SD. *p<0.05.

[0060] FIG 4O shows U937 SLC19A1- / --tet-SLC7A1 (WT, single or double mutant)-Flag cells were incubated with or without 1 μg / mL doxycycline (dox) for 24 h. Cells were then treated with 21 nM3H-Arginine for 1 min (n = 1-2 biological replicates, as indicated), and radioactivity was measured using a scintillation counter and normalized to protein amount.

[0061] FIG 5A shows U937 SLC19A1- / --tet-SLC7A1-Flag cells were incubated with or without 1 µg / mL doxycycline (dox) for 24 h. Cells were then treated with 21 nM3H- Arginine and different concentrations of 2’3’-cGAMP and L-Lysine for 1 min (n = 2 biological replicates), and radioactivity was measured using a scintillation counter and normalized to protein amount.

[0062] FIGs 5B & 5C show activated mouse CD3+T cells treated with 75 µM cGAMP for 3 h, with or without 2 mM of L and D stereoisomers of Lysine and Arginine, and signaling was assessed by Western blot (n = 1 -2 biological replicates, as indicated). One representative Western blot from is shown in FIG 5C and quantification of multiple blots from the two groups are shown in FIG 5B.

[0063] FIG 5D shows activated human CD3+T cells treated with 75 µM cGAMP for 2 h, with or without 1 mM of L and D stereoisomers of Lysine, and signaling was assessed by Western blot (n = 2 biological replicates). One representative Western blot is shown in FIG 5C.

[0064] FIG 5E shows activated mouse CD3+T cells treated with 2’3’-cGAMP for 24h in the presence of ENPP1 inhibitor, STF-1623, with or without the addition of L and D stereoisomers of Lysine and Arginine into supraphysiological-CAA control media (0.05 mM L-Arg and L-Lys) with 2 mM L-Citrulline. Relative viability was measured using Cell Titer Glo Assay (n = 4 biological replicates).

[0065] FIG 5F shows activated human CD3+T cells treated with 2’3’-cGAMP for 24h in the presence of ENPP1 inhibitor, STF-1623, either in RPMI SILAC (0 mM L-Arg and L-Lys) or RPMI (1.15 mM L-Arg and 0.22 mM L-Lys), both supplemented with 2 mM L- Citrulline. Relative viability was measured using Cell Titer Glo Assay (n = 3 biological replicates).Attorney Docket No.: ARCI-003WO

[0066] FIG 6A & 6B shows U937 SLC19A1- / --tet-SLC7A2-Flag cells were incubated with or without 1 μg / mL doxycycline (dox) for 24 h. Cells were then treated with 50 μM cGAMP for 2 h, and signaling was assessed by Western blot (n = 3 biological replicates). One representative Western blot is shown in FIG 6A and quantification of multiple blots is shown in FIG 6B. Data are shown as mean + / - SD.

[0067] FIG 6C & 6D shows U937 SLC19A1- / --tet-SLC7A2-Flag cells were incubated with or without 1 μg / mL doxycycline (dox) for 24 h. Cells were then electroporated with 100 nM cGAMP for 2 h, and signaling was assessed by Western blot (n = 3 biological replicates). One representative Western blot is shown in FIG 6C and quantification of multiple blots is shown in FIG 6D. Significance calculated by unpaired, two-tailed t-test. Data are shown as mean + / - SD.

[0068] FIG 6E shows U937 SLC19A1- / --tet-SLC7A2-Flag cells were incubated with or without 1 μg / mL doxycycline (dox) for 24 h. Cells were then incubated with increasing concentrations of 2’3’-cGAMP for 24 h in the presence of ENPP1 inhibitor, STF-1623. Relative cell count was measured using Attune NxT Flow Cytometer (n = 4 biological replicates). Data are shown as the mean + / - SD.

[0069] FIG 6F shows U937 SLC19A1- / - -tet-SLC7A2-Flag cells were incubated with or without 1 μg / mL doxycycline (dox) for 24 h. Cells were then incubated with 10 μM cGAMP at 37°C for 15 min in the presence of ENPP1 inhibitor STF-1623 (n = 5-6 biological duplicates, as indicated. One extreme outlier with value 29.810-7mol / g protein, from the dox-treated group was omitted after performing Grubb’s test). Cells were then washed in cold PBS, lysed in T-PER lysis buffer, and intracellular cGAMP level was determined using cGAMP ELISA kit and normalized to protein amount. Significance calculated by unpaired, two-tailed t-test. Data are shown as the mean + / - SD. *p<0.05.

[0070] FIG 6G shows AlphaFold-predicted structure of human SLC7A2 with cGAMP docked into a pocket on the extracellular-facing surface. Residues mutated are shown as sticks; residues validated to have an effect on cGAMP transport shown in purple.

[0071] FIG 6H & 6I shows U937 SLC19A1- / --tet-SLC7A2 (WT, E234A, S356M, K61A or D406A)- Flag cells were incubated with or without doxycycline (dox) for 24 h. Cells were then treated with 50 μM cGAMP, and signaling was assessed by Western blot (n = 3 biological replicates). One representative Western Blot is shown in (H), and quantificationAttorney Docket No.: ARCI-003WO of multiple blots is shown in (I). Significance calculated by unpaired, two-tailed t-test. Data are shown as mean + / - SD. *p<0.05

[0072] FIG 6J shows U937 SLC19A1- / --tet-SLC7A2 (WT, E234A, S356M, K61A or D406A)- Flag cells were incubated with or without doxycycline (dox) for 24 h. Cells were then treated with 21 nM3H-Arginine for 1 min (n = 3 biological replicates), and radioactivity was measured using a scintillation counter and normalized to protein amount. Significance calculated by unpaired, two-tailed t-test. Data are shown as mean + / - SD.

[0073] FIG 6K shows human transcript database adopted from Schmiedel et al., 2018 for SLC7A2 expression in different immune cell subsets.

[0074] FIG 6L shows U937 SLC19A1- / --tet-SLC7A2-Flag cells were incubated with or without 1 μg / mL doxycycline (dox) for 24 h. Cells were then treated with 21 nM3H- Arginine for 1 min (n = 3 biological replicates), and radioactivity was measured using a scintillation counter and normalized to protein amount. Data are shown as the mean + / - SD.

[0075] FIG 6M & 6N U937 SLC19A1- / --tet-SLC7A2-Flag cells were incubated in the absence of all cationic amino acids, and with or without 1 μg / mL doxycycline (dox) for 24 h. Cells were then treated with 75 μM cGAMP for 2 h, and signaling was assessed by Western blot (n = 2 biological replicates). One representative Western blot is shown in FIG 6M and quantification of multiple blots is shown in FIG 6N. Data are shown as mean + / - SD.

[0076] FIG 6O shows differential gene analysis (DESeq2) of U937 SLC19A1- / --tet- SLC7A2-Flag cells incubated with or without 1 μg / mL doxycycline (dox) for 24 h (n = 2 biological duplicate). Significantly enriched or depleted coding mRNAs and known extracellular cGAMP / STING signaling elements are annotated.

[0077] FIG 7A, 7B, & 7C shows activated human primary CD3+ T cells electroporated with Cas9 protein and non-targeting (scramble) or SLC7A1-targeting sgRNAs. Cells were treated with 2’3’-cGAMP (FIG 7A), 2’3’-CDAS(FIG 7B), or 3’3’-cGAMP (FIG 7C) at varying concentration four days later, and relative viability was assessed 24 h later using Cell Titer Glo Assay (n = 3 biological replicates). Indel score was determined by ICE (Synthego).

[0078] FIG 7D & 7E shows knockdown of SLC7A1 using three sgRNAs together and further supports that SLC7A1 is not only a cGAMP transporter in but also the transporterAttorney Docket No.: ARCI-003WO of cGAMP analog STING agonists (i.e., 2’3’-CDAs). Lane 1 and 2 in each Donor is cGAMP and Lane 3 and 4 is 2’3’-CDAs.

[0079] FIG 8A shows U937 SLC19A1- / --tet-SLC7A2 (WT, K61A or D406A)-Flag cells were incubated with or without doxycycline (dox) for 24 h. Cells were then treated with 2 μM 2’3’-CDAS(ADU-S100) 24 h in the presence of ENPP1 inhibitor, STF-1623. Relative viability was measured using Cell Titer Glo Assay (n = 3 biological replicates).

[0080] FIG 8B & 8C shows U937 SLC19A1- / --tet-SLC7A2-Flag cells incubated with or without doxycycline (dox) for 24 h. Cells were then treated with 50 µM 2’3’-cGAMP, 15 µM 2’3’-cGSASMP, 125 µM 3’3’-cGAMP, 15 µM 2’3’-CDASor 150 µM 3’3’-CDA for 2 h, and signaling was assessed by Western blot (n = 2 biological replicates). One representative Western blot is shown in (B) and quantification of multiple blots is shown in (C). Data are shown as mean + / - SD.

[0081] FIG 9 shows a gene therapy approach whereby endogenous SLC7A1 is knocked out in a cell, and the cell subsequently transduced / transfected with a construct comprising (a) SLC7A2 comprising either a K61A or D406A mutant, and (b) a CAR. The SLC7A2 mutant facilitates arginine uptake by the cell, but inhibits the uptake of cGAMP and prevents cellular toxicity, while preserving the ability of the CAR to target a tumor associated antigen on a cancer cell (such as a solid tumor cancer cell). DETAILED DESCRIPTION OF THE INVENTION I. Definitions

[0082] Unless otherwise defined, all terms of art, notations, and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this application pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art.

[0083] The singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells,Attorney Docket No.: ARCI-003WO comprising mixtures thereof. “A and / or B” is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B”.

[0084] The terms “administration” and “administering”, as used herein, refer to the delivery of a bioactive composition or formulation by an administration route comprising, but not limited to, intranasal, transdermal, intravenous, intra-arterial, intramuscular, intranodal, intraperitoneal, subcutaneous, intramuscular, oral, intravaginal, and topical administration, or combinations thereof. The term includes, but is not limited to, administering by a medical professional and self-administering.

[0085] The term “effective amount”, “therapeutically effective amount”, or “pharmaceutically effective amount” of a composition of the disclosure, e.g., nucleic acid constructs, srRNAs, recombinant cells, and / or pharmaceutical compositions, generally refers to an amount sufficient for the composition to accomplish a stated purpose relative to the absence of the composition (e.g., achieve the effect for which it is administered, stimulate an immune response, prevent or treat a disease, or reduce one or more symptoms of a disease, disorder, infection, or health condition). An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.” A “reduction” of a symptom means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s). The exact amount of a composition including a “therapeutically effective amount” will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols.1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0086] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.Attorney Docket No.: ARCI-003WO

[0087] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. If the degree of approximation is not otherwise clear from the context, “about” means either within plus or minus 10% of the provided value, or rounded to the nearest significant figure, in all cases inclusive of the provided value. In some embodiments, the term “about” indicates the designated value ± up to 10%, up to ± 5%, or up to ± 1%.

[0088] The term “pharmaceutically acceptable excipient” as used herein refers to any suitable substance that provides a pharmaceutically acceptable carrier, additive, or diluent for administration of a compound(s) of interest to a subject. As such, “pharmaceutically acceptable excipient” can encompass substances referred to as pharmaceutically acceptable diluents, pharmaceutically acceptable additives, and pharmaceutically acceptable carriers. As used herein, the term “pharmaceutically acceptable carrier” includes, but is not limited to, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds (e.g., antibiotics and additional therapeutic agents) can also be incorporated into the compositions.

[0089] As used herein, a “subject” or an “individual” includes animals, such as human (e.g., human individuals) and non-human animals. In some embodiments, a “subject” or “individual” is a patient under the care of a physician. Thus, the subject can be a human patient or an individual who has, is at risk of having, or is suspected of having a health condition of interest (e.g., rabies infection) and / or one or more symptoms of the health condition. The subject can also be an individual who is diagnosed with a risk of the health condition of interest at the time of diagnosis or later. The term “non-human animals” includes all vertebrates, e.g., mammals, e.g., rodents, e.g., mice, non-human primates, and other mammals, such as e.g., sheep, dogs, cows, chickens, and non-mammals, such as amphibians, reptiles, etc.

[0090] It is understood that aspects and embodiments of the disclosure described herein include "comprising", "consisting", and "consisting essentially of" aspects and embodiments. As used herein, "comprising" is synonymous with "including", "containing", orAttorney Docket No.: ARCI-003WO "characterized by", and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of" excludes any elements, steps, or ingredients not specified in the claimed composition or method. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method. Any recitation herein of the term "comprising", particularly in a description of components of a composition or in a description of steps of a method, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or steps.

[0091] All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species for which the compositions and methods disclosed herein are applicable. Thus, the terms include, but are not limited to genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates. Thus, for example, for the genes or gene products disclosed herein, which in some embodiments relate to mammalian nucleic acid and amino acid sequences, are intended to encompass homologous and / or orthologous genes and gene products from other animals including, but not limited to other mammals, fish, amphibians, reptiles, and birds. In some embodiments, the genes, nucleic acid sequences, amino acid sequences, peptides, polypeptides and proteins are human. The term “gene” is also intended to include variants thereof.

[0092] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub- combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.Attorney Docket No.: ARCI-003WO II. Introduction

[0093] Elucidating the cell type-specific cGAMP transporter(s) acting in human T cells could provide a means to selectively diminish the negative effects of cGAMP and other STING agonists on T cell survival and function, while maintaining their anti-cancer effects on other cell types in the tumor microenvironment. The LRRC8A:C heteromeric channel was reported to induce mouse T cell death by acting as a cGAMP channel to facilitate STING- P53 activation20. However, it is not known whether the LRRC8A:C channel transports cGAMP in human T cells, or whether it is in fact the dominant or only cGAMP transporter in mouse T cells.

[0094] In this study, we show that, despite its role in mice, the LRRC8A:C channel does not play a role in transporting cGAMP in activated human Jurkat T cells or activated primary human T cells. Instead, we identified the solute carrier protein SLC7A1 as the human T cell-specific cGAMP transporter. SLC7A1 is highly upregulated in activated T cells to transport the essential amino acid nutrient arginine, but also transports endogenous cGAMP secreted by cancer cells and synthetic cGAMP analogs. Therefore, this upregulation renders activated T cells more susceptible to cGAMP toxicity than resting T cells. Finally, we discovered that the SLC7A1 binding sites for arginine and cGAMP are distinct, suggesting that we can potentially inhibit cGAMP transport without disrupting transport of the essential amino acid arginine. Together, our study demonstrates that SLC7A1 is a potential drug target for sparing tumor infiltrating T cells from collateral damage of therapeutic STING agonists or ionizing radiation, which is crucial for the curative effect of these therapies.

[0095] In this study, we show that, the cationic amino acid transporter SLC7A1 is the dominant T cell-specific cGAMP transporter in activated mouse and human T cells. SLC7A1 is highly upregulated in activated T cells to transport the essential amino acid, arginine, but also transports endogenous cGAMP secreted by cancer cells and synthetic cGAMP analogs. Therefore, this upregulation renders activated T cells more susceptible to cGAMP toxicity than resting T cells. In addition, we discovered that the SLC7A1 binding sites for arginine and cGAMP are distinct and that cGAMP transport can be perturbed without impacting arginine transport. Together, our study demonstrates that SLC7A1 is a potential target for small molecule drug development and gene editing for sparing tumor infiltrating T cells from collateral damage of therapeutic STING agonists or ionizing radiation, which is crucial for the curative effect of these therapies.Attorney Docket No.: ARCI-003WO III. Methods of inducing toxicity in a T cell

[0096] In one aspect, the invention provides a method for inducing toxicity in a T cell, the method comprising contacting the T cell with an effective amount of: a) a compound capable of binding to amino acid residue S354 of SLC7A1; or b) a compound having a structure according to Formula I or II: (I) wherein R1and R2 2and Y are each independently O or S, thereby inducing toxicity in the T cell. In an exemplary embodiment, the compound is selected from the group consisting of: i) the compound of Formula (I), wherein R1is guanine, R2is adenine, Y1is O, and Y2is O; ii) the compound of Formula (I), wherein R1is guanine, R2is adenine, Y1is S, and Y2is S; iii) the compound of Formula (I), wherein R1is adenine, R2is adenine, Y1is S, and Y2is S; iv) the compound of Formula (II), wherein R1is guanine, R2is adenine, Y1is O, and Y2is O; v) the compound of Formula (II), wherein R1is adenine, R2is adenine, Y1is O, and Y2is O; and vi) the compound of Formula (II), wherein R1is guanine, R2is guanine, Y1is O, and Y2is O. In an exemplary embodiment, the T cell is an activated T cell. In an exemplary embodiment, the T cell is a CD8+T cell. In an exemplary embodiment, the T cell is a CD4+T cell. In an exemplary embodiment, the compound inhibits arginine transport in the T cell. In an exemplary embodiment, the compound does not inhibit transport of cGAMP or analogs thereof in the T cell. In an exemplary embodiment, the compound is a) and is a peptide, a small molecule, or an antibody.Attorney Docket No.: ARCI-003WO IV. Methods of inducing toxicity in a T cell population

[0097] In one aspect, the invention provides a method for inducing toxicity in a T cell population, the method comprising contacting the T cell population with an effective amount of: a) a compound capable of binding to amino acid residue S354 of SLC7A1; or b) a compound having a structure according to Formula I or II: (I) wherein R1and R2 2and Y are each independently O or S, thereby inducing toxicity in the T cell population. In an exemplary embodiment, the compound is selected from the group consisting of: i) the compound of Formula (I), wherein R1is guanine, R2is adenine, Y1is O, and Y2is O; ii) the compound of Formula (I), wherein R1is guanine, R2is adenine, Y1is S, and Y2is S; iii) the compound of Formula (I), wherein R1is adenine, R2is adenine, Y1is S, and Y2is S; iv) the compound of Formula (II), wherein R1is guanine, R2is adenine, Y1is O, and Y2is O; v) the compound of Formula (II), wherein R1is adenine, R2is adenine, Y1is O, and Y2is O; and vi) the compound of Formula (II), wherein R1is guanine, R2is guanine, Y1is O, and Y2is O. In an exemplary embodiment, the T cells are activated T cells. In an exemplary embodiment, the T cells are CD8+T cells. In an exemplary embodiment, the T cells are CD4+T cells. In an exemplary embodiment, the compound inhibits arginine transport in the T cells. In an exemplary embodiment, the compound does not inhibit transport of cGAMP or analogs thereof in the T cells. In an exemplary embodiment, the compound is a) and is a peptide, a small molecule, or an antibody.Attorney Docket No.: ARCI-003WO V. Methods of treating a solid cancerous tumor in a subject

[0098] In one aspect, the invention provides a method of treating a solid cancerous tumor in a subject, comprising: administering to the subject not otherwise in need of treatment thereof, a therapeutically effective amount of an agent that binds to the cGAMP binding pocket of SLC7A1, thereby treating the solid cancerous tumor in the subject. In an exemplary embodiment, the agent binds to one or more amino acid residues of SLC7A1 selected from R59, R130, and / or D404. In an exemplary embodiment, the method further comprises administering one or more additional compounds to the subject to treat the solid cancerous tumor. In an exemplary embodiment, the invention further comprises administering ionizing radiation to the subject. In an exemplary embodiment, the solid cancerous tumor is selected from the group consisting of adrenal, liver, kidney, bladder, breast, colon, gastric, ovarian, cervical, uterine, esophageal, colorectal, prostate, pancreatic, lung (both small cell and non-small cell), thyroid, carcinomas, sarcomas, glioblastomas, melanoma and various head and neck tumors. In an exemplary embodiment, the subject is a human. In an exemplary embodiment, the agent does not interfere with arginine transport in T cells of the subject. In an exemplary embodiment, the agent inhibits transport of cGAMP or analogs thereof in T cells of the subject. In an exemplary embodiment, the agent enhances T cell infiltration into the solid cancerous tumor of the subject. VI. Methods of reducing toxicity in a T cell population

[0099] In one aspect, the invention provides a method for reducing toxicity in a T cell population, the method comprising contacting the T cell population with an effective amount of an agent that binds to the cGAMP binding pocket of SLC7A1, under conditions effective to reduce toxicity in the T cell population. In an exemplary embodiment, the agent binds to one or more amino acid residues of SLC7A1 selected from R59, R130, and / or D404. In an exemplary embodiment, the toxicity is induced by cGAMP. In an exemplary embodiment, the T cell population comprises activated T cells. In an exemplary embodiment, the T cell population comprises CD8+T cells. In an exemplary embodiment, the T cell population comprises CD4+T cells. In an exemplary embodiment, the T cell population is one T cell. In an exemplary embodiment, the agent does not interfere with arginine transport into the T cells. In an exemplary embodiment, the agent inhibits transport of cGAMP or analogs thereof into the T cells. In an exemplary embodiment, the toxicity is induced by ionizing radiation. In an exemplary embodiment, the toxicity is induced cGAMP.Attorney Docket No.: ARCI-003WO VII. Methods of inhibiting cGAMP binding to SLC7A1 on a T cell

[0100] In one aspect, the invention provides a method of inhibiting cGAMP binding to SLC7A1 on a T cell, the method comprising contacting the T cell with an effective amount of an agent that binds to the cGAMP binding pocket of SLC7A1, under conditions effective to inhibit the cGAMP binding to SLC7A1 on the T cell. In an exemplary embodiment, the agent binds to one or more amino acid residues of SLC7A1 selected from R59, R130, and / or D404. In an exemplary embodiment, the T cell is an activated T cell. In an exemplary embodiment, the T cell is a CD8+T cell. In an exemplary embodiment, the T cell is a CD4+T cell. In an exemplary embodiment, the agent does not interfere with arginine transport in the T cells. VIII. Methods of identifying an agent that inhibits SLC7A1

[0101] In one aspect, the invention provides a method of identifying an agent that inhibits SLC7A1, the method comprising: contacting a cell overexpressing wildtype SLC7A1 with a compound and detecting cGAMP induced toxicity in the cell. In an exemplary embodiment, a decrease in cGAMP induced toxicity identifies the agent as an inhibitor of SLC7A1. In an exemplary embodiment, no change in arginine transport identifies the agent as an inhibitor of SLC7A1. In an exemplary embodiment, the cell is a T cell. In an exemplary embodiment, further comprising overexpressing a mutant SLC7A1 in a cell and contacting said cell with the agent. IX. Agents that bind amino acid residues of SLC7A1

[0102] In one aspect, the invention provides an agent that binds to the cGAMP binding pocket of SLC7A1. In one aspect, the invention provides an agent that binds to one or more amino acid residues of SLC7A1 selected from R59, R130, and / or D404. In an exemplary embodiment, the agent inhibits transport of cGAMP or analogs thereof in T cells. In an exemplary embodiment, the agent does not interfere with arginine transport in T cells. In an exemplary embodiment, the agent comprises a peptide, a small molecule, or an antibody. In an exemplary embodiment, the agent reduces T cell toxicity induced by cGAMP or analogs thereof. X. Methods of inhibiting cGAMP binding to SLC7A2 on a T cell

[0103] In one aspect, the invention provides a method of inhibiting cGAMP binding to SLC7A2 on a T cell, the method comprising contacting the T cell with an effective amount of an agent that binds to the cGAMP binding pocket of SLC7A2, under conditions effectiveAttorney Docket No.: ARCI-003WO to inhibit the cGAMP binding to SLC7A2 on the T cell. In an exemplary embodiment, the agent binds to one or more amino acid residues of SLC7A2 selected from K61 or D406. In an exemplary embodiment, the T cell is an activated T cell. In an exemplary embodiment, the T cell is a CD8+T cell. In an exemplary embodiment, the T cell is a CD4+T cell. In an exemplary embodiment, the agent does not interfere with arginine transport in the T cells. XI. Methods of identifying an agent that inhibits SLC7A2

[0104] In one aspect, the invention provides a method of identifying an agent that inhibits SLC7A2, the method comprising: contacting a cell overexpressing wildtype SLC7A2 with a compound and detecting cGAMP induced toxicity in the cell. In an exemplary embodiment, a decrease in cGAMP induced toxicity identifies the agent as an inhibitor of SLC7A2. In an exemplary embodiment, no change in arginine transport identifies the agent as an inhibitor of SLC7A2. In an exemplary embodiment, the cell is a T cell. In an exemplary embodiment, further comprising overexpressing a SLC7A2 (such as a mutant SLC7A2) in a cell and contacting said cell with the agent. XII. Agents that bind amino acid residues of SLC7A2

[0105] In one aspect, the invention provides an agent that binds to the cGAMP binding pocket of SLC7A2. In one aspect, the invention provides an agent that binds to one or more amino acid residues of SLC7A2 selected from K61 or D406. In an exemplary embodiment, the agent inhibits transport of cGAMP or analogs thereof in T cells. In an exemplary embodiment, the agent does not interfere with arginine transport in T cells. In an exemplary embodiment, the agent comprises a peptide, a small molecule, or an antibody. In an exemplary embodiment, the agent reduces T cell toxicity induced by cGAMP or analogs thereof. XIII. Proteins

[0106] In an exemplary embodiment, the invention comprises a SLC7A1 protein with a diminished ability to bind cGAMP, as compared to wild-type SLC7A1. Wild-type SLC7A1 protein has a sequence according to SEQ ID NO: 9. In an exemplary embodiment, the SLC7A1 protein comprises a sequence according to SEQ ID NO: 9, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 9. In an exemplary embodiment, the invention comprises a SLC7A1 protein with a diminished ability to bind cGAMP, and with an essentially undiminished ability to bind arginine, as compared to wild-type SLC7A1. InAttorney Docket No.: ARCI-003WO an exemplary embodiment, the SLC7A1 protein comprises a mutation at R59, R130, and / or D404, relative to wild-type SLC7A1. In an exemplary embodiment, the SLC7A1 protein comprises a R59A, R130A, and / or D404A mutation, relative to wild-type SLC7A1. In an exemplary embodiment, the SLC7A1 protein comprises a sequence according to SEQ ID NOs: 10, 11, 12, or 13, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOs: 10, 11, 12, or 13. In an exemplary embodiment, the SLC7A1 protein comprises a sequence according to SEQ ID NOs: 10, 11, 12, or 13, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOs: 10, 11, 12, or 13, wherein the sequence is not SEQ ID NO: 9. In an exemplary embodiment, the SLC7A1 protein comprises a sequence according to SEQ ID NOs: 10, 11, 12, or 13, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOs: 10, 11, 12, or 13, wherein the sequence is not a wild-type SLC7A1 protein, such as SEQ ID NO: 9. In an exemplary embodiment, the invention provides an engineered cell comprising (such as expressing) the SLC7A1 protein described herein. In an exemplary embodiment, the engineered cell comprising (such as expressing) the SLC7A1 protein described herein is an immune cell. In an exemplary embodiment, the engineered immune cell comprising (such as expressing) the SLC7A1 protein described herein is a T cell. In an exemplary embodiment, the engineered T cell comprising (such as expressing) the SLC7A1 protein described herein is a tumor-infiltrating CD3+ T lymphocyte, including CD8+ cytotoxic T lymphocyte (CTL) and CD4+ helper T cell. In an exemplary embodiment, the engineered T cell can be an allogeneic T cell or an autologous T cell. In an exemplary embodiment, the engineered cell further comprises (such as expresses) an antibody, antigen- binding fragment, receptor, e.g., Chimeric Antigen Receptor (CAR), which recognizes a tumor associated antigen on a cancer cell.

[0107] In an exemplary embodiment, the invention comprises a SLC7A2 protein with a diminished ability to bind cGAMP, as compared to wild-type SLC7A2. Wild-type SLC7A2 protein has a sequence according to SEQ ID NO: 14. In an exemplary embodiment, the SLC7A2 protein comprises a sequence according to SEQ ID NO: 14, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 14. In an exemplary embodiment, the invention comprises a SLC7A2 protein with a diminished ability to bind cGAMP, and withAttorney Docket No.: ARCI-003WO an essentially undiminished ability to bind arginine, as compared to wild-type SLC7A2. In an exemplary embodiment, the SLC7A2 protein comprises a mutation at K61 or D406, relative to wild-type SLC7A2. In an exemplary embodiment, the SLC7A2 protein comprises a K61A or D406A mutation, relative to wild-type SLC7A2. In an exemplary embodiment, the SLC7A2 protein comprises a sequence according to SEQ ID NOs: 15 or 16, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOs: 15 or 16. In an exemplary embodiment, the SLC7A2 protein comprises a sequence according to SEQ ID NOs: 15 or 16, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOs: 15 or 16, wherein the sequence is not SEQ ID NO: 14. In an exemplary embodiment, the SLC7A2 protein comprises a sequence according to SEQ ID NOs: 15 or 16, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOs: 15 or 16, wherein the sequence is not a wild-type SLC7A2 protein, such as SEQ ID NO: 14. In an exemplary embodiment, the invention provides an engineered cell comprising (such as expressing) the SLC7A2 protein described herein. In an exemplary embodiment, the engineered cell comprising (such as expressing) the SLC7A2 protein described herein is an immune cell. In an exemplary embodiment, the engineered immune cell comprising (such as expressing) the SLC7A2 protein described herein is a T cell. In an exemplary embodiment, the engineered T cell comprising (such as expressing) the SLC7A2 protein described herein is a tumor-infiltrating CD3+ T lymphocyte, including CD8+ cytotoxic T lymphocyte (CTL) and CD4+ helper T cell. The engineered T cell can be an allogeneic T cell or an autologous T cell. In an exemplary embodiment, the engineered cell further comprises (such as expresses) an antibody, antigen-binding fragment, receptor, e.g., Chimeric Antigen Receptor (CAR), which recognizes a tumor associated antigen on a cancer cell.

[0108] In an exemplary embodiment, the CAR comprises an extracellular and intracellular domain. The extracellular domain comprises a tumor associated antigen binding element otherwise referred to as an antigen binding moiety. The intracellular domain or otherwise the cytoplasmic domain comprises a costimulatory signaling region and a zeta chain portion. The costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. Costimulatory molecules are cellAttorney Docket No.: ARCI-003WO surface molecules other than antigen receptors or their ligands that are required for an efficient response of lymphocytes to antigen.

[0109] Between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR, there may be incorporated a spacer domain. As used herein, the term “spacer domain” generally means any oligo- or polypeptide that functions to link the transmembrane domain to, either the extracellular domain or, the cytoplasmic domain in the polypeptide chain. A spacer domain may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids.

[0110] CARs of the disclosure can target solid tumors via any tumor associated antigen to target and eliminate cancer cell. Exemplary tumor associated antigens include mesothelin (MSLN), HER2, EGFR, GD2, PSMA, and claudin 18.2. Other targets include MUC1, CEA, ROR1, and GPC3. CARs of the disclosure can target tumor associated antigens known to those skilled in the art.

[0111] A variety of assays are known for assessing binding affinity, equilibrium dissociation constant (KD), equilibrium association constant (KA), EC50, on-rate (association rate constant; kon or ka; units of 1 / Ms or M-1s-1) and the off-rate (dissociation rate constant; koff or kd; units of 1 / s or s-1) and / or determining whether a binding molecule (e.g., an antibody or fragment thereof, or a protein such as SLC7A1 or SLC7A2) specifically binds to a particular ligand (e.g., an antigen, such as a tumor associated antigen, or cGAMP or arginine). One can determine the binding affinity of a binding molecule by using any of a number of binding assays that are well known. For example, in some embodiments, a BIAcore® instrument can be used to determine the binding kinetics and constants of a complex between two proteins, using surface plasmon resonance (SPR) analysis (see, e.g., Scatchard et al., Ann. N.Y. Acad. Sci.51:660, 1949; Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res.53:2560, 1993; and U.S. Patent Nos.5,283,173, 5,468,614, or the equivalent).

[0112] SPR measures changes in the concentration of molecules at a sensor surface as molecules bind to or dissociate from the surface. The change in the SPR signal is directly proportional to the change in mass concentration close to the surface, thereby allowing measurement of binding kinetics between two molecules. The dissociation rate constant (koff or kd), the association rate constant (konor ka) and / or equilibrium dissociation constant (Kn)Attorney Docket No.: ARCI-003WO and / or equilibrium association constant (KA) for the complex can be determined by monitoring changes in the refractive index with respect to time as buffer is passed over the chip. Other suitable assays for measuring the binding of one protein to another include, for example, immunoassays such as enzyme linked immunosorbent assays (ELISA) and radioimmunoassays (RIA), or determination of binding by monitoring the change in the spectroscopic or optical properties of the proteins through fluorescence, UV absorption, circular dichroism, or nuclear magnetic resonance (NMR). Other exemplary assays include, but are not limited to, Western blot, ELISA, analytical ultracentrifugation, spectroscopy, flow cytometry, sequencing, genetic reporter assays, flow cytometry, and other methods for detection of expressed nucleic acids or binding of proteins.

[0113] In an exemplary embodiment, the invention provides a SLC7A1 protein comprising a sequence according to SEQ ID NOs: 10, 11, 12, or 13, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOs: 10, 11, 12, or 13. In an exemplary embodiment, the invention provides a SLC7A2 protein comprising a sequence according to SEQ ID NOs: 15 or 16, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOs: 15 or 16. In an exemplary embodiment, the invention provides an engineered cell comprising the protein of claim 1 or 2 or a SLC7A2 protein comprising a sequence according to SEQ ID NO: 14, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 14. In an exemplary embodiment, the engineered T cell which is tumor-infiltrating CD3+ T lymphocyte. In an exemplary embodiment, the engineered cell essentially has no endogenous SLC7A1 activity. In an exemplary embodiment, the engineered cell further comprising a CAR which recognizes a tumor associated antigen. In an exemplary embodiment, the invention provides a method of making the engineered cell described herein, comprising incorporating nucleic acids encoding a SLC7A2 or mutant SLC7A1 described herein into the cell. In an exemplary embodiment, the method further comprises incorporating nucleic acids encoding a chimeric antigen receptor described herein into the cell. XIV. Nucleic acids

[0114] In an exemplary embodiment, the invention comprises a sequence of nucleic acids encoding a SLC7A1 described herein, such as SEQ ID NOs: 1, 2, 3, 4, or 5, or a sequence of nucleic acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,Attorney Docket No.: ARCI-003WO 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOs: 1, 2, 3, 4, or 5. A sequence of nucleic acids encoding wild-type SLC7A1 protein is according to SEQ ID NO: 1. In an exemplary embodiment, the sequence of nucleic acids encoding wild-type SLC7A1 protein is according to SEQ ID NO: 1, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1. In an exemplary embodiment, the invention comprises a sequence of nucleic acids encoding a SLC7A1 protein with a diminished ability to bind cGAMP, as compared to wild-type SLC7A1. In an exemplary embodiment, the sequence of nucleic acids encode a SLC7A1 protein comprising a mutation at R59, R130, and / or D404, relative to wild-type SLC7A1. In an exemplary embodiment, the sequence of nucleic acids encode a SLC7A1 protein comprising a R59A, R130A, and / or D404A mutation, relative to wild-type SLC7A1. In an exemplary embodiment, the invention comprises a sequence of nucleic acids encoding a SLC7A1 described herein, such as SEQ ID NOs: 2, 3, 4, or 5, or a sequence of nucleic acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOs: 2, 3, 4, or 5. In an exemplary embodiment, the invention comprises a sequence of nucleic acids encoding a SLC7A1 described herein, such as SEQ ID NOs: 2, 3, 4, or 5, or a sequence of nucleic acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOs: 2, 3, 4, or 5, wherein the sequence is not SEQ ID NO: 1. In an exemplary embodiment, the invention comprises a sequence of nucleic acids encoding a SLC7A1 described herein, such as SEQ ID NOs: 2, 3, 4, or 5, or a sequence of nucleic acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOs: 2, 3, 4, or 5, wherein the sequence is not a wild-type SLC7A1 protein, such as SEQ ID NO: 1. In an exemplary embodiment, the invention provides a vector comprising a sequence of nucleic acids described in this paragraph.

[0115] In an exemplary embodiment, the invention comprises a sequence of nucleic acids encoding a SLC7A2 described herein, such as SEQ ID NOs: 6, 7, or 8, or a sequence of nucleic acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOs: 6, 7, or 8. A sequence of nucleic acids encoding wild-type SLC7A2 protein is according to SEQ ID NO: 6. In an exemplary embodiment, the sequence of nucleic acids encoding wild-type SLC7A2 protein is according to SEQ ID NO: 6, or a sequence that exhibits at least 85%, 86%, 87%,Attorney Docket No.: ARCI-003WO 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 6. In an exemplary embodiment, the invention comprises a sequence of nucleic acids encoding a SLC7A2 protein with a diminished ability to bind cGAMP, as compared to wild-type SLC7A2. In an exemplary embodiment, the sequence of nucleic acids encoding a SLC7A2 protein comprises a mutation at K61 or D406, relative to wild-type SLC7A2. In an exemplary embodiment, the sequence of nucleic acids encoding a SLC7A2 protein comprises a K61A or D406A mutation, relative to wild-type SLC7A2. In an exemplary embodiment, the invention comprises a sequence of nucleic acids encoding a SLC7A2 described herein, such as SEQ ID NOs: 7 or 8, or a sequence of nucleic acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOs: 7 or 8. In an exemplary embodiment, the invention comprises a sequence of nucleic acids encoding a SLC7A2 described herein, such as SEQ ID NOs: 7 or 8, or a sequence of nucleic acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOs: 7 or 8, wherein the sequence is not SEQ ID NO: 6. In an exemplary embodiment, the invention comprises a sequence of nucleic acids encoding a SLC7A2 described herein, such as SEQ ID NOs: 7 or 8, or a sequence of nucleic acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOs: 7 or 8, wherein the sequence is not a wild-type SLC7A2 protein, such as SEQ ID NO: 6. In an exemplary embodiment, the invention provides a vector comprising a sequence of nucleic acids described in this paragraph.

[0116] The nucleic acid sequence described herein can be prepared from an amino- acid sequence of the SLC7A1 or SLC7A2 protein of interest by a conventional method. A nucleotide sequence encoding an amino acid sequence can be obtained using the tools provided to the public by the National Center for Biotechnology Information (NCBI), e.g., from NCBI RefSeq IDs or accession numbers of GenBank for an amino acid sequence of each domain. The nucleic acid of the present invention can be prepared using a standard molecular biological or chemical procedure. In some embodiments, based on the nucleotide sequence, portions of the nucleic acid are synthesized. In some embodiments, the nucleic acid of the present invention is prepared by combining DNA fragments which are obtained from a cDNA library using the polymerase chain reaction (PCR).

[0117] In some embodiments, a nucleic acid encoding the SLC7A1 or SLC7A2 protein of interest is inserted into a vector, and the vector is introduced into a cell. In someAttorney Docket No.: ARCI-003WO embodiments, the vector is a viral vector such as a retroviral vector (including an oncoretroviral vector, a lentiviral vector, and a pseudo-type vector), an adenoviral vector, an adeno-associated virus (AAV) vector, a simian virus vector, a vaccinia virus vector, a Sendai virus vector, an Epstein-Barr virus (EBV) vector, or a herpes simplex virus (HSV) vector. In some embodiments, a viral vector lacking the replicating ability so as not to self-replicate in an infected cell is used.

[0118] In some embodiments, retroviral particles are prepared using a packaging cell line. In such embodiments, a suitable packaging cell line based on the LTR sequence, and the packaging signal sequence possessed by the viral vector is selected. Examples of the packaging cell lines include PG13 (ATCC CRL-10686), PA317 (ATCC CRL-9078), GP+E- 86, GP+envAm-12, and Psi-CRIP. In some embodiments, retroviral particles are prepared using the HEK293 cell line or the HEK293t cell line having high transfection efficiency. One of skill in the art is aware of many kinds of retroviral vectors and packaging cell lines that are commercially available. XV. Adoptive Cell Therapies

[0119] By constructing a T-cell with a nucleic acid encoding the SLC7A1 mutant or SLC7A2 protein of interest, a T-cell is generated with a reduced capacity for cGAMP uptake, and thus reduced T-cell toxicity. Such a T-cell can be utilized in adoptive cell therapies. In some embodiments, expression of endogenous SLC7A1 is downregulated in a cell, and a mutant SLC7A1 or SLC7A2 of the disclosure is introduced into such a cell.

[0120] In brief summary, the expression of natural or synthetic nucleic acids encoding mutant SLC7A1, SLC7A2 and / or, CARs is typically achieved by operably linking a nucleic acid encoding the SLC7A1, SLC7A2 and / or, CAR polypeptide or portions thereof to a promoter, and incorporating the construct into an expression vector. The vectors can be suitable for replication and integration in eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence. In some embodiments, two vectors are introduced into a T cell. One vector can comprise a nucleic acid encoding an SLC7A1 or SLC7A2 of the disclosure. Another vector can comprise a nucleic acid encoding a CAR of the disclosure.

[0121] Any nucleic acid of the disclosure (e.g., a nucleic acid encoding SLC7A1, SLC7A2 and / or, CARs) can be cloned into a number of types of vectors. For example, theAttorney Docket No.: ARCI-003WO nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. In some embodiments the vector comprising a nucleic acid encoding SLC7A1 and / or SLC7A2 does not comprise a nucleic acid encoding a CAR.

[0122] The nucleic acids of the disclosure can be operably linked to a promoter. The term “operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. In some embodiments a vector comprises a nucleic acid encoding a SLC7A1 or SLC7A2 of the disclosure are operably linked to one promoter and a nucleic acid encoding a CAR of the disclosure is operably linked to a second promoter.

[0123] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is Elongation Growth Factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0124] Vectors of the disclosure may comprise additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located inAttorney Docket No.: ARCI-003WO the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

[0125] In order to assess the expression of a SLC7A1, SLC7A2, or CAR polypeptide or portions thereof as described herein in a cell of the disclosure, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.

[0126] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene. In some embodiments the reporter gene is tNGFR (i.e., truncated nerve growth factor receptor). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5′ flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription. Methods for downregulating expression of endogenous SLC7A1

[0127] In some embodiments, the T cell of the disclosure comprises a downregulated or knocked-out endogenous SLC7A1. Methods of modifying gene expression, such as downregulating gene expression (e.g., via knock-out) can be achieved by methods that are readily available and well-known in the art. Gene editing methods, such as CRISPR-Cas9, orAttorney Docket No.: ARCI-003WO any other CRISPR-based gene editing system known in the art may be used to downregulate expression of, or knock-out, endogenous SLC7A1 from a cell described herein. CRISPR- Cas9-mediated gene editing can involve the use of at least 1, 2, 3, 4, 5 or 6 guide RNAs (sgRNAs). In some embodiments, CRISPR-Cas9-meditated gene editing involves the use of three guide RNAs.

[0128] Additional gene editing methods as known in the art can also be used in knocking-out endogenous SLC7A1. Some examples include gene editing approaching involve zinc finger nuclease (ZFN), transcription activator-like effector nucleases (TALEN), restriction endonucleases, meganucleases homing endonucleases, and the like.

[0129] Any nucleic acids disclosed herein to be delivered to a cell of the disclosure (including, but not limited to, gene editing systems for knocking-out endogenous SLC7A1 and / or nucleic acid constructs encoding mutant SLC7A1 or SLC7A2 and a CAR) may be delivered using a vector system, including, but not limited to, plasmid vectors, DNA minicircles, retroviral vectors, lentiviral vectors, adenovirus vectors, poxvirus vectors; herpesvirus vectors and adeno-associated virus vectors, and combinations thereof.

[0130] Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding nucleases and / or guide RNAs into cells (e.g., T cells). Non- viral vector delivery systems include DNA plasmids, DNA minicircles, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. Methods of non-viral delivery of nucleic acids include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, naked RNA, capped RNA, artificial virions, and agent-enhanced uptake of DNA. Sonoporation can also be used for delivery of nucleic acids. XVa. Chimeric Antigen Receptor (CAR) T Cell Therapy

[0131] A CAR-T cell (or a CAR-NK cell) binds to a tumor associated antigen via the CAR, whereby a signal is transmitted into the cell, and the cell is activated. The activation of the cell expressing the CAR is varied depending on the kind of the cell type and the intracellular domain of the CAR. Activation of the cell can be confirmed based on, for example, release of a cytokine, any improvement of a cell proliferation rate, a change in any cell-surface molecule, or the like. Furthermore, the release of cytotoxic cytokines (IFNy,Attorney Docket No.: ARCI-003WO TNFa, etc.) from the activated CAR-T cell (or CAR-NK cell) causes destruction of a target cell expressing an antigen which can be detected or measured. In addition, release of a cytokine or change in a cell-surface molecule results in detectable or measurable stimulation of other immune cells, for example, B cells, dendritic cells, NK cells, and macrophages.

[0132] In certain embodiments, the invention provides a T-cell with a nucleic acid encoding the SLC7A1 mutant or SLC7A2 protein of interest (such as a SLC7A2 mutant described herein), along with a CAR. Without being bound by theory, CAR T cell therapy to target solid tumors may suffer because of the cytotoxic effects of STING signaling on T cells. Efficacy of CAR T cell therapy may be improved by pairing the CAR with a SLC7A1 mutant or SLC7A2 protein of interest (such as a SLC7A2 mutant described herein).

[0133] In some embodiments, the cell expressing the SLC7A1 mutant or SLC7A2 protein of interest (such as a SLC7A2 mutant described herein), along with the CAR is used as a therapeutic agent for a disease. The therapeutic agent comprises the cell expressing the SLC7A1 mutant or SLC7A2 protein of interest, along with the CAR.

[0134] In one embodiment, the invention comprises T-cells engineered with chimeric antigen receptors to target cancer cells overexpressing a tumor associated antigen of interest.

[0135] The T-cells of the invention can be effectively used to target the tumor associated antigen of interest in certain positive cells and tumors (such as solid tumors). The T cells of the invention can be used clinically against tumor cells, tumors, and cancer stem cells that are resistant to chemotherapy and form aggressive tumors.

[0136] The nucleic acids disclosed herein can be used for generating CAR-T cells. The present invention provides T cells of the disclosure modified to express the SLC7A1 mutant or SLC7A2 of interest, along with the CAR.

[0137] The CAR-expressing cells disclosed herein can be autologous cells and allogenic cells.

[0138] FIG 9 shows an approach for generating an engineered cell of the invention.

[0139] In an exemplary embodiment, the invention provides an engineered cell (such as a T cell) comprising a protein described herein (such as SLC7A1 mutant or SLC7A2 of interest). In an exemplary embodiment, the engineered cell (such as a T cell) essentially has no endogenous SLC7A1 activity. In an exemplary embodiment, the engineered cell (such as a T cell) further comprises a CAR which recognizes a tumor associated antigen.Attorney Docket No.: ARCI-003WO

[0140] In an exemplary embodiment, the invention provides a method of making the engineered cell described herein, comprising incorporating nucleic acids encoding a mutant SLC7A1 or SLC7A2 described herein into the cell. In an exemplary embodiment, the method further comprises incorporating nucleic acids encoding a chimeric antigen receptor described herein into the cell.

[0141] In an exemplary embodiment, the invention provides a method of treating a disease (such as cancer) in a subject in need of treatment thereof, comprising administering an engineered cell described herein to the subject, thereby treating the disease. In an exemplary embodiment, the disease is associated with a tumor specific antigen described herein.

[0142] All publications and patent applications mentioned in this disclosure are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0143] No admission is made that any reference cited herein constitutes prior art. The discussion of the references states what their authors assert, and the Applicant reserves the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of information sources, including scientific journal articles, patent documents, and textbooks, are referred to herein; this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0144] The discussion of the general methods given herein is intended for illustrative purposes only. Other alternative methods and alternatives will be apparent to those of skill in the art upon review of this disclosure, and are to be included within the spirit and purview of this application.

[0145] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims. EXAMPLES

[0146] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art.Attorney Docket No.: ARCI-003WO Such techniques are explained fully in the literature, such as Sambrook, J., & Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russel, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (jointly referred to herein as “Sambrook”); Ausubel, F. M. (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (including supplements through 2014); Bollag, D. M. et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, M. G. et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, K. B., Ferré, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, E. A. (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, S. L. et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements through 2014); and Makrides, S. C. (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences B.V., the disclosures of which are incorporated herein by reference.

[0147] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims. EXAMPLE 1 Materials and Methods Material Source and / or ID Reference Recombinant DNA pLVX-TetOne-Flag Cordova et al 20219N / A pLVX-TetOne-SLC7A1-Flag Herein N / A pLVX-TetOne-SLC7A1 R59A-Flag Herein N / A pLVX-TetOne-SLC7A1 D404A-Flag Herein N / A pLVX-TetOne-SLC7A1 R130A-Flag Herein N / A pLVX-TetOne-SLC7A1 S354M-Flag Herein N / A pLVX-TetOne-SLC7A1 T153A-Flag Herein N / A pLVX-TetOne-SLC7A1 E162A-Flag Herein N / AAttorney Docket No.: ARCI-003WO pLVX-TetOne-SLC7A1 K406A-Flag Herein N / A pLVX-TetOne-SLC7A1 R59A Herein N / A D404A-Flag pLVX-TetOne-SLC7A1 R59A Herein N / A R130A-Flag pLVX-TetOne-SLC7A1 D404A Herein N / A R130A-Flag pLVX-TetOne-SLC7A1 R59A D404A Herein N / A R130A-Flag pLVX-TetOne-SLC7A2-Flag Herein N / A pLVX-TetOne-SLC7A2 K61A-Flag Herein N / A pLVX-TetOne-SLC7A2 D406A-Flag Herein N / A pLVX-TetOne-SLC7A2 S356M-Flag Herein N / A pLVX-TetOne-SLC7A2 E234A-Flag Herein N / A pTB146 His-SUMO T. Bernhard, Harvard N / A Medical School pTB146 His-SUMO-DnCV Herein N / A pcDNA3.1 ThermoFisher Cat #: V79020 lentiCRISPR v2 Addgene RRID: Addgene_52961 pHDM-G Harvard PlasmID EvNO00061606 pHDM-Hgmp2 Harvard PlasmID EvNO00061607 pHDM-tat1b Harvard PlasmID EvNO00061608 pRC / CMV-rev1b Harvard PlasmID EvNO00061616 pMCB320 Addgene RRID: Addgene_89359 pcDNA3.1-FLAG-SLC7A1 Herein N / A Chemicals and reagents Direct-zol RNA extraction kit Zymo Research Cat #: R2050 Gibson Assembly HiFi DNA Master NEB Cat #: E2621 Mix Pierce Anti-DYKDDDDK Affinity ThermoFisher Cat #: A36803 Resin FLAG peptide MedChemExpress Cat #: HY-P0223 HisPur Cobalt Resin ThermoFisher Cat #: 89966 cOmplete protease inhibitor tablets Roche Cat #: 11697498001 2'3'-cGAMP (in-house synthesis) Ritchie et al 2019293'3'-cGAMP Invivogen Cat #: tlrl-nacga-2.5 2'3'-cGAM(PS)2 (Rp / Sp) (2'3'- Invivogen Cat #: tlrl-nacga2srs-05 cGSASMP) ADU-S100 (2'3'-CDAs) Invivogen Cat #: tlrl-nacda2r-1 3'3'-CDA Invivogen Cat #: tlrl-nacda-5 3'3'-CDG Invivogen Cat #: tlrl-nacdg-5 STF-1623 Carozza et al 20206Attorney Docket No.: ARCI-003WO Doxycycline Hydrochloride Sigma-Aldrich Cat #: D3447 Sulfasalazine Sigma-Aldrich Cat #: S0883 DCPIB Tocris Cat #: 1540 Folinic acid calcium salt hydrate Sigma-Aldrich Cat #: F7878 Methotrexate hydrate Sigma-Aldrich Cat #: A6770 Phorbol 12-myristate 13-acetate Cayman Chemical Cat #:10008014 (PMA) Ionomycin calcium salt Cayman Chemical Cat #:11932 L-Citrulline TCI Chemical Cat #: C0372 L-Lysine Sigma-Aldrich Cat #: W384720 Dynabeads™ Human T-Activator ThermoFisher Cat #: 11131D CD3 / CD28 Dynabeads™ Mouse T-Activator ThermoFisher Cat #:11452D CD3 / CD28 Human IL-2 Recombinant Protein Peprotech Cat #: 200-02 (ThermoFisher) UltraComp eBeads™ Plus ThermoFisher Cat #: 01-3333-41 Compensation Beads ArC™ Amine Reactive Compensation ThermoFisher Cat #: A10346 Bead Kit T-PER™ Tissue Protein Extraction ThermoFisher Cat #: 78510 Reagent 6-Chloropurine-riboside-5'- Jena Bioscience Cat #: NU-1109L triphosphate Guanosine 5′-triphosphate sodium salt Sigma Aldrich Cat #: G8877 hydrate Triethylamine Sigma Aldrich Cat #: 471283 2-[3-(but-3-yn-1-yl)-3H-diazirin-3- AxisPharm Cat #: AP15093 yl]ethan-1-amine Arginine Monohydrochloride L-[2,3,4- Perkin Elmer Cat #: NET1123250UC3H]-, 250μCi (9.25MBq) ([3H] L- (Revvity) Arginine) Critical Commercial Assays CellTiter-Glo® 2.0 Cell Viability Promega Cat #: G9241 Assay NextSeq 500 / 550 mid output v2 kit Illumina Cat#: FC-404-2001 Pierce BCA Protein Assay Kit Thermo Scientific Cat #: 23227 RosetteSep Human T Cell Enrichment StemCell Cat #: 15061 Cocktail Technologies MojoSort Mouse CD4 T Cell Isolation BioLegend Cat #: 480033 Kit MojoSort Mouse CD8 T Cell Isolation BioLegend Cat 3:480035 Kit 2'3'-cGAMP ELISA kit Cayman Chemical Cat #: 501700Attorney Docket No.: ARCI-003WO Antibodies for Western Blot α-tubulin (DM1A), Mouse mAb Sigma-Aldrich Cat#: T6199; RRID: AB_477583 STING (D2P2F) Rabbit mAb Cell Signaling Cat#: 13647; RRID: Technologies AB_2732796 IRF-3 (D83B9) Rabbit mAb Cell Signaling Cat# 4302; RRID: Technologies AB_1904036 DYKDDDDK (D6W5B) Rabbit mAb Cell Signaling Cat#: 14793 Technologies RRID:AB_2572291 Phospho-IRF-3 (Ser396) (D6O1M) Cell Signaling Cat# 10327; RRID: Rabbit mAb Technologies AB_2773013 Phospho-STING (Ser366) (D7C3S) Cell Signaling Cat# 19781; RRID: Rabbit mAb Technologies AB_2737062 Goat anti-Mouse IgG (H+L) IRDye LiCOR Biosciences Cat# 926-68070; 680RD RRID: AB_10956588 Goat anti-Rabbit IgG (H+L) IRDye LiCOR Biosciences Cat# 925-32211; 800CW RRID: AB_2651127 Antibodies for Flow Cytometry TruStain FcX (anti-mouse CD16 / 32) BioLegend Cat #: 101319 Antibody LIVE / DEAD™ Fixable Blue Dead ThermoFisher Cat #: L34962 Cell Stain Kit FITC anti-mouse CD3ε (145-2C11) BioLegend Cat #: 100306 Alexa Fluor 594 anti-mouse CD8a BioLegend Cat #: 100758 (53-6.7) Alexa Fluor® 700 anti-mouse CD45 BioLegend Cat #: 103127 (30-F11) BUV805 Rat Anti-Mouse CD4 BD Biosciences Cat #: 612900 (GK1.5) Bacterial Strains E. coli Rosetta (DE3) Sigma-Aldrich Cat#: 79054 E. coli Stbl3 ThermoFisher Cat #: C737303 Mammalian cell lines U937 SLC19A1- / - Ritchie et al 201929HEK 293T ATCC ATCC CRL-3216; RRID: CVCL_0063 Jurkat ATCC ATCC TIB-152; RRID: CVCL_036 TIME ATCC Cat# CRL-4025; RRID: CVCL_0047 Biological samples Buffy coat from donors Stanford Blood N / A CenterAttorney Docket No.: ARCI-003WO Software and algorithms PyMoL The PyMOL Molecular Graphics System, Version 2.5.2., Schrodinger, LLC Maestro Schrodinger GraphPad Prism 10 GraphPad Software Biorender Biorender ImageJ 2.30 ImageJ ICE Synthego DESeq2 Love et al 201436casTLE Morgens et al 201637Cell Culture

[0148] HEK 293T cells used for lentivirus generation were maintained in DMEM with L-glutamine, 4.5 g / L glucose, and sodium pyruvate (Corning) supplemented with 10% FBS (Atlanta Biologicals) and 1% penicillin-streptomycin (GIBCO). Telomerase- immortalized human microvascular endothelial (TIME) cells were maintained in vascular cell basal media supplemented with microvasculature endothelial cell growth kit-VEGF (ATCC) and 0.1% penicillin-streptomycin (GIBCO). U937 cells, Jurkat cells and Human CD3+primary T cells were maintained in RPMI (Corning) supplemented with 10% heat-inactivated FBS (Atlanta Biologicals) and 1% penicillin-streptomycin (GIBCO). Mouse CD4+and CD8+primary T cells were maintained in RPMI (Corning) supplemented with 10% heat-inactivated FBS (Atlanta Biologicals), 1% penicillin-streptomycin (GIBCO), and 50 µM 2- Mercaptoethanol (Sigma Aldrich). Both human and mouse T cells were also supplemented with 300 U / mL rIL-2 (PeproTech). Cells were maintained in a 5% CO2 incubator at 37°C. Expi293 cells were maintained in FreeStyle medium (Gibco) supplemented with 33% Expi293 Expression medium (Gibco) in an 8% CO2 incubator at 37°C. Recombinant DNA

[0149] A plasmid containing the CDS of human SLC7A2 (pDONR221_SLC7A2) was purchased from Addgene. A custom plasmid (pTwist-CMV) containing the coding sequences of human SLC7A1 was purchased from Twist Bioscience. custom plasmid (pTB146 His-SUMO-DncV) containing full-length Vibrio cholerae DncV cDNA sequence38 was purchased from Twist Bioscience. To generate doxycycline inducible lentiviral plasmids, the transporter CDS was amplified from the appropriate plasmid and cloned into anAttorney Docket No.: ARCI-003WO EcoRI / BamHI linearized pLVX-TetOne-FLAG-Blast plasmid by isothermal Gibson assembly. To introduce point mutations in SLC7A1, the CDS was amplified with primers carrying the corresponding base substitutions and cloned into linearized pLVX-TetOne- FLAG-Blast plasmid by isothermal Gibson assembly. To generate plasmid for SLC7A1 protein expression, the SLC7A1 CDS was amplified with an N-terminal FLAG tag and a HRV-3C protease sequence linker, and cloned into pcDNA3.1 plasmid (ThermoFisher) by isothermal Gibson Assembly. Primers used for cloning can be made available upon request. Reagents and antibodies

[0150] 2’3’-cyclic-GMP-AMP (cGAMP) and soluble STING were synthesized and purified in-house as previously described29.2’3’-bisphosphorothioate-cyclic-GMP-AMP (2’3’-cGSASMP), 2’3’-cyclic-di-AMP (2’3’-CDA), 2’3’-bisphosphorothioate-cyclic-diAMP (2’3’-CDAS), 3’3’-cyclic-GMP-AMP (3’3’-cGAMP), and 3’3’-cyclic-di-AMP (3’3’-CDA) were purchased from Invivogen and reconstituted in endotoxin-free water. Rabbit polyclonal antibodies against, IRF3 (1:1000), pIRF3 (S396, 1:1000), STING (1:1000), pSTING (S366, 1:100), pSTING (pS365, 1:100), and Flag (1:1000) were purchased from Cell Signaling Technology. Mouse monoclonal anti-a-tubulin (1:4000) was purchased from Sigma-Aldrich. Mouse Models

[0151] Mice were maintained at Stanford University in compliance with the Stanford University Institutional Animal Care and Use Committee (IACUC) regulations. All procedures were approved by the Stanford University Administrative Panel on Laboratory Animal Care (APLAC). Flow Cytometry Analysis of Tumors

[0152] Tumor analysis was done as previously described9. The 7−9-week-old female BALB / c mice (Jackson Laboratories) were inoculated with 5 × 1044T1-luciferase cells suspended in 50 μL of PBS. The cells were injected into the right fifth mammary fat pad. When tumor volume reached 100 ± 20 mm3, tumors were irradiated with 12 Gy using a 225 kVp cabinet X-ray irradiator with a 0.5 mm Cu filter (IC-250, Kimtron Inc.). Mice were anesthetized with a mixture of 80 mg / kg ketamine (VetaKet) and 5 mg / kg xylazine (AnaSed) prior to irradiation and were shielded with a 3.2 mm lead shield with 15 mm × 20 mm apertures to expose the tumors. Mice were then intratumorally injected with 100 μL of 100 μM neutralizing STING or nonbinding STING 24 h after irradiation. Mice were euthanized 24 h later, and the tumors were extracted. Following tumor extraction, the tumors were incubated at 37 °C for 30 min in 10 mL of RPMI supplemented with 10% heat-inactivatedAttorney Docket No.: ARCI-003WO FBS and 1% penicillin-streptomycin as well as 20 μg / mL DNase I type IV (Millipore) and 1 mg / mL collagenase from Clostridium histolyticum (Sigma-Aldrich). The samples were then passed through a 100 μm cell strainer (Sigma-Aldrich) to form a single-cell suspension. Red blood cells were lysed in 155 mM NH4Cl, 12 mM NaHCO3, and 0.1 mM EDTA for 5 min at room temperature. Samples were stained with LIVE / DEAD Fixable Blue Dead Cell Stain (Invitrogen) for 30 min. Samples were then fixed and permeabilized with either eBioscience Foxp3 / Transcription Factor Staining Buffer Set (Invitrogen) or Fixation / Permeabilization Solution Kit (BD Biosciences). Samples were Fc blocked for 10 min using TruStain fcX (BioLegend) and then stained for 1 h. All samples were run on an Aurora analyzer (Cytek). Isolation of Primary T cells

[0153] For human CD3+T cells, buffy coat (Stanford Blood Center) was diluted 1:3 with PBS with 2% heat-inactivated FBS. Human T cells were then isolated from a diluted buffy coat using RosetteSep Human T Cell Enrichment Cocktail (StemCell Technologies) and SepMate tubes (StemCell Technologies) per manufacturer’s protocol. Isolated cells were cryopreserved in liquid nitrogen until further use. Purity of isolated cells was determined by flow cytometry to be >90%.

[0154] For mouse CD4+and CD8+T cells, spleens from 8-12 weeks old C57BL / 6J mice were isolated and mashed through 70 μM strainer. Cell suspension was then pelleted and treated with 1X RBC Lysis Buffer (Invitrogen) for 15 min. Following lysis, cells were washed with PBS, and mouse CD4+and CD8+T cells were isolated using MojoSort Mouse CD4 T Cell Isolation Kit (BioLegend) and MojoSort Mouse CD8 T Cell Isolation Kit (BioLegend), respectively, per manufacturer’s protocol. Isolated cells were immediately activated for further use. Purity of isolated cells was determined by flow cytometry to be >90%. Activation of Jurkat and primary T cells

[0155] Jurkat cells (1-2 x 106cells / mL) were activated with 5 ng / mL Phorbol 12- myristate 13-acetate (Cayman Chemical) and 500 ng / mL Ionomycin (Cayman Chemical) for 6 h. Primary human CD3+T cells (1 x 106cells / mL) were activated with Dynabeads Human T-Activator CD3 / CD28 (ThermoFisher) at 1:1 cell-to-bead ratio for 4 days. Primary mouse CD4+and CD8+T cells (1 x 106cells / mL) were activated with Dynabeads™ Mouse T- Activator CD3 / CD28 (ThermoFisher) at 4:3 cell-to-bead ratio for 2 days.Attorney Docket No.: ARCI-003WO CDN Killing Assay

[0156] For some experiments, U937 SLC19A1- / -cell lines were pre-treated with 1 μg / mL of Doxycycline (Sigma-Aldrich) for 24 h to induce expression of SLC7A1 or SLC7A2 protein. U937 SLC19A1- / -cell lines (5 x 105 cells / mL) and primary human CD3+T cells (3 x 105cells / mL) were treated with the indicated concentrations of CDN for 24 h, in the presence of 500 nM STF-16236. To measure relative viability, cells were diluted 1:3 in PBS, and 10 μL of CellTiter-Glo (Promega) were added directly to 10 μL of diluted cells in a 384-well plate. The mixture was shaken gently and incubated at RT for 20 min. The luminescence was measured on a Tecan Spark with a 500 ms integration time. To measure relative count, cells were diluted 1:3 in PBS with 2 mM EDTA and 1:1000 DAPI (ThermoFisher). Cells were counted using Forward Scatter (FSC) and Side Scatter (SSC) gating, and the absence of DAPI staining on an AttuneNxt Flow Cytometer. CDN Stimulation

[0157] For some experiments, U937 SLC19A1- / -cell lines (1 × 106 cells / mL), Jurkat cell lines (1 × 106cells / mL), and human and mouse primary T cells (1 × 106cells / mL) were pretreated with methotrexate (Sigma-Aldrich), folinic acid (Sigma-Aldrich), sulfasalazine (Sigma-Aldrich), DCPIB (Tocris Bioscience), for 10 min at the indicated concentration. For some experiments, U937 SLC19A1- / -cell lines were pre-treated with 1 μg / mL of Doxycycline (Sigma-Aldrich) for 24 h to induce expression of SLC7A1 or SLC7A2 protein. Cells were treated with the indicated concentration of CDN for 2 h in a 5% CO2incubator at 37°C, unless otherwise indicated. Following treatments, cells were collected, lysed with Laemmli Sample Buffer, and run on SDS-PAGE gels for Western blot analysis. Jurkat CRISPR knockout library generation

[0158] Jurkat CRISPR knockout library line was created as previously described37. Briefly, a whole-genome library of exon-targeting sgRNAs were designed, with the goal of minimizing off-target effects and maximizing gene disruption. The top 10 sgRNA sequences for each gene were included in the library, along with thousands of safe-targeting and non- targeting negative controls. The library was cloned into a lentiviral vector, pMCB320, which also expresses mCherry and a puromycin resistance cassette. Jurkat cell line was transduced with a BFP-tagged Cas9 plasmid, selected clonally and Cas9 function was confirmed by the efficiency of knocking out GFP expression using a sgRNA sequence against GFP confirmed by using a sgRNA sequence against GFP. Jurkat-Cas9 cells were infected with the lentiviral library, and then selected with puromycin.Attorney Docket No.: ARCI-003WO Live / Dead CRISPR screen

[0159] The Jurkat CRISPR knockout library line was activated with 5 ng / mL Phorbol 12-myristate 13-acetate (Cayman Chemical) and 500 ng / mL Ionomycin (Cayman Chemical) for 6 h. Cells were then grown in 4 spinner flasks (1 L), with 2 flasks serving as untreated controls and 2 flasks receiving cGAMP treatment. Throughout the screen all the samples were split daily to keep the cell density at 250 million cells per 200 mL, which corresponded to 1,000 cells per guide in the untreated samples. The experimental samples were treated with 2’3’-cGAMP starting at day 2 with enough cGAMP to reduce cell viability by 30% each day as compared to the control samples for 10 days. The initial treatment was 100 μM and was increased steadily to 300 μM by the final treatment day. At the end of the screen, the genomic DNA was extracted using a QIAGEN Blood Maxi Kit. The library was sequenced using a NextSeq 500 / 550 Mid Output v2 kit (Illumina). One experimental sample was dropped out of analysis due to failed sequencing QC. One experimental and two control samples were compared using casTLE37. The algorithm determines the likely effect size for each gene, as well as the statistical significance of this effect. Generation of SLC7A1 Knock-Out Jurkat Line

[0160] LentiCRISPR v2 (Addgene) was used as the 3rd-generation lentiviral backbone for all knockout lines. A guide sequence targeting SLC7A1 (5’- GGGTGCTGGTGTCTACGTCC-3’) was cloned into the lentiviral backbone using the Lentiviral CRISPR Toolbox protocol from the Zhang Lab at MIT39,40. Lentiviral packaging plasmids (pHDM-G, pHDM-Hgmp2, pHDM-tat1b, and pRC / CMV-rev1b) were purchased from Harvard Medical School. To generate lentivirus, 500 ng of the lentiviral backbone plasmid containing the guide sequence and 500 ng of each of the packaging plasmids were transfected into HEK293T cells using FuGENE 6 transfection reagent (Promega).

[0161] Cell supernatant containing lentivirus was harvested after 48 h, and passed through a 0.45 μm filter.1 mL of filtered supernatant was supplemented with 8 mg / mL Polybrene (Sigma-Aldrich) and added to 1 x 106Jurkat cells in a 12-well plate. Cells were spun at 700 x g for 30 min, and left to incubate overnight at 37°C. The next day, the virus containing media was removed and cells were resuspended in fresh media. Cells were selected 48 h later with 1 μg / ml Puromycin (Sigma-Aldrich) alongside control cells (uninfected) until all control cells died. Heterozygous knock-out population was assayed for CDN stimulation within 1 week after antibiotic selection.Attorney Docket No.: ARCI-003WO CRISPR / Cas9 KO of Primary T Cells

[0162] For human T cells, negative (non-targeting) control and SLC7A1 (5’- GGGTGCTGGTGTCTACGTCC-3’) sgRNAs were purchased from Synthego and resuspended to 100 μM in TE buffer. Cas9 RNPs were formed by adding 1 μL of 61μM Alt- R S.p. Cas9 Nuclease V3 (IDT) to 1 μL of 100 μM sgRNA and incubating for 30 min at room temperature. Freshly activated CD3+T cells were washed once with PBS, then resuspended in P3 Primary Cell nucleofector solution (Lonza) to a density of 1.5 x 106cells / 20 μL. A volume of 20 μL of resuspended T cells was then added to the Cas9 RNPs, transferred to a Nucleocuvette stripwell, and nucleofected using program EO-115 on a 4-D Nucleofector X unit (Lonza).

[0163] Immediately after nucleofection, 150 μL of warm media was added to the cells. Cells were then then transferred to a 24-well plate containing 1 mL of RPMI, 10% heat- inactivated FBS, 1% penicillin-streptomycin and 300 U / mL rIL-2. At 24 h after nucleofection, cells were pelleted and resuspended in 2 mL of fresh media. At 72 h after transfection, cells were used for CDN stimulation assays and genomic DNA was isolated to measure the knockout efficiency. The knockout efficiency was determined by amplifying the region of genomic DNA surrounding sgRNA target sites, performing Sanger sequencing, and using the sequencing trace to estimate knockout efficiency through ICE (Synthego) analysis.

[0164] For mouse T cells, Rosa26 and SLC7A1 (5’- ATTTTCACGGGCCACGGCAC-3’) sgRNAs were purchased from Synthego and resuspended to 100 μM in TE buffer. Cas9 RNPs were formed by adding 0.6 μL of 61μM Alt-R S.p. Cas9 Nuclease V3 (IDT) to 1 μL of 100 μM sgRNA and incubating for 30 min at room temperature. Freshly activated CD4+and CD8+T cells were washed once with PBS, then resuspended in P3 Primary Cell nucleofector solution (Lonza) to a density of 1.5 x 106cells / 20 μL. A volume of 20 μL of resuspended T cells was then added to the Cas9 RNPs, transferred to a Nucleocuvette stripwell, and nucleofected using program DN-100 on a 4-D Nucleofector X unit (Lonza). Immediately after nucleofection, 150 μL of warm media was added to the cells. Cells were then then transferred to a 24-well plate containing 1 mL of RPMI, 10% heat-inactivated FBS, 1% penicillin-streptomycin, 50 μM 2-Mercaptoethanol, 2 mM L-Citrulline (TCI Chemicals), and 300 U / mL rIL-2. At 24 h after nucleofection, cells were pelleted and resuspended in 2 mL of fresh media. At 72 h after transfection, cells were used for CDN stimulation assays and genomic DNA was isolated to measure the knockout efficiency. The knockout efficiency was determined by amplifying the region of genomicAttorney Docket No.: ARCI-003WO DNA surrounding sgRNA target sites, performing Sanger sequencing, and using the sequencing trace to estimate knockout efficiency through ICE (Synthego) analysis. Electroporation of 2’3-cGAMP

[0165] U937 SLC19A1- / -cell lines were pelleted and resuspended in nucleofector solution (90 mM Na2HPO4, 90 mM NaH2PO4, 5 mM KCl, 10 mM MgCl2, 10 mM sodium succinate) with 100 nM 2’3’-cGAMP to a density of 1 × 106cells / mL. A volume of 100 μL of cells was then transferred to a 0.2 cm electroporation cuvette and electroporated with program U-013 on a Nucleofector IIb device. Immediately after nucleofection, 500 μL of warm media was added to the cells. Cells were then transferred to a 24-well plate containing an additional 500 μL of media and incubated at 37°C for 2 h. Following this, cells were collected, lysed with Laemmli Sample Buffer, and run on SDS-PAGE gels for Western blot analysis.

[0166] Mouse primary CD4+and CD8+T cells were pelleted and resuspended in P3 Primary Cell Nucleofector Solution (Lonza) with 150 nM 2’3’-cGAMP to a density of 1 × 106cells / mL. A volume of 20 μL of cells was then transferred to the Nucleocuvette stripwell and electroporated with program DN-100 on a 4-D Nucleofector X unit (Lonza). Immediately after nucleofection, 150 μL of media was added to the cells. Cells were then transferred to a 24-well plate containing an additional 850 μL of media and incubated in a 5% CO2incubator at 37 °C for 3 h. Following this, cells were collected, lysed with Laemmli Sample Buffer, and run on SDS-PAGE gels for Western blot analysis. SLC7A1 Protein Purification

[0167] To express SLC7A1 protein, Expi293 cells were cultured at a density of 3 × 106cells / mL. Cells were then transfected with pcDNA3.1-FLAG-HRV3C-SLC7A1 plasmid was transfected with FectoPro transfection reagent (VWR) and supplemented with 4 g / L of D-glucose (Sigma Aldrich) and 3 mM of valproic acid (Sigma-Aldrich).

[0168] To purify SLC7A1 protein, Expi293 cells were harvested at 72 h post- transfection, snap-frozen in liquid nitrogen, and thawed on ice in 20 mL of hypotonic buffer (10 mM HEPES pH 7.5, 25 mM NaCl). Cell mixture was then mechanically dounced, and 1 eq. volume of 2x isotonic buffer (50 mM HEPES pH 7.5, 600 mM NaCl) and EDTA-free cOmplete protease inhibitor cocktail (Roche) were added. Mixture was spun at 100,000 xg for 40 min, and the resulting pellet (membrane fraction) was solubilized for 1 h at 4oC in solubilization buffer (25 mM HEPES pH 7.5, 300 mM NaCl, 1% DDM / 0.1% CHS). ProteinAttorney Docket No.: ARCI-003WO mixture was then spun at 100,000 xg for 40 min, and the resulting supernatant containing membrane proteins was collected. Anti-DYKDDDDK affinity resin (ThermoFisher) were incubated overnight with the membrane proteins, washed with washing buffer (25 mM HEPES pH 7.5, 500 mM NaCl, 2 mM CaCl2, 0.1% DDM / 0.01% CHS), and SLC7A1 proteins were eluted from the resin with 200 μg / mL FLAG Peptide (MedChemExpress) in elution buffer (25 mM HEPES pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.1% DDM / 0.01% CHS). Eluted SLC7A1 proteins were dialyzed into PBS with 0.04% DDM / 0.004% CHS. Samples from every purification steps were run on SDS-PAGE gel and stained with Coomasie total protein stain or blotted for anti-Flag on Western Blot. Synthesis of DncV

[0169] Rosetta cells expressing pTB146-His-SUMO-DncV were grown in 2xYT medium with 100 mg / mL ampicillin and induced with 0.5 mM IPTG when OD600reached 1, and then were grown overnight at 16°C. All subsequent procedures using proteins and cell lysates were performed at 4°C. Cells were pelleted and lysed in 20 mM HEPES pH 7.5, 400 mM NaCl, 10% glycerol, 10 mM imidazole, 1 mM DTT, and EDTA-free, cOmplete protease inhibitor cocktail (Roche). Cell lysate was then cleared by ultracentrifugation at 50,000 x g for 1 h. The cleared supernatant was incubated with HisPur cobalt resin (ThermoFisher Scientific; 1 mL resin per 1 L bacterial culture) for 1 hour. Cobalt resin was then washed with 20 mM HEPES pH 7.5, 1 M NaCl, 10% glycerol, 10 mM imidazole, and 1 mM DTT. Protein was eluted from resin with 300 mM imidazole in 20 mM HEPES pH 7.5, 400 mM NaCl, and 1 mM DTT. Fractions containing His-SUMO-DncV were pooled, concentrated, and dialyzed against 20 mM HEPES pH 7.5, 400 mM NaCl, 1 mM DTT, and then snap-frozen in aliquots for future use. Synthesis of 3’3’-cGAMP diazirine probe

[0170] To synthesize 3’3’-cGAMP diazirine probe, 1 mM of 6’Cl ATP (Jena Biosciences) and 1 mM of GTP (Sigma-Aldrich) were mixed in 20 mM of Tris-HCl at pH 7.4 and 20 mM of MgCl2. Subsequently, 200 nM of purified DncV was added, and the reaction mixture was stirred vigorously at 37°C for 1h. The reaction’s progress was monitored via Thin Layer Chromatography (TLC) and LC-MS (Waters). The product 6’-Cl-2’3’-cG(A)MP was purified using HPLC on a PFP Poroshell 120 column (Agilent Technologies, Inc.) with Acetonitrile / 0.1% Formic Acid as the mobile phase. Purified product was subsequently lyophilized and resuspended in water.6’-Cl-2’3’-cG(A)MP was then mixed with 5 eq. of amino diazirine alkyne and 20 eq. of triethylamine, and the reaction was stirred at 40°CAttorney Docket No.: ARCI-003WO overnight. The product was purified with HPLC as previously described in 2’3’-cGAMP synthesis step29. Diazirine crosslinking competition assay

[0171] cGAMP diazirine probe (500 μM) and 2’3’- and 3’3’-cGAMP (500 μM for low concentration, 2 mM for high concentration; water for controls) were mixed in binding buffer (20 mM Tris-HCl pH 7.4, 200 mM NaCl, 1 mM MgCl2).0.1 mg / mL of purified human SLC7A1 protein was subsequently added to a volume of 10 μL, and the mixture was incubated for 10 min at RT. The mixture was then crosslinked at 365 nm with UVP crosslinker CL-3000 (Analytik Jena US) on ice for 20 minutes. To a crosslinked reaction mixture, 0.1 mM of R110 azide (MedChemExpress) was added into a pre-mixed complex of 1 mM of CuSO4 and 5 mM BTTAA (Sigma-Aldrich), which were mixed with 5 mM of sodium ascorbate to a total volume of 15 μL. The click reaction mixture was incubated for 1- 2 h at RT. The reaction was quenched with the addition of 15 μL 2x reducing Laemmli Sample Buffer. Sample was then assayed by Western blot for R110 fluorescence and Flag signal. Generation of Doxycycline Inducible Cell Lines

[0172] Lentiviral packaging plasmids (pHDM-G, pHDM-Hgmp2, pHDM-tat1b, and RC / CMV-rev1b) were purchased from Harvard Medical School. To generate lentivirus, 500 ng of lentiviral plasmid encoding doxycycline inducible transporters and 500 ng of each of the packaging plasmids were transfected into HEK 293T cells with FuGENE 6 transfection reagent (Promega). Cell supernatant containing lentivirus was harvested after 48 h, and passed through a 0.45 μm filter. To create the inducible cell lines in U937 SLC19A1− / −,291 mL of filtered supernatant was supplemented with 8 mg / mL Polybrene (Sigma-Aldrich) and added to 5 × 105cells in a 12-well plate. Cells were spun at 700g for 30 min, and left to incubate overnight at 37 °C. The next day, the virus containing media was removed and cells were resuspended in fresh media. Cells were selected 48 h later with 5 μg / ml Blasticidin (Sigma-Aldrich) alongside control cells (uninfected) until all control cells died. [3H]-Arginine Uptake Assay

[0173] U937 SLC19A1- / -cell lines (1 x 10 cells) were treated with 21 nM [3H]-L- Arginine (Perkin Elmer) in RPMI SILAC buffer (ThermoFisher) for 1 min. For some experiments, L-Lysine (Sigma-Aldrich) or 2’3’-cGAMP was added at the same time as [3H]- L-Arginine at the indicated concentrations. Cold PBS was then added to cells to halt import. Following two more washes in cold PBS, cells were lysed in 500 mL 400 mM NaOH andAttorney Docket No.: ARCI-003WO heated at 65°C for 45 min to completely dissolve the lysate. Then, 400 mL of cell lysate was used to count3H signal on a scintillation counter and 25 mL were used in a bicinchoninic acid assay (Thermo Scientific) to quantify protein levels for normalization. Bulk RNA-Seq

[0174] U937 SLC19A1- / -cell lines (5 x 106cells) were treated with 1 μg / mL of Doxycycline (Sigma-Aldrich) for 24 h to induce expression of SLC7A1 or SLC7A2 protein. Cells were then harvested and lysed in TRIzol reagent (ThermoFisher). Total RNA was then purified using Direct-zol RNA purification kit (Zymo Research). RNA from different samples (n = 2 biological replicates) were then shipped to sequencing service provider Innomics for QC testing and sequencing on DNBSEQ platform. FASTQs were demultiplexed, mapped onto human reference genome (GRCh38.p13), and differential gene expression were generated using the DESeq2 method36by sequencing service provider Innomics. Modeling the cGAMP and Arginine Binding Sites in SLC7A1 / 2

[0175] AlphaFold structure of human SLC7A1 (UniProt ID P30825) was minimized using Schrödinger Maestro using standard protein preparation parameters at all default settings at pH 7.4. Binding site detection was run to identify possible pocket sites on SLC7A1 and the predicted pocket scoring highest and occurring on the extracellular-facing region of the structure was selected for receptor grid preparation (all standard parameters). All possible conformations of 2’,3’-cGAMP at pH 7.4 were prepared using LigPrep, and the set of structures docked against the SLC7A1 receptor grid using Glide at standard precision.

[0176] The minimized SLC7A1 structure was aligned to the crystal structure of arginine-bound Geobacillus LAT (PDB: 6F34) and the ligand was used to create a composite SLC7A1–arginine structure specifying a candidate amino acid binding and transport site. The site was similarly used for receptor grid preparation, and re-docked with prepared arginine conformations for validation of the model, before cGAMP structures were separately docked. cGAMP Uptake Experiment

[0177] U937 SLC19A1- / -tet-SLC7A2-Flag cells (1 x 107cells) were treated with 1 μg / mL of Doxycycline (Sigma-Aldrich) for 24 h. Cells were then treated with 10 μM cGAMP at 37°C for 15 min in serum-free RPMI SILAC with 500 nM of STF-1623. Cold PBS was then added to cells to halt import. Following 3 more washes, cells were lysed in T- PER protein extraction buffer (ThermoFisher), and heated at 80°C for 10 min to releaseAttorney Docket No.: ARCI-003WO STING-bound cGAMP. Intracellular cGAMP level was measured using 2’3’-cGAMP ELISA kit (Cayman Chemical) and normalized to protein level from the resulting supernatant. Quantification and Statistical Analysis

[0178] All statistical analyses were performed using GraphPad Prism 10. All p values were calculated using an unpaired t test with Welch’s correction. For all experiments involving Western blots, densitometric measurements of protein bands were made using ImageJ 2.30.

[0179] STING signaling in tumor-infiltrating myeloid cells exerts anti-cancer effects, but kills activated T cells, counteracting the effects of immunotherapy or ionizing radiation. cGAMP and its analogs utilize different transporters in different cell types and as a result have different STING activating efficacy towards different cell types. While they activate myeloid cells similarly using known transporters, we observed that cGAMP is ~10 fold less toxic to human CD3+T cells than 2’3’-cGsAsMP and 2’3’-CDAs, suggesting cGAMP and its analogs use a yet unknown transporter(s) in T cells. In this study, we identified SLC7A1, which is cationic amino acid (CAA) transporter, as a cGAMP transporter in T cells. Because proliferating T cells require CAA as an energy source, activated naïve CD4+and CD8+T cells express 10–30-fold more SLC7A1 than other immune cells types and resting T cells, rendering SLC7A1 essential for them. Partial knockout of SLC7A1 in both activated mouse and human CD4+and CD8+T cells significantly reduced transporter-dependent cGAMP- STING signaling. Using molecular modeling validated by mutagenesis studies, we identified the cGAMP binding pocket as well as the arginine binding pocket that are >16 Å away from each other. The activated T cell-specific expression and separate binding pocket for transporting arginine, together, suggest that the cGAMP binding pocket on SLC7A1 is a potential target for alleviating T cell toxicity.

[0180] STING agonists are promising innate immune therapies and can synergize with adaptive immune checkpoint blockade therapies for cancer treatment, but their effectiveness is limited by the toxicity to activated T cells. An important class of STING agonists are analogs of the endogenous STING agonist, cGAMP, and while transporters for these small molecules are known in some cell types, how they enter and kill T cells remains unknown. Here, we identify the cationic amino acid transporter SLC7A1 as the dominant transporter of cGAMP and its analogs in activated primary mouse and human T cells. T cells upregulate this transporter upon activation and rapid proliferation to meet their high metabolic demand, but this comes at the cost of enabling increased transport and toxicity ofAttorney Docket No.: ARCI-003WO cGAMP. To circumvent the essentiality of SLC7A1 to proliferating T cells, we found that the residues responsible for cGAMP transport are separate from the arginine binding pocket allowing us to perturb cGAMP transport and STING-activation mediated killing without impacting arginine transport. These results suggest that SLC7A1 is a potential target for alleviating T cell toxicity associated with cGAMP and its analogs. Human T cell killing by extracellular cGAMP is not mediated by the known transporters

[0181] Although cGAMP and its analogs kill mouse T cells, given the clinical implications, we first wanted to test the response of primary human T cells (FIG 1Q, 1H). Indeed, cGAMP weakly killed activated primary human CD3+ T cells from two unique donors with an IC50 of ~20-30 μM (FIG 1R).2’3’-cGSASMP and 2’3’-CDAS were both more potent than the natural cGAMP second messenger, each displaying IC50 values of ~2-5 μM – even though all three molecules share similar binding affinities for STING13,14. We also tested several bacterial cyclic dinucleotides, which have been shown to activate human STING with weaker affinity26,27, in cells from one of the two donors. Surprisingly, we found that 3’3’-cGAMP kills human T cells with a comparable IC50 value to 2’3’-cGAMP, whereas 3’3’-cyclic di-AMP (CDA) and 3’3’-cyclic di-GMP (CDG) did not lead to any appreciable death at the highest tested concentration. Taken together, we hypothesize that differential import efficiencies of each compound may help explain the incongruence between its reported affinity for STING and toxicity toward T cells17,23.

[0182] We have previously shown that ionizing radiation induces extracellular cGAMP production which enters tumor infiltrating myeloid cells to activate their STING signaling6,9. Even though cGAMP is ~10 times less potent at killing T cells than its clinical analogs, we next tested whether clinically relevant doses of ionizing radiation could produce enough extracellular cGAMP to kill infiltrating T cells. Indeed, we observed dose-dependent killing of tumor-infiltrating T cells when we treated mammary tumor-bearing mice with ionizing radiation at 20 Gy, but not 8 Gy (FIG 1B). To specifically study the role of extracellular cGAMP in this process, we used a cell-impermeable neutralizing STING agent (Neu) to sequester extracellular cGAMP, or its non-binding mutant as a control (NB)6,9. Strikingly, when we depleted extracellular cGAMP in irradiated tumors by intratumoral injection of Neu, we observed increased tumor-infiltrating T cell numbers. This increased T cell infiltration surpassed the level we observed in non-irradiated tumors injected with NB, indicating that extracellular cGAMP generated in response to high dosage ionizing radiationAttorney Docket No.: ARCI-003WO attracts, but also kills, T cells. These two opposing effects of endogenous extracellular cGAMP at high concentrations mirrored previous reports with the non-hydrolyzable cGAMP analog 2’3’-CDAS.17,23

[0183] We next investigated whether primary human T cells transport cGAMP using any of the previously identified transporters: LRRC8A:C / E channels used by endothelial cells8, fibroblasts28, and mouse T cells20, or the myeloid cell transporters SLC19A1 and SLC46A1-39,29. According to published human mRNA expression data30, activated primary human T cells express high levels of the LRRC8A:C channel, followed by low to moderate levels of SLC19A1, SLC46A3 and SLC46A1 (FIG 1I). We treated primary human T cells with inhibitors of these known transporters: methotrexate (MTX) and reduced folic acid (RFA) for SLC19A1, Sulfasalazine (SSZ) as a non-specific inhibitor for SLC19A1 and SLC46A1-3, and DCPIB as an inhibitor for LRRC8A:C / E channels. While SSZ exhibited high toxicity that precluded further analysis, none of the remaining inhibitors reduced phosphorylation of STING in primary human (FIG 1D, 1E, 1K) or mouse (FIG 1L, 1M, 1N, 1O) T cells. DCPIB is known to be less effective in the presence of serum, so we also tested it in serum free media and observed no inhibitory effect (FIG 1T, 1U), suggesting that both human and mouse primary T cells use a previously unidentified transporter to import cGAMP.

[0184] To further test this hypothesis, we performed a structure-activity relationship (SAR) analysis of cGAMP and its analogs in a different cell type with known cGAMP transporters. We previously reported that TIME cells, a telomerase-immortalized human microvascular epithelial cell (HMVEC) cell line, use LRRC8A:C as its dominant transporter8. SAR of four cGAMP analogs and their cellular toxicity across TIME cells, and primary human T cells showed that the LRRC8A:C-dependent cell line exhibited very similar SAR patterns (FIG 1P, 1Q), while primary human T cells showed a distinct signature (FIG 1R). In TIME cells, 2’3’-cGSASMP is >2-fold more potent than 2’3’-CDAS. However, in primary human T cells, 2’3’-CDAS and 2’3’-cGSASMP exhibited similar levels of toxicity. Consistent with our findings from the inhibitor screen, this result suggests that LRRC8A:C is not the main transporter in primary CD3+ T cells, and that the cGAMP transporter in human T cells is still at large.Attorney Docket No.: ARCI-003WO An unidentified transporter mediates human T cell killing by extracellular cGAMP

[0185] We have previously shown that ionizing radiation induces extracellular cGAMP production which enters tumor infiltrating myeloid cells to activate their STING signaling6,9. In this study, we first tested whether extracellular cGAMP induced by high dosage ionizing radiation could kill T cells like cGAMP analogs. Indeed, we observed dose- dependent killing of tumor-infiltrating T cells when we treated mammary tumor-bearing mice with ionizing radiation at 20 Gy, but not 8 Gy (FIG 1A, 1B). To specifically study the role of extracellular cGAMP in this process, we used a cell-impermeable neutralizing STING agent (Neu) to sequester extracellular cGAMP, or its non-binding mutant as a control (NB)6,9. Strikingly, when we depleted extracellular cGAMP in irradiated tumors by intratumoral injection of Neu, we observed increased tumor-infiltrating T cell numbers without any radiation-induced T cell toxicity. This increased T cell infiltration surpassed the level we observed in non-irradiated tumors injected with NB, indicating that extracellular cGAMP generated in response to high dosage ionizing radiation attracts, but also kills, T cells. These two opposing effects of endogenous extracellular cGAMP at high concentrations mirrored previous reports with the non-hydrolyzable cGAMP analog 2’3’-CDAS.17,23

[0186] We asked whether cGAMP and non-hydrolyzable cGAMP analogs of clinical interest also kill primary human T cells, which has not been reported before. Indeed, cGAMP weakly killed activated primary human CD3+T cells from two unique donors with an IC50 of ~30-50 µM (FIG 1C, 1H).2’3’-cGSASMP and 2’3’-CDASwere both more potent than the natural cGAMP second messenger, each displaying IC50 values of ~2-5 µM (FIG 1C, 1H) – even though all three molecules share similar binding affinities for STING13,14. We also tested several bacterial cyclic dinucleotides, which have been shown to activate human STING with weaker affinity26,27, in cells from one of the two donors. Surprisingly, we found that 3’3’-cGAMP kills human T cells with a comparable IC50 value to 2’3’-cGAMP, whereas 3’3’-cyclic di-AMP (CDA) and 3’3’-cyclic di-GMP (CDG) did not lead to any appreciable death at the highest tested concentration. Taken together, we hypothesize that differential import efficiencies of each compound may help explain the incongruence between its reported affinity for STING and toxicity toward T cells.

[0187] We next investigated whether primary human T cells transport cGAMP using any of the previously identified transporters: LRRC8A:C / E channels used by endothelial cells8, fibroblasts28, and mouse T cells20, or the myeloid cell transporters SLC19A1 and SLC46A1-39,29. According to published human mRNA expression data30, activated primaryAttorney Docket No.: ARCI-003WO human T cells express high levels of the LRRC8A:C channel, followed by low to moderate levels of SLC19A1, SLC46A3 and SLC46A1 (FIG 1I). We treated primary human T cells with inhibitors of these known transporters: methotrexate (MTX) and reduced folic acid (RFA) for SLC19A1, Sulfasalazine (SSZ) as a non-specific inhibitor for SLC19A1 and SLC46A1-3, and DCPIB as an inhibitor for LRRC8A:C / E channels. While SSZ exhibited high toxicity that precluded further analysis, none of the remaining inhibitors reduced phosphorylation of STING in primary human (FIG 1D, 1E) or mouse (FIG 1L, 1M, 1N, 1O) T cells, suggesting that human primary T cells use a distinct transporter to import cGAMP.

[0188] Building on the idea that transport efficiency influences relative toxicity of each cGAMP analog, we reasoned that conducting a structure-activity relationship (SAR) analysis across different cell types could illuminate patterns that would reflect the identity of each cell type’s dominant transporter. We previously reported that U937 cells, a human cancerous monocytic cell line, use SLC19A1 as their dominant cGAMP transporter, and switch to LRRC8A:C when SLC19A1 is absent8. Meanwhile, a telomerase-immortalized human microvascular epithelial cell (HMVEC) cell line (TIME cells) uses LRRC8A:C as its dominant transporter8. SAR of four cGAMP analogs and their cellular toxicity across U937 SLC19A1- / -, TIME cells, and primary human T cells showed that the two LRRC8A:C- dependent cell lines exhibited very similar SAR patterns (FIG 1F, 1G, 1P), while primary human T cells showed a distinct signature (FIG 1C). In U937 SLC19A1- / -and TIME cells, 2’3’-cGSASMP is >2-fold more potent than 2’3’-CDAS. However, in primary human T cells, 2’3’-CDASand 2’3’-cGSASMP exhibited similar levels of toxicity. Consistent with our findings from the inhibitor screen, this result suggests that LRRC8A:C is not the main transporter in primary CD3+T cells, and that the cGAMP transporter in human T cells is still at large. CRISPR screen identifies SLC7A1 as a positive regulator of extracellular cGAMP signaling in T cells

[0189] To identify the unknown T cell transporter, we aimed to conduct a genome- wide CRISPR knockout screen. First, to verify a screen-compatible model system, we tested whether the immortal human Jurkat CD4+T cell line is also susceptible to cGAMP toxicity and uses the same transporter(s) as primary human T cells. Mirroring our results in primary human CD4+T cells, none of the transporter inhibitors had significant effects on STING signaling in either resting Jurkat (FIG 2I, 2J) or Jurkat activated with PMA / ionomycin (FIGAttorney Docket No.: ARCI-003WO 2A, 2B). Furthermore, SAR of cGAMP analogs in both resting and activated Jurkat T cells are similar to that observed with primary human T cells (FIG 2C, 2D, 2K, 2L). Therefore, we proceeded to use Jurkat as a model cell line for a genome-wide survival screen (FIG 2E), reasoning that knockout of factors involved in cGAMP-mediated cell death – including the cGAMP transporter – would allow a cell to survive treatment with a lethal dose of cGAMP. We treated a pool of 200 million cells carrying the 200,000 sgRNA library (10 guides per gene) with a LD30dose of cGAMP, a concentration that kills 30% of the cells every day. Following 10 days of treatment, we measured sgRNA enrichment among the surviving population to identify genes contributing to cGAMP-mediated toxicity.

[0190] As expected, genes encoding STING, TBK1, and IRF3 had among the highest casTLE score and casTLE effect, which are established parameters used to represent the statistical significance and fold enrichment, respectively (FIG 2F). The gene encoding solute carrier protein SLC7A1, which is characterized as a cationic amino acid transporter that transports substrates such as L-arginine31, emerged as a top candidate transporter. Activated T cells rapidly expand and increase their metabolic consumption by upregulating SLC7A1 mRNA levels by ~30 fold (FIG 2M) and protein levels by ~10 fold (FIG 2N) to take in more arginine. SLC7A1 knockout in T cells has previously been shown to arrest cell cycle progression and diminish cytokine release32, and indeed we observed that Jurkat SLC7A1- / -cell viability decreased after multiple passages. In order to assess the phenotype of SLC7A1 knockout despite this viability limitation, we measured extracellular cGAMP-mediated IRF3 phosphorylation in a heterogeneous pool of Jurkat cells immediately following SLC7A1 knockout without selection. We achieved SLC7A1 knockout in ~60-70% of cells, which led to a 40% reduction in pIRF3 in the resting cellular pool upon cGAMP treatment (FIG 2O, 2P). In the same cellular pool, we observed a 30% reduction of pIRF3 in stimulated cells (FIG 2G, 2H). These results suggest that SLC7A1 plays a role in extracellular cGAMP signaling in Jurkat T cells, nominating it as a possible cGAMP transporter.

[0191] As expected, genes encoding STING, TBK1, and IRF3 arose to have the highest statistical significance and fold enrichment (FIG 2F). The gene encoding solute carrier protein SLC7A1 emerged as a top candidate transporter. It has been previously characterized as a cationic amino acid transporter that transports substrates such as L- arginine31. Activated T cells rapidly expand and increase their metabolic consumption by upregulating SLC7A1 mRNA levels by ~30 fold (FIG 2M) and protein levels by ~10 fold (FIG 2N) to take in more arginine. SLC7A1 knockout in T cells has previously been shownAttorney Docket No.: ARCI-003WO to arrest cell cycle progression and diminish cytokine release32, and indeed we observed that Jurkat SLC7A1- / - cell viability decreased after multiple passages. To circumvent this viability limitation, we measured extracellular cGAMP-mediated IRF3 phosphorylation in a heterogeneous pool of Jurkat cells immediately following SLC7A1 knockout. We achieved SLC7A1 knockout in ~60-70% of cells, which led to a 40% reduction in pIRF3 in the resting cellular pool upon cGAMP treatment (FIG 2O, 2P). In the same cellular pool, we observed a 30% reduction of pIRF3 in stimulated cells (FIG 2G, 2H). These results suggest that SLC7A1 plays a role in extracellular cGAMP signaling in Jurkat T cells, nominating it as a possible cGAMP transporter. SLC7A1 is important for cGAMP transport and STING signaling in primary human and mouse T cells

[0192] Next, we investigated whether SLC7A1 is also involved in extracellular cGAMP signaling in primary T cells. We followed the same strategy to create heterogeneous SLC7A1 knockout pools of primary human T cells from two donors, and treated the cells with cGAMP during the brief 72 hours window before significant cell death was observed from arginine starvation. Partial knockout of SLC7A1 in donor 1’s primary human CD3+by 48% reduced pSTING levels by ~25% compared to the non-targeting control guide, whereas 73% knockout of SLC7A1 in cells from donor 2 led to a ~40% reduction of pSTING levels (FIG 3A, 3B, 3G). Likewise, the heterogenous pool of SLC7A1 knockout mouse primary CD4+cells (~30% indel score) responded 25% less and CD8+cells (~60% indel score) responded 55% less to extracellular cGAMP-induced p-IRF3 than their WT counterparts (FIG 3C, 3D, 3H). Importantly, these differences were abolished when cGAMP was electroporated to bypass the need for transport (FIG 3E, 3F). We then performed a direct binding assay with purified flag-tagged full-length SLC7A1 and 3’3’-cGAMP photoaffinity probe (FIG 3K). If SLC7A1 binds to the probes, it can be covalently crosslinked with the probes via diazirine upon UV irradiation at 365 nm and visualized in gel by conjugating the alkyne handle of the probe to fluorescence tag (R110-N3) via click chemistry (FIG 3I). Indeed, the probe successfully crosslinked with SLC7A1 in a UV crosslinking dependent manner, as evidenced by the fluorescent signal as distinct bands corresponding to the FLAG bands (FIG 3L). Utilizing this probe, we performed competition experiment and observed that both 2’3’-cGAMP and 3’3’-cGAMP inhibited the probe from labeling SLC7A1 with 3’3’-cGAMP expectedly being a more potent competitor (FIG 3J). Together, our dataAttorney Docket No.: ARCI-003WO demonstrates that SLC7A1 mediates STING signaling induced by extracellular cGAMP in primary T cells by facilitating cGAMP import. cGAMP and arginine bind distinct sites on SLC7A1

[0193] Puzzled by the fact that SLC7A1 can transport substrates that are not only structurally distinct, but also different in their charges, we hypothesized that it might bind different substrates at different sites. Molecular modeling predicted a cGAMP binding site on the extracellular surface of SLC7A1, >16 Å away from the arginine binding site modeled in the interior33(FIG 4A). To experimentally test the molecular model, we performed mutagenesis studies in an SLC7A1 overexpression system. SLC7A1 can only be lowly expressed when introduced exogenously into U937 cells, which yielded slight but significant decrease in STING signaling and cell viability 24h after induction (FIG 4B, 4E, 4F). These small increases are not due to expression changes in the STING pathway components or other cGAMP transporters as expressions of these genes are unaltered upon doxycycline-induction (FIG 4M). As expected, SLC7A1 expression also increased [3H]L-arginine intake in these cells (FIG 4G) which also basally uptake arginine (FIG 4H). By introducing point mutations in this exogenous SLC7A1 expression system, we observed that the predicted arginine binding site mutation (S354M) completely abolished arginine transport without affecting cGAMP-mediated cell killing (FIG 4B, 4C). We then performed single and double mutations around the predicted cGAMP binding site and observed that R59A / D404A double mutant and R130A single mutant all decreased cGAMP-mediated cell killing without affecting arginine transport. The R59A / R130A double mutant and D404A / R130A double mutant also decreased cGAMP-mediated cell killing without affecting arginine transport. Remarkably, the R59A / D404A / R130A triple mutant completely abolished the effect of 30 μM extracellular cGAMP without affecting arginine transport. Remarkably, the R59A / D404A / R130A triple mutant completely abolished the effect of 20 μM extracellular cGAMP without affecting arginine transport (FIG 4I, 4J). The identification of the triple mutant triangulated the cGAMP binding pocket on SLC7A1 and validated our molecular modeling results (FIG 4D). Interestingly, while the triple mutant also abolished 2’3’-CDAs and 2’3’-cGsAsMP induced cell death via SLC7A1, it did not completely abolish that of ‘3’3-cGAMP, suggesting that 3’3’-cGAMP engages with additional residues explaining why it is a better substrate (FIG 4K). Finally, cGAMP does not compete with direct arginine uptake by SLC7A1, while lysine does (FIG 4L). Together, these biochemical assays definitively demonstrated that SLC7A1 is a cGAMP transporter and uses separate binding sites for different substrates.Attorney Docket No.: ARCI-003WO Lysine and arginine unidirectionally inhibit cGAMP intake and STING signaling in primary T cells

[0194] Given that the arginine binding pocket sits lower in the transporter, we hypothesize that arginine binding might block the passageway that cGAMP uses and might inhibit cGAMP transport but not vice versa. We first tested whether cGAMP blocks arginine transport.3H-L-arginine uptake could not be inhibited by 2 mM cGAMP (at ~200,000 molar excess), whereas it could be inhibited by L-lysine (FIG 5A). However, the presence of cationic amino acid (CAA) inhibited cGAMP-mediated STING signaling (FIG 5B, 5C, 5D) and T death (FIG 5E, 5F) in both primary mouse and human CD3+T cells. The fact that the cGAMP binds a site on the extracellular surface distinct from the CAA binding site in the interior core suggests that cGAMP transport by SLC7A1 can potentially be blocked by pharmacological inhibitors without depriving cells of arginine. We reasoned that we could potentially develop cGAMP selective SLC7A1 inhibitors by synthesizing and identifying cGAMP analogs that can bind SLC7A1 at the cGAMP binding site, but cannot be transported by it; or can be slowly transported by SLC7A1, therefore acting like a cGAMP transport blocker, but cannot activate STING once inside the cells.3’3’-cGAMP, 3’3’-cyclic-di-GMP, and 3’3’-cyclic-di-AMP are much worse human STING activators, but can still be internalized by SLC7A1. Homolog SLC7A2 also transports cGAMP

[0195] SLC7A1 is a member of the larger SLC7 family, of which SLC7A2 is the most closely related: sharing 58% sequence identity and 71% sequence similarity with SLC7A1. SLC7A2 is also a cationic amino acid transporter, but has a different cell type expression profile and is notably absent from immune cells (FIG 6K). Although SLC7A2 is not expressed by T cells, we reasoned that it might also transport cGAMP given its close homology to SLC7A1. To test this hypothesis, we set up an analogous dox-inducible, FLAG- tagged system for heterologous SLC7A2 expression in SLC19A1- / - U937 cells. Dox induction led to robust protein expression – significantly higher than the expression we achieved with SLC7A1 (FIG 4E) – with smearing consistent with its predicted glycosylation (FIG 6A). A large increase in3H-arginine uptake after SLC7A2 overexpression confirmed its functionality in this heterologous system (FIG 6L). SLC7A2 overexpression greatly increased extracellular cGAMP-mediated pIRF3 signaling (FIG 6A, 6B) but had no effect when the need for cGAMP transport was bypassed by electroporating cGAMP directly into cells (FIG 6C, 6D). This increase in pIRF3 was not due to any effect of altered amino acidAttorney Docket No.: ARCI-003WO uptake, as we saw a similar increase in pIRF3 when we cultured cells in media lacking the SLC7A2 amino acid substrates arginine, lysine, histidine, and ornithine (FIG 6M, 6N). This increase in pIRF3 was also not due to expression changes in the STING pathway components or other cGAMP transporters as expressions of these genes are unaltered upon doxycycline- induction (FIG 6O). Similar to SLC7A1, overexpression of SLC7A2 drastically shifted the dose response curve of cGAMP-mediated U937 cell killing (FIG 6E). Most importantly, overexpression of SLC7A2 directly increased cGAMP transport into the cells (FIG 6F). Molecular modeling predicted a cGAMP binding site in SLC7A2 similar to that in SLC7A1, far away from the Arginine binding site (FIG 6G). Our mutagenesis studies showed that K61A and D406A mutant each individually abolished cGAMP transport activity without abolishing arginine transport activity (FIG 6H, 6I, 6J). Interestingly, the E234A mutant increased cGAMP transport without affecting arginine transport, likely because the glutamate at 234 position is repulsing the electrons in the guanine ring. Finally, the S356M mutant abolished arginine transport activity without affecting cGAMP transport demonstrating that SLC7A2 also has a separate binding pocket for cGAMP than for arginine. Together, our data suggest that SLC7A2, like SLC7A1, is a cGAMP transporter. But unlike SLC7A1 which is highly expressed on activated T cells, SLC7A2 has a broad expression profile across different tissues and cancer types. Future studies are warranted to explore the physiological relevance of SLC7A2’s cGAMP transport activity. Engineered T cell Results

[0196] FIG 7A, 7B, & 7C show that the knockout of SLC7A1 in T cells partially rescues toxicity due to 2’3’-cGAMP, 2’3’-CDAS(ADU-S100) and 3’3’-cGAMP. This further supports that SLC7A1 is a relevant cGAMP transporter to target in this cell type.

[0197] FIG 8A, 8B, & 8C show that cell death is decreased with the SLC7A2 mutants and that pIRF3 signal increase in +dox condition (where SLC7A2 expression is induced) relative to -dox condition (no SLC7A2). This shows that SLC7A2 can mediate transport for all the cyclic dinucleotides tested, including the 2’3’ and 3’3’-variants. Discussion:

[0198] In this study, we identified SLC7A1 as the highly sought after transporter responsible for STING agonist-induced T cell toxicity. Our screening approach, modified from our previous work8,29, succeeded in identifying the T cell transporter because we first identified a model cell line, Jurkat T cells, which share the same cyclic dinucleotide substrateAttorney Docket No.: ARCI-003WO selectivity and import inhibitor profiles as primary human T cells. In addition, Jurkat T cells can be activated like primary T cells and, when activated, are also susceptible to toxicity caused by cGAMP and its analogs. Our previous whole genome CRISPR screens in U937 cells identified SLC19A1 as the first cGAMP transporter29and LRRC8A:C and LRRC8A:E channels as the cGAMP transporters in endothelial cells and fibroblasts respectively8, but did not identify SLC7A1 due to its lower expression level in U937 cells.

[0199] Previous work reported that LRRC8C is a cGAMP transporter in mouse T cells and mediates mouse T cell death by cGAMP-STING-induced p53 activation20. Since we previously observed differences between mouse and human transporters in their ability to import cGAMP9,29, we prioritized testing the relative importance of SLC7A1 and LRRC8A:C channels in activated primary T cells from both species. We conclude that SLC7A1 is the dominant T cell transporter in both mouse and human T cells, given that knockout of SLC7A1 impaired response to imported cGAMP similarly in both mouse and human T cells. Two factors likely contribute to SLC7A1’s functional dominance compared to the LRRC8A:C channel in activated primary T cells. First, SLC7A1 is upregulated to a level much higher than that of the LRRC8A:C channel when T cells are activated and start proliferating due to their increased demand for cationic amino acids. Second, SLC7A1 is a solute carrier, and it transports cGAMP down its electrochemical gradient. When cGAMP analogs are used therapeutically, the extracellular concentrations are much higher than intracellular concentrations. The LRRC8A:C channel, however, is only somewhat active at isotonic conditions. The pore is opened by hypotonicity, which occurs less frequently in T cells than in endothelial cells where it is the dominant cGAMP transporter8.

[0200] With the addition of SLC7A1, the complexity and diversity of cGAMP transporters continues to grow and suggests a finely tuned response mechanism to cGAMP when it is secreted as an immunotransmitter. The biological importance of each cGAMP transporter depends on its relative expression levels on the cell type of interest, its cGAMP transport efficiency, and its function transporting other substrates. SLC7A1’s cGAMP transport activity is weak compared to its arginine transport activity, so its upregulation in primary T cells has a concentration window where it promotes T cell proliferation without cGAMP toxicity. A microenvironment is a mixture of stromal cells, myeloid cells, and T cells. We hypothesize that the strong cGAMP transporters in stromal cells and myeloid cells ensure that virally induced or cancer secreted cGAMP activates STING in the stroma and myeloid cells to produce type-I interferon and mount potent anti-viral and anti-cancerAttorney Docket No.: ARCI-003WO immunity. This is because compared to SLC7A1 which is lowly expressed in naive T cells, SLC46A2 is highly expressed by primary human monocytes and transports cGAMP more efficiently in these cells; the LRRC8A:C complex is highly expressed by endothelial cells and LRRC8A:E complex is highly expressed by fibroblasts and both transport cGAMP efficiently basally but much more so with cell swelling8,10,34,35. However, we hypothesize that the increase of SLC7A1 expression in T cells as they become activated with rapid proliferation allows cGAMP-mediated T cell killing once they have performed their effector functions to avoid autoimmunity. In a way, SLC7A1’s cGAMP transport activity in T cells could function as an adaptive immune checkpoint. Future studies are warranted to test these hypotheses.

[0201] In the case of cancer immunotherapy, blocking this adaptive immune checkpoint could potentially prolong the lifespan of activated T cells. This could be used during treatments with exogenous cGAMP or cGAMP analogs, or when high concentrations of endogenous extracellular cGAMP are generated after high dose ionizing radiation. Since arginine and cGAMP transport by SLC7A1 are functionally separable, specifically inhibiting cGAMP but not arginine transport would be a viable strategy to block only cGAMP analog- mediated T cell toxicity without affecting T cell metabolism. This would also permit STING activation in other cell types essential for antiviral and anti-cancer immunity. SLC7A1 is the first cGAMP transporter that is an actionable drug target for boosting the efficacy of cancer immunotherapy and other treatment mechanisms that rely on T cell functions.

[0202] In addition, we show that SLC7A1’s homolog SLC7A2, which is not expressed in T cells, also imports cGAMP in a manner that is separable from arginine import. SLC7A2 is highly expressed in cancer cells and its physiological role there needs to be explored. Since these transporters are conserved in sequence and molecular function, it would be important to consider the impact of any potential inhibitors on both transporters, including other therapeutic contexts for targeting SLC7A2 or undesired consequences of inhibiting both transporters.

[0203] STING agonist induced T cell toxicity was a known phenomenon for many years and the T cell transporter is highly sought after. In this study, we successfully identified this transporter using activated T cells for CRISPR screen. The screen gave a low casTLE effect likely because SLC7A1 is essential for T cell proliferation and complete knockout of SLC7A1 cells die over time. Even with SLC7A1 being a positive regulator of T cell proliferation and that Jurkat T cells are not nearly as sensitive to cGAMP-STING mediatedAttorney Docket No.: ARCI-003WO toxicity as primary T cells, we were able to identify it as a top positive regulator of cGAMP- induced cell death in Jurkat T cells, demonstrating the robustness of the screen. Other known transporters did not show up as hits suggesting SLC7A1 is the dominant transporter. Indeed, knockout studies demonstrated that SLC7A1 is used by both mouse and human primary T cells. We were able to overcome toxicity due to Arginine deprivation using Citrulline rescue when knocking out SLC7A1 in mouse CD4+and CD8+T cells explaining why we observed bigger effects in diminishing extracellular cGAMP-induced IRF-3 phosphorylation.

[0204] We proceeded to demonstrate that SLC7A1 is the direct cGAMP transporter in human and mouse T cells. A gold standard in testing whether we have identified a direct target of cGAMP in T cells is by making cGAMP specific mutations that do not affect other functions of the target. Indeed, we were able to identify a R59A / D404A / R130A triple mutation combination in SLC7A1 that completely abolished extracellular cGAMP signaling without affecting its activity in transporting Arginine. These data demonstrated that SLC7A1 does not indirectly control cGAMP activity through Arginine transport, but directly transports cGAMP.

[0205] After demonstrating that SLC7A1 is a direct cGAMP transporter used in human T cells, two lines of evidence support that SLC7A1 is also the dominant cGAMP transporter. First, incomplete knockout of SLC7A1 in primary mouse and human T cells lead to significant reduction in extracellular cGAMP signaling, but not when cGAMP is electroporated. Second, the SAR of cGAMP analogs in Jurkat T cells where SLC7A1 was identified matched their SAR in primary activated human T cells.

[0206] cGAMP transport activity of SLC7A1 is weak compared to its arginine transport activity with a IC50 of 30-50 µM in primary human T Cells. In contrast, SLC46A2 expressed by macrophages transports cGAMP with an IC5025 µM in these cells and the LRRC8A:C complex expressed by endothelial cells also transports cGAMP efficiently. We hypothesize that the strong cGAMP transporters in these cell types ensure that virally induced or cancer secreted cGAMP activate STING in myeloid cells and endothelial cells to produce type-I interferon and mount potent anti-viral and anti-cancer immunity. However, we hypothesize that the weak T cell transporter allows T cell killing when extracellular cGAMP reaches a certain threshold to avoid autoimmunity.

[0207] It was previously shown that synthetic cGAMP analog 2’3’-CDASkills mouse activated T cells much more efficiently than the endogenous second messenger. Indeed, weAttorney Docket No.: ARCI-003WO show here that in Jurkat T cells and primary human T cells that also use SLC7A1 as the cGAMP transporter, 2’3’-CDASis ~10 fold more toxic than cGAMP in the presence of an ENPP1 inhibitor that render the two substrates equally stable. SLC7A1 inhibitors should also be developed to alleviate the T cell toxicity when high concentrations of endogenous extracellular cGAMP are generated from high dosage ionizing radiation. SLC7A1 inhibitors that specifically inhibit cGAMP but not Arginine transport could block only cGAMP analogs mediated T cell toxicity without affecting T cell metabolism and STING activation in cell types essential for antiviral and anti-cancer immunity. 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[0208] In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0209] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and / or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one havingAttorney Docket No.: ARCI-003WO skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0210] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0211] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0212] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0213] Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of theAttorney Docket No.: ARCI-003WO invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

[0214] The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 112 (f) or 35 U.S.C. §112(6) is not invoked.

Claims

Attorney Docket No.: ARCI-003WO WHAT IS CLAIMED IS:

1. A SLC7A1 protein comprising a sequence according to SEQ ID NOs: 10, 11, 12, or 13, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOs: 10, 11, 12, or 13.

2. A SLC7A2 protein comprising a sequence according to SEQ ID NOs: 15 or 16, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOs: 15 or 16.

3. An engineered cell comprising the protein of claim 1 or 2 or a SLC7A2 protein comprising a sequence according to SEQ ID NO: 14, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:

14.

4. The engineered cell of claim 3, which is an engineered T cell which is tumor- infiltrating CD3+ T lymphocyte.

5. The engineered cell of claim 3, which essentially has no endogenous SLC7A1 activity.

6. The engineered cell of any one of claims 3-5, further comprising a CAR which recognizes a tumor associated antigen.

7. A method of making the engineered cell of any one of claims 3-6, comprising incorporating nucleic acids encoding a SLC7A2 or mutant SLC7A1 described herein into the cell.

8. The method of claim 7, further comprising incorporating nucleic acids encoding a chimeric antigen receptor described herein into the cell.Attorney Docket No.: ARCI-003WO 9. A method for inducing toxicity in a T cell, the method comprising contacting the T cell with an effective amount of: a) a compound that binds to amino acid residue S354 of SLC7A1; or b) a compound having a structure according to Formula I or II: wherein R1and R2and2Y are each independently O or S thereby inducing toxicity in the T cell.

10. The method of claim 9, wherein the compound is selected from the group consisting of: i) the compound of Formula (I), wherein R1is guanine, R2is adenine, Y1is O, and Y2is O; ii) the compound of Formula (I), wherein R1is guanine, R2is adenine, Y1is S, and Y2is S; iii) the compound of Formula (I), wherein R1is adenine, R2is adenine, Y1is S, and Y2is S; iv) the compound of Formula (II), wherein R1is guanine, R2is adenine, Y1is O, and Y2is O; v) the compound of Formula (II), wherein R1is adenine, R2is adenine, Y1is O, and Y2is O; and vi) the compound of Formula (II), wherein R1is guanine, R2is guanine, Y1is O, and Y2is O.

11. The method of claim 9 or 10, wherein the T cell is an activated T cell.Attorney Docket No.: ARCI-003WO 12. The method of claim 9 or 10, wherein the T cell is a CD8+T cell.

13. The method of claim 9 or 10, wherein the T cell is a CD4+T cell.

14. The method of any one of claims 9 to 13, wherein the compound inhibits arginine transport in the T cell.

15. The method of any one of claims 9 to 14, wherein the compound does not inhibit transport of cGAMP or analogs thereof in the T cell.

16. The method of any one of claims 9 to 15, wherein the compound is a) and is a peptide, a small molecule, or an antibody.

17. A method for inducing toxicity in a T cell population, the method comprising contacting the T cell population with an effective amount of: a) a compound that binds to amino acid residue S354 of SLC7A1; or b) a compound having a structure according to Formula I or II: wherein R1and R2and Y2are each independently O or S thereby inducing toxicity in the T cell population.Attorney Docket No.: ARCI-003WO 18. The method of claim 9, wherein the compound is selected from the group consisting of: i) the compound of Formula (I), wherein R1is guanine, R2is adenine, Y1is O, and Y2is O; ii) the compound of Formula (I), wherein R1is guanine, R2is adenine, Y1is S, and Y2is S; iii) the compound of Formula (I), wherein R1is adenine, R2is adenine, Y1is S, and Y2is S; iv) the compound of Formula (II), wherein R1is guanine, R2is adenine, Y1is O, and Y2is O; v) the compound of Formula (II), wherein R1is adenine, R2is adenine, Y1is O, and Y2is O; and vi) the compound of Formula (II), wherein R1is guanine, R2is guanine, Y1is O, and Y2is O.

19. The method of claim 9 or 10, wherein the T cells are activated T cells.

20. The method of claim 9 or 10, wherein the T cells are CD8+T cells.

21. The method of claim 9 or 10, wherein the T cells are CD4+T cells.

22. The method of any one of claims 9 to 21, wherein the compound inhibits arginine transport in the T cells.

23. The method of any one of claims 9 to 22, wherein the compound does not inhibit transport of cGAMP or analogs thereof in the T cells.

24. The method of any one of claims 9 to 23, wherein the compound is a) and is a peptide, a small molecule, or an antibody.

25. A method of treating a solid cancerous tumor in a subject, comprising: administering to the subject not otherwise in need of treatment thereof, a therapeutically effective amount of an agent that binds to the cGAMP binding pocket of SLC7A1, thereby treating the solid cancerous tumor in the subject.

26. The method of claim 25, wherein the agent binds to one or more amino acid residues of SLC7A1 selected from R59, R130, and / or D404.

27. The method of claim 25-26, further comprising administering one or more additional compounds to the subject to treat the solid cancerous tumor.

28. The method of claim 25-27, further comprising administering ionizing radiation to the subject.Attorney Docket No.: ARCI-003WO 29. The method of claim 25-28, wherein the solid cancerous tumor is selected from the group consisting of adrenal, liver, kidney, bladder, breast, colon, gastric, ovarian, cervical, uterine, esophageal, colorectal, prostate, pancreatic, lung (both small cell and non-small cell), thyroid, carcinomas, sarcomas, glioblastomas, melanoma and various head and neck tumors.

30. The method of claim 25-29, wherein the subject is a human.

31. The method of claim 25-30, wherein the agent does not interfere with arginine transport in T cells of the subject.

32. The method of claim 25-31, wherein the agent inhibits transport of cGAMP or analogs thereof in T cells of the subject.

33. The method of claim 25-32, wherein the agent enhances T cell infiltration into the solid cancerous tumor of the subject.

34. A method for reducing toxicity in a T cell population, the method comprising contacting the T cell population with an effective amount of an agent that binds to the cGAMP binding pocket of SLC7A1, under conditions effective to reduce toxicity in the T cell population.

35. The method of claim 34, wherein the agent binds to one or more amino acid residues of SLC7A1 selected from R59, R130, and / or D404.

36. The method of claims 34-35, wherein the toxicity is induced by cGAMP.

37. The method of claims 34-36, wherein the T cell population comprises activated T cells.

38. The method of any one of claims 34-37, wherein the T cell population comprises CD8+T cells.

39. The method of any one of claims 34-37, wherein the T cell population comprises CD4+T cells.

40. The method of any one of claims 34-39, wherein the T cell population is one T cell.

41. The method of any one of claims 34-40, wherein the agent does not interfere with arginine transport into the T cells.Attorney Docket No.: ARCI-003WO 42. The method of any one of claims 34-41, wherein the agent inhibits transport of cGAMP or analogs thereof into the T cells.

43. The method of any one of claims 34-42, wherein the toxicity is induced by ionizing radiation.

44. A method of inhibiting cGAMP binding to SLC7A1 on a T cell, the method comprising contacting the T cell with an effective amount of an agent that binds to the cGAMP binding pocket of SLC7A1, under conditions effective to inhibit the cGAMP binding to SLC7A1 on the T cell.

45. The method of claim 44, wherein the agent binds to one or more amino acid residues of SLC7A1 selected from R59, R130, and / or D404.

46. The method of claim 44 or 45, wherein the T cell is an activated T cell.

47. The method of claim 44 or 45, wherein the T cell is a CD8+T cell.

48. The method of claim 44 or 45, wherein the T cell is a CD4+T cell.

49. The method of any one of claims 44-40, wherein the agent does not interfere with arginine transport in the T cells.

50. A method of identifying an agent that inhibits SLC7A1, the method comprising: contacting a cell overexpressing wildtype SLC7A1 with a compound and detecting cGAMP induced toxicity in the cell.

51. The method of claim 50, wherein a decrease in cGAMP induced toxicity identifies the agent as an inhibitor of SLC7A1.

52. The method of claim 50, wherein no change in arginine transport identifies the agent as an inhibitor of SLC7A1.

53. The method of any one of claims 50 to 52, wherein the cell is a T cell.

54. The method of any one of claims 50 to 53 further comprising overexpressing a mutant SLC7A1 in a cell and contacting said cell with the agent.Attorney Docket No.: ARCI-003WO 55. A method of identifying an agent that binds the cGAMP binding pocket of SLC7A1, the method comprising: contacting a cell overexpressing wildtype SLC7A1 with cGAMP, contacting the cell with a compound and detecting the displacement of the cGAMP.

56. An agent that binds to the cGAMP binding pocket of SLC7A1.

57. An agent that binds to one or more amino acid residues of SLC7A1 selected from R59, R130, and / or D404.

58. The agent of claim 56 or 57, wherein the agent inhibits transport of cGAMP or analogs thereof in T cells.

59. The agent of any one of claims 56-58, wherein the agent does not interfere with arginine transport in T cells.

60. The agent of any one of claims 56-59, wherein the agent comprises a peptide, a small molecule, or an antibody.

61. The agent of any one of claims 56-60, wherein the agent reduces T cell toxicity induced by cGAMP or analogs thereof.

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