Treatment of solid tumors with implantable polymers with small molecules or cells

Abstract

Disclosed are implantable polymers that release therapeutic agents in proximity to a tumor in a patient. In some embodiments, a polyethylene glycol hydrogel can release a STING agonist and / or a CAR-T cell in proximity to a phosphatase and tensin homolog (PTEN)-null triple negative breast cancer (TNBC) in the patient.

Description

TREATMENT OF SOLID TUMORS WITH IMPLANTABLE POLYMERS WITH SMALL MOLECULES OR CELLS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 596,837, filed on November 7, 2023, and U.S. Provisional Application No. 63 / 669,112, filed on July 9, 2024, the entire contents of which are incorporated herein by reference.

[0002] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.GOVERNMENT INTERESTS

[0004] This invention was made with government support from the National Cancer Institute / NIH / DHHS grant R01CA258688. The government has certain rights in the invention.FIELD

[0005] Disclosed are implantable biocompatible polymers that continuously release therapeutic agents (e.g., STING agonists, CAR-T cells) in the body to treat cancers.BACKGROUND

[0006] Injectable delivery of certain therapeutic agents to tumors in a subject can be toxic to immune cells (e.g., T cells) that attack the tumors. Other modes of delivery in these instances are sought.SUMMARY

[0007] Disclosed herein are implantable, biocompatible polymers (e.g., polyethylene glycol- based hydrogels) that can release therapeutic agents, including STING agonists and CAR-T cells in a subject to treat a cancer. In some embodiments, a biocompatible polymer that contains a STING agonist can be implanted in proximity to a solid tumor in a subject. After implantation,in some embodiments, CAR-T cells can be injected into the implanted biocompatible polymer. In some embodiments, the cancer can be a breast cancer. In some embodiments, the cancer can be a phosphatase and tensin homolog (PTEN) null, triple negative breast cancer (TBNC). In some embodiments, the cancer can have functioning PTEN.

[0008] Disclosed are methods of delivering a therapeutic agent to a subject to treat a solid tumorby implanting a biocompatible polymer that includes a therapeutic agent into the subject. The biocompatible polymer continuously releases the therapeutic agent in the subject over time. In some embodiments, the cancer treated can be a breast cancer, including TNBC, including PTEN-null TBNC. In some embodiments, the implanted biocompatible polymer can contain a STING agonist and / or CAR-T cell.

[0009] In some embodiments, the implantable biocompatible polymer can be a biopsy marker. In some embodiments, the biopsy marker can have a core of poly(dioxanone) (PDO), polylactide (PLA), polyglycollide (PGA), polycaprolactone (PCL), or copolymers thereof; and a sealant surrounding the core, comprising a polyethylene glycol (PEG)-based hydrogel. In some embodiments, the biopsy marker can be a HydroMARK™ biopsy marker.

[0010] In some embodiments, disclosed herein is a method for treating a breast cancer in a patient by hydrating a dehydrated biopsy marker with a STING agonist and / or a CAR-T cell specific for a molecule on a surface of a cell from the breast cancer and implanting the hydrated biopsy marker into the patient in proximity to the cells of the breast cancer. The dehydrated biopsy marker can include a polyethylene glycol (PEG)-based hydrogel.

[0011] In some embodiments, disclosed herein is a method for treating a PTEN-null, TNBC in a subject by, without resecting the breast cancer, implanting a hydrated HydroMARK™ biopsy marker having a STING agonist in proximity to the breast cancer; and injecting a CAR-T cell cell into the implanted biopsy marker subsequent to the implanting. In some embodiments, the biopsy marker having a STING agonist and CAR-T cells can be used to treat breast cancers, including TNBC, that have functioning PTEN.

[0012] Disclosed herein are HydroMARK™ biopsy markers that have a STING agonist and / or a CAR-T cell.BRIEF DESCRIPTION OF THE FIGURES

[0013] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0014] Figure 1. Defective STING trafficking renders TNBC cells hypersensitive to STING agonism. Figure 1A shows images depicting STING staining of a TNBC tissue microarray (TMA) (n=63) with scoring criteria. 0 = no staining in tumor cells; 1+ = faint staining in >10% of tumor cells; 2+ = moderate staining in >10% of tumor cells; 3+ = strong staining in >10% of tumor cells. Figure IB depicts images from two cases showing core to core discrepancies in staining intensity. Case 24 with score of 3+ and 0; Case 89 with score of 2 and 0. Figure 1C shows representative images of PTEN staining of TNBCs with STING 3+ Figure ID shows an immunoblot of a panel of TNBC cell lines for PTEN and STING. Figure IE shows an immunoblot of MDAMB231 unaltered (Parent) or containing CRISPR control vector (Scramble) or PTEN knockout vectors (Sgl, Sg2, Sg3) for PTEN and STING. Cells were treated with PBS as control or ADU at lOpM or 50pM for 48h. Supernatants were then analyzed for CXCL10 expression by ELISA (n=3). *, P < 0.05; **, P < 0.005; ***, P < 0.0002; ****, P < 0.0001 by two-way ANOVA with Tukey’s MCT. Figure IF shows an immunoblot of MDA- MB231 Parent or containing CRISPR control vector (Scramble) or Rab7 knockout vectors (Sgl, Sg2) for Rab7 and STING. Cells were treated with ADU at 50pM for 48h, and supernatants were then analyzed for CXCL10 expression by ELISA (n=2). *, P < 0.05; ***, P< 0.0002; ****, P < 0.0001 by ANOVA with Tukey’s MCT. Figure 1G shows immunofluorescence staining for STING (Green) and lysosome (Red) colocalization was performed after MDAMB231 cells were treated with 50pM ADU or lOOnM CID1067700 or in combination for Ih and 6h (representative of n = 3 independent experiments). Co-localization was quantified using Image! (n=4). **, P < 0.005; ***, P < 0.0002 by two-way ANOVA with Tukey’s MCT. Figure IH shows a growth inhibition assay of PTEN null and PTEN WT cell lines over 6 weeks. MDA-MB468 and HCC70 were treated twice-weekly with lOpM or 50pM ADU. MDAMB231 and HCC1806 were treated twice-weekly with lOpM or 50pM ADU + / - lOOnM CID 1067700. Cell growth was visualized with crystal violet staining and then quantified using absorbance of 590nm (n=3) (Figure II). Figure 1 J shows a growth inhibition assay where MDA-MB231 Scramble and PTEN KO cell lines (Sgl, Sg3) were treated similarly as in Figure IH, stained for visualization with crystal violet and then quantified using absorbance of 590nm (n=3). *, P < 0.05; **, P < 0.005; ***, P <0.0002; ****, P < 0.0001 by ANOVA with Tukey’s MCT (Figure IK). Figure IL shows an immunoblot of MDMAB468 containing CRISPR control vector (Scramble) or Rab7 knockout vectors (Sgl, Sg2) for Rab7 and STING. Figure IM shows immunofluorescence staining for STING (Green) and lysosome (Red) colocalization was performed after MDAMB231 cells were treated with 50pM ADU, lOOnM CID1067700, or in combination for Ih and 6h. Co-localization was quantified using Imaged (n=4). **, P < 0.005; ***, P < 0.0002 by two-way ANOVA with Tukey’s MCT. Figure IN shows a growth inhibition assay. Figure IO shows effects of various treatments on CXCL10 production. Figure IP shows H-score analysis of STING levels in the PTEN negative (n=38) and PTEN positive (n=2) subsets. Six PTEN low positive TNBCs were included in the PTEN negative subset. Figure IQ shows quantitative analysis and frequency distribution of H-score of STING expression in PTEN negative subset (n=38). H-score <30 indicates low to negative expression. H-score >75 indicates high expression.

[0015] Figure 2. Impaired STING turnover enhances STING agonist induced T cell migration. Figure 2A shows a Luminex cytokine profiling performed for scramble and Rab7 knockout cells using PTEN WT cell lines MDAMB231 and HCC1806, and the PTEN null cell line MDAMB468. Log-2 -fold change cytokine / chemokine differences between Rab7 knockout, Scramble treated with ADU, and Rab7 knockout treated with ADU relative to untreated Scramble cells are shown (n=2-4). Figure 2B shows CXCL10, IL-8, and IL-6 expression measured by ELISA in culture media (CM) of samples from the experiment described in Figure 1 A. *, P < 0.05; **, P < 0.005; ***, P < 0.0002; ****, P < 0.0001 by ANOVA with Tukey’s MCT. Figure 2C depicts a schematics of CD8+ T cell migration assay utilizing a 3D microfluidic device. Figure 2D shows images of T cell (yellow) migration towards MDAMB231 spheroids (DAPI). MDAMB231 Scramble and Rab7 knockout spheroids were pretreated with 50pM ADU for 18h prior to loading the 3D microfluidic device. Migrated CD8+ T cells were quantified after 48h (n=8- 9). ***, P < 0.0002; ****, p < 0.0001 by ANOVA with Tukey’s MCT. Figure 2E shows images of T cell migration towards HCC1806 spheroids treated similarly as in Figure ID. Migrated CD8+ T cells were quantified after 48h (n=7-10). *, P < 0.05; **, P < 0.005 by ANOVA with Tukey’s MCT. Figure 2F shows CXCL10 quantification in MDAMB23 1 and HCC1806 cells treated with 50pM or lOpM ADU in the presence or absence of increasing doses of CID1067700 for 48h. Supernatants were analyzed for CXCL10 by ELISA (n=3-5). *, P < 0.05; ***, P < 0.0002; ****, P < 0.0001 by ANOVA with Tukey’s MCT. Figure2G depicts a schematic of a Jurkat cell migration assay utilizing a 3D microfluidic device. Representative images of Jurkat cells (pink) migrating towards MDA-MB231 spheroids (DAPI) after treatment with lOOnM CID 1067700 + / - 50pM ADU-S100. Migrated Jurkat cells were quantified after 48h (n=4). *, P < 0.05; ***, P < 0.0002; ****, P < 0.0001 by ANOVA with Tukey’s MCT. Figure 2H shows CCL5 expression assessment by ELISA in the culture media (CM) used in Figure 1A for HCC1806. ***, P < 0.0002; ****, P < 0.0001 by ANOVA with Tukey’s MCT. Figure 21, Representative images of Jurkat cells (pink) migrating towards MDAMB23 1 spheroids (DAPI) after treatment with lOOnM CID1067700 + / - 50pM ADU. Migrated Jurkat cells were quantified after 48h (n=4). **, P < 0.005; ***, P < 0.0002; ****, P < 0.0001 by ANOVA with Tukey’s MCT. Figure 2 J shows multiplexed cytokine profiling of control PTEN null cell line MDAMB468 and PTEN WT cell lines MDAMB231 and HCC1806, compared with Rab7 KO - / + ADU treatment. Heatmap shows control cell normalized log-2 fold change (L2FC) of key STING-IRF3 and STING-NFKB cytokine / chemokines. Figure 2K is immunoblot of HCC1806 Parent or containing CRISPR control vector (Acramble) or Rab7 knockout vectors (Sgl, Sg2).

[0016] Figure 3. Impregnation of STING agonist in a PEG hydrogel biopsy marker result in its local delivery into TNBC. Figure 3A shows an image of desiccated radiographic biopsy markers and their corresponding image after hydrating for 2 hrs. Figure 3B shows an image of implanted PEG marker in a mouse on Day 0 and 4, with magnification views of the marker on Day 8 and 25. Figure 3C depicts a schematic of THP-lassay. Known amount of ADU-S100 is added to THP1 cells followed by ELISA assay to determine corresponding CXCL10 levels. Figure 3D shows average values of triplicates from Figure 3C that were plotted to generate a standard curve. Figure 3E shows a schematic of the assay used to determine the amount of ADU released from a PEG marker with lOOpg of ADU over time. Figure 3F(a) shows CXCL10 ELISA values corresponding to Figure 3E that were used to determine the amount of ADU released at each time point utilizing the standard curve generated in Figure 3D. Figure 3F(b) shows CXCL10 ELISA values corresponding to Figure 3E that were used to determine the amount of ADU released at each time point utilizing the standard curve generated in Figure 3D. Figure 3G shows the amount of ADU-S100 released from the various biopsy markers after each marker was soaked with lOOpg ADU over time. Average values of triplicates are shown. Figure 3H shows the size of tumors developed in mice after 4T1 cells (10k) wereinoculated into the mammary fat pad of BALB / c mice followed by implantation of PEG marker with PBS or lOOpg ADU on Day 4. Individual tumor volume measurements are shown (n=5 mice per group). Figure 31 shows the size of tumors developed in mice after 4T1 cells (100k) were inoculated into the mammary fat pad of BALB / c mice. At tumor size of 150mm3 on Day 6, mice were implanted with PEG marker soaked with PBS or lOOpg ADU. Individual tumor volume measurements are shown (n=7 mice per group). Figure 3 J shows images of a 4T1 mouse from Figure 31 treated with PEG+PBS on Day 25. Dissection of the tumor shows an intact PEG marker with a visible metal clip. Figure 3K (Left) shows the volume of the PEG marker at each time point after tumor implantation in the mouse. (Right) shows the amount of ADU released from the various biopsy markers embedded with lOOpg of ADU.

[0017] Figure 4. STING agonist monotherapy is toxic to T cells and does not result in T cell clonality. Figure 4A shows percentages of CD45+ leukocytes, lymphocytes, and T cells (CD3+, CD4+, CD8+) in primary human TNBC explants that were treated with 50pM ADU or PBS for 24h, followed by dissociation into single cell suspensions and analyzed by flow cytometry. Figure 4B shows percentages of cell viability in human CD8+ T cells that were treated with various dosages of ADU for 24h; cell viability was determined by CellTiter-Glo (CTG) assay at 24h (n=3). ****, P < 0.0001 by ANOVA with Tukey’s MCT. Figure 4C percentage of viability in CD3+, CD4+, and CD8+ T cells. Splenocytes were harvested from C57BL / 6 mice and treated with various dosages of ADU for 24h. Viability of CD3+, CD4+, and CD8+ T cells (n=2) was determined by flow cytometry. *, P < 0.05; **, P < 0.005; ***, P < 0.0002 by ANOVA with Tukey’s MCT. Figure 4D (Left) shows percentage of viable cells after human CD8+ T cells were treated with conditioned media and collected at various timepoints from PEG soaked with lOOpg ADU as in Figure 3E. Figure 4D (Right) shows CD8+ T cells that were treated with direct addition of PBS, direct addition of lOOpg ADU, PEG+PBS, or PEG+ADU embedded with lOOpg ADU. Cell viability was determined by CTG assay at 24h (n=3). The first panel shows treatment for 5 days with no media changes and cell viability was determined by CTG assay (N=3). The second panels shows cells treated the same way with surviving cells after ADU treatment replated at Day 5 and then stimulated with IL-2, IL-7, and IL-15, followed by CTG assay on Day 10 (n=3). Figure 4E shows the size of tumors after 4T1 cells (10k) were inoculated into the mammary fat pad of BALB / c mice, and at Day 4 a PEG marker with PBS or withlOOpg of ADU was implanted. On Day 30, another PEG marker withPBS or lOOpg of ADU was implanted. Individual tumor volume measurements are shown (PBS n=6, ADU n=10). * Denotes mice used for single-cell RNA-sequencing (scRNA-seq) and T-cell receptor- sequencing (TCR-seq). Figure 4F depicts a schematic of the experimental design used in Figure 4E. Figure 4G shows UMAP plots displaying unsupervised clustering of scRNA-seq data for PBMCs collected from PEG+PBS and PEG+ADU treated 4T1 tumors from E. Cell assignment was based on comparison to cluster gene expression signatures from established literature. Activated T cells was the main cluster to exhibit any associated clonotypes. Figure 4H shows UMAP plots displaying clusters with any associated TCR clonotypes. Figure 41, Pie charts demonstrating percentage of cells with unique TCR clonotypes of frequency=l, >=2, and >=5 in for PBS and ADU treatment groups. Figure 4J shows primary human TNBC explants treated with 50 pm ADU or PBS for 24h, followed by dissociation into single cell suspensions. Flow cytometry was performed to analyze percentages of live CD45+ leukocytes, CD3+ lymphocytes, and T cells (CD4+, CD8+) as depicted by the pie graph.

[0018] Figure 5. CAR-T with STING agonist as combination therapy for TNBC. Figure 5A depicts immunoblots of PTEN WT (HCC1143, HCC1806, MDAMB231) and PTEN null (MDAMB468, HCC1937, HCC70) TNBC showing mesothelin expression (MSLN). Figure 5B shows flow cytometry charts confirming expression of MSLN in HCC1806 and HCC70 cells using HCC1143 cells as a control. Flow cytometry representative of n = 2 independent experiments. Figure 5 C and D show the percentage of dead cells in cell lines that were cocultured as a 2D monolayer with CD19 or MSLN CAR-T cells for 24h (n=3) at the indicated EffectorTarget (E:T) ratios. Cell death was determined by CTG assay. ****, P < 0.0001 by ANOVA with Tukey’s MCT. Figure 5E and F show images of HCC1806 (Figure 5E) and HCC70 (Figure 5F) that were co-cultured as 3D spheroids with CD19 or MSLN CAR-T cells in a microfluidic device for 72h (n=2-5 ). Staining as indicated was performed for live and dead cells. Figure 5G shows quantification of percentage of cell death (n=2-5). ****, p < 0.0001 by two-way ANOVA with Tukey’s MCT. Figure 5H shows IFNy, Granzyme B, and TNFa quantification using ELISA in HCC1143 (black bars), HCC1806 (red bars) and Hcc70 (blue bars) (n=2-6). **, P < 0.005; ***, P < 0.0002; ****, P < 0.0001 by two-way ANOVA with Tukey’s MCT. Figure 51 shows images of MSLN CAR-T migration assays with HCC70 and HCC1806 spheroids after 48h and utilizing the 3D microfluidic device. Live and dead cells stained as indicated. Quantification of migrated CAR-Ts and percentage of cell death (n=5-6). *,P < 0.05; **, P <0.005 by t test. Figure 5J shows images of MSLN CAR-T migration assays with HCC1806 Scramble and Rab7 knockout spheroids after 48h and utilizing the 3D microfluidic device described above. Spheroids were pretreated with 50pM ADU for 18h prior to loading. MSLN CAR-T cells were loaded to the side channel of the device. Quantification of migrated CAR-Ts and percentage of cell death (n=4-7). Conditioned media analyzed for IFNy and Granzyme B by ELISA (n=3-5). *, P < 0.05; **, P < 0.005; ***, P < 0.0002; ****, P < 0.0001 by ANOVA with Tukey’s MCT. Figure 5K depicts CAR-T transduction efficacy. Figure 5L depicts the results of a Luminex profiling of HCC70 using the indicated conditions.

[0019] Figure 6. Impregnation of CAR-T in a PEG hydrogel biopsy marker results in its local delivery into TNBC. Figure 6A shows bioluminescent imaging of PEG marker soaked with PBS or Luc-MSLN CAR-T. Figure 6B showsbioluminscenct imaging of PEG markers that were first hydrated with 250pl of ddH20, and then followed by injection of PBS or Luc- MSLN CAR-T before obtaining the bioluminescent images. Figure 6C depicts a schematic of MSLN CAR-T cells soaked into various biopsy markers and then co-cultured with HCC70-Luciferased cells (HCC70-Luc). Cell death was determined by CTG assay. Figures 6D, E, F, and G show the percentage of death for HCC70-Luc cells co-cultured with no biopsy marker or with various biopsy markers embedded with BCMA or MSLN CAR-T at the indicated E:T ratios. Cell death was determined by luciferase assay at 24h (n=3). **, P < 0.005; ***, P < 0.0002; ****, P < 0.0001 by two-way ANOVA with Tukey’s MCT. Figure 6H shows a growth curve representing the size of tumors developed after NSG mice with established HCC70 tumors at 250 mm3were implanted with PEG+PBS or PEG+MSLN CAR-T (4* 106). Figure 61 shows human CD3 IHC performed on HCC70 tumors harvested on Day 70 from the mice described in the experiment corresponding to Figure 6H. Figure 6J shows CD3 IHC performed on HCC70 tumors on Day 14. Figure 6K shows the bioluminescent distribution of CAR-T cells at the indicated time points in NSG mice with established HCC70 tumors at 150 mm3that were implanted with PEG+Luc-CD19 or PEG+Luc-MSLN CAR-T (4>< 106). Figure 6L shows individual tumor volume measurements (n=6 mice per group). Figure 6M shows a Kaplan-Meier survival curve of treated mice. Statistical significance calculated using the log- rank Mantel-Cox test. **, P < 0.005. Figure 6N depicts CAR-T transduction efficacy. Figure 60 shows MSLN IHC performed on remnant tumors collected from Figure 6K. Figure 6P shows MSLN IHC performed on CD 19 and MSLN CAR-T treated tumors.

[0020] Figure 7. Local delivery of STING agonist and CAR-T through a PEG hydrogel biopsy marker obliterates TNBC. Figure 7A shows NSG mice with established HCC70 tumors at 150 mm3 were treated with ADU + 4xl06NanoLuc-MSLN CAR-T cells via direct intratumoral injection without a PEG, or placement of 2 PEGs, or 1 PEG with ADU soaked in (Day -7) followed by injection of CAR-T (Day 0). Bioluminescent distribution of CAR-T cells at indicated time points is shown. Figure 7B shows individual tumor volume measurements (n=5 mice per group). Figure 7C shows a Kaplan-Meier survival curve of treated mice. Statistical significance was calculated using the log-rank Mantel-Cox test. **, P < 0.005. Figure 7D shows bioluminescent distribution of CAR-T cells at the indicated time points. NSG mice with established HCC70 tumors were implanted with PEG+PBS or PEG+ADU (lOOpg). 7 days later, 2* 106 or 4>< 106 Luc-MSLN CAR-T cells were injected into the PEG marker previously impregnated with PBS or ADU. Control cells were were treated with PEG+PBS followed by injection of 4xl06CD19 CAR-T cells. Bioluminescent distribution of CAR-T cells was measured at indicated time points shown in Figure 7D. Figure 7E shows tumor volume measurements for each treatment group shown in Figure 7D (n=5 mice per group). Figure 7F, Kaplan-Meier survival curve of treated mice with significance calculated using the log-rank Mantel-Cox test. **, P < 0.005. Figure 7G shows a graph depicting quantification of human IFNy in mouse serum at Day 7 after CAR-T cell administration. **, P < 0.005 by ANOVA with Tukey’s MCT. Figure 7H shows graphs depicting quantification of human CD3+ T cells identified by flow cytometry in samples from mouse peripheral blood obtained at 7, 14, 21 and 28 days after CAR-T administration. CD3 T cell counts are shown for each individual animal. Figure 71 shows a graph depicting statistical analysis performed for the various groups at Day 14. *, P < 0.05 by ANOVA with Tukey’s MCT. Figure 7J shows bioluminescent distribution of CAR-T cells at indicated time points. NSG mice with established HCC70 tumors were injected with PBS or ADU and, 7 days later, 4x 106Luc-CD19 CAR-T cells were injected into the mice treated with PBS and 4x 106Luc-MSLN CAR-T cells were injected into the mice treated ADU (Injection group in Figure 7 J). NSG mice with established HCC70 tumors were implanted with either PEG+ADU and PEG+4x 106Luc-MSLN CAR-T or PEG+PBS and PEG+4x 106Luc-CD19 CAR-T (2 PEG group in Figure 7 J). Figure 7K shows the bioluminescent distribution of CAR- T cells with stringent scale applied at min=8.00xl06and max=5.00xl08; previous scale was=7.00xl0:>and max=5.00xl08. Figure 7L shows graphs depicting individual tumor volumemeasurements corresponding to mice from the experiment (n=5 mice per group). Figure 7M shows graphs depicting quantification of human IFNy in mouse serum obtained at Day 14 and Day 21 after CAR-T cell administration, ns, Not significant by ANOVA with Tukey’s MCT. Figure 7N shows graphs depicting quantification of human TNFa in mouse serum obtained at Day 7, 14, and 21 after CAR-T cell administration. *, P < 0.05; **, P < 0.005 by ANOVA with Tukey’s MCT. Figure 70 shows MSLN IHC performed on all remnant tumors collected from Figure 7A in the animals treated via 1 PEG marker. Figure 7P shows MSLN IHC performed on all remnant tumors treated with PBS, ADU, or ADU+MSLN CAR-T.

[0021] Figure 8. Local delivery of STING agonist and CAR-T through a PEG hydrogel biopsy marker results in extravasion of CAR-T cells. Figure 8A shows NSG mice with established HCC1806 tumors at 150 mm3that were treated with ADU + 4xl06NanoLuc-MSLN CAR-T cells via placement of PEG with ADU in (Day -7) followed by injection of CAR-T (Day 0). NSG mice with established HCC1806 at 150 mm3were implanted with PEG+PBS or PEG+ADU (lOOpg). 7 days later, 4>< 106Luc-MSLN CAR-T cells were injected into the PEG marker previously impregnated with PBS or ADU. Control cells were treated with PEG+PBS followed by injection of 4xl06CD19 CAR-T cells. Bioluminescent distribution of CAR-T cells at indicated time points is shown (Figure 8A). Figure 8B shows tumor volume measurements for each treatment group shown in Figure 8A (n=5 mice per group). Figure 8C shows individual tumor volume measurements (n=5 mice per group). Figure 8D (top) shows graphs depicting quantification of human IFNY in mouse serum obtained at Day 7, Day 14, Day 21 and Day 28 after CAR-T cell administration, ns, Not significant by ANOVA with Tukey’s MCT. Figure 8D (bottom) shows graphs depicting quantification of human TNFa in mouse serum obtained at Day 7, Day 14, Day 21 and Day 28 after CAR-T cell administration. *, P < 0.05; **, P < 0.005 by ANOVA with Tukey’s MCT. Figure 8E (top) shows graphs depicting quantification of human CD3+ T cells identified by flow cytometry in samples from mouse peripheral blood obtained at 7, 14, 21 and 28 days after CAR-T administration. CD3 T cell counts are shown for each individual animal. Figure 8E (bottom) shows comparison of data from Figure 8E (top) at day 7. Figure 8F shows graphs depicting quantification of human IFNY (top) or human TNFa (bottom) for each individual in mouse serum obtained at Day 7, Day 14, Day 21 and Day 28 after CAR-T cell administration. *, P < 0.05; **, P < 0.005 by ANOVA with Tukey’s MCT.

[0022] Figure 9 shows identifying target antigens for a panel of triple negative breast cancers (e g., MSLN, Trop2, R0R1 and MUC1-C in TNBC cell lines). Figure 9A shows an immunoblot of a panel of TNBC cell lines for MSLN, Trop2, ROR1 and MUC1-C. Figure 9B shows confirmation by cell surface expression of MSLN, Trop2, ROR1 and MUC1-C in PTEN WT (HCC1143, HCC1806, MDAMB231) and PTEN null (MDAMB468, HCC1937, HCC70) TNBC cell lines).

[0023] Figure 10 shows example intratumoral CAR-T expansion and tumor efficacy. PEG hydrogel was implanted adjected to the tumor site. A, B: Tumor-adjacent hydrogel was injected with control (CD 19) or mesothelin (MSLN4) CAR-T cells, and in vivo T cell expansion visualized by bioluminescence. C, D: Tumor growth inhibition observed for all mice in both HCC70 and HCC1806 breast cancer tumor models following MSLN CAR-T treatment (red lines).

[0024] Figure 11 shows example preconditioning of the tumor microenvironment enhances CAR-T expansion and prevents relapse. Left: STING agonist ADU-S100 injected into tumor- adjacent hydrogel 7 days prior to hydrogel injection of MSLN CAR-T. Middle: Preconditioning with ADU-S100 enhances CAR-T expansion. Right: Preconditioning with ADU-S100 (blue lines) prevents tumor relapse.

[0025] Figure 12 shows example preconditioning of the tumor microenvironment promotes CAR-T extravasation from the tumor microenvironment. Measurement of circulating CAR-T cells followed injection into tumor-adjacent hydrogel. Graph right top: No TME preconditioning shows lack of circulating CAR-T cells. Graph right bottom: TME preconditioning with STING agonist ADU-S100 promotes extravasation of CAR-T cells into circulation. The data indicate that this platform can be efficacious against metastatic tumors.

[0026] Figure 13 shows example CAR-T expansion at a secondary tumor site. PEG hydrogel was implanted adjected to the primary tumor in the mammary fat pad. Secondary tumor was established in the flank. The data indicate that preconditioning of the tumor microenvironment enhances CAR-T expansion at secondary tumor site.

[0027] Figure 14 shows example data indicating the immunotherapy platform is efficacious against metastatic lesions. PEG hydrogel was implanted adjacent to the primary tumor in the mammary fat pad. Secondary tumor was established in the flank.

[0028] Figure 15 shows example data indicating the immunotherapy platform is efficacious in blocking formation of new metastatic lesions. PEG hydrogel was implanted adjacent to the primary tumor in the mammary fat pad. Secondary tumor was injected 7 days post-CAR-T treatment.

[0029] Figure 16 shows multiple example treatment options (embodiments) for metastatic tumors.

[0030] Figure 17 shows an example of overcoming resistance due to tumor heterogeneity and antigen loss. (Top) Is an analysis of breast cancer cells from a patient showing heterogeneity in antigen expression, complicating treatment options. (Top left) Primary TNBC sample was stained with CD45 to gate on CD45 negative cells enriched for tumor cells. (Top right) Costaining with fluorescently conjugated antibodies targeting MUC1, TROP2, R0R1 and MSLN to quantify cell surface expression reveals MUC1 and especially TROP2 positivity of this particular sample. (Bottom) shows tumor cells that escape treatment often lack CAR-T target expression. Relapsing mouse tumors treated with MSLN CAR-T no longer show MSLN expression.

[0031] Figure 18 shows an example of overcoming resistance due to tumor heterogeneity and antigen loss.

[0032] Figure 19 shows an example of an integrated immunotherapy platform for the treatment and cure of solid tumors.

[0033] Figure 20 shows examples of priming the tumor microenvironmen with STING agonists prior to CAR-T delivery to further boost anti -tumor activity and CAR-T expansion in mice treated with the immunotherapy platform (also see Figure 3G).

[0034] Figure 21 shows an example that the immunotherapy platform is efficacious in metastatic cancer models. The data show robust increase in circulating Human CD3+ T cells in mice treated with ADU + CAR-T cells.

[0035] Figure 22 shows an example indicating that any site that can be biopsied can be potentially treated.

[0036] Figure 23 shows an example application of CAR-T-PET platform to a patient sample.(A) Fresh patient tissue undergoes FACS for antigen profiling, expansion using PDX model, and(B) creation of PDOTS for 3D microfluidic culture. (C) NSG mice with established tumors (150 mm3) are treated as shown. (D) Given tumor heterogeneity, Trop2 CAR-T escapers can betreated with a second CAR. The process shown can be repeated until tumor obliteration is achieved.

[0037] Figure 24 shows an example of cancer treatment using the systems disclosed herein. Figure 24 is similar to Figure 23.

[0038] Figure 25 shows example data obtained from the systems described herein.

[0039] Figure 26 shows example data obtained from the systems described herein.

[0040] Figure 27 shows example data obtained from the systems described herein.

[0041] Figure 28 shows aditional studies using STING agonist in PEG hydrogel biopsy markers. (A) shows a study that did not use the PEG hydrogel. In established 4T1 tumors, on day 6, PBS or 100 pg ADU was injected intratumorally. Mean tumor volume measurements are shown (n=4-5 mice / group). (B) shows representative IHC images of pSTATl and pTBKl staining of 4T1 tumors treated with PEG+PBS or PEG+100 pg ADU for 4 days. Scale bar, 100 pm. (C) shows a schematic of orthotopic implantation of 100K 4T1 cells followed by PEG + PBS or 100 pg ADU implantation. ScRNA-seq and TCR-seq were performed with 4 pooled tumors / group 14 days later. (D) (Left) UMAP plots displaying unsupervised clustering of scRNA-seq data for CD45+cells collected from Figure 28C. (D) (Right) Volcano plot showing overview of differentially expressed genes (DEGs) in PEG+ADU versus PEG+PBS treated tumors in the cluster of APC + Macrophage in (D). Red dots indicate significantly upregulated genes. Blue dots indicate significantly downregulated genes. (E) and (F) shows gene set enrichment analysis (GSEA) based on the expression fold-change values of ADU versus PBS treated tumors in the cluster of APC + Macrophage. GSEA plots for IFNy and IFNa response are shown in (E). Bar plot represents normalized enrichment scores (NES) for the top upregulated Hallmark gene sets (F). Quantitative data are represented as mean ± SEM; p values were calculated by two-way ANOVA followed by Sidak’s / xzsZ hoc test (A), ns, not significant; ****, p < 0.0001.

[0042] Figure 29 shows (A) Bioluminescent imaging of PEG marker soaked with 20 pL PBS or 4x 106NanoLuc-MSLN CAR T for 30 minutes in 60mm dish. (B) Bioluminescent imaging of hydrated PEG marker injected with PBS or 4* 106NanoLuc-MSLN CAR T in 50 pL. (C) Schematic of MSLN CAR T cells soaked into various biopsy markers and then co-cultured with firefly luciferase (ffLUC) expressing HCC70 (ffLUC-HCC70) spheroids in 24-well plate. (D, E) ffLUC-HCC70 spheroids formed from 0.5* 106single cells co-cultured with PEG markerssoaked (D) or injected (E) with BCMA or MSLN CAR T at indicated E:T ratios. Tumor cell death was determined by luciferase assay at 24h (n=3-4). (F, G) NSG mice with established HCC70 tumors (250 mm3) were implanted with PEG marker soaked with PBS or 4* 106MSLN CAR Ts. Tumors were harvested on Day 14 (F) and Day 42 (G). Representative IHC images of human CD3 staining are shown. Scale bar, 100 pm. (H) Image of a HCC70 tumor-bearing mouse implanted with a PEG marker on Day 35. Dissection of the tumor shows an intact PEG marker with a visible metal clip. (I) Schematic of MSLN CAR T cells co-cultured with PBS / ADU soaked biopsy markers after implantation. NSG mice with established HCC70 tumors were implanted with PEG+PBS or PEG+100 pg ADU. 7 days later, PEG markers were extracted and cocultured with MSLN CAR T cells for 24h. (J) MSLN CAR T cell viability from (I) was determined by CTG assay (n=2-3). (K) Schematic of experimental design for sequential delivery PEG marker. NSG mice with established HCC70 tumors (400 mm3) were implanted with PEG+PBS or PEG+100 pg ADU, followed by 50K MSLN CAR T cells in 50 pL injection into the PEG marker 7 days later. (L) Representative IHC images of human CD3 staining on Day 2 from (K). Scale bar, 100 pm (left). Quantification of CD3+cells per high-power field (HPF) (n=4-5 mice / group) (right). (M, N) HCC70 tumors were established and treated as in (K).Digested tumors were analyzed for human CD3 by flow cytometry on day 2 (M). Quantifications of CD3+cells are shown (N) (n=5 mice / group). Quantitative data are represented as mean ± SEM; p values were calculated by two-way ANOVA followed by Sidak’s post hoc test (D and E), one way ANOVA followed by Tukey’s post hoc test (J) and unpaired Student’s t test (L and N). ns, not significant; *, p < 0.05; ***, p < 0.001; ****, p < 0.0001.

[0043] Figure 30 shows (A) NSG mice with established HCC70 tumors at 150 mm3were treated with 100 pg ADU + 4* 106NanoLuc labelled CAR T cells via direct intratumoral injection, via dual PEG delivery (one PEG for ADU and one PEG for CAR T cells implanted adjacent to the tumor simultaneously), or sequential delivery PEG method as shown in Figure 5K (one PEG with ADU soaked in on Day -7, followed by injection of CAR T cells on Day 0). Bioluminescent distribution of CAR T cells at indicated time points is shown. (B) Total flux of bioluminescent signals from ADU+MSLN CAR T groups from (A) was quantified at indicated time points. (C) NSG mice with established HCC70 tumors were implanted with PEG+PBS or PEG+100 pg ADU on Day -7. On Day 0, indicated doses of NanoLuc-CD19 / MSLN CAR T cells (50 pL) were injected into the PEG marker with PBS or ADU. MSLN2, 2* 106MSLN CART cells; MSLN1,1 106MSLN CAR T cells; MSLN0.5, 0.5x 106MSLN CAR T cells. Mean tumor volume measurements are shown (n=3-4 mice / group). (D) Bioluminescent distribution of CAR T cells from (C) is shown at indicated time points. (E) NSG mice with established HCC70 tumors were treated similarly as in (C). On Day 0, 2* 106 / 4* 106MSLN CAR T (MSLN2 or MSLN4) or 4><106CD19 CAR T cells were injected into the PEG marker following PBS or ADU (100 pg). Bioluminescent distribution of CAR T cells at indicated time points is shown.(F) Individual tumor volume measurements from (E) are shown (n=5 mice / group). Significance was calculated at Day 91 during tumor regrowth for MSLN2, PBS versus ADU. (G) Kaplan- Meier relapse free survival curves from (F) are shown (n=5 mice / group). Relapse was defined when tumor regrew to size > 500mm3. (H) Human fFNy levels in mouse serum at Day 7, 14, and 21 after indicated doses of CAR T cell administration for PBS or ADU treated individual animals are shown. (I) Representative IHC images of MSLN staining of PBS+CD19 control tumor and three different relapsed tumors from PBS+MSLN2 group from (F). Scale bar, 100 pm. Quantitative data are represented as mean ± SEM; p values were calculated by two-way ANOVA followed by Tukey’s post hoc test (C), Sidak’s post hoc test (F) and log-rank Mantel-Cox test(G). ns, not significant; *, p < 0.05; **, p < 0.01; ****, p < 0.0001.

[0044] Figure 31 shows (A) Human CD3+T cell count by flow cytometry in peripheral blood at Day 7, 14, 21, and 28 after indicated doses of MSLN CAR T administration from PBS or ADU (100 pg) treated animals from Figure 6E are shown (n=5 mice / group). (B) Schematic of PEG marker implantation in dual tumor model. (C, D) NSG mice with established dual HCC70 tumors were implanted with PEG+PBS or PEG+100 pg ADU adjacent to the mammary fat pad tumor on Day -7. On Day 0, 2x 106 / 4x 106MSLN CAR T or 4x 106CD19 CAR T cells were injected into the PEG marker. Mean tumor volume measurements of primary and flank tumors are shown with two independent experiments (Experiment #1 and Experiment # 2, n=3-4 mice / group). (E) Bioluminescent distribution of CAR T cells from primary and flank tumor sites of dual tumor model from Experiment #1 in (C, D) at indicated time points is shown. (F, G) Kaplan-Meier survival curves from dual tumor model are shown (n=7-8 mice / group, 2 cohorts of animals were combined). (H) Schematic of PEG marker implantation in metastasis prevention model. (I) NSG mice with established mammary fat pad HCC70 tumors were implanted with PEG+PBS or PEG+100 pg ADU on Day -7. On Day 0, 2x 106MSLN CAR T or 4x 106CD19 CAR T cells were injected into the PEG marker. 7 days later, HCC70 tumor cells were injectedinto the ipsilateral flank of NSG mice for metastasis prevention model. Mean tumor volume measurements of primary and flank tumors are shown (n=4 mice / group). (J) Kaplan-Meier survival curves from metastasis prevention model are shown (n=4 mice / group). Quantitative data are represented as mean ± SEM; p values were calculated by two-way ANOVA followed by Tukey’s post hoc test (C and D) or Sidak’s post hoc test (I), and log-rank Mantel-Cox test (F, G and J), ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.DETAILED DESCRIPTION

[0045] Disclosed herein are methods for administering STING agonists to solid tumors (e.g., breast cancer) at doses that avoid negative effects to T cells, but that are still efficacious to treat the solid tumor (e.g., immune-mediated clearance of STING-positive TNBCs). First, disclosed herein is that certain breast cancers, including phosphatase and tensin homolog (PTEN)-null, triple-negative breast cancers (TNBCs), can be hypersensitive to STING agonism, in some embodiments, due to defective Rab7 trafficking, and / or can result in enhanced T cell migration to the cancer. Second, we have discovered that certain biocompatible polymers containing anticancer therapeutics (e.g., STING agonists, CAR-T cells), implanted in proximity to a solid tumor in a subject, can continuously release the therapeutic agents over time. In the case of STING agonists, its release from the biocompatible polymers can avoid the harsh effects on T cells observed when STING agonists are injected intratumorally.

[0046] In some embodiments, the biocompatible polymer can be an expandable solid shape of polyethylene glycol (PEG) based hydrogel. The PEG can be dry / dessicated and expandable with fluid contact. In some embodiments, the polymers can be part of a biopsy site marker or biopsy clip. The biopsy site marker can include a metallic marker component. In some embodiments, a biopsy site marker can include a HydroMARK™ device.

[0047] In some embodiments, this platform provides for local delivery of both STING agonists and CAR-T cells into a tumor microenvironment of solid cancers.

[0048] In some embodeiments, the methods disclosed herein can be used to treat tumors in humans.Definitions

[0049] Detailed descriptions of one or more embodiments are provided herein. It is to be understood, however, that the present invention may be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in any appropriate manner.

[0050] The singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0051] Wherever any of the phrases “for example,” “such as,” “including” and the like are used herein, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. Similarly, “an example,” “exemplary” and the like are understood to be nonlimiting.

[0052] The term “substantially” allows for deviations from the descriptor that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term “substantially” even if the word “substantially” is not explicitly recited.

[0053] The terms “comprising” and “including” and “having” and “involving” (and similarly “comprises”, “includes,” “has,” and “involves”) and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of “comprising” and is therefore interpreted to be an open term meaning “at least the following,” and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, “a process involving steps a, b, and c” means that the process includes at least steps a, b and c. Wherever the terms “a” or “an” are used, “one or more” is understood, unless such interpretation is nonsensical in context.

[0054] The term “about” is used herein to mean approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).Biocompatible Polymers

[0055] In some embodiments, the polymers disclosed herein are biocompatible. In some embodiments, the polymers are implantable. The polymers can retain a therapeutic agent, which therapeutic agent can be released from the polymer (e.g., diffuse out from the polymer) after the polymer is implanted in a subject. In some embodiments, the therapeutic agent can be released from the polymer continuously over a period of time in the subject (e.g., sustained release).

[0056] In some embodiments, the therapeutic agent can adsorb to the polymer. When implanted, the therapeutic agent can de-adsorb from the polymer. In some embodiments, the therapeutic agent can be absorbed by the polymer. For example, a therapeutic agent can be dissolved in a solvent (e.g., water), which solvent can be used to hydrate a dehydrated polymer (e.g, aqueous-expandable polymer)(ab sorption). When implanted, the therapeutic agent can move out of the hydrated polymer with the solvent as bodily fluids displace the solvent.

[0057] In some embodiments, a biocompatible polymer can be a biodegradable polymer, a non-biodegradable polymer or a natural polymer. In some embodiments, a biodegradable polymer can be a poly(dioxanone) (PDO), polyethylene glycol (PEG), poly(glycolic acid) (PGA), poly(lactic acid) (PLA), poly(lactic-co-glycolytic acid) (PLGA), poly(caprolactone) (PCL), or combinations thereof.

[0058] In some embodiments, a non-biodegradable polymer can be a poly(siloxane) (silicone) or poly(ethylene-vinyl acetate).

[0059] In some embodiments, a natural polymer can be an albumin, cellulose, chitosan, collagen elastin, gelatin, a milk protein, silk, and the like.

[0060] In some embodiments, the biocompatible polymer can be hydrophilic. In some embodiments, the biocompatible polymer can be a hydrogel. In some embodiments, the biocompatible polymer can be a PEG-containing hydrogel. A hydrogel can be chemically linked (e.g., covalent cross-linking bonds), physically-linked (e.g., non-covalent bonds), or a combination thereof.

[0061] In some embodiments, the biocompatible polymer can have poly(dioxanone) (PDO), polylactide (PLA), polyglycollide (PGA), polycaprolactone (PCL), copolymers thereof and the like.

[0062] In some embodiments, the biocompatible polymer can have a core that can be surrounded by a core-surrounding layer. In some embodiments, the core-surrounding layer can be a sealant. In some embodiments, the core can have poly(dioxanone) (PDO), polylactide(PLA), polyglycollide (PGA), polycaprolactone (PCL), and / or copolymers thereof. In some embodiments, the core can be surrounded by a sealant layer. The sealant layer can be a polyethylene glycol-based hydrogel.

[0063] In some embodiments, the biocompatible polymer (e.g., the core) can have a metal marker. In some embodiments, the marker can be titanium or stainless steel.

[0064] In some embodiments, the biocompatible polymer can have pores of pores of 10-500 pm in size. Spacing between pores in the polymer can be from about 5 to 100 nm.Biopsy Markers

[0065] In some embodiments, the biocompatible polymers described herein can be part of a biopsy marker or biopsy clip. The biopsy marker can have a metal marker, like a titanium or stainless-steel marker. Biopsy site markers can be used to mark areas of the body from which biopsies are taken. Implanted biopsy markers can remain indefinitely and be used for preoperative localization when the biopsy identifies cells that must be removed, or for future identification of the site when pathology indicates the biopsi ed cells are benign.

[0066] Herein, any biopsy marker can be used for delivery of therapeutic agents. In some embodiments, the biopsy markers can have a PEGylated substance, collagen, P-glucan or polylactic-co-glycolic acid. In some embodiments, the biopsy marker can be a HydroMARK™, MammoMARK®, MammoSTAR® or SecurMark® biopsy marker. In some embodiments, HydroMARK™ biopsy markers are marketd by Mammotome, Devicor Medical Products, Inc., which is part of Leica Biosystems, USA..

[0067] The HydroMARK™ device can be made of an expandable solid cylinder of polymerized and desiccated polyethylene glycol (PEG)-based hydrogel. The device can expand with fluid contact, and the implanted device can be visible under ultrasound for 6 weeks to 12 months. The device can contain an embedded metallic component, for example titanium or stainless steel, which can be permanently visible by X-ray and MR imaging.

[0068] In some embodiments, the biopsy markers can be dehydrated or dessicated (FIG. 3A). In some embodiments, the biopsy markers can be hydrated. Hydrated markers, that eventually are implanted into a subject, can be approximately 7, 9, 11, 13, 15, 17, or 19 mm in length.

[0069] In some embodiments, disclosed herein are biopsy markers that have been “loaded” with one or more therapeutic agents. In some embodiments, disclosed herein areHydroMARK™ biopsy markers that have been loaded with a STING agonist and / or a CAR-T cell.Cancers

[0070] A wide variety of cancers can be treated using the compositions and methods disclosed herein. In some embodiments, the cancers can be solid tumors. In some embodiments, the cancers can be primary solid tumors or metastatic solid tumors. In some embodiments, the cancer can be a carcinoma, lymphoma, melanoma or sarcoma. In some embodiments, the cancer can be a bladder, bone, brain, breast, cervical, colorectal, gastrointestinal, kidney, liver, lung, ovarian, pancreatic, prostate, skin or thyroid cancer. In some embodiments, the cells of the cancer can have no or reduced amounts of functioning phosphatase and tensin homolog (PTEN). In some embodiments, PTEN can be inactivated or null. In some embodiments, cells of the cancer can have functioning PTEN.

[0071] In some embodiments, the cancer treated can be breast cancer. The breast cancer can include ductal carcinoma in situ (DCIS), HER2-positive breast cancer, invasive ductal carcinoma (IDC), invasive lobular carcinoma (ILC), mixed ductal and lobular carcinoma, inflammatory breast cancer or triple-negative breast cancer (TNBC). In some embodiments, the cells of the breast cancer can have no or reduced amounts of functioning phosphatase and tensin homolog (PTEN). In some embodiments, PTEN can be inactivated or null. In some embodiments, cells of the cancer can have functioning PTEN.

[0072] In some embodiments, the cancer can be one that overexpresses STING. STING (Stimulator of interferon genes) is a transmembrane protein encoded by TMEM173. STING is a regulator of the innate immune response to cytoplasmic double stranded DNA (dsDNA) in cancer. Following its activation, STING protein can undergo Ras-related protein Rab-7a (Rab7)- mediated lysosomal turnover. In some cells, including triple-negative breast cancer (TNBC) cells, this physiologic STING degradation can be disrupted by maintenance of Rab7 in a hyperphosphorylated inactive state.

[0073] We recently reported that tumor cell STING expression is elevated in TNBCs, whereas Luminal A and B subtypes are largely STING negative. In addition, when STING expression was examined in a tumor microarray (TMA) of 64 human TNBCs, there was a distinct trend of STINGhigh and STINGlow TNBCs, with approximately 61% of the TNBCs expressing STING.

[0074] In TNBC, phosphatase and tensin homolog (PTEN) inactivated or null cells maintain Rab7 in an inactive state. Trafficking of STING to lysosomes in impaired in these cells and the cells are hypersensitive to STING. Rab7 inhibitors exist and can be used to increase sensitivity to STING in cells that are not PTEN deficient.Therapeutic Agents

[0075] The therapeutic agents released by the implanted biocompatible polymers can be of a variety of types. The therapeutic agents can be small molecules, large molecules, cells or a combination thereof.

[0076] In some embodiments, a therapeutic agent can be a small molecule. In some embodiments, a small molecule can be a STING agonist. A STING agonist can cause increase activity of STING. In some examples, STING agonists can cause increases in inflammatory cytokines that can remodel the tumor microenvironment and can increase anti-tumor T cell responses.

[0077] Herein, any STING agonist can be used. In some embodiments, the STING agonist can include ADU-S100, MK-1454, MK-2118, SB11285, GSK3745417, BMS-986301, BISTING, E7766, TAK-676, SNX281, SYNB1891, and the like, or combinations thereof.

[0078] In some embodiments, a therapeutic can be a small molecule that is an estrogen receptor-target therapy. The estrogen receptor targeted therapy can be a selective estrogen receptor modulator (SERM). In some embodiments, the SERM can be tamoxifen, raloxifene, or the like.

[0079] In some embodiments, a therapeutic can be a large molecule. The large molecule can be a cytokine, chemokine, interleukin, or the like. In some embodiments, an interleukin can be IL-2, IL- 12, IL- 15 or combinations thereof.

[0080] In some embodiments, the therapeutic agent can be a cell. In some embodiments, the cell can be a chimeric antigen receptor (CAR) T cell (CAR-T cell). The CAR-T cell used herein can be specific for a molecule on a cell of a solid tumor. In some embodiments, the molecule can include MUC-1, TROP2, HER2 / ERRB2, EGFR / ERRB1, HGFR / cMET, ROR1, AXL, MUC1, MSLN, CD70, CD133, CD44v6, EpCAM, CSGP4, ICAM1, TEM8, TROP2, FRalpha, GD2, NGK2D ligand, CEA and the like. In some embodiments, CAR-T cells used in thesystems and methods disclosed herein can expand when released from the implantable polymers disclosed herein and into tissue surrounding the implantable polymers.

[0081] In some embodiments, the therapeutic agent can be a Rab7 inhibitor.

[0082] In some embodiments, the biocompatible polymers can release two or more of a small molecule, large molecule and cell.Methods

[0083] In some embodiments, the biocompatible polymers can be “loaded” with a therapeutic agent. The therapeutic agents can be loaded into the biocompatible polymers before and / or after the polymers have been implanted into the body of a subject.

[0084] In some embodiments, prior to loading of a therapeutic agent, the biocompatible polymer can be dehydrated. A therapeutic agent can be loaded into the polymer by rehydrating the polymer with a solution that contains the therapeutic agent. The polymer can be hydrated using various volumes of solution containing the therapeutic agent. In some embodiments, the biocompatible polymer can be hydrated with a volume less than about 20 pl.

[0085] In some embodiments, the biocompatible polymer can be implanted into a body of a subject. The biocompatible polymer can be implanted in proximity to the tumor or cancer that is desired to be treated. The biocompatible polymer can be implanted within within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mm of the solid tumor. The biocompatible polymer can be implanted within the solid tumor. The biocompatible polymer can be implanted in the periphery of the solid tumor. The biocompatible polymer can remain in the body of the subject for various periods of time. In some embodiments, the polymer can remain in the body of the subject for days, weeks or months.

[0086] In some embodiments, the tumor is not resected prior to / during implantation of the biocompatible polymer. In some embodiments, the tumor can be resected prior to / during implantation of the biocompatible polymer.

[0087] Implanting the biocompatible polymer can include surgical implanting or implanting using a large-bore needle. In some embodiments, multiple biocompatible polymers (i.e., plugs) can be implanted.

[0088] In some embodiments, the biocompatible polymer can be loaded with a therapeutic agent after the polymer has been implanted. This can be done using a syringe containing thetherapeutic agent to be loaded. A needle of the syringe can be inserted through the skin of the subject, and into the implanted polymer. The syringe can deliver the therapeutic agent (e.g., STING agonist, CAR-T cells) to the implanted polymer. In some embodiments, other substances can be injected into the implanted biocompatible polymer. For example, substances like cytokines, chemokines, interleukins, and the like, can be injected. In some embodiments, a dehydrated biocompatible polymer (e.g., a biopsy marker) can be loaded with a therapeutic agent (e.g., STING agonist) and implanted into a subject. Subsequently, another therapeutic agent and / or substance (e.g., CAR-T cells) can be injected into the implanted biocompatible polymer.

[0089] In some embodiments, a large-bore needle can be used to insert a dehydrated biocompatible polymer (e.g., biopsy marker) through the skin and place it in proximity to a tumor in a subject. In some embodiments, another syringe containing a therapeutic agent can then inserted through the skin and into the placed biocompatible polymer. The therapeutic agent can be delivered to the placed biocompatible polymer using the syringe.

[0090] Implanted biocompatible polymers that provide a therapeutic agent to a solid tumor, as disclosed herein, can be used alone to treat a subject. In some embodiments, this treatment can be used in combination with other treatments. In some embodiments, the treatments disclosed herein can be used in combination with conventional therapy. In some embodiments, conventional therapy can include chemotherapy, endocrine therapy, and the like.

[0091] In some embodiments, preconditioning of a tumor microenvironment (e g., using a STING agonist) can enhance CAR-T cell treatment. In some embodiments, both the STING agonist and the CAR-T cells can be delivered using an implantable polymer. In some embodiments, such preconditioning can promote of CAR-T cells from the tumor microenvironment into the circulation (extravasation). The extravasation can result in the CAR- T cells having efficacy against tumors elsewhere in the body (e.g., metastatic cancers).

[0092] In some embodiments, the effect of the PEG / STING / CAR-T cell therapies described herein can be evaluated using standard methods described in the art. In some embodiments, the effect of the therapy can be assessed by measuring tumor and / or metastasis size. In some embodiments, the effect of the therapy can be assessed by measuring the levels of TNFa and / or IFNy. In some embodiments, the effect of the therapy can be assessed by measuring the amount of CD3+T cells in blood (e.g., systemic circulation). In some embodiments, the CD3+T cells are human CD3+T cells.

[0093] Tn some embodiments, any of the STING / CAR-T cell combination therapies delivered through a PEG hydrogel described herein can be used to treat a subject (e.g., animal). In some embodiments, the subject can be a human. In some embodiments, the subject has a cancer. In embodiments, the cancer can be a triple negative breast cancer (TNBC). In some embodiments, the subject’s TNBC is cutaneous disease. In some embodiments, the subject’s TNBC is a chest wall disease.

[0094] In some embodiments, a biopsy of the disease present in the skin and / or in the chest wall of a subject can be performed to detect the presence of a target molecule (e.g., tumor antigen). In some embodiements the target molecule can be recognized by a CAR-T cell (CAR- T+target cell). In some embodiments, the CAR-T+ target cell can recognize molecules present in the tumor (tumor antigen / target molecule). In some embodiments, the tumor antigens can include mesothelin (MSLN), TROP2, c-Met, MUC1, ROR1, and NKG2DL, or combinations thereof. Any other suitable tumor antigens described in the art can also be used.

[0095] In some embodiments, the presence of any of the tumor antigens in a biopsy can be assessed using immunoblots, flow cytometry or any other suitable technique described in the art. In some embodiments, tumor antigen(s) present / expressed in a subject’s biopsy can be selected as a target(s) for the production of CAR-T cells (CAR-T+target) that recognize said target(s) (they recognize and interact with the target molecule present in the subject’s tumor and / or biopsy(ies).

[0096] CAR-T+target cells can be produced using standard CAR-T cell methods of production described in the art. In some embodiments, the CAR-T+ target cells can be lab-grade CAR-T+target cells. In some embodiments, the CAR-T+target cells can be treatment-grade CAR-T+target. In some embodiments, CAR-T+target cells can be produced from T cells enriched from donor leukapheresis. In some embodiments, the donor can be the subject. In some embodiments, T cells from the subject can be activated.

[0097] In some embodiments, a CAR-T that recognizes the target molecule can be introduced into the subject’s T cells using any of the methods described in the art (e.g., transduced with a lentiviral vector). In some embodiments, CAR-T+target cells can be expanded before formulation and use. See Dickinson et al., Cancer Discov (2023) 13 (9): 1982-1997.

[0098] In some embodiments, CAR-T cells can be selected based on the CAR-T cell ability to treat cancer. In some embodiments, the ability of CAR-T+target cells to treat cancer can beassessed using any of the methods described in the art. In some embodiments, the ability of CAR-T+target cells to treat cancer can be assessed (Zurowski D. et al., SLAS Discovery 28 (2023) 65-72).

[0099] In some embodiments, CAR-T cells can be selected by exposing the developed CAR- T+target cells to spheroids comprising a target molecule and isolating CAR-T+target cells capable of interacting with and / or reducing the diameter of a spheroid comprising the target molecule. In some embodiments, the CAR-T+target cells can reduce the diameter of the spheroid to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% percent of the diameter of the spheroid before exposing the speroid to the CAR-T+target cell. In some embodiments, CAR- T+target cells can be evaluated at various CAR-T+target cell to target cell ratios.

[0100] In some embodiments, one or more hydrated HydroMARK™ biopsy markers having one or more STING agonists (PEG+STING) can be implanted in proximity to the cancer (day 0). In some embodiments, the STING can be any of the STINGs described herein, or a combination thereof. In some embodiments, the STING can be any STING agonist described in the art. In some embodiments, the PEG+STING can be implanted into one or more primary tumors. In some embodiments, the PEG+STING can be implanted in one or more metastatic cancers and / or lymph nodes. In some embodiments, the PEG+STING can be implanted into one or more primary tumors and / or into one or more metastatic cancers and / or lymph nodes. In some embodiments, the PEG+STING can be implanted simultaneously and / or sequentially into the one or more primary tumors and / or one or more metastatic cancers and / or lymph nodes. In some embodiments, the PEG can be embedded with any of the STING agonists described herein, or a combination thereof, before implantation. In some embodiments, the STING agonists described herein, or a combination thereof, can be injected into the PEG after implantation of the PEG into the subject. In some embodiments, the STING agonists described herein, or a combination thereof, can be slowly released from the PEG.

[0101] In some embodiments, one or more selected CAR-T+target cells can be injected simultaneously or sequentially into the PEG plug. In some embodiments, the injected CAR- T+target cell can recognize any of target molecules ROR-1, Trop-2, Muc-1, and MSLN. In some embodiments, the injected CAR-T+target cell can recognize any tumor antigen (target molecules) described herein. In some embodiments, the injected CAR-T+target cell can recognize any tumor antigen (target molecules) described in the art.

[0102] In some embodiments, a selected CAR-T+target cell can be injected into a PEG plug containing any of the STING agonists described herein, or a combination thereof. In some embodiments, the CAR-T+target cell agonist can be injected into the PEG plug at any time after implantation of the plug (implantable polymer). In some embodiments, the CAR-T+target cell can be injected into the PEG plug at any time after injection of the PEG. In some embodiments, the CAR-T+target cell can be injected at day 7, or seven days after implantation of the PEG+STING. In some embodiments, the CAR-T+target cell can be added multiple times after the first application. In some embodiments, the CAR-T+target cell can be applied multiple times until the cancer is not longer detectable or cured (using standard technologies described in the art for the detection of breast cancer).

[0103] In some embodiments, the CAR-T+target cell can be injected into a PEG+STING implanted next to a primary tumor. In some embodiments, the CAR-T+target cell can be injected into a PEG+STING implanted next to a metastatic cancer and / or a lymph node. In some embodiments, the CAR-T+target cell can be injected into a one or more PEG+STING implanted next to one or more primary tumors and / or next to one or more metastatic cancers and / or lymph nodes. In some embodiments, the isolated CAR-T+target cell can be injected into a HydroMARK™ biopsy marker having one or more STING agonists at 1, 2, 3, 4, 5, 6, 7, 8, 25, 20, 30, 40, 60, 100 days after implantation of the marker.

[0104] In some embodiments, the cancer can recur (e.g., reappear in the same spot or part of the subject’s body). In some embodiments, a new biopsy can be perfomed in the new spot or at the area of a recurrent cancer. In some embodiments, a new target can be identified in a biopsy of a recurrent cancer. In some embodiments, a new CAR-T+target cell that can specially interact with the new target can be developed and evalualed for selection using the methods described above (for the first target). In some embodiments, the new spot or recurrent cancer can be treated using the new CAR-T+target that recognized the new target using the methods described above for the fist target.

[0105] In some embodiments, the subject can be monitored for changes in the tumor and / or metastasis using any of the methods described herein. In some embodiments, the subject can be monitored for the presence of new onset tumors, metastatic tumors, or affected lymph nodes using any of the methods described herein.

[0106] In some embodiments, the procedure described above, from biopsy to the assessmentof the effect of CAR-T+target cells injected into the PEG+STING can be repeated until the cancer (primary or metastatic) can be considered to be undetectable and / or eliminated.Compositions

[0107] Disclosed herein are implantable biocompatible polymers that include a therapeutic agent. Embodiments of these biocompatible polymers plus therapeutic agents that can be combined with the biocompatible polymers have been described throughout this application.

[0108] In some embodiments, compositions of a biocompatible polymer that can include a metal marker or clip, and a therapeutic agent are disclosed. The biocompatible polymer that has the therapeutic agent can be hydrated.

[0109] In some embodiments, the biocompatible polymer can be a polymer core that includes the metal marker, surrounded by a polyethylene glycol (PEG)-based hydrogel. In some embodiments, the polymer core can be a poly(dioxanone) (PDO), polyethylene glycol (PEG), poly(glycolic acid) (PGA), poly(lactic acid) (PLA), poly(lactic-co-glycolytic acid) (PLGA), poly(caprolactone) (PCL), or combinations of these polymers. The PEG-based hydrogel surrounding the core can be called a “sealant”. In some embodiments, the metal marker or clip can be titanium, stainless-steel, and the like.

[0110] In some embodiments, the biocompatible polymer can be a biopsy marker. In some embodiments, the biopsy marker can be a combination biodegradable polymer encompassed by a hydrogel. In various embodiments, the biopsy marker can be of many different types, including HydroMARK™, MammoMARK®, MammoSTAR®, SecurMark®, and the like.

[0111] The therapeutic agents associated with the biocompatible polymer can be absorbed into and / or adsorbed onto the biocompatible polymer. In some embodiments, a dehydrated biocompatible polymer can be hydrated in a solution or suspension containing a therapeutic agent. The biocompatible polymer can have one or more associated therapeutic agents.

[0112] In some embodiments, the therapeutic agents can be small molecules. An example small molecule can be a STING agonist, as described herein. Example STING agonists can ADU-S100, MK-1454, MK-2118, SB11285, GSK3745417, BMS-986301, BI-STING, E7766, TAK-676, SNX281, SYNB1891, and other molecules that have the same or similar function.

[0113] In some embodiments, the therapeutic agents can be cells. In some embodiments, the calls can be CAR-T cells. The CAR-T cells can be specific for molecules on the surface of a cell in a tumor. In some embodiments, the tumor can be a solid tumor. In some embodiments, theCAR-T cells can be specific for molecules on the surface of a breast cancer cell. In some embodiments the CAR-T cells can be specific for TROP2, c-Met, MUC1, R0R1, and NKG2DL, or combinations thereof.Local Delivery of Therapeutic Agents; Delivery of Therapeutic Agents to Metastatic Cancers

[0114] In some embodiments, the implantable, biocompatible polymers disclosed herein can release therapeutic agents that can treat tumors or cancers that are in proximity to the implanted polymer. In some embodiments, the implanted polymer can release therapeutic agents (e.g., CAR-T cells) that can treat cancers that may not be in proximity to the implanted polymer. While not wishing to be bound by any theory, a therapeutic agent (e.g., a CAR-T cell) released from an implanted polymer can leak into surrounding tissue (i.e., extravasation). In some embodiments, such a therapeutic agent can enter the bloodstream and / or lympathic system. Therapeutic agents (e.g., CAR-T cells) that enter the bloodstream or lymphatic system can be transported to other parts of the body. In some embodiments, the therapeutic agents transported by these systems, the bloodsteam for example, can move from the bloodstream to surrounding tissue (e.g., extravasation) such that the therapeutic agent can contact tumors or cancers (e.g., metastatic cancers) that are nearby, but not in proximity to the implanted polymer that released the therapeutic agent. In some embodiments, CAR-T cells originally released from an implantable biopolymer that enter the bloodstream and then leave the bloodstream to infuse into a tissue elsewhere in the body (i.e., secondary site) can expand at the secondary site. These CAR-T cells can be efficacious against tumors in the secondary locations. In some embodiments, such CAR-T cells that leave the bloodstream can prevent formation of tumors at secondary locations in the body.EXAMPLES

[0115] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.EXAMPLE 1Defective STING trafficking renders TNBC cells hypersensitive to STING agonism.

[0116] A STING immunohistochemistry study was performed on a TNBC tissue microarray (TMA) derived from treatment-naive patients (n=63) with clinicopathologic characteristics described in Table 1. A breast pathologist visually scored the samples using an intensity scale: 0 = no staining in tumor cells with intact expression in internal controls using endothelial and inflammatory cells; 1+ = faint staining in >10% of tumor cells; 2+ = moderate staining in >10% of tumor cells; 3+ = strong staining in >10% of tumor cells (Fig. 1A). More than half of the TNBCs showed STING expression, and four samples showed high variability ranging from 0 to 2+ / 3+ staining with similar histology in both the high and low STING expressing areas (Fig. IB). Among the STING 3+ TNBCs, 5 out of 6 specimens were available for PTEN staining and were either PTEN IHC negative or low. No association of heterogeneity with age, lymph node status, tumor size or grade was seen (Table 1).

[0117] Table 1. Clinicopathologic characteristics of triple negative breast cancer patients.1-2

[0118] 'American Joint Committee on Cancer (8thedition) pT and pN categories

[0119] 2N / A is not applicable.

[0120] It was previously found that PTEN (phosphatase and tensin homolog; a tumor suppressor) null TNBCs maintain the lysosomal protein, Rab7 in an inactive state and, therefore, trafficking of STING to the lysosome can be impaired, resulting in baseline upregulation of STING protein and increased sensitivity to STING agonism (12). PTEN IHC was therefore performed on the TMA and confirmed this inverse relationship between PTEN and STING (Fig. 1C). Analysis of the larger TMA showed that PTEN null status had a tendency towards elevated tumor cell STING levels (Fig. IP and IQ). Similarly, using a panel of TNBC cell lines, PTEN null lines (MDAMB468, HCC1937, HCC70) showed higher STING protein expression than PTEN WT lines (HCC1143, HCC1806, MDAMB231) (Fig. ID). Furthermore, when PTEN was knocked out in MDAMB231, STING protein was upregulated and became sensitized to ADU- S100, producing higher levels of the interferon regulatory factor 3 (IRF3)-regulated chemokine CXCL10 (Fig. IE).

[0121] To broaden the use of STING agonists to include PTEN WT TNBCs, we targeted Rab7 directly through both protein knock out and the use of the Rab7 inhibitor (CID1067700), which acts as a competitor for the GTP and GDP binding site in Rab7, thus preventing nucleotide exchange and activation (24, 25). Rab7 knockout in MDAMAB231 resulted in a marked increase in STING protein and sensitized response to ADU-S100 with a marked increase in CXCL10 (Fig. IF). As a control, Rab7 knockout of PTEN WT MDAMB468 cells did notalter STING protein levels (Fig. IL).

[0122] To explore the immediate effects of Rab7 inhibition, MDAMB231 cells were treated with ADUS100, the Rab7 inhibitor CID1067700, and in combination. Immunofluorescence staining for STING and lysosome was then performed to monitor for colocalization. After ADU- S100 stimulation, there was distinct colocalization of STING to the lysosomes for degradation that was disrupted in the presence of Rab7 inhibition. (Fig. 1G; Fig IM). In a prior growth assay, chronic ADU- SI 00 treatment of the PTEN null cell lines MDAMB468 and HCC70 resulted in significant growth inhibition with low dose ADU-S100 at lOpM that was not seen with MDAMB231 and HCC1806. Addition of Rab7 inhibitor was able to sensitize both MDAMB23 1 and HCC1806 to ADU-S100 with a more significant effect in the latter (Fig. 1H) that is quantified by crystal violet absorbance (Fig.ll).

[0123] Since MDAMB231 had a less robust response to Rab7 inhibitor with ADUS100 at lOpM, MDAMB231 PTEN knockout cells were treated with ADU-S100 + / - Rab7 inhibitor and it was found that PTEN loss sensitized the cells to the lower concentration of ADU- SI 00 (lOpM) and that addition of Rab7 inhibitor further enhanced this effect (Fig. 1 J and Fig. IK; Fig. IN). Moreover, combination treatment also increased CXCL10 production in MDMAMB23 1 and HCC1806 but not in MDMAB468 in which Rab7 is already inactivated and a robust increase in CXCL10 has already been achieved with ADU-S100 treatment alone (Fig. 10).

[0124] These data show that PTEN null TNBCs have a dysregulation of STING that prevents its trafficking to the lysosomes for degradation, and in PTEN WT TNBCs this can be recapitulated with Rab7 knockout or addition of a Rab7 inhibitor.EXAMPLE 2Impaired STING turnover enhances STING agonist induced T cell migration.

[0125] Prior data showed upregulation of CXCL10 with Rab7 knockout. To better profile these resultant cytokines, a Luminex Assay was performed on MDAMB231 and HCC1806, using MDAMB468 as a control. In addition, scramble control cells and RAB7 knockout cells (Fig. 2K) for each cell line were treated with 50pM ADU-S100 to assess if cytokine levels could be further augmented. The key cytokines that were induced were TBK1-IRF3 regulated CXCL10 and CCL5 and the NF-KB- regulated cytokines IL-8 and IL-6 (Fig. 2A and 2J). Both CXCL10 and CCL5 have been implicated in T cell recruitment whereas IL-6 and IL-8 have beenassociated with both T-cell recruitment and immunosuppression (26). ELISA was then performed to confirm the upregulation of CXCL-10, IL-8, and IL-6 (Fig. 2B). The MDMAMB468 control did not show any further increases in cytokine production from Rab7 knockout as Rab7 is already inactivated in PTEN null cells, and the addition of ADU- SI 00 to scramble cells and Rab7 knockout cells, did not show any additive effects. However, MDA- MB231 cells with Rab7 knockout not only led to increased CXCL10 production but also increases in IL-8 and IL-6, both at baseline and in response to ADU-S100 stimulation. Similarly, HCC1806 Rab7 knockout cells showed an increased in CXCL10 level which was further enhanced with ADU-S100. There was also a significant increase in CCL5 (Fig. 2H), IL-8, and IL-6 with ADU-S100 stimulation of Rab7 knockout HCC1806 cells versus scramble.

[0126] Given that activating cytotoxic T lymphocytes depends on successful trafficking and infiltration to tumor tissue (27) and the cytokines that are upregulated by Rab7 knockout are largely involved in T cell trafficking, the potential in vivo implications for immune cell recruitment was modeled utilizing a previously described 3D microfluidic T-cell migration assay (Fig. 2C) (28). MDA-MB231 scramble and Rab7 knockout spheroids were pretreated with 50pM ADU-S100 and loaded into the migration zone of the 3D microfluidic device. Primary human CD8+ T cells were then introduced to a side channel, and T cell trafficking into the migration zone was evaluated after 48 hours. As shown in Fig. 2D, minimal T cells migrated towards the MDAMB231 scrambled spheroids even with ADU-S100 treatment. Since Rab7 knockout in PTEN WT TNBCs increases CXCL10 levels, significant T cell migration towards the MDAMB231 Rab7 knockout spheroids was observed, and ADU-S100 treatment further increased T cell trafficking into the migration zone. HCC 1806 showed a similar T cell migration pattern (Fig. 2E).

[0127] To assess whether these findings could be duplicated using Rab7 inhibitor CID1067700, it was first confirmed that addition of Rab7 inhibitor increases CXCL10 levels in both untreated and ADU-S100 treated MDAMB231 and HCC1806 cells. Next, the dosage of the Rab7 inhibitor with ADU- S100 was optimized to achieve the highest increase in CXCL10 levels for MDAMB231 and HCC 1806 (Fig. 2F). Using MDAMB231, spheroids were treated with lOOnM Rab7 inhibitor in the presence or absence of 50pM ADU-S100 in the 3D microfluidic device and then CXCR3-expressing Jurkat cells were introduced from a side channel. Significant migration of Jurkat cells was observed with ADU-S100 pretreatment that was additionallyenhanced by the Rab7 inhibitor (Fig. 2G; Fig 21).

[0128] Taken together, these findings identify PTEN null or PTEN WT with Rab7 knockout / inhibition as a genomic context in TNBCs for which STING agonists can be applied to not only induce cell intrinsic toxicity but to recruit T cells to the TME.EXAMPLE 3Impregnation of STING agonist in a PEG hydrogel biopsy marker results in its local delivery into TNBC.

[0129] In human clinical trials, intratumoral administration is seen as a barrier to therapeutic efficacy (14). In current clinical practice, after a patient has a breast biopsy, a metal marker is deployed to mark the area of biopsy. More recently, the metallic markers have been embedded in hydrogels that swell to optimize their visualization on ultrasound. These markers are FDA approved and can be made from polyethylene glycol, collagen, and beta-glucan. Once hydrated, these markers can fully expand within 6 hours, with the PEG hydrogel increasing proportionately the most in size (Fig. 3A). In an example, dehydrated PEG measures 5x1x1 mm and at maximum expansion it is 14x4x4 mm and can absorb at least 350 l of ddH20, which can be used to deliver an amount of desired drug.

[0130] We embedded ADU-S100 into PEG hydrogel markers in attempt to retain it in the TME. To ensure that the PEG will expand as expected, the PEG was implanted into the subcutaneous tissue of a Balb / c mouse. Significant expansion of the PEG was observed by Day 4, maximum expansion between Days 11-15, and by Day 25 there was some decrease in the size of the PEG (Fig. 3B, Fig. 3K).

[0131] In human breast tissue, the PEG is noted to be visible even after 6-12 months. To assess whether the PEG hydrogel can release ADU-S100 in a controlled fashion, a THP-1 cellbased reporter assay was developed. THP-1 cells were first treated with phorbol 12-myristate 13- acetate to differentiate into macrophages that are sensitive to STING agonist treatment and secrete CXCL10. We first established a standard curve for which the amount of ADU-S100 secreted from the PEG can be deduced from the corresponding amount of CXCL10 released. THP-1 cells were treated with a known amount of ADU-S100 and after 24 hours, supernatants were harvested for CXCL10 ELISA analysis (Fig. 3C) to generate a standard curve (Fig. 3D). Next, lOOpg of ADU-S100 was reconstituted into 20 pl of ddH20 and the PEG hydrogel was allowed to absorb the drug Alternatively, the drug was injected into the hydrogel, vs soaking upthe drug into the PEG. The PEG+ADU-S100 was then covered by collagen to mimic the in vivo tissue barrier with 1 ml of media placed on top and replaced with fresh media every 24 hours for a continuous 5-day collection. The harvested conditioned media at the 5 time points was then added to differentiated THP-1 cells for CXCL10 analysis (Fig. 3E).

[0132] The amount of ADU-S100 released at the various time points was deduced (Fig. 3F) by using the standard curve previously generated in Fig. 3D. As expected, the PEG hydrogel soaked in the drug and the one injected with the drug were able to release ADU-S100 in a slow sustained fashion with release of 70pg of the embedded lOOpg by Day 5. Unlike the PEG hydrogel, the collagen and beta-glucan breast markers could not release ADU-S100 (Fig. 3G).

[0133] Next, the TNBC 4T1 model described above was treated with PEG hydrogel embedded with lOOpg of ADU-S100 versus PBS. Given the aggressiveness of the 4Tlmouse model, the PEG hydrogels were first implanted in a low tumor burden model at Day 4 after injecting 10K 4T1 cells into the 4th mammary fat pad. PEG+ADU-S100 prevented the 4T1 cells from establishing a tumor whereas PEG+PBS showed robust tumor growth (Fig. 3H). However, when PEG+ADU-S100 was implanted adjacent to an established 4T1 tumor, growth was inhibited initially, and by Day 20 there was exponential growth. (Fig. 31). At Day 25, the PEG marker could be easily identified adjacent to the tumor, and resection showed that the PEG marker was still intact (Fig. 3J).

[0134] These data show that the PEG hydrogel marker is a new drug delivery method for ADU-S100.

[0135] In another experiment, we compared the results shown in Fig. 3H and 31 with direct intratumoral injection of ADU-S 100 and found minimal response in tumor growth compared to PEG delivery (Fig. 28A).EXAMPLE 4STING agonist monotherapy is toxic to T cells and does not result in T cell clonality.

[0136] T cells can become hyperresponsive to STING agonist, activating a distinct set of genes that results in apoptosis (34, 35). To investigate this potential toxicity, both a primary human TNBC explant and human CD8+ T cells were used. Explants were made from a patient’s TNBC as previously described (35) and were treated with PBS or 50pM ADU-S100 for 24 hours. The TNBC were examined by flow cytometry and showed a slight increase in CD45+ leukocytes and a small decrease in lymphocytes with ADU-S 100. A marked decrease in CD3+,CD4+ and CD8+ T cells was observed with ADU-S100 treatment (Fig. 4A and 4J). Treatment of primary human CD8+ T cells with increasing concentrations of ADU-S100 showed a corresponding increase in T cell death (Fig. 4B).

[0137] Next, splenocytes were obtained from wild-type mice and the T population was sorted by flow cytometry into CD3+, CD4+, and CD8+ subpopulations. Each subpopulation was then treated with increasing concentrations of ADU-S100 for 24 hours and showed a corresponding increase in cell death, with CD8+ T cells being the most sensitive to treatment (Fig. 4C)

[0138] Given that PEG hydrogel can deliver STING agonist in a controlled sustained fashion (Fig. 3F), it was hypothesized that embedding ADU-S100 in the hydrogel can limit T cell toxicity. For example, if lOOpg of ADU-S100 is injected, the T cells are immediately exposed to the full dose of the drug which would likely result in immediate toxicity, whereas if the lOOpg is delivered over time in a controlled fashion that toxicity will likely be minimized. As such, human CD8+ T cells were plated in triplicate and then were treated with direct addition of PBS, direct addition of ADU- SI 00, PEG+PBS, or PEG+ADUS100. T cell toxicity was significantly higher with the direct ADU-S100 treatment versus embedding the drug in the PEG (Fig. 4D).EXAMPLE 5CAR-T with STING agonist as combination therapy for TNBC.

[0139] Evaluation of TNBC cell lines for endogenous mesothelin (MSLN) expression showed that HCC1806 (PTEN WT) and HCC70 (PTEN null) are positive for MSLN expression. HCC 1143 (PTEN WT) was negative for MSLN and, therefore, was selected as a control (Fig. 5A). Flow cytometry was performed to confirm MSLN positivity of over 70% in both HCC70 and HCC1806 cells, whereas HCC1143 was negative for MSLN (Fig. 5B).

[0140] Luciferase tagged MSLN CAR-T cells and luciferase tagged CAR-T cells targeting a non-relevant antigen CD 19 (as a control) were generated. Flow cytometry of the transduced T cells revealed expression of the MSLN / CD19 CAR constructs marked by LssmOrange in approximately 83.3% and 90.9% of cells respectively (Fig. 5K).

[0141] These CAR-T cells were cultured with HCC1143, HCC1806 and HCC70 target cells at different EffectorTarget Cell (E:T) ratios. For HCC 1143 cells, which do not express MSLN, there was no distinct difference in targeting efficacy between MSLN and CD 19 CAR-T cells (Fig. 5C). When MSLN CAR-T were co-cultured with the MSLN positive cell lines, HCC 1806and HCC70, there was significant cell death even at the lowest E:T ratio. As the E:T ratio increased to 5: 1, MSLN CAR-T cells achieved complete eradication of HCC1806 and HCC70 in 2D culture, whereas CD 19 CAR-T only exhibited moderate non-specific targeting efficacy (Fig. 5D), supporting the specificity of the MSLN CAR-T cells.

[0142] Next, a microfluidic assay was used (12) to assess the cytotoxic killing effects of CAR-T cells in 3D culture. HCC1806 cells were grown in ultra-low attachment plates to create spheroids. The MSLN CAR-T cells were then mixed with the HCC1806 spheroids at a 1 : 1 E:T ratio and placed into the central channel of the device. Three days later, cytotoxicity was evaluated by staining cells with DAPI and Calcein for live cells and Draq7 for dead cells. As shown in Fig. 5E, MSLN CAR-T cells effectively targeted the spheroids, with the majority staining positive for Draq7. Co-culturing HCC1806 with CD 19 CAR-T cells showed that the majority of the spheroids were alive. Similarly, HCC70 showed the same patterns of staining (Fig. 5F). Quantification of cell death percentage showed that MSLN CAR-T cells achieved nearly 100% cell death when co-cultured with both HCC1806 and HCC70 but not in our control HCC1143 (Fig. 5G). Conditioned media was then analyzed for interferon-y (IFNy), Granzyme B, and tumor necrosis factor-a (TNFa), showing the on-target effects of MSLN CAR-T (Fig. 5H)

[0143] Next, a 3D microfluidic assay as described in Example 2 (Fig. 2C) was used to assess the ability of MSLN CAR-T cells to migrate to the central channel and kill the MSLN+ spheroids at 1 : 1 E:T ratio. As described in Example 2 above (Fig. 2), PTEN null TNBCs, like HCC70, have high baseline levels of CXCL10 from impaired STING turnover, unlike PTEN WT TNBCs (e.g., HCC1806). The basal level of CXCL10 for HCC1806 is approximately 25 pg / ml (Fig. 2B) and that for HCC70 is approximately 200 pg / ml (Fig. 5L). Therefore, MSLN CAR-T cells introduced through the side channel of the device are quickly drawn into the central channel of the HCC70 spheroids resulting in killing. On the other hand, PTEN WT TNBCs rely on Rab7 knockout / inhibition to prevent STING degradation. As such, HCC1806 spheroids were not able to attract the MSLN CAR-T cells into the central channel (Fig. 51). However, once spheroids were generated from HCC1806 Rab7 knockout cells, MSLN CAR-T cells were able to migrate to the central zone which was further enhanced by pretreatment with ADU-S100. This resulted in cell death of the HCC1806 Rab7 knockout spheroids and upregulation of IFNy and Granzyme B (Fig. 5J)

[0144] Taken together, the 3D migration assay described herein shows that combination STING agonist and MSLN CAR-T therapy can be a viable strategy to target PTEN null MSLN positive TNBC spheroids.EXAMPLE 6Association of CAR-T Cells in a PEG hydrogel biopsy marker results in its local delivery into TNBC.

[0145] CAR-T therapy is generally used to target cancers in the blood. CAR-T therapy can be less effective against solid tumors. However, we assessed whether CAR-T therapy would be effective in this system.

[0146] To test whether a PEG hydrogel biopsy marker associates with CAR-T cells, the PEG marker was soaked with luciferase labeled MSLN CAR-T cells or PBS and then the markers were imaged using the Xenogen Bioluminescence System. While the PEG+PBS showed background signal, the PEG + MSLN CAR-T displayed strong luciferase signal, confirming that the PEG binds CAR-T cells (Fig. 6A, 29A). Then, to assess whether CAR-T cells can be injected into a hydrated PEG marker, two PEG markers were hydrated with 300pl of ddH20 and then injected with either PBS or luciferase-MSLN CAR-T cells and imaged, confirming that CAR-T cells can be retained in the hydrated marker after injection (Fig. 6B, 29B).

[0147] Next, to ensure that the CAR-T cells delivered by the PEG marker can result in killing, we generated MSLN CAR-T cells and B-cell maturation antigen (BCMA) CAR-T cells as a control with lentiviral vectors containing vex-GFP. Flow cytometry of the transduced T cells for vex-GFP revealed expression of the MSLN / BCMA CAR constructs in approximately 49.5% and 40.3% of cells respectively (Fig. 6N).

[0148] Luciferase tagged HCC70 (HCC70-Luc) spheroids were treated with either MSLN CAR-T cells or BCMA CAR-T cells associate with various biopsy markers (PEG, Collagen, or Beta-glucan). After 24 hours, HCC70-Luc cells were harvested for luciferase assay to analyze for viability (Fig. 6C). Consistent with previous results, direct co-culture of MSLN CAR-T cells with HCC70 spheroids resulted in significant cell death compared to BCMA CAR-T (Fig. 6D). MSLN CAR-T+PEG resulted in specific targeting as the percentage of HCC70-Luc cell death correlated with increasing E:T ratios (Fig. 6E). Although a high E:T ratio of 12: 1 was needed to achieve complete elimination, the same number of BCMA CAR-T cells resulted in only 20% cell death. Both the collagen marker and the beta-glucan marker also showed some efficacy indelivering CAR-T cells (Fig. 6F and 6G). However, given these markers could not release ADU- S100 (Fig. 3G), we chose the PEG marker for subsequent in vivo studies.

[0149] HCC70 cells were injected into the fourth mammary fat pad of NSG mice and, once tumors were established, a PEG+PBS or PEG+MSLN CAR-T was implanted immediately adjacent to the cancer. After 10 days of treatment, the MSLN CAR-T treated cancer began to shrink in size (Fig. 6H). Tumors were harvested at Day 70 and IHC staining for human CD3 demonstrated intensive infiltration of T cells into the treated tumor only (Fig. 61 and 6J or 29F).

[0150] Given these results, we proceeded to treat a larger group (n=6 per group) of NSG mice bearing established HCC70 tumors (size 150 mm3) with PEG+MSLN CAR-T (4* 106cells) or PEG+CD-19 CAR-T. In the MSLN CAR-T group, 4 out of 6 mice showed consistent intratumoral CAR-T expansion. MSLN2 had a migrated plug that was palpated at Week 1, and MSLN6 despite appropriate implantation only showed low levels of CAR-T expansion (Fig.6K). The two mice (MSLN3 and MSLN4) which showed strong luciferase signals at Week 2 had tumors that were no longer palpable by Week 3. MSLN4 demonstrated continued CAR-T expansion and tumor-free survival in the subsequent weeks whereas MSLN3 had recurrent growth at Week 7 and luciferase signals became detectable again at Weeks 8-12 which stabilized its growth. All mice receiving CD 19 CAR-T cells were euthanized because of tumor progression within 9 weeks, whereas 3 mice were still alive in the MSLN treated group at the study endpoint of 12 weeks. Overall, PEG+MSLN CAR-T treated mice had smaller tumors (Fig. 6L) and a better probability of survival (Fig. 6M).

[0151] The two mice (MSLN3 and MSLN4) which showed strong luciferase signals at Week 2 had tumors that were no longer palpable by week 3. MSLN4 demonstrated continued CAR-T expansion and tumor-free survival in the subsequent weeks. MSLN3, however, had recurrent growth at week 7 and luciferase signals became detectable again at Weeks 8-12 which stabilized its growth. IHC staining of this tumor outgrowth showed absence of MSLN (Figure 60 and 6P), confirming the on-target effects of the CAR-T. Taken together, these data confirm the extended capability of this PEG marker platform to retain and release CAR-T cells intratum orally.EXAMPLE 7Local delivery of STING agonist and CAR-T through a PEG hydrogel biopsy marker obliterates TNBC.

[0152] Now that we have established that our PEG marker can deliver both STING agonistand CAR-T cells locally into the TME, we proceeded to test this combination therapy in our HCC70 mouse model. Three delivery methods were assessed in a HCC70 mouse model by (1) directly injecting ADU-S100 into a tumor and then, 7 days later, direct injection of the CAR-T cells; (2) implanting two PEG markers with one containing ADU-S100 and the other containing CAR-T cells; and (3) implanting one PEG marker soaked with ADU-S100 followed by injection of CAR-T 7 days later (Fig. 3F shows that the majority of ADU-S100 has been secreted by Day 5) into the now hydrated marker. Within 4 weeks, it was evident that the latter approach resulted in the most robust expansion of intratumoral CAR-T cells (Fig. 7A, Fig 7 J), requiring us to apply a more stringent luminescence scale than previously used for the MSLN CAR-T treatment group (Fig. 6K). When we applied this stringent scale to our soaked MSLN CAR-T monotherapy group (Fig. 7K), combination therapy with injection of MSLN CAR-T into the hydrated marker resulted in more robust and uniform expansion of the CAR-T cells. With this approach, 2 out of 5 mice were tumor free (#1 and #3) and corresponded to the two mice demonstrating early and persistent CAR-T expansion. Mouse #2 showed significant CAR-T expansion initially with the cancer shrinking to less than 100 mm3, however the tumor began to regrow starting at week 5 when there was loss of luciferase signaling. Reassuringly, by week 8 there was recurrent luciferase signaling that persisted to week 12 which appeared to contain the growth of the tumor. Mouse #2 had a small tumor at study endpoint which was MSLN negative (Fig. 70 and 7P), confirming the on-target activity of the CAR-T. Mouse #4 had excellent luciferase signaling throughout treatment but then its associated tumor began to grow. This tumor was filled with serous fluid suggesting a partial response with a heterogenous MSLN staining pattern (Fig. 70). Comparatively, mice in the PBS and ADU-S100 groups were euthanized because of tumor progression by week 7-8. The ADUS100 monotherapy group showed growth inhibition initially but this response was not durable (Fig. 7B). The combination treatment group had overall smaller tumors and probability of survival was much greater (Fig. 7B and 7C).

[0153] Having optimized the implantation technique, we conducted another independent experiment to ensure that these results were reproducible and to see if a lower dose of MSLN CAR-T (2><106compared with 4* 106) could be implemented. Similarly, HCC70 TNBC xenografts were grown to a tumor size of 150 mm3and PEG markers (soaked with PBS or 100 pg ADU-S100) were then implanted adjacent to the palpable tumors. A week later, 2*106or4* 106NanoLuc tagged MSLN CAR-T cells were injected into the now hydrated PEG markers previously containing PBS or ADU-S100. As a negative control, 4><106NanoLuc tagged CD19 CAR-T cells were injected into the PEG marker previously soaked with PBS. Between weeks 1- 3, luciferase signaling did not appear to be intensified by ADU-S100 as there was robust signaling among all the MSLN CAR-T treated animals. However, by week 3, tumor growth of the mice receiving ADU-S100 with MSLN CAR-T at 2* 106was comparable to the mice treated with MSLN at 4* 106(Fig. 7D, 7E, Fig. 7L with individual tumor growth), showing the additive effects of combination therapy. By week 5, 3 mice out of 5 from the PBS+CD19 group had to be sacrificed given marked tumor growth. In PBS+MSLN2, 1 mouse was free of tumor and the remaining animals had small tumors. The PBS+MSLN4 and the ADU+MSLN2 groups had 3 animals that were tumor free and 2 animals with small tumors. The ADU+MSLN4 group had the most impressive response with 4 animals that were tumor free by week 4 and 1 animal with a small tumor. By week 6, 5 out of 5 mice treated in the ADU+MSLN4 group were tumor free, 4 out of 5 mice in the PBS+MSLN4 and ADU+MSLN2 were tumor free, and 2 out of 5 mice in the PBS+MSLN2 were tumor free. At study endpoint, the PBS+MSLN2 animals all had regrowth of small tumors (68.8-183.8 mm3), whereas the PBS+MSLN4 (52.7 mm3, 91.9 mm3) and the ADU+MSLN2 (66.3 mm3, 87.4 mm3) had 2 animals with tumors, and the ADU+MSLN4 (58.2 mm3, 72.5 mm3) had 2 animals with very early tumor regrowth. All treatment groups had smaller tumors, and all treated mice were alive (Fig. 7E and 7F). Taken together, the addition of ADU-S100 can compensate for a lower dosage of MSLN CAR-T and resulted in earlier shrinkage of the tumors. However, it was the ADU+MSLN4 that showed the most robust response by obliterating all tumors by week 6.

[0154] To ensure that the tumor inhibitory effects seen are on target, we obtained peripheral blood from all five groups at Day 7, 14, 21, and 28 after CAR-T treatment to measure IFNy and TNFa levels. By Day 7, there was a trend towards higher levels of IFNy and TNFa among all treatment groups. However, it was only the ADU+MSLN4 group that was statistically significant for both IFNy and TNFa (Fig. 7G, 7M and 7N). The ADU+MSLN2 group trended towards significance with a p value of 0.06 for IFNy. Next, to see if CAR-T cells can extravasate into systemic circulation, flow cytometry was performed to analyze the levels of CD3+ T cells in blood among the 5 groups. Combination therapy resulted in striking elevations in CD3+ T cells at both treatment doses of CAR-T (Fig. 7H) that was most evident at Day 14 unlike themonotherapy groups which showed only marginal increases. The ADU+MSLN2 group, however, had more variability in CD3+ T cell extravasation with Mouse #2 having a delayed elevation at Day 21 and Mouse #3 with no increase levels, and as such it was only the ADU+MSLN4 group that showed significance with almost all groups uniformly having elevated CD3+ T cell levels at Day 14 (Fig. 71). Taken together, these data show that the sequential delivery of ADU-S100 followed by CAR-T from the PEG marker results in a more uniform response and earlier obliteration of HCC70 tumors. Most importantly, whereas MSLN monotherapy did not lead to extravasation of CD3+ T cells, there was a robust increase in CD3+ T cells in the peripheral blood with combination treatment suggesting a novel immune mediated pathway to clear the primary tumor and likely circulating tumor cells that can potentiate distal metastatic disease.EXAMPLE 8

[0155] In some embodiments, disclosed herein is a therapeutic strategy (e.g., for TNBC patients) that is a combination therapy with STING agonism and CAR-T cells. Our platform addresses three hurdles to successfully deliver CAR-T cells into the TME of solid tumors (22). Firstly, when CAR-T is delivered via intravenous infusion, CAR-T cells trafficking into the TME of the solid tumor are rarely seen. Herein, the disclosed PEG marker physically maintains the CAR-T cells in the TME and is then successful at releasing the CAR-T cells.

[0156] Secondly, some have proposed that, even if CAR-T cells can traffic into the TME, their activity is not preserved given the immunosuppressive environment of the TME. Herein, we showed that pretreatment with ADU-S100 induces a more favorable environment for CAR-T cells, as shown in the more robust intratumoral expansion of CAR-T cells seen with the ADU- S100 combination treatment compared to CAR-T cell monotherapy. We also showed that although CAR-T cell monotherapy was successful at obliterating the primary tumor, by using the PEG marker, STING agonist can first be given to prime the TME, and later, when the STING agonist has been released by the PEG and can no longer induce T cell toxicity, CAR-T cells can be injected into the hydrogel and then released into a TME that has been optimized for its activity.

[0157] Thirdly, solid tumors can be heterogeneous and, as such, treatment with one CAR may not eradicate disease. Disclosed herein is that, using just one PEG marker, we can first release ADU-S100 in a controlled fashion and then, a week later, inject CAR-T cells. Given thatthe PEG hydrogel remains in human breast tissue for 6-12 months, multiple courses of CAR-T cell therapy (+ / - STING agonist or other types of immunostimulants that are hydrophilic) targeting different antigens can be introduced by injecting them into the PEG marker that is embedded adjacent to the primary tumor. If the cancer location is superficial such as that seen with cutaneous metastatic disease, the PEG marker can be palpated and injected. Otherwise, given the PEG marker contains a metal clip, ultrasound imaging at the bedside can identify the hydrogel for additional injections. Alternatively, given the ease of implanting these PEG markers, multiple markers can also be implanted at the same time or sequentially to deliver combinations of CAR-T therapy. There are numerous CARs in clinical development for TNBC, including those targeting mesothelin, TR0P2, c-Met, MUC1, R0R1, and NKG2DL (45).

[0158] Fourthly, we have identified PTEN null as a genomic state to apply this combination therapy in TNBC because STING levels are already upregulated. For PTEN WT TNBCs, a Rab7 inhibitor could be used.

[0159] The PEG hydrogel marker disclosed here is part of a biopsy system that is FDA approved and used internationally to mark breast biopsy sites. Given the small size of the PEG when it is dehydrated, it is conventionally delivered with a large bore needle by a radiologist at the bedside and does not require surgery. Also, distal sites such as the lung, liver, and brain can be treated, as can any site where a biopsy (and PEG hydrogel placement) can be performed. The longevity (6-12 months) of the PEG hydrogel in human tissue can be relatively long. Furthermore, in a histologic study using 88 breast tissue and 36 lymph node samples to analyze the clip site of the same PEG hydrogel marker used herein, there are specific characteristics seen such as the formation of a pseudocyst lined by layers of epithelioid histiocytes and there is the absence of lymphocytes with minimal or no fibrosis (46). The PEG hydrogel marker does not result in a non-specific lymphocytic infiltrate that can crowd out CAR-T cells. Furthermore, the PEG hydrogel can result in a rim of histocytes. While not wishing to be bound by theory, and given that STING agonists have been implicated in conversion of macrophages from M2 to Ml to induce an immune supportive environment (37), the PEG can not only serve as a reservoir for controlled release of STING agonist and CAR-T, but can also contribute to disrupting the immune-suppressive TME. Once the PEG hydrogel marker is absorbed at 6-12 months, it leaves behind a permanent titanium clip, which can be used for future imaging surveillance. Additionally, while implanation of other materials can require a resected tumor bed, or lowtumor burden, PEG hydrogels could obliterate an established tumor and induce extravasation of CAR-T cells without this. Therefore, this method can be used to treat distal metastatic disease. Although a TNBC model was used herein, this therapeutic platform can be used for other types of solid cancers.

[0160] For patients with metastatic PTEN null / PDL1+ TNBC, example situations where the treatment disclosed herein include the following. ADU-S100+CAR-T PEG marker can be implanted adjacent to a cutaneous metastatic implant or in the axilla if there is an involved lymph node concurrently with IV Pembrolizumab administration. In patients with recurrent disease after KEYNOTE-522, ADU-S100+ CAR-T therapy delivered by our PEG system can be similarly given.EXAMPLE 9Extravasation of CAR-T cells loaded in a PEG hydrogel.

[0161] The ability of CAR T cell injected into the PEG to extravasate to systemic circulation was assessed using the HCC1806 mouse model.

[0162] HCC1806 TNBC xenografts were grown to a tumor size of 150 mm3and PEG markers (soaked with PBS or 100 pg ADU-S100) were then implanted adjacent to the palpable tumors. A week later, 4* 106NanoLuc tagged MSLN CAR T cells were injected into the now hydrated PEG markers previously containing PBS or ADU-S100. As a negative control, 4x 106NanoLuc tagged CD19 CAR-T cells were injected into the PEG marker previously soaked with PBS. Within 4 weeks, it was observed that combination therapy with injection of MSLN CAR T into the hydrated marker resulted in more robust and uniform expansion of the CAR T cells.With this approach, at week 4, 2 out of 5 mice were tumor free (Fig. 8A and B) in the ADU+MSLN4 group. Comparatively, mice in the PBS+CD19 and ADU+CD19 groups were euthanized because of tumor progression by week 3-4.

[0163] To ensure that the tumor inhibitory effects seen are on target, we obtained peripheral blood from all five groups at Day 7, 14, 21, and 28 after CAR-T treatment to measure IFNy and TNFa levels. By Day 14, there was a trend towards higher levels of IFNy and TNFa among the PBS+MSLN and ADU+MSLN treatment groups. However, it was only the ADU+MSLN group that was statistically significant for TNFa (Fig. 8D).

[0164] Next, to see if CAR-T cells can extravasate into the systemic circulation, flow cytometry was performed to analyze the levels of CD3+ T cells in blood among the 4 groups.Combination therapy group ADU+MSLN resulted in elevations in CD3+ T (Fig. 8E) that, in this study, was most evident at Day 14 when compared to PBS+MSLN. The PBS+CD19 and ADU+CD19 groups showed no significant increases. Taken together, these data show that the sequential delivery of ADU-S100 followed by CAR-T from the PEG marker resulted in a more uniform response and earlier obliteration of HCC1806 tumors. Whereas MSLN monotherapy did not lead to significant extravasation of CD3+ T cells, there was a robust increase in CD3+ T cells in the peripheral blood with combination (ADU+MSLN) treatment, indicating that the treatment of the primary tumor can have an effect on distal metastatic disease.EXAMPLE 10Extravasation of CAR-T cells to treat metastatic disease.

[0165] As described above, CAR-T cells injected at a primary tumor can extravasate into systemic circulation indicating that the treatment of the primary tumor could have an effect on distal metastatic disease.

[0166] The tumor models described herein (e.g., HCC1086, HCC70, and the like) and the methods described above, can then be used to assess the effect(s) in a distant metastasis of treating a primary tumor.

[0167] TNBC xenografts can be grown to a tumor size of 150 mm3and PEG markers (soaked with PBS or 100 pg ADU-S100) can then be implanted adjacent to primary tumor(s). Tumor cells can be injected in a distant part of the mice body (e.g., flank tumor placement) either at the same time of PEG implantation at the primary tumor site, or some time later (e.g., 1, 2, 3, 4 weeks). Next, 4* 106(2x 106or any other suitable quantity) of NanoLuc tagged MSLN CAR-T cells can be injected into the hydrated PEG markers containing PBS or ADU-S100. As a negative control, NanoLuc tagged CD 19 CAR-T cells can be injected into a PEG marker previously soaked with PBS.

[0168] Given the ease of implanting the PEG markers described herein, multiple markers can be implanted, at the same time or sequentially, in one or multiple primary tumors and / or one or multiple metastasis (including lymph nodes) to deliver any of the therapies described herein.

[0169] The size of the primary tumors and the size of the metastases can be assessed to evaluate whether treatment of the primary tumor results in a reduction and / or inhibition of the metastasis.

[0170] Other parameters including presence of CAR-T cells at the primary tumor andmetastases, CAR-T cell extravasation, among others, can also be assessed to evaluate effectiveness of the treatment.[001711 Other STING agonists, or combinations thereof, in combination with any of the CAR- T cells, or combinations thereof, described herein can be used to assess the effect of any of the combination therapies described herein in any of the models described herein. There are numerous CARs in clinical development for TNBC, including those targeting mesothelin, TROP2, c-Met, MUC1, ROR1, and NKG2DL.

[0172] PTEN null was described herein as a genomic state to apply the combination therapy described herein, in TNBC, because STING levels are already upregulated. For PTEN WT TNBCs, a Rab7 inhibitor can be used. Immunoblot analysis and flow cytometry analysis of PTEN null TNBCs HCC1143, HCC1806 and MDA-MB231, and PTEN WT TNBCs MDAMB468, HCC1937 and HCC70, showed that TROP2, c-Met, MUC1, ROR1, and NKG2DL are expressed in cells from these TNBCs (Fig.9 A and B). Therefore, CAR-T cells including mesothelin, TROP2, c-Met, MUC1, ROR1, and NKG2DL, or combinations thereof can be developed.

[0173] Experimental protocols for the evaluation of STING agonist (e.g., ADU-S100) in combination with CAR-T cells are described in Example 7 and Fig. 7. Using these protocols, STING agonists ADU-S100, MK-1454, MK-2118, SB11285, GSK3745417, BMS-986301, BISTING, E7766, TAK-676 and SNX281, SYNB1891, or combinations thereof, can be incorporated into the PEG hydrogel described herein. Next, combination therapy using any of the mouse models described herein (e.g., HCC1086, HCC70, and the like) can be used to evaluate the effect of delivery of any of the STING agonists described above together with any of the CAR-T cells described herein.

[0174] In other experiments, multiple markers can be implanted, at the same time or sequentially, in one or multiple primary tumors and / or one or multiple metastasis (including lymph nodes) to deliver any of the therapies using any of the STING agonists, or combination thereof, described herein.

[0175] The size of the primary tumors and the size of the metastases can be assessed to evaluate whether treatment of the primary tumor resulted in a reduction and / or inhibition of the metastasis. Other parameters including presence of CAR-T cells at the primary tumor and metastases, CAR-T cell extravasation, among others, can also be assessed to evaluateeffectiveness of the treatment.

[0176] The effect of addition of interleukins (e.g., IL-2, IL-12, IL-15), or combinations thereof to any of the STING / CART cell combination therapy models described herein can also be assessed.

[0177] Experimental protocols for the evaluation of STING agonist (e.g., ADU-S100) combined with CART cells (e.g., MSLN) are described in Example 7 and Fig. 7. Similar protocols can be used to assess the local delivery through a PEG hydrogel biopsy marker of any of the interleukins described herein (e.g., IL-2, IL- 12, IL-IL- 15), or a combination thereof, in combination with any of the CART cells and / or any of the STING agonists described herein, or a combination thereof.

[0178] Any of these combinations of STING agonists plus CART cells plus interlukins can be evaluated in any of the primary tumor and / or metastasis models, and experimental protocols described herein.

[0179] Any of the STING / CAR-T cell methods described herein can be used to treat human subjects with cutaneous an / or chest wall disease.

[0180] First, a biopsy of a cancer present in a subject can be performed, and expression of a tumor antigen (also called herein target molecule) candidate for CAR-T production can be identified. The tumor antigen(s) can include mesothelin (MSLN), TROP2, c-Met, MUC1, R0R1, and NKG2DL, or combinations thereof, and they can be assessed in tissue obtained from the biopsy(ies) using western blot, flow cytometry, or any other suitable technique described in the art. See Fig. 9A and B.

[0181] Once a putative CAR-T cell target is selected, lab grade CAR-T cells expressing the target can be developed (CAR-T+target). For example, using traditional manufacturing, T cells can be enriched from donor leukapheresis, activated, and transduced with a lentiviral vector including the target to produce the CAR-T+target cell. CAR-T+target cells can then be expanded before formulation and use. See Dickinson et al., Cancer Discov (2023) 13 (9): 1982-1997.

[0182] The efficacy of the CAR-T+target cells can be assessed using the spheroids as describe, for example, in Zurowski D. et al., SLAS Discovery 28 (2023) 65-72, by evaluating, for example, CART+target cell-mediated killing of 3D tumor spheroids, at various CAR-T+taget cell E:T ratios.

[0183] After selection and assessment of a CAR-T+target, the PEG polymers, including anyof the STING or other therapeutic agents described herein can be implanted into a subject (Day 0). PEG+STINGs can be implanted into one or more primary tumors and / or into one or more metastases and / or lymph nodes. Seven days after implanation of the PEG+STING(s) device(s), at “Day 7”, the previously selected CAR-T+target can be injected into the PEG plug.

[0184] Subjects can be monitored and observed in search of any response to the treatment (e.g., tumor size, and the like), or regrowth of the cancer. If regrowth of the cancer is detected, a new biopsy of the cancer can be performed and analyzed for the expression of the same or a new putative CAR-T+ target.

[0185] When a new target is identified, one or more new CAR-T+target cells expressing the new target(s) can be developed and assesses using the methods and speroids as described above. The newly selected CAR-T+target, can be injected into the primary tumors and / or into the metastases, and / or into the lymph nodes using the same PEG device(s) already implanted in the subject. Without wishing to be bound by theory, this method can be used as an efficient and effective way to treat any primary tumor an its metastases without the need for further quirugical procedures.

[0186] The procedure described above, from biopsy to the assessment of the effect of CAR-T cells injected into the PEG can be repeated until the cancer (primary or distal) can be undetectable and / or eliminated.EXAMPLE 11

[0187] Additional data are described in this Example.

[0188] Fig. 10 shows example intratumoral CAR-T expansion and tumor efficacy.

[0189] Fig. 11 shows example preconditioning of the tumor microenvironment enhances CAR-T expansion and prevents relapse.

[0190] Fig. 12 shows example preconditioning of the tumor microenvironment promotes CAR-T extravasation from the tumor microenvironment.

[0191] Fig. 13 shows example CAR-T expansion at a secondary tumor site.

[0192] Fig. 14 shows example data indicating the immunotherapy platform is efficacious against metastatic lesions.

[0193] Fig. 15 shows example data indicating the immunotherapy platform is efficacious in blocking formation of new metastatic lesions.

[0194] Fig. 16 shows multiple example treatment options (embodiments) for metastatic tumors.

[0195] Fig. 17 shows an example of overcoming resistance due to tumor heterogeneity and antigen loss.

[0196] Fig. 18 shows an example of overcoming resistance due to tumor heterogeneity and antigen loss.

[0197] Fig. 19 shows an example of an integrated immunotherapy platform for the treatment and cure of solid tumors.

[0198] Fig. 20 shows examples of priming the tumor microenvironment with STING agonists prior to CAR-T delivery to further boost anti-tumor activity and CAR-T expansion in mice treated with the immunotherapy platform.

[0199] Fig. 21 shows an example that the immunotherapy platform is efficacious in metastatic cancer models.

[0200] Fig. 22 shows an example indicating that any site that can be biopsied can be potentially treated.

[0201] Fig. 23 shows an example application of CAR-T-PET platform to a patient sample.

[0202] Fig. 24 shows an example of cancer treatment using the systems disclosed herein.

[0203] Fig. 25 shows example data obtained from the systems described herein.

[0204] Fig. 26 shows example data obtained from the systems described herein.

[0205] Fig. 27 shows example data obtained from the systems described herein.

[0206] Regarding Fig. 26 and 27, those figures show metastatic animal model data. In such studies, tumor cells were injected at both the fourth mammary fat pad and the flank. When tumors were established at both sites, the tumor at the fourth mammary fat pad was treated using the methods described herein (the tumor at the flank was not treated). The data show that, even though the flank tumor was not treated, the flank tumors were obliterated by the fourth mammary fat pad treatment.

[0207] In an additional animal study, tumors were established in the fourth mammary fat pad. The tumors at the fat pad were treated using the methods described herein. Tumor cells were injected into the flank. The data showed that tumors were not established at the flank, due to the treatment at the fat pad.EXAMPLE 12

[0208] Based upon data discussed in Example 3, we continued investigation of the potential of the PEG marker to deliver ADU-S100 in vivo using the well-established TNBC 4T1 syngeneic model (Aslakson, Cheryl J., and Fred R. Miller, Cancer research 52.6, 1992, 1399-1405).

[0209] To determine whether the decrease in tumor size coincided with on-target tumor intrinsic effects of ADU-S100 being delivered by the PEG in this model, treated 4T1 mouse tumors were stained for pTBKl and pSTATl at Day 4, revealing evidence of on target STING activation (Fig. 28B). Next, to better characterize the TME effects of ADU-S100 delivered by the PEG, we performed single cell RNA sequencing (scRNA-seq) on CD45+immune cells from 4T1 tumors 14 days after treatment (Fig. 28C). Uniform Manifold Approximation and Projection (UMAP) clustering revealed 5 different immune cell types (Fig. 28D), and we found that antigen presenting cell (APC) and macrophage clusters exhibed robust upregulation of IFN-induced genes by ADU-S100, includings well as significant enrichment for hallmark IFN gene signatures (Figs. 28E and 28F). Taken together, these findings confirmed effective PEG marker delivery of ADU-S100 into the TME.EXAMPLE 13PEG biopsy marker mediated local delivery of CAR T cells into TNBC.

[0210] To test the potential of the PEG marker to deliver CAR T cells, we next soaked the PEG with NanoLuc-MSLN CAR T cells (4xl06cells) or PBS, and imaged markers using the Xenogen Bioluminescence System. Whereas the control PEG showed a background signal, the PEG+NanoLuc-MSLN CAR T displayed a strong luciferase signal, confirming that the PEG can indeed absorb CAR T cells (Fig. 29A). Since concurrent STING agonist / CAR T loading could be toxic to T cells, we next explored whether this system could be useful for sequential CAR T delivery via injection into a hydrated PEG marker. Two PEG markers were hydrated with ddFEO, injected with either PBS or NanoLuc-MSLN CAR T cells (4xl06cells), and then imaged, confirming that CAR T cells can also be retained in the hydrated marker after injection (Fig. 29B). To then ensure that the PEG hydrogel was not toxic to the CAR T cells, we soaked or injected 2 million and 4 million Nano-luc tagged CAR Ts into the PEG marker and compared the luciferase signal of increasing amounts of CAR Ts in a comparable volume of collagen under bioluminescence imaging. We found that CAR T cells showed significantly higher signaling when embedded in the PEG versus collagen, which further highlights that the PEG hydrogel does not negatively affect CAR T cell viability. Next, to ensure that soaked or injected CAR Tcells can be released by the PEG marker and result in killing, we generated MSLN versus control CAR T cells using lentiviral vectors containing vex-GFP for facile CAR-T detection. Flow cytometry of the transduced T cells for vex-GFP revealed expression of the MSLN or control B- cell maturation antigen (BCMA) CAR constructs in approximately 49.5% and 40.3% of cells respectively. Direct co-culture of MSLN CAR T cells with firefly luciferase tagged HCC70 (ffLUC-HCC70) spheroids resulted in significant cell death compared to control CAR T cells. Next, ffLUC-HCC70 spheroids were treated with either MSLN CAR T cells or control CAR T cells soaked into various biopsy markers (PEG, Collagen, or Beta-glucan) at different E:T ratios. After 24 hours, ffLUC-HCC70 spheroids were harvested for luciferase assay to analyze for viability (Fig. 29C). MSLN-CAR T cells delivered by the PEG marker, especially when injected, resulted in potent and specific targeting compared to control CAR T cells (Fig. 29D and 29E). Both the collagen marker and the beta-glucan marker also showed some efficacy in delivering CAR T cells; however, since these markers could not release ADU-S100 and have suboptimal expansion properties for injection of CAR T cells in vivo, we chose the PEG marker for subsequent in vivo studies.

[0211] To test whether this system can deliver CAR T cells in vivo, we orthotopically established HCC70 tumor xenografts in the fourth mammary fat pad of NSG mice and implanted PEG+PBS or PEG+MSLN CAR T immediately adjacent to the cancer. We first performed a short term in vivo experiment, measuring human CD3 IHC expression on Day 14 to ensure that CAR T cells could migrate out of the PEG into the tumor. Indeed, CAR T cells were successful at trafficking into both the tumor interior and periphery (Fig. 29F). We then repeated this experiment to examine longer term durability and found that the MSLN CAR T cells controlled growth of the HCC70 tumor xenograft by Day 20 (Figure S5E), with persistent evidence of CAR T infiltration by Day 42 (Fig. 29G). Given these results, we proceeded to treat a larger group (n=6 per group) of NSG mice bearing established HCC70 tumor xenografts (size 150 mm3) with PEGs soaked with NanoLuc tagged MSLN CAR T (4* 106cells) or NanoLuc tagged control CD 19 CAR T control cells delivered in equal number, using in vivo imaging to detect CAR T cell expansion. In contrast to MSLN CAR T cells, we observed no significant intratumoral expansion of control CD19 CAR T cells. All mice receiving CD19 CAR T cells were euthanized because of tumor progression within 9 weeks, whereas 3 mice were still alive in the MSLN treated group at the study endpoint of 12 weeks. Overall, PEG+MSLN CAR T treated mice hadsmaller tumors and a better probability of survival. The two MSLN CAR-T treated mice (#3 and #4) that showed strong luciferase signals at Week 2 had tumors that were no longer palpable by week 3. While mouse #4 demonstrated continued CAR T expansion and tumor-free survival in the subsequent weeks, mouse #3 exhibited recurrent tumor growth at week 7, with reappearance of luciferase signals at weeks 8-12. Interestingly, in contrast to CD19 CAR T treated tumors, IHC staining of this tumor outgrowth showed absence of MSLN, confirming escape due to loss of antigen and thus on-target effects of the MSLN CAR T cells.

[0212] We next explored sequential delivery of ADU-S100 followed by CAR T cells. Unlike other polymers which degrade within days of implantation, the PEG marker remained intact and was easily palpated adjacent to the tumor even at later time points well beyond 35 days (Fig. 29H), allowing for a substantial therapeutic window where multiple injections of drugs into the PEG can be performed. To ensure that ADU-S100 is completely released from the PEG marker by Day 7, PEGs hydrated with ADU-S100 or PBS were implanted into mice and after 7 days, these PEGs were retrieved and co-cultured with MSLN CAR T cells in vitro, resulting in no significant cytotoxicity to CAR T cells (Fig. 291 and 29J).

[0213] We therefore tested the feasibility of implanting PEG markers hydrated with PBS or ADU-S100 adjacent to HCC70 tumors in mice 7 days prior to MSLN CAR Ts being injected into the PEG, henceforth referred to as the Sequential Delivery PEG Method or the sd-PEG Method (Fig. 29K). In some embodiments, this approach allows for ADU-S100 priming of tumors while limiting STING agonist induced T cell toxicity. Indeed, following ADU-S100 pretreatment, there was a significant increase in CAR T infiltration within 2 days as evidenced by increased human CD3 IHC staining of the HCC70 tumors (Fig. 29L) and by flow cytometry (Fig. 29M and 29N). Taken together, in some embodiments, these data confirm the extended capability of this PEG marker platform to prime tumors with STING agonism and then allow for robust infiltration of CAR T cells intratumorally.EXAMPLE 14Sequential local delivery of STING agonist followed by CAR T cells through a PEG hydrogel biopsy marker results in long term durable control of TNBC growth.

[0214] Having established that the PEG marker can deliver both STING agonists and CAR T cells locally and that priming of tumors with ADU-S100 first results in increased CAR T cell infiltration, we proceeded to further test this combination therapy in the same HCC70 xenograftmodel. We examined multiple potential co-delivery methods to compare the Sequential Delivery PEG Method (sd-PEG Method) (Fig. 29K) to (1) directly injecting ADU-S100 into the tumor and then 3 days later directly injecting CAR T cells without a PEG, and (2) implanting two PEG markers with one containing ADU-S100 and the other containing CAR T cells. Within 4 weeks, it was evident that our initial approach with sequential delivery using one PEG marker resulted in good expansion of intratumoral CAR T cells (Fig. 30A and 30B). As such, we proceeded to treat a larger group of HCC70 mice with the sd-PEG Method using different concentrations of MSLN CAR T (Fig. 30C) with ADU-S100 held constant at 100 pg, with a starting tumor size of 150 mm3. The impact of ADU-S100 release was apparent following downtitration of MSLN CAR T cells to as low as 0.5X106T cells per PEG, resulting in greater control of HCC70 tumor growth (Fig. 30C), greater intratumoral CAR T cell expansion (Fig. 30D) and higher levels of IFNy in the peripheral blood.

[0215] Given these results with combination therapy using the sd-PEG Method, we performed a large-scale study using two different MSLN CAR T cell concentrations (2xl06and 4xl06) with ADU-S100 priming (100 pg), to further explore the ability of this platform to durably control the growth of the aggressive HCC70 model (Fig. 30E). By week 5, 3 mice out of 5 from the control PBS+CD19 group had to be sacrificed given marked tumor growth, with no evidence of CAR T expansion by luciferase signaling. Comparatively, all PBS or ADU-S100 primed tumors responded to MSLN CAR T cells at the 4x 106cell dose (MSLN4), and we also observed robust CAR T expansion and efficacy using the 2* 106cell dose (MSLN2).Furthermore, while the higher dose of CAR T cells (MSLN4) itself led to durable tumor control, downtitrating the injected MLSN CAR T dose to 2* 106cells (MSLN2) unveiled the ability of ADU-S100 priming to prevent tumor escape and promote durable eradication of these TNBC xenografts up to 120 days (Fig. 30F). Relapse-free survival (RFS) at 120 days was markedly improved among all the MSLN CAR T treated groups versus the CD 19 CAR T group (Fig. 30G). Furthermore, with the lower dosage (2* 106) of MSLN CAR T, ADU-S100 priming was able to achieve a 100% RFS at 120 days versus 40% with MSLN CAR T monotherapy, further supporting the additive effects of this combination therapy.

[0216] To ensure that the tumor inhibitory effects were on-target, we obtained peripheral blood from all five groups on Day 7, 14, 21, and 28 after CAR T treatment to measure IFNy levels (Fig. 30H). By Day 7, there was a trend towards higher levels of IFNy among all treatmentgroups which was enhanced by ADU-S100 priming. We also performed MSLN IHC staining of the three tumors that regrew with PBS+MSLN (2X106) monotherapy and found that all the tumors exhibited weak or absent MSLN staining relative to control, again revealing loss of tumor antigen as a mechanism of escape and confirmed the on-target effects of our MSLN CAR T (Fig. 301).EXAMPLE 15Local delivery of STING agonist followed by CAR T cells with the sd-PEG Method results in effective control of distant TNBC growth and prevention of metastasis.

[0217] Since STING agonism can also promote vascular remodeling and T cell adhesion, we sought to evaluate whether priming of tumors with ADU-S100 in the xenografted TME before the addition of CAR T cells increases their ability to extravasate by measuring circulating CAR T cells in our HCC70 mouse model. Flow cytometry was performed to analyze the levels of CD31T cells in peripheral blood between animals receiving PBS+MSLN CAR T versus ADU+MSLN CAR T. Regardless of the dosage of MSLN CAR T (0.25X106, 0.5X106, 2X106, and 4X106), combination therapy with ADU-S100 resulted in striking elevation of circulating CD3+T cells by flow cytometry, versus MSLN CAR T monotherapy (Fig. 31A).

[0218] Since orthotopic disease was eradicated by this sequential PEG delivery platform, and we observed enhanced extravasation by ADU-S100 priming, we therefore sought to evaluate the potential of this platform to treat distant metastatic disease. We first tested the Dual Tumor Model by establishing HCC70 tumor xenografts (150 mm3) at both the fourth mammary fat pad and also the ipsilateral flank in NSG mice (Fig. 31B). Using the sd-PEG Method, the NSG mice were first given ADU-S100 or PBS and 7 days later they were treated with MSLN CAR T cells (2X106and 4X106) or CD 19 CAR T cells. We observed that treatment of the primary site with ADU-S100 and MSLN CAR T cells was able to not only obliterate disease locally but also contained the growth of the distal flank tumor (Fig. 31C and 31D). Although mice treated with PBS and MSLN CAR T cells showed growth inhibitory effects of both the primary and flank tumors, this was less consistent than that seen with ADU-S100 priming, which resulted in more robust intratumoral CAR T cell expansion in the ipsilateral flank (Fig. 31E). Additionally, while MSLN CAR T treated groups (+PBS or +ADU) exhibited disease control at both the primary and distal sites in surviving animals, there was a drastic difference in probability of survival at 120days that favored the MSLN CAR T groups that had received ADU-S100 priming (Fig. 31F and 31G)

[0219] We next sought to evaluate whether obliteration of the primary tumor could result in memory response whereby distant metastatic disease might be prevented from forming at a later time point (Metastasis Prevention Model, Fig. 31H). Using this model, we explored whether extravasation of CAR T cells could prevent distant metastasis from forming following treatment of the primary tumor site. HCC70 tumor xenografts (150 mm3) were treated orthotopically (fourth mammary fat pad) as per the sd-PEG Method for ADU-S100 versus PBS, followed by MSLN (2X106)versus CD19 CAR T cell therapy. Seven days following CAR T injection into the PEG, the ipsilateral flank was injected with 3X106HCC70 cells. Whereas the PBS+CD19 animals exhibited significant growth of both primary and flank tumors, the ADU+MSLN2 treated animals exhibited minimal flank tumor growth in addition to obliteration of the primary tumor (Fig. 311). The probability of survival was also much greater among the ADU+MSLN2 group than the PBS+CD19 group (Fig. 31J). Taken together, these data show that local delivery of STING agonism followed by CAR T cell therapy has the potential to not only eradicate primary TNBC disease but also treat distant metastasis.References

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[0267] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

Claims

CLAIMSWhat is claimed:

1. A method for delivering a therapeutic agent to a subject to treat a solid tumor, comprising implanting into the subject a biocompatible polymer including the therapeutic agent, wherein the biocompatible polymer (plug) continuously releases the therapeutic agent in the subject over time.

2. The method of claim 1, wherein the solid tumor is in a human.

3. The method of claim 2, wherein the solid tumor comprises a cancer.

4. The method of claim 3, wherein the cancer comprises a carcinoma, lymphoma, melanoma or sarcoma.

5. The method of claim 3, wherein the cancer comprises bladder, bone, brain, breast, cervical, colorectal, gastrointestinal, kidney, liver, lung, ovarian, pancreatic, prostate, skin or thyroid cancer.

6. The method of claim 5, wherein the breast cancer comprises ductal carcinoma in situ (DCIS), HER2-positive breast cancer, invasive ductal carcinoma (IDC), invasive lobular carcinoma (ILC), mixed ductal and lobular carcinoma, inflammatory breast cancer or triple-negative breast cancer (TNBC).

7. The method of claim 5, wherein the TNBC has a mutated or absent phosphatase and tensin homolog (PTEN).

8. The method of claim 2, wherein the solid tumor is a primary solid tumor or a metastatic solid tumor.

9. The method of claim 2, wherein the solid tumor is not resected prior to the implanting.

10. The method of claim 9, wherein the biocompatible polymer is implanted in proximity to the solid tumor.

11. The method of claim 10, wherein the biocompatible polymer is implanted within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mm of the solid tumor.

12. The method of claim 9, wherein the biocompatible polymer is implanted within the solid tumor.

13. The method of claim 9, wherein the biocompatible polymer is implanted in the periphery of the solid tumor.

14. The method of claim 9, wherein the biocompatible polymer is implanted at a distance from the solid tumor.

15. The method of claim 2, wherein the solid tumor is resected prior to the implanting.

16. The method of claim 1, wherein the biocompatible polymer comprises a biodegradable polymer, a non-biodegradable polymer or a natural polymer.

17. The method of claim 16, wherein the biodegradable polymer comprises a poly(dioxanone) (PDO), polyethylene glycol (PEG), poly(glycolic acid) (PGA), poly(lactic acid) (PLA), poly(lactic-co-glycolytic acid) (PLGA), poly(caprolactone) (PCL), or combinations thereof.

18. The method of claim 16, wherein the non-biodegradable polymer comprises a poly(siloxane) (silicone) or poly(ethylene-vinyl acetate).

19. The method of claim 16, wherein the natural polymer comprises albumin, cellulose, chitosan, collagen elastin, gelatin, a milk protein or silk.

20. The method of claim 16, wherein the biocompatible polymer comprises a biodegradable polymer that remains in the subject for days, weeks, or months.

21. The method of claim 1, wherein the biocompatible polymer comprises a poly (urethane).

22. The method of claim 1, wherein the biocompatible polymer is hydrophilic.

23. The method of claim 1, wherein the biocompatible polymer comprises a hydrogel.

24. The method of claim 23, wherein the hydrogel comprises pores of 10-500 pm.

25. The method of claim 24, wherein spacing between pores in the hydrogel is from about 5 to about 100 nm.

26. The method of claim 23, wherein the hydrogel comprises a chemically-linked hydrogel or a physically-linked hydrogel.

27. The method of claim 23, wherein the biocompatible polymer comprises a PEG-based hydrogel.

28. The method of claim 1, wherein the biocompatible polymer comprises: a core comprising poly(dioxanone) (PDO), polylactide (PLA), polyglycollide (PGA), polycaprolactone (PCL), or copolymers thereof; and a sealant surrounding the core, comprising a polyethylene glycol (PEG)-based hydrogel.

29. The method of claim 28, wherein the core additionally comprises a titanium or stainless-steel marker.

30. The method of claim 1, wherein the biocompatible marker comprises a biopsy marker.

31. The method of claim 30, wherein the biopsy marker comprises a PEGylated substance, collagen, P-glucan or polylactic-co-glycolic acid.

32. The method of claim 31, wherein the biopsy marker comprises an aqueous-expandable polymer.

33. The method of claim 30, wherein the biopsy marker comprises a HydroMARK™ biopsy marker.

34. The method of claim 30, wherein the biopsy marker is selected from the group consisting of a MammoMARK®, MammoSTAR® and SecurMark® biopsy marker.

35. The method of claim 1, wherein the therapeutic agent comprises a small molecule, large molecule, cell or combinations thereof.

36. The method of claim 35, wherein the therapeutic agent comprises a small molecule.

37. The method of claim 36, wherein the small molecule comprises a STING agonist.

38. The method of claim 37, wherein the STING agonist comprises ADU-S100, MK-1454, MK- 2118, SB11285, GSK3745417, BMS-986301, BI-STING, E7766, TAK-676, SNX281, SYNB1891, or combinations thereof.

39. The method of claim 36, wherein the small molecule comprises an estrogen receptor- targeted therapy.

40. The method of claim 39, wherein the estrogen-receptor targeted therapy comprises a selective estrogen receptor modulator (SERM).41 . The method of claim 40, wherein the SERM comprises tamoxifen or raloxifene.

42. The method of claim 35, wherein the therapeutic agent comprises a large molecule.

43. The method of claim 42, wherein the large molecule comprises a cytokine, a chemokine or an interleukin.

44. The method of claim 43, wherein the interleukin comprises IL-2, IL-12, IL-15 or combinations thereof.

45. The method of claim 35, wherein the therapeutic agent comprises a cell.

46. The method of claim 45, wherein the cell comprises a CAR-T cell.

47. The method of claim 46, wherein the CAR-T cell is specific for a molecule on a cell of a solid tumor.

48. The method of claim 47, wherein the cell of the solid tumor comprises a breast cancer cell.

49. The method of claim 47, wherein the molecule comprises mesothelin, MUC-1, TROP2, HER2 / ERRB2, EGFR / ERRB1, HGFR / cMET, R0R1, AXL, MUC1, MSLN, CD70, CD 133, CD44v6, EpCAM, CSGP4, ICAM1, TEM8, TROP2, FRalpha, GD2, NGK2D ligand, CEA50. The method of claim 35 wherein a single biocompatible polymer plug comprises two or more of a small molecule, large molecule, or cell.

51. The method of claim 1, wherein the biocompatible polymer is approximately 17, 15, 13, 11, 9 or 7 mm long when hydrated.

52. The method of claim 1, wherein the implanting comprises surgical implanting or implanting using a large-bore needle.

53. The method of claim 1, wherein multiple biocompatible polymer plugs are implanted.

54. The method of claim 2, wherein the method is used in combination with a conventional therapy.

55. The method of claim 54, wherein conventional therapy comprises chemotherapy or endocrine therapy.

56. A method for treating a solid tumor in a human subject, comprising: a. hydrating a dehydrated biopsy marker with a therapeutic agent; and b. implanting the hydrated biopsy marker in proximity to the solid tumor in the subject or at a site distant from the solid tumor.

57. The method of claim 56, wherein the biopsy marker comprises a metal clip embedded in a biocompatible polymer.

58. The method of claim 56, wherein the biopsy marker comprises: a. a core comprising poly(dioxanone) (PDO), polylactide (PLA), polyglycollide (PGA), polycaprolactone (PCL), or copolymers thereof; and b. a sealant surrounding the core comprising a polyethylene glycol (PEG)-based hydrogel.

59. The method of claim 56, wherein the biopsy marker comprises a HydroMARK™ biopsy marker.

60. The method of claim 56, wherein the therapeutic agent comprises a small molecule, a large molecule, a cell, or combinations thereof.

61. The method of claim 60, wherein the small molecule comprises a STING agonist.

62. The method of claim 60, wherein the cell comprises a CAR-T cell.

63. The method of claim 56, additionally comprising injecting a substance into the implanted biopsy marker subsequent to the implanting.

64. The method of claim 63, wherein the injected substance comprises a cytokine, a chemokine or an interleukin.

65. The method of claim 63, wherein the injected substance comprises a cell.

66. The method of claim 65, wherein the cell comprises a CAR-T cell.

67. The method of claim 56, wherein the therapeutic agent comprises a STING agonist and, additionally comprising injecting a CAR-T cell into the implanted biopsy marker subsequent to the implanting.

68. A method for treating a breast cancer in a human patient, comprising: hydrating a dehydrated biopsy marker with a STING agonist and / or a CAR-T cell specific for a molecule on a surface of a cell from the breast cancer; wherein the dehydrated biopsy marker comprises a polyethylene glycol (PEG)- based hydrogel; and implanting the hydrated biopsy marker into the patient in proximity to the cells of the breast cancer.

69. The method of claim 68, wherein the dehydrated biopsy marker comprises a HydroMARK™ biopsy marker.

70. A method for loading a biopsy marker with a therapeutic agent, comprising hydrating the biopsy marker with an aqueous solution comprising the therapeutic agent to obtain a hydrated combination.71 . The method of claim 70, wherein the biopsy marker is hydrated with a volume of aqueous solution of less than about 20 pl.

72. The method of claim 70, additionally comprising implanting the hydrated combination into a human subject for sustained release of the therapeutic agent.

73. The method of claim 70, wherein the biopsy marker comprises a combination biodegradable polymer encompassed by a dehydrated hydrogel.

74. The method of claim 70, wherein the biopsy marker comprises a HydroMARK™ biopsy marker.

75. The method of claim 70, wherein the therapeutic agent comprises a small molecule, large molecule, cell or combinations thereof.

76. The method of claim 75, wherein the small molecule comprises a STING agonist.

77. The method of claim 75, wherein the cell comprises a CAR-T cell.

78. A method for treating a phosphatase and tensin homolog (PTEN)-null, triple negative breast cancer (TNBC), comprising: without resecting the breast cancer, implanting a hydrated HydroMARK™ biopsy marker having a STING agonist in proximity to the breast cancer; and injecting a CAR-T cell into the implanted biopsy marker subsequent to the implanting.

79. The method of claim 78, wherein the implanted HydroMARK™ biopsy marker additionally has a Rab7 inhibitor.

80. A HydroMARK™ biopsy marker comprising a STING agonist.

81. A HydroMARK™ biopsy marker comprising a CAR-T cell.

82. A composition, comprising: a biocompatible polymer that includes a metal marker; and a therapeutic agent absorbed into and / or adsorbed onto the biocompatible polymer.

83. The composition of claim 82, wherein the biocompatible polymer is hydrated.

84. The composition of claim 82, wherein the biocompatible polymer comprises a polymer core that includes the metal marker, surrounded by a polyethylene glycol (PEG)-based hydrogel.

85. The composition of claim 84, wherein the polymer core comprises a poly(dioxanone) (PDO), polyethylene glycol (PEG), poly(glycolic acid) (PGA), poly(lactic acid) (PLA), poly(lactic-co- glycolytic acid) (PLGA), poly(caprolactone) (PCL), or combinations thereof.

86. The composition of claim 82, wherein the metal marker comprises a titanium or stainless- steel marker.

87. The composition of claim 82, wherein the biocompatible polymer comprises a biopsy marker.

88. The composition of claim 87, wherein the biopsy marker comprises a HydroMARK™, MammoMARK®, MammoSTAR® or SecurMark® biopsy marker.

89. The composition of claim 87, wherein the biopsy marker comprises a combination biodegradable polymer encompassed by a hydrogel.

90. The composition of claim 89, wherein the biopsy marker comprises a HydroMARK™ biopsy marker.

91. The composition of claim 82, wherein the therapeutic agent comprises a small molecule.

92. The composition of claim 91, wherein the small molecule comprises a STING agonist.

93. The composition of claim 92, wherein the STING agonist comprises ADU-S100, MK-1454, MK-2118, SB11285, GSK3745417, BMS-986301, BI-STING, E7766, TAK-676, SNX281, SYNB1891, or combinations thereof.

94. The composition of claim 82, wherein the therapeutic agent comprises a cell.

95. The composition of claim 94, wherein the cell comprises a CAR-T cell.

96. The composition of claim 95, wherein the CAR-T cell is specific for a molecule on a cell of a solid tumor.

97. The composition of claim 96, wherein the cell of the solid tumor comprises a breast cancer cell.

98. A method for treating a cancer in a patient, comprising implanting into the patient the biocompatible polymer of claim 82.

99. The method of claim 98, wherein the implanting is distant from a tumor that is treated by the therapeutic agent.

100. The method of claim 98, wherein the therapeutic agent migrates from the biocompatible polymer into the bloodstream of the patient.

101. The method of claim 100, wherein the therapeutic agent extravasates from the bloodstream into surrounding tissue.

102. The method of claim 101, wherein the therapeutic agent treats a tumor in the surrounding tissue.

103. A method for treating a cancer in a patient, comprising: implanting into the patient a HydroMARK™ biopsy marker; and subsequent to the implanting, infusing a therapeutic agent into the biopsy marker.

104. The method of claim 103, wherein a first infusion is a STING agonist and a second infusion is a CAR-T cell.

105. The method of claim 104, wherein the second infusion occurs after the first infusion has preconditioned a tumor microenvironment.

106. A method for treating a cancer in a human subject, comprising:(a) performing one or more biopsies of the cancer;(b) determining the presence of one or more target molecules in the one or more biopsies;(c) developing a CAR-T+target cell capable of interacting with the one or more target molecules;(d) injecting the CAR-T+target cell into a HydroMARK1Mbiopsy marker, wherein the biopsy marker is implanted into the subject;(e) monitoring the subject for a response; and(f) if after step (e), the cancer is still present and / or has regrown, repeating steps (a)- (e).

107. The method of claim 106, wherein the target molecule comprises ROR-1, Trop-2, Muc-1, MSLN, or a combination thereof.

108. The method of claim 106, wherein the one or more biopsies are obtained from a tumor, a metastasis, a lymph node, or a combination thereof.

109. The method of claim 106, wherein developing the CAR-T+target comprises exposing the CAR-T+target cell to spheroids comprising a target molecule.

110. The method of claim 106, wherein the CAR-T+target cell recognizes any of target molecules ROR-1, Trop-2, Muc-1, or MSLN.

111. The method of claim 106, wherein prior to step (d), a tumor microenvironment into which the biopsy marker has been implanted has been preconditioned by a STING agonist.

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