Nanocarrier compositions and methods for enhanced car-t therapy

Abstract

Methods for enhancing CAR-T or CAR-M cell activity by modulating (e.g., activating or inhibiting) cytotoxic activity of the CAR cells over time to speed up or delay killing activity of the cells. Enhanced CAR-T or CAR-M cells prepared according to methods. Therapeutic compositions and treatment methods, such as for treating cancers, using the enhanced CAR-T or CAR-M cells. The enhanced CAR-T or CAR-M cells comprise drug-loaded nanoparticles or nanocarriers comprising a cell-penetrating peptide (WTAS or rp-182) or a fragment thereof and a modified poly(P-amino ester) (PBAE) polymer associated or assembled with one or more cell modulating agents. The cell modulating agents are released into the cytoplasm to thereby modulate the activity of the enhanced CAR-T cells or CAR-M cells.

Description

[0001] NANOCARRIER COMPOSITIONS AND METHODS FOR ENHANCED CAR-T THERAPY

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] The present application claims the priority benefit of U.S. Provisional Patent Application Serial No. 63 / 711,297, filed October 24, 2024, and U.S. Provisional Patent Application Serial No. 63 / 683,368, filed August 15, 2024, each entitled NANOCARRIER COMPOSITIONS AND METHODS FOR ENHANCED CAR-T THERAPY, and each of which is incorporated by reference in its entirety herein.

[0004] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under CA284065 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] SEQUENCE LISTING

[0007] The following application contains a sequence listing submitted electronically as a Standard ST.26 compliant XML file entitled "SequenceListing_60210.xml," created on April 8, 2025, as 13,251 bytes in size, the contents of which are incorporated herein.

[0008] BACKGROUND

[0009] Technical Field

[0010] The present disclosure relates to improved approaches for chimeric antigen receptor immunotherapies.

[0011] Description of Related Art

[0012] Chimeric antigen receptor (CAR) immunotherapy has become a revolutionary therapeutic strategy for various cancers. CAR-T cell therapy is a type of cancer immunotherapy that uses immune cells to attack cancer cells. CAR-T cells combine the cytolytic potency of a T cell with the tumor specificity of an antibody. Autologous therapy involves altering a patient’s own T cells to express a CAR specific for one or several of the patient’s tumor antigens, then infusing the modified cells into the patient. Researchers are also working towards developing allogeneic CAR- T cells from donor T cells. In either case, once administered to the patient, the CAR-Ts bind to antigens on the cancer cells allowing the T cells to kill them.

[0013] CAR-T cell therapy, although promising, has many challenges. For example, the optimal potency of CAR-T cell therapy for solid tumors, especially Glioblastoma Multiforme (GBM), has not been achieved. In general, antitumor activity of CAR-T cells against solid tumors, like GBM, remains insufficient. GMB is the most common malignant brain tumor in adults, and current treatment involves maximal safe resection, followed by adjuvant chemoradiation. Although this treatment is life prolonging, it is never curative. Five year survival is less than 7 % and improved therapies remain urgently needed.

[0014] Further, immunotherapies in general suffer from potential side effects implicated whenever a patient’s own immune system is being used in the treatment. For example, when the CAR-T cells encounter their antigen targets, they are rapidly activated. At this point, numerous inflammatory cytokines, including interleukin-6 (IL-6), tumor necrosis factor-alpha (TNFa) and interferon gamma (IFNy), are released. The large amounts of cytokines produced and then released by the activated immune system can cause a collection of mild to potentially life-threatening symptoms in the patient via this overactivation of the immune system. Similar problems are encountered when the immune system remains activated for too long and the normal resolution of the inflammatory response does not resume.

[0015] At the other end, some CAR-T cell therapies fail to result in sufficient antitumor activity due to repressive elements in the tumor microenvironment (TME). Recent clinical trial experiences of CAR-T cells in solid tumors have identified the immune-suppressive TME as a major barrier to clinical success. Further, certain CAR-T cells exhibit suppression in response to various types of cancers and fail to persist in the TME. One immune-suppressive endogenous negative regulator in GBM TME is TGF- P, which triggers CAR-T cell suppression and has been identified as barrier to CAR-T tumor eradication. It has been well documented that the TGF- signaling pathway promotes cancer progression by concomitantly enhancing tumor growth, drug resistance and metastases, while inhibiting the host immune response. Likewise, an elevated level of TGF-P in cancer patient sera correlates with drug resistance, tumor progression, and poor prognosis. A variety of approaches are being studied to overcome T cell dysfunction, and developing additional treatments that can improve CAR-T cell effectiveness, such as functionality, persistence, and infiltration into tumor sites.

[0016] In recent work, small molecule inhibitors of the TGFp receptor as well as TGFp resistant CAR-T cells have been developed and tested clinically. For example, some have proposed that knocking out the endogenous TGF- receptor II (TGFBR2) in CAR-T cells with CRISPR / Cas9 technology could reduce the induced Treg conversion and prevent the exhaustion of CAR-T cells. Again, however, as these approaches target the patient’s own immune system, they have found limited success due to systemic toxicity and other immune system side effects when the endogenous TGF- signaling pathway is impaired. For example, these approaches have shown systemic toxicity in clinical trials, including one case of a patient’s death in a prostate cancer trial involving a CAR-T encoding a dominant-negative TGF-P Receptor. Toxicity has also been reported with bintrafusp alpfa, a bifunctional fusion protein targeting TGF-P and PD-L1.

[0017] Accordingly, there remains a need for improved CAR-T cell therapies, particularly against difficult-to-treat solid tumors, such as GBM, as well as techniques which are able to more precisely enhance CAR-T cell therapies in a more targeted and time-limited manner to minimize patient side effects or long-term implications. There also remains a need to be able to efficiently deliver modulating drugs and other active agents into CAR-T cells to both enhance their activity, as well as to even downregulate T cell activity.

[0018] There also remains a need for improved immunotherapies targeting blood cancers and other cancers with less toxicity, as well as new approaches for modulating the TME. More recently, efforts have been directed as reprogramming macrophages and other monocyte cell types using CAR to yield CAR-M cells (i.e., CAR macrophages or CAR monocytes). However, these techniques can suffer similar drawbacks as CAR-T. The present disclosure is aimed and leveraging CAR and improving on these platforms to provide modified and improved CAR-based immunotherapies that can be tailored for a variety of different situations and cancers.

[0019] SUMMARY

[0020] In one aspect, described herein is a platform technology for enhancing CAR-T cells as well as CAR macrophages or CAR monocytes (“CAR-M”). In one aspect, the platform technology is used to load CAR-T cells with active agents via nanocarriers, thus enhancing the therapeutic window of CAR-T cells and addressing CAR-T exhaustion, suppression, and / or overactivity, which is one of the major obstacles to CAR-T cells in brain tumors. The platform can also be used to load CAR-M cells (CAR macrophages or CAR monocytes) with active agents to reprogram the cell and modify its phenotype into an anti-tumor cell. For ease of reference, the nanocarrier constructs are referred to herein sometimes as “drug-loaded nanoparticles,” even though the active agent that may be loaded into the nanocarrier is not a small-molecule drug, but may be a biologic or a nucleic acid, etc. Further, the term “nanoparticle” refers to the polymeric nanocarriers comprising up 250 repeat units having the active agent loaded thereon via cleavable linkages, alternatively having the polymer backbone decorated with dendrimers upon which the active agent can be loaded (via the cleavable linkages).

[0021] In one aspect, described herein are formulations of nanocarriers, which may be drug-loaded nanoparticles or nanocarriers for various active agents for enhancing CAR-T cell activity, such as through modulating CAR-T cell activity. In one aspect, described herein are formulations of active agent-nanocarrier conjugates for enhancing CAR-M cell activity, such as through modulating CAR-M cell activity. As used herein, references to “modulating” activity refer to increasing or decreasing the intensity of cell activity, and / or prolonging or shortening the duration of activity, and / or promoting or inhibiting one or more cell receptors and / or signaling molecules. Modulating may also refer to changing the phenotype of the cell.

[0022] In one aspect, the drug-loaded nanoparticles each comprise a cell -penetrating or targeting peptide (WTAS) or a fragment thereof and a modified poly(P-amino ester) (PBAE) polymer associated or assembled with one or more cell modulating agents (active agents). In one aspect, the drug-loaded nanoparticles each further comprise a detectable moiety such as a fluorescent dye or other tracer. Other cell targeting or penetrating peptides, such as CD206 targeting peptides (rp- 182) for macrophages or monocyte uptake are also described herein.

[0023] In one aspect, methods of modulating CAR-T cell activity are described. The methods comprise incubating the nanocarriers loaded with active agents with T cells for a sufficient period of time to allow the nanocarriers loaded with active agents to be taken up by the T cells ex vivo. The resulting transfected T cells comprise the nanocarriers loaded with active agents within the cytoplasm wherein the activity modulating agent is released to thereby modulate the activity of the transfected T cell.

[0024] In one aspect, methods of modulating CAR-M cell activity are described. The methods comprise incubating the nanocarriers loaded with active agents with monocyte cells for a sufficient period of time to allow the drug-loaded nanoparticles to be taken up by the monocyte cells ex vivo. The resulting transfected monocyte cells comprise the drug-loaded nanoparticles within the cytoplasm wherein the activity modulating agent is released to thereby modulate the activity of the transfected monocyte cell.

[0025] In one aspect, the active agent or activity modulating agent is a small molecule drug. In one aspect, the activity modulating agent is a biologic drug. In one aspect, the activity modulating agent is a kinase-blocker. In one aspect, the activity modulating agent is a TGFp receptor inhibitor. In one aspect, the activity modulating agent is a Protein Kinase C agonist class family such as Ingenol class: Ingenol, Ingenol-3-Angelate (Ingenol-mebutate), Ingenol-3 -hexanoate, GSK445A, Bryostatin-1; Ingenol diterpenes: gnidimacrin, SJ23B; Di acylglycerol (DAG)-like Agonists: DAG lactone compounds: LMC03, LMC07; Phorbol ester compounds: PMA, Prostratin, and DPP). In one aspect, the activity modulating agent is a PI3K / mT0R class inhibitor such as paxalisib, Alpelisib (BYL719), Copanlisib (BAY 80-6946), Duvelisib (IPI-145), Idelalisib (GS-1101), Buparlisib, Umbralisib, Leniolisib. In one aspect, the activity modulating agent is a cell supplement / nutrient or energy resource, such as amino acid(s), fatty acid(s), and / or carboxylic acid(s).

[0026] In one aspect, treatment methods with enhanced CAR-T or CAR-M cells are described herein. The methods generally comprising administering to a subject in need thereof an effective amount of enhanced CAR-T or CAR-M cells prepared according to the embodiments described herein. In one aspect, the CAR-T or CAR-M cells may be autologous cells derived from the subject and enhanced ex vivo using methods of the invention. In one aspect, the CAR-T or CAR-M cells may be allogeneic cells from a donor or cell line, and enhanced ex vivo / iii vitro using methods of the invention. As demonstrated in the working examples, the enhanced CAR-T or CAR-M cells are particularly effective as against heterogeneous tumors, which are more representative of in vivo cancers and tumor burdens.

[0027] In one aspect, methods of modulating CAR-T or CAR-M cells are described. The methods comprise administering to a subject the inventive nanocarriers as described herein. The nanocarriers are loaded with the activity modulating agent for the T or M cells, as well as nucleic acid that encodes the CAR target for the cells (e.g., CAR encoding mRNA or CAR encoding plasmid DNA). The nanocarriers are taken up into the cells efficiently delivering CAR mRNA for producing transient CAR-T or CAR-M cells in vivo bypassing the need for ex vivo cell manipulation. The nanocarriers can advantageously also deliver the activity modulating agent to the cells, further enhancing their effect as outlined herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] 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.

[0029] FIG. 1 shows a structure of a PBAE nanocarrier having vactosertib linked via a urethane linkage, and showing the conjugation to the cell penetrating WTAS peptide.

[0030] FIG. 2 shows a TEM image of PBAE nanocarrier and the zeta potential (surface charge) of the nanocarrier.

[0031] FIG. 3 shows data demonstrating that the WTAS peptide bound to the PBAE enhances uptake by the T cells as compared to PBAE alone as measured by rhodamine B labeled PBAE and flow cytometry.

[0032] FIG. 4 show (A) Immunofluorescent and Hoechst Nuclear Counter Stain of T cells images after 24-hour incubation with WTAS-PBAE; (B) T cells loaded at 0-1 mg / mL WTAS-PBAE-NC have uptake at 98% and higher after 24-hour incubation with WTAS-PBAE; and (C) T cell viability of 97% or higher after 24-hour incubation with WTAS-PBAE.

[0033] FIG. 5 is a graph showing T cell proliferation and viability after being transduced with vacto-NC-10 conjugate as compared to untreated cells, but decrease in proliferation at 72 hours. NC is nanocarrier only. NC+Vac is mixed and unconjugated. Vactosertib (Vac) alone lum, mixed NC+Vac, or conjugated Vacto-NC-10. T cells were plated a 3 million cells per mb and counted / viability at 72 hours on Muse Guava Analyzer.

[0034] FIG. 6 is an immunoblot showing that WTAS-PBAE (NC) conjugated Vactosertib (Vac- NC-10) conjugated at 10% w / v Vactosertib and incubated with human T cells at 24 hrs, 7 days, 21 days. Vac-NC-10 demonstrates persistent inhibition of phosphor-SMAD at 21 days, after 30 min. TGF-beta activation. Vac alone or unconjugated loses the inhibition ability by day 7.

[0035] FIG. 7 shows a PBAE nanocarrier conjugated with Vactosertib via an amide linkage.

[0036] FIG. 8A shows that CAR-T cell activation markers are not impeded by nanocarrier or conjugated nanocarrier. Of note, the treated CAR-T cells showed higher activation of CD69 as compared to untreated CAR-T cells.

[0037] FIG. 8B shows that the T cells transduced with PBAE nanocarrier conjugated with Vactosertib does not impede the CAR T kill activity. T cells (mock or CAR) were co-plated with U87 tumor cells at a 2: 1 E:T ratio. After 72 hours coincubation, cells were collected using trypsin and stained for flow cytometry. One way ANOVA analysis was done for statistical comparison.

[0038] FIG. 9 shows that T cell exhaustion is mitigated after transduction with PBAE nanocarrier conjugated with Vactosertib as compared to untreated cells or cells transduced only with nanocarrier.

[0039] FIG. 10A shows that EGFR CAR T cell tumor killing was enhanced with addition of the vactosertib-conjugated nanocarriers. The graph of EGFR CAR T cells plated with T98-luciferase labeled cells (GBM model) at 1:2.5 E:T ratio and evaluated for luminescent activity at Day 2 followed by 3 additional tumor rechallenges and interval luminescent detection at Day 5, 7, and 9. Statistically significant tumor killing by the enhanced CAR T cells was seen.

[0040] FIG. 10B shows that EGFR CAR T cell tumor killing was enhanced with addition of the vactosertib-conjugated nanocarriers. The graph of EGFR CAR T cells plated with T98-luciferase labeled cells at 1 : 10 E:T ratio and evaluated for luminescent activity at Day 2 followed by 3 additional tumor rechallenges and interval luminescent detection at Day 5, 7, and 9. Statistically significant tumor killing by the enhanced CAR T cells was seen.

[0041] FIG. 10C shows parallel experiments plating at E:T ratio 3: 1 for cytokine bead-based ELISA for Granzyme A. Statistically significant difference in T cell cytokine secretion by the enhanced CAR T cells was seen.

[0042] FIG. 10D shows parallel experiments plating at E:T ratio 3: 1 for cytokine bead-based ELISA for IFN-gamma. Statistically significant difference in T cell cytokine secretion by the enhanced CAR T cells was seen.

[0043] FIG. 11A shows the results for CD19 CAR T cells loaded with paxalisib-conjugated nanocarriers demonstrated a significant reduction in cytokine release without reduction in lymphoma killing. CD19 CAR T cells or mock control were loaded with empty nanocarriers (NC) or paxalisib-conjugated nanocarrier (CAR+Pax Conj) and incubated with luciferase labeled Raji lymphoma cells. Luciferase detection was assessed at indicated days and interval Raji cells were added in tumor rechallenge format in the interval days.

[0044] FIG. 11B shows the results for CD19 CAR T cells loaded with paxalisib-conjugated nanocarriers demonstrated a significant reduction in IL-2 release without reduction in lymphoma killing (FIG. 11A).

[0045] FIG. 11C shows the results for CD19 CAR T cells loaded with paxalisib-conjugated nanocarriers demonstrated a significant reduction in IFN-gamma release without reduction in lymphoma killing (FIG. 1 1 A).

[0046] FIG. 11D shows the results for CD19 CAR T cells loaded with paxalisib-conjugated nanocarriers demonstrated a significant reduction in TNF-alpha release without reduction in lymphoma killing (FIG. 11A).

[0047] FIG. 12 shows the results of CD19 CAR T cells loaded with paxalisib-conjugated nanocarriers against luciferase labeled Raji lymphoma cells, with Pax loaded at 0.25 mg / ml as compared to mock controls and free drug. CD 19 CAR T Cells loaded with NC-Pax show delayed killing of tumor cells and able to eliminate tumor by 21 days.

[0048] FIG. 13 shows parallel results as FIG. 12, but with the Pax loaded at 0.35 mg / ml.

[0049] FIG. 14A show immunoblot images at day 5 showing that paxalisib-conjugated nanocarriers preferentially disrupts PI3K-mTOR downstream signaling phosphor-S6. Western blot was run for pS6 (Ser235 / 236) with Actinin as loading control.

[0050] FIG. 14B shows full data from the S6 study at Day 1 for Pax loaded at 0.25 mgl / ml and 0.35 mg / ml. Loading of Pax-NC at 0.35mg / mL on T Cells inhibits S6 phosphorylation on Day?.

[0051] FIG. 14C shows full data from the S6 study at Day 7 for Pax loaded at 0.25 mg / ml and 0.35 mg / ml.

[0052] FIG. 15A shows that the CD3 T Cells treated with Pax-NC exhibit higher Central Memory Phenotype (CCR7+ CD45RO+) 11 days after treatment as compared to DMSO treated T Cells.

[0053] FIG. 15B shows the raw data for FIG. 15B.

[0054] FIG. 16A shows 13 A enhances antigen independent killing (Green tumor cells) by EGFR CAR T Cells. 2500 EGFR positive (RFP-red) cells were plated in a well with untransduced (Mock) or EGFR CAR T Cells with or without 150nM. 72 hours post co-culture, fluorescent images were taken and number of live RFP and GFP cells quantified and plotted. Data is represented as Mean ± S.D. and statistical analysis done using one-way ANOVA.

[0055] FIG. 16B 13 A enhances antigen independent killing (Green tumor cells) by EGFR CAR T Cells. 2500 EGFR Knock Out(CFSE -green) Tumor (MDA-MB-231) cells were plated in a well with untransduced (Mock) or EGFR CAR T Cells with or without 150nM. 72 hours post coculture, fluorescent images were taken and number of live RFP and GFP cells quantified and plotted.

[0056] Data is represented as Mean ± S.D. and statistical analysis done using one-way ANOVA.

[0057] FIG. 17 shows images of the cells from FIG. 16A and 16B. FIG. 18A shows IL-6 results for Day 2 and Day 9 showing that CAR T cells loaded with conjugated NC-I3A (NC-I3A-Conj) enhances the secretion of IL -2 over 9 days of co culture as compared to free I3A or unconjugated I3A (+NC+I3A). Data is represented as Mean ± S.D. and statistical analysis done using two-way ANOVA.

[0058] FIG. 18B shows TNF-alpha results for Day 2 and Day 9 showing that CAR T cells loaded with conjugated NC-I3A (NC-I3A-Conj) enhances the secretion of TNF-alpha over 9 days of co culture as compared to free I3A or unconjugated I3A (+NC+I3A).

[0059] FIG. 18C shows Granzyme B results for Day 2 and Day 9 showing that CAR T cells loaded with conjugated NC-I3 A (NC-I3A-Conj) enhances the secretion of Granzyme B over 9 days of co culture as compared to free I3A or unconjugated I3A (+NC+I3A).

[0060] FIG. 18D shows sFasL results for Day 2 and Day 9 showing that CAR T cells loaded with conjugated NC-I3A (NC-I3A-Conj) enhances the secretion of sFasL over 9 days of co culture as compared to free I3A or unconjugated I3A (+NC+I3A).

[0061] FIG. 19 shows that CAR T cells can be loaded with a combination of agonist and inhibitor drugs using two different release kinetics to modulate CAR T activity as desired over time. CAR T cells were loaded with slow-release agonist (NC-PCK0Ag(amide)) and fast-release inhibitor Paxalisib (NC-PI3KInhibitor(-COOH)), which demonstrated enhanced and delayed cytokine activity as compared to the CAR T cells treated with slow-release NC-PCK0Ag(amide) alone. MDA-MD231 cells were co-cultured 50:50 EGFR KO and mixed at 3: 1 E:T ratio with EGFR- IgG4EQ-41 lB-CD3z CAR T cells. Tumor challenge was done at Day 3, 6, and 10, and cytokines collected at the given days for media cytokine analysis. Two-Way ANOVA.

[0062] FIG. 20 shows that Intracranial injection of PCKOAg(amide) or NC-PCK0Ag does not demonstrate toxicity in immunocompetent tumor bearing mice.

[0063] FIG. 21 shows survival curve for mice bearing homogenous tumors treated with EGFR CAR as compared to no treatment.

[0064] FIG. 22 shows survival curves for treatment of mice having heterogenous tumors and showing that EGFR CAR T cells or CD19CAR T cells had no survival benefit in the heterogenous model, while the mice treated with the NC-PCK0Ag (13 A) enhanced EGFR CAR T cells had an increased survival benefit.

[0065] FIG. 23A shows imaging of tumor burden in mice treated in the working examples with NC-PCK0Ag(amide) or NC-PCK0Ag(-COOH). The PKCOAgAMIDE CD19 CAR-T cells have statistically significant reduced tumor burden at Day 42 compared to CAR T only group. 1 e6 Raji- Luc cells (50% CD19 KO-Luc) were injected IP. After confirmation of BLI engraftment, Tumor only and CAR T (41bb-CD3z costimulation) groups were also injected IP and tumor burden monitored by weekly BLI imaging. BLI images demonstrates tumor clearance in 8 of 8 loaded CAR T cells at day 42.

[0066] FIG. 23B show the results for the PKCQAgAMlDF CD 19 CAR-T cell treatment in the heterogenous model.

[0067] FIG. 23C shows the mouse weights in the treatment groups over the course of the ongoing study. Mouse weights demonstrates loss of weight only in the Mock T cell treated mice.

[0068] FIG. 24A shows I3A loaded NC is efficiently taken up by human PBMC derived monocytes and macrophages, in particular, successful dose-dependent uptake of nanocarrier(NC)- I3A by MO, Ml, and M2 macrophages, resulting in potent repolarization of M2 into Ml-type macrophages.

[0069] FIG. 24B shows downregulation of M2 markers following NC-I3A treatment of human PBMC derived monocytes and macrophages resulting in potent repolarization of M2 into Ml- type macrophages.

[0070] FIG. 25 A shows CD206-targeted 13 A nanocarrier treatment decreases phenotypic M2 marker CD 163.

[0071] FIG. 25B shows that CD206-I3 A NC treatment stimulates secretion of anti-tumor soluble factors and cytokines, TNF-alpha.

[0072] FIG. 25C shows that CD206-I3A NC treatment stimulates secretion of anti-tumor soluble factors and cytokines, Granzyme B.

[0073] FIG. 25D shows that CD206-I3 A NC treatment stimulates secretion of anti-tumor soluble factors and cytokines, IL- 17a.

[0074] FIG. 26 shows that viral transduction produces EGFR CAR macrophage with high CAR positivity and that combinatorial EGFR CAR-M and I3A robustly kills GBM cells in vitro.

[0075] FIG. 27 shows that combinatorial EGFR CAR-M and I3A robustly kills GBM cells in vitro.

[0076] FIG. 28 shows a reaction scheme for improved nanotargeting chimeras, i.e., nanocarriers with the PBAE backbone and targeting moieties (WTAS, etc.), with PPI dendrimers and attached drug (I3A shown for example).

[0077] FIG. 29 is an exemplary reaction scheme using I3A to show how different chemistry approaches can be used to synthesize different linkages for tethering the drug to the nanocarrier for faster or slower release kinetics of the active agent to be delivered to the CAR cells.

[0078] FIG. 30 is an example showing a PBAE backbone with a CD206 targeting moiety (for targeting CAR M cells) as opposed to WTAS, with I3A being attached via an esterase-cleavable linker.

[0079] DETAILED DESCRIPTION

[0080] Herein we propose a platform technology to effectively introduce active agents into T cells and M cells via novel nanocarriers loaded with the active agents. The technology can be used to effectively introduce a wide variety of active agents into T cells and M cells, and in particular CAR-T or CAR-M cells, thus enhancing the therapeutic window of CAR immunotherapies and addressing issues like off-target effects, CAR cell fatigue, and the like. The inventive nanocarriers can be used to load various active agents having different effects on the modified cells to modulate the activity of the modified cell. For example, active agents that have an inhibitory effect on the cells can be used to suppress certain T or M cell activities. Alternatively, agonists can be used to activate the cells. Advantageously, the nanocarrier constructs are designed so that the release and elution of the different active agents can be tailored for slow or fast release of the active agent, depending on the desiring timing, duration, and effect of the agent on the modified cell. It will be appreciated, for example, that inhibitors that inhibit release of cytotoxic cytokines from T cells can be introduced into CAR-T cells via a nanocarrier construct that uses a fast cleaved tether, such as a carboxylic acid, such that the modified CAR-T cells will be suppressed in their killing activity until they reach their destination. This would be advantageous for systemically delivered CAR-T cells and reduce off-target or systemic toxicity effects. Over time, as the inhibitor drug is eluted, its effects on the CAR-T cells wears off and they are able to resume their normal killing function.

[0081] At the same time, the CAR-T cells can be simultaneously loaded with activator compound that increase or enhances the killing or activity of the T cells. These can be tethered to the nanocarrier via slower release linkages, such as amide bonds. In this away, the active agent remains tethered for a period of time until the modified CAR-T cells reach their target, and the active agent begins to be released thereby activating the CAR-T cells in the vicinity of their target. Similarly, active agents can be delivered to CAR-M cells to reprogram or repolarize M cells from an antiinflammatory to a more pro-inflammatory (anti -turn or) phenotype. Thus, it will be appreciated that active agents can be selected to preferentially turn the CAR cells “on” or “off’ depending on the therapeutic modality, the mode or location of administration, and other therapeutic considerations (as the clinician may deem appropriate).

[0082] In one or more embodiments, the technology demonstrates the ability to transiently disrupt the TGF-pi pathway to overcome immunosuppression in the TME while minimizing broader or long-term immune side effects with reduced toxicity and increased efficacy (FIG. 10). In another formulation, the technology demonstrates that modified CAR-T cells can be “throttled-down” to reduce toxic side-effects, such as Cytokine-Release-Syndrome (CRS). In this formulation, the CAR-T-cells are loaded, for example, with a small-molecule PI3-Kinase-mTOR inhibitor (paxalisib), which can potently reduce the production and release of CRS related cytokines, thus improving the tolerance and safety -profile of CAR-T cells. Importantly, the paxalisib-loaded CAR-T cells exemplified in the working examples have a reduction in cytokine release without a reduction in tumor killing (FIG. 11).

[0083] In some embodiments, the currently described nanoplatform technology proposes to utilize CAR-T cells as a small-molecule drug-vehicle, dvCAR-T cells. The nanocarrier (NC) comprises a cell-penetrating peptide called WTAS or a fragment thereof as a primary nanocarrier and a modified poly(P-amino ester) (PBAE) polymer as a secondary nanocarrier. The nanocarrier can self-assemble with one or more active agents for delivery into CAR-T cells, for example, via covalent bonds or through non-covalent interactions such as ionic bonding, hydrogen bonding, as well as Van der Waals, electrostatic attraction, physical entanglement, and the like.

[0084] WTAS & WTAS-fragments The preliminary data demonstrates that WTAS-PBAE significantly enhances PBAE penetration into T cells compared to PBAE alone (FIG. 3). The WTAS peptide or fragment facilitates transport of the nanocarrier across the cell membrane. The PBAE polymers can further be functionalized with a wide variety of targeting sequences (“targeting chimeras” or “TC”) beyond WTAS exemplified here. For example:

[0085] Drug

[0086] In more detail, the targeting sequence can be a signaling sequence. Signaling sequences can be “eat me” sequences, “don’t eat me” sequences, targeting sequences, detectable labels, and / or combinations thereof. The signaling sequence can, for example, be a signaling sequence for CD44 comprising sHPWSYLWTQQAs (SEQ ID NO: 1); a plectin-targeting sequence comprising KTLLPTPG (SEQ ID NO:2); a signaling sequence for EGFR comprising sYHWYGYTPENVIGsG (SEQ ID NO:3); a signaling sequence for LDLR comprising sCMPRLRGCGAGsG (SEQ ID NO:4); a signaling sequence for CD46 comprising sLPGTICKRTMLDGLNDYCTGsG (SEQ ID NO:5); a signaling sequence for SIRPa “don’t eat me” comprising kGNYTCEVTELSREGKTVIELKsG (SEQ ID NO:6); a signaling sequence for SIRPa “don’t eat me” comprising kGNYTCEVTELSREGKTVIELKk (SEQ ID NO:7); a signaling sequence for SIRPa “eat me” comprising 4N1 : RFYWMWK (SEQ ID NO:8) and 7N3 FIRVVMYEGKK (SEQ ID NO:9); a signaling sequence for CD206 “shut down M2 polarization” (rp-182) comprising KFRKAFKRFFGsG (SEQ ID NO: 10); a signaling sequence for Mucosal Membrane: WTAS comprising PLKWPGKKKKGKPGKRKEQEKKKRRTRG (SEQ ID NO: 11).

[0087] They can also be modified with detectable moieties such as fluorescent dyes and other tracers. This can be used to confirm loading of the nanocarriers onto the CAR-T cells. In particular, as seen in imaging in the data, the PBAE-WTAS nanocarrier self-assembled with a plasmid DNA engineered to express Green Fluorescent Protein (GFP), which is then observable. The PB AE polymer, a nontoxic and biodegradable polymer, is used to improve the stability of WTAS peptide while facilitating the transportation into cells. In particular, as a polymer / dendrimer-based nanocarrier, the PBAE polymer comprises monomers facilitating not only assembly of the polymer into a supramolecular assembly but also ultimate breaking apart of the structure therefore improving transportation of the active agents into the cells.

[0088] The PBAE polymer described herein can include an esterase or protease cleavable linker so that the conjugated drug can be released into the cytoplasm once taken up by the cells. The nanocarrier can be used to conjugate to any drug or other active agent with an amine, hydroxyl, or carboxylic acid group. The PBAE polymer is particularly well-suited for delivering nucleic acids. In particular, DNA and RNA form complexes with the PBAE polymer with a high binding constant

[0089] (KB>100,000 M1). Even more surprising, the PBAE polymer exhibits a very high binding constant (KB>200,000 M’1) to dsDNA such that the dsDNA when complexed with the PBAE polymer is protected from enzymatic degradation. For example, the nanocarrier can be used to introduce mRNA encoding for the CAR to carry out in vivo engineering of the CAR-T or CAR-M cells, in addition to loading the activity modulating agent into the CAR cells.

[0090] An exemplary platform is illustrated below.

[0091] The WTAS-PBAE nanocarrier will self-assemble with the drug or active agent and / or plasmid carrying genetic material that is wished to be introduced into the cell. WTAS and its fragments are peptides that have been demonstrated to penetrate cell nuclei within a few minutes after exposure, which makes it an ideal candidate to transport genetic materials into the nucleus of cells. The PBAE polymers can be synthesized to have primary, secondary, or tertiary amines and cleavable ester bonds to facilitate carrying a wide variety of active agents. For example, the PBAE polymers can be used as nanocarriers for therapeutically active agents such as DCLK1 (Doublecortin Like Kinase 1) inhibitors, including those having a structure comprising Formula 1 or 1A: or wherein A is a 5 or 6 membered heteroaryl group, such as

[0092] R1 and R2 are independently hydrogen, alkyl, alkaryl, or R1 and R2 together with the carbons they are attached to form a fused 5- or 6-membered ring; X is -C(O)-, -NHC(O)-, -C(0)NH-, or - NHC(0)NH-; and J 1 and J2 are independently CH or N.

[0093] Examples include

[0094]

[0095] In one or more embodiments, the PBAE polymers can be used as nanocarriers for therapeutically active agents such as MK2 inhibitors, such as PF-3644022, MK2-IN-1, or MK2-IN-4:

[0096] Other active agents include targeted cytotoxic compounds, such as lethality inducers against p53 deficient cells, such as analogs of KU-D2F:

[0097] KU-D2F* differs from the parent KU-D2F by the presence of a phenolic group instead of a phenyl-methyl-ether.

[0098] Other active agents include MIF inhibitors:

[0099] 4-yl)-N-(4-methylbenzo[d]thiazol-2-

[0100] N-(benzo[c][l,2,5]thiadiazol-4-yl)-2-(2-((5- yl)acetamide; KUO 180931; F5950-0872 chloropyridin-2-yl)amino)thiazol-4- yl)acetamide; KU0180903; F5950-0730

[0101]

[0102] N-(benzo[d]thiazol-2-yl)-4-(indolin-l- (5-(2,3-dihydrobenzo[b][l,4]dioxin-6- ylsulfonyl)benzamide; KU0168339; F0725- yl)isoxazol-3-yl)methyl 3-(furan-2-yl)-lH-

[0103] 0226 pyrazole-5-carboxylate; KU0171533; F2496- 3431

[0104] (isopropylthio)phenyl)-l,3,4-oxadiazol-2- yl)i soxazole-3 -carboxamide; KUO 178573; 2-(3-(3 -benzyl- 1 ,2,4-oxadiazol-5-yl)azetidin- 1 - F5773-3144 yl)-l-(p-tolyl)ethan-l-one; KU0181099; F6036- 1941

[0105] 2-(benzo[d]thiazol-2-yl(methyl)amino)-N-(4-(furan-2-yl)thiazol-2-yl)acetarnide; KUO 182093;

[0106] F6225-0003

[0107] Other active agents include PI3K Inhibitors, such as paxalisib (which is a dual PI3K / mTOR inhibitor), idelalisib, copanlisib, duvelisib, umbralisib, and buparlisib. Paxalisib is a particularly preferred compound due to is ongoing clinical trials for GBM.

[0108]

[0109] Paxalisib: Dual PI3K / mT0R inhibitor IC50 ranging from 0.3 to 1.1 pM. Brain penetrant

[0110] Maximum tolerated dose in humans is 60mg (Img / kg)

[0111] For example, the present data (FIG. 10 and FIG. 11) exemplifies deposit of a kinase-blocker (TGFbRII or PI3K-mT0R specific) within the CAR-T cells. With regards to TGFbRII blockade, this platform addresses a suitable strategy against CAR-T fatigue by blocking the phosphorylation of SMAD. If SMAD is undergoing TGF-b triggered phosphorylation in tumor-infiltrated CAR-T cells, rapid CAR-T cells exhaustion is observed. We were able to extend the activity phase of CAR-Ts in solid tumor tissue from 3-4 days in CAR-T cells without enhancement to 21 days when the CAR-T cells are enhanced with the nanocarriers loaded with TGFbRII kinase blocker. The enhanced CAR-T cells containing the kinase blocker vactosertib are resistant to TGFpRII mediated T cell suppression, making them less susceptible to suppression by TGF-P secreted in the tumor microenvironment. The present data also exemplifies deposit of a PI3K- mTOR inhibitor within the CAR-T cells, specifically a PI3K class of inhibitors that has been shown to enhance T cell memory phenotypes important for long term immune response, whilst also reducing the production and release of cytokines from CAR-T cells — potentially significantly improving the toxicity profile of CAR-T cells.

[0112] Advantageously, it has been demonstrated that the nanocarrier successfully transfected brain tumor cells with high specificity, which shows the potential this polymer could have to cross the blood brain barrier. Beyond, GBM, diffuse intrinsic pontine glioma (DIPG) and other H3K27M-mutated diffuse midline gliomas (DMGs) are universally lethal pediatric tumors of the central nervous system. Previous work has shown that disial oganglioside GD2 is highly expressed on H3K27M-mutated glioma cells and have demonstrated promising preclinical efficacy of GD2- directed chimeric antigen receptor (CAR) T cells, providing the rationale for a first-in-human phase I clinical trial (NCT04196413). Because CAR-T cell-induced brainstem inflammation can result in obstructive hydrocephalus, increased intracranial pressure and dangerous tissue shifts, neurocritical care precautions were incorporated. The present work is based on clinical experience from the first four patients with H3K27M-mutated DIPG or spinal cord DMG treated with GD2- CAR-T cells at dose level 1 (l x 106GD2-CAR-T cells per kg administered intravenously). Patients who exhibited clinical benefit were eligible for subsequent GD2-CAR-T cell infusions administered intracerebroventricularly. Toxicity was largely related to the location of the tumor and was reversible with intensive supportive care. On-target, off-tumor toxicity was not observed. Three of four patients exhibited clinical and radiographic improvement. Pro-inflammatory cytokine levels were increased in the plasma and cerebrospinal fluid. Transcriptomic analyses of 65,598 single cells from CAR-T cell products and cerebrospinal fluid elucidate heterogeneity in response between participants and administration routes. These early results underscore the promise of this therapeutic approach using CAR-T cells for patients with H3K27M-mutated DIPG or spinal cord DMG.

[0113] However, lack of CAR-T persistence and the immune-suppressive tumor microenvironment remains major hurdles to cure solid tumors in the central nervous system.

[0114] It will be appreciated that the present nanocarriers are advantageous in that they provide alternatives to using viral vectors for gene therapy. These compositions can act as gene delivery nanocarriers. In such embodiments, the PBAE backbone can be engineered with an additional esterase-cleavable group for better release of DNA, mRNA, miRNA, and / or siRNA. This addresses issues when DNA or RNA are too slowly released from the nanocarrier after it has been taken up by endocytosis or micropinocytosis, followed by rapid escape from early endosomes into the cytoplasm. In original versions of the constructs, this led to lower-than-desirable transfection rates with various cell types (cancer and non-cancer) and nucleic acids (plasmid DNA, mRNA, or miRNA) combinations. The addition of an additional esterase-cleavage site in the backbone leads to faster enzymatic carrier breakdown, followed by a faster release of the nucleic acids.

[0115] Further, we have changed the structure of the attached small dendrimer that can be used to bind to nucleic acids. In other PBAEs, small dendrimers with -(CH2)2-bridges between the tertiary and primary amine groups were used. We advanced to using three methylene bridges (i.e., - (CH2)3-bridges) now which was inspired by micellar transfection systems (e.g. lipofectamine).

[0116] Dendrimers with 3 methylene bridges

[0117] We found that the -(CH2)3-bridges are an excellent match for the distance of the phosphate groups in the nucleic acid sugar-phosphate backbone. This allows a better neutralization of the negative charges during membrane crossing, thus leading to significantly higher transfection rates. Whereas micellar systems cannot be targeted towards individual solid tumors of CAR-T cells, our novel PBAE systems can. It should be noted that small and larger dendrimers with -(CH2)3-brid es are not commercially available. This enables us to create new PBAE-derived nanopolymers with not (yet) commercially available units (only generation 1 propylenimine dendrimer is commercially available). Because the addition reactions of C3 -amine groups are modular, larger generations of propylenimine starburst dendrimers (G2, G3, G4 etc.) can be added as well to further improve transfection. In one or more embodiments, the nanocarriers incorporate dendrimer-like branched or hyperbranched polymeric structures of 800 Da or less in size or 10 nm or less in largest dimension (e g., diameter). In one or more embodiments, the dendrimers can comprise three or more maleimide groups, three or more carboxylate groups, three or more amide groups, or three or more hydroxyl groups as illustrated by Formula A or B:

[0118] where n is 2 to 5, preferably 2 to 3, preferably 3; and Z is a maleimide, -NH2, -COOH, or -OH. In some cases, Z itself represents a nitrogen with further branching. In the above structure, additional backbone elements include di / tri-saccharides with alpha- glycosidic bonds can be used for activation in fast metabolizing cells. Similarly, di / tri-saccharides with beta-glycosidic bonds can be used for activation in hypoxic zones by bacteria. Targeting peptides can be used. Peptides that can be metabolized (e.g., glutamate). Fatty acids can also be included.

[0119] The dendrimer can be linked to the backbone as shown above to the terminal ends of the polymer, with the drug being bound to the polymer backbone via the -OH group at the polymer side chain or to the terminal double bonds of PBAE via Michael addition. In other embodiments, the dendrimer can instead be bonded to the -OH group at the polymer side chain (and in fact a plurality of dendrimers can be attached along the polymer backbone and used to decorate the backbone), with the drug then being attached to the -NH2 of the dendrimer.

[0120] In this manner, multiple active agents can be attached to the nanocarrier construct, with multiple active agents being bound to the multiple -NH2 sites of each dendrimer, and multiple dendrimers decorating the polymer carrier. Exemplary dendrimers that can be linked to the PBAE backbone and used for conjugating to the active agents include:

[0121] Hyperbranched dendrimers with repeating units are also envisioned including:

[0122] where is n and Z are defined as above and m is 1-5. Examples of such hyperbranched polymeric cores include, without limitation:

[0123]

[0124]

[0125] As illustrated above, a variety of branched polymeric cores can be envisioned, having 3 or more terminal amine groups. In such embodiments, the active agent (e.g., the terminal -COOH group of a therapeutic peptide sequence) can be linked to the terminal -NH2 to form a very stable amide bond at each arm of the core. The skilled person will recognize that the -NH2 groups will be converted into amides with attached peptide chains. Again, the branched dendrimers can be synthesized with arms terminating in other suitable functional groups, including COOH as also illustrated, or maleimide groups, amide groups, or hydroxyl groups, for covalent bonding to the active agents. Other envisioned modifications to the PBAE backbone include carboxylamides allowing permitting very slow release, as well as acetals, which allow release in response to cancer metabolic properties. For example, lactose or maltose units can be incorporated into the PBAE backbone design, which can be readily cleaved by alpha-glucose converting glycosidases that are present in glucose metabolizing cells, such as cancer cells. Similarly, cellobiose could be incorporated as it has a beta-glycosidic bond that can be metabolized by microbes, which usually associate with hypoxic areas of tumors. Moreover, the WTAS peptide could be modified such as by cyclic peptides or a retro-inverso peptides.

[0126] Advantageously, the PBAE backbone is amenable to a variety of modifications. As explained herein and demonstrated in the working examples, the release kinetics of the selected active agent can be adjusted by selecting a different chemistry for the tether. Thus, linkages for the active agent can be selected from urethanes, ureas, thioureas, amides, amines, ethers, thioesters, and the like. Active agents can also be associated with the polymer nanocarrier via non-covalent bonding such as electrostatic interactions, hydrogen bonds or hydrophobic interactions, and the like.

[0127] As shown in the preliminary results (FIG. 11), drug-loaded CAR-T cells may reduce toxicity in CAR-T in blood cancer. We have also conducted experiments using Paxalisib (Pax) and Vactosertib (Vacto) loaded CAR-T cells. The additional data presented herein also shows persistence and lack of fatigue of the enhanced CAR-T cells, even when subjected to multiple tumor re-challenges. Data is also provided demonstrating the ability to change the phenotype of macrophages and monocytes into anti-tumor macrophages, as well as enhancement of the memory phenotype of the modified cells.

[0128] Thus, in one or more embodiments, the present technology and advancement concerns formulations of nanocarriers loaded with active agents (e.g., vactosertib, paxalisib, or PKC agonists such as Ingenol-3-Angelate loaded nanoparticle nanocarriers) which can be used to provide enhanced CAR-T cells ex-vivo. In one or more embodiments, methods of producing enhanced CAR-T cells are described herein, such as methods for modulating CAR-T activity. In the methods, a plurality of nanocarriers loaded with active agents according to any of the embodiments described herein, are mixed with the T cells during ex vivo CAR-T cell manufacturing. Advantageously, it will be recognized that the present technology is CAR-T agnostic, and can be used to enhance (by increasing, decreasing, or otherwise modulating) activity of any CAR-T cell. For example, a paxalisib-loaded nanocarrier may benefit not just CD19-28z constructs, but potentially all CAR-T constructs, especially CAR-T constructs targeting CD 19 tumor antigen (for lymphoma), and BCMA antigen (for myeloma). Without wishing to be bound by theory, the current data and experiments suggest that paxalisib-loaded CAR-Ts will be relevant for CAR-T cells targeting any hematologic malignancies including, without limitation, lymphomas, leukemia, and myeloma. Thus, the technology could be applied to a specific CAR-T cell targeting any tumor-antigen in any specific cancer, as the problems of TGFp suppression or CRS are common throughout the field.

[0129] Thus, enhanced CAR-T cells and formulations thereof are contemplated herein, which comprise an active agent loaded via the WTAS-PBAE nanocarrier that increases, decreases, or modulates T cell activity, and in some cases modulates the activity in a time-dependent manner. Methods contemplated herein include methods of obtaining T cells from a patient or donor, transfecting the cells with a nanocarrier conjugated with at least one active agent, as described herein, wherein the active agent is introduced into the cytoplasm of the T cells thereby enhancing or modulating activity of the CAR-T cells. As used herein, conjugation can refer to a covalent (but cleavable) linkage or it can relate to non-covalent interactions, provided the active agent is otherwise held in association with the nanocarrier to be transported into the cell.

[0130] It will also be appreciated that the nanocarriers can be used to introduce a variety of modulating or enhancing elements (active agents) into the CAR-T or CAR-M cells. For example, the nanocarriers can be used to introduce energy resources, such as amino acids (glutamine, glutamate), omega-hydroxic fatty acids, alpha-keto-dicarboxylic acids and the like, into the CAR- T or CAR-M cells before administering to patients to provide the CAR-T or CAR-M cells with sufficient resources to initially survive in a hostile TME. It will be appreciated that as tumor systems evolve defense mechanisms, a wide variety of response tactics can be employed to enhance CAR-T or CAR-M cells to address and overcome the hurdles of the TME. Other examples of compounds or biologies that can be loaded into CAR-T or CAR-M cells to enhance their activity, time their activity and regulate their activity, without limitation. For example, some immunotherapies have to be delayed when the patient is otherwise on corticosteroids for brain tumor related cerebral edema, which can interfere with CAR immunotherapy. Thus, the CAR cells could be loaded with corticosteroid receptor antagonists such as mifepristone (RU-486), ORIC- 101, Relacorilant (CORT125134), and the like to make the CAR cells resistant to the corticosteroids, and avoid the need to delay the CAR immunotherapy for the patient.

[0131] An additional advantage is that the uptake of the nanocarriers by the T cells leads to a significantly better protection of the cells during freeze-thaw cycles, which provides a significant practical improvement in preparing, storing, and shipping therapeutic formulations.

[0132] Again, enhanced transfection properties of novel PBAE-derivatives will enable the replacement of lentiviral constructs for the creation of CAR-T or CAR-M cells. This again, will be a considerable improvement of CAR technologies (more adaptable and much safer than viral vectors). Thus, CAR cells prepared according to the invention can be cryopreserved for storage. For use, the cells are thawed and prepared for infusion.

[0133] Depending on the condition being treated and in particular the location of the cancer or tumor, the modified CAR cells or the nanocarriers can be formulated for various routes of delivery and modalities of treatment. Therapeutic compositions are described herein which comprise a modified CAR-T or CAR-M cell (or a plurality of CAR-T or CAR-M cells) dispersed in a suitable delivery vehicle or carrier for administration to a subject. For in vivo engineering, compositions comprise a plurality of nanocarriers as described herein dispersed in a suitable delivery vehicle or carrier for administration to a subject.

[0134] The therapeutic compositions comprise a therapeutically effective amount of CAR-T or CAR-M cells (or nanocarriers with loaded active agent and mRNA) dispersed or suspended in a pharmaceutically acceptable carrier. In one or more embodiments, the compositions can comprise a mixture of two or more different types of modified CAR cells. For example, it is contemplated that CAR cells could be co-administered with other CAR cells carrying detectable labels for monitoring and visualizing delivery to the targeted area.

[0135] A pharmaceutically acceptable carrier or vehicle is any carrier suitable for in vivo administration. As used herein, the term “pharmaceutically acceptable” means not biologically or otherwise undesirable, in that it can be administered to a subject without excessive toxicity, irritation, or allergic response, and does not cause unacceptable biological effects or interact in a deleterious manner with any of the other components of the composition in which it is contained. A pharmaceutically-acceptable carrier would be selected to minimize any degradation of the CAR cells or nanocarriers (at least during storage) and to minimize any adverse side effects in the subject.

[0136] Pharmaceutically-acceptable ingredients include those acceptable for veterinary use as well as human pharmaceutical use. For example, pharmaceutically-acceptable carriers suitable for injection and / or infusion include cryoprotectants (e.g., DMSO, albumin, polyethylene glycol, dextran, chitosan, and the like ), interleukin-6 antagonists (e.g., tocilizumab) and corticosteroids, to help manage potential side effects. Additional components of the compositions may suitably include excipients such as stabilizers, preservatives, diluents, emulsifiers, and lubricants. It is primarily envisioned that the compositions will be formulated for either direct local injection and / or infusion. Other ingredients may be included in the composition, such as adjuvants, other active agents, preservatives, buffering agents (e.g., histidine, phosphates), salts, and other pharmaceutically-acceptable ingredients. In one or more embodiments, the pharmaceutically- acceptable carrier comprises a combination of one or more of the above-described vehicles, and preferably is configured for enhancing delivery via the desired route.

[0137] Advantageously, the nanocarriers can be pre-formulated in a variety of solid or liquid forms. For example, the nanocarriers can advantageously be dried, e.g., lyophilized, spray-dried, etc., to create as shelf-stable powder that can be reconstituted as a practical option for later preparing engineered CAR-T or CAR-M cells.

[0138] As used herein, a “therapeutically effective” amount or “therapeutic dose” refers to the amount or dosage that will elicit the biological or medical response of a tissue, system, or subject that is being sought by a researcher or clinician, such as to elicit some desired therapeutic or prophylactic effect as against the disease or condition depending upon the active agent delivered. One of skill in the art recognizes that an amount may be considered therapeutically “effective” even if the disease, condition, or symptom is not totally eradicated or prevented, but it or its symptoms and / or effects are improved or alleviated partially in the subject (e.g., reduction in tumor volume, etc.).

[0139] The disclosure is also directed to a method of treating a disease or disorder in a subject in need thereof comprising administering the modified CAR cells or loaded nanocarrier (CAR therapy) disclosed herein in a therapeutic dose to the patient. The CAR therapy be administered by one or more dosages, which dosages can be administered, by the same or different route, to achieve the desired prophylactic or therapeutic effect.

[0140] The methods of treating a disease or disorder described herein, wherein the disease or disorder is colon cancer, cholangiocarcinoma, a genitourinary cancer, a gynecologic cancer, a gastrointestinal cancer, lymphoma, melanoma or skin cancer, a head and neck cancer, lung cancer, intestinal cancer, kidney cancer, pancreatic cancer, breast cancer, stomach cancer, leukemia, bone cancer, thyroid cancer, brain cancer, a glioma, idiopathic pulmonary fibrosis (IPF), head and neck fibrosis, osteosarcoma, or bladder cancer; further, wherein the disease or disorder is colon cancer; additionally, wherein the disease or disorder is idiopathic pulmonary fibrosis (IPF); and also, wherein the disease or disorder is osteosarcoma.

[0141] In some embodiments, contemplated herein are kits comprising the nanocarrier along with instructions for reconstituting the nanocarrier and using it for transducing CAR cells to introduce active agents into the cells. The nanocarrier can be pre-loaded with a variety of active agents, including small molecule drugs for modulating CAR cell activity, as well as with nucleic acids for in vivo engineering of CAR cells.

[0142] Additional advantages of the various embodiments of the invention will be apparent to those skilled in the art upon review of the disclosure herein and the working examples below. It will be appreciated that the various embodiments described herein are not necessarily mutually exclusive unless otherwise indicated herein. For example, a feature described or depicted in one embodiment may also be included in other embodiments, but is not necessarily included. Thus, the present invention encompasses a variety of combinations and / or integrations of the specific embodiments described herein.

[0143] As used herein, the phrase "and / or," when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed. For example, if a composition is described as containing or excluding components A, B, and / or C, the composition can contain or exclude A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.

[0144] The present description also uses numerical ranges to quantify certain parameters relating to various embodiments of the invention. It should be understood that when numerical ranges are provided, such ranges are to be construed as providing literal support for claim limitations that only recite the lower value of the range as well as claim limitations that only recite the upper value of the range. For example, a disclosed numerical range of about 10 to about 100 provides literal support for a claim reciting "greater than about 10" (with no upper bounds) and a claim reciting "less than about 100" (with no lower bounds).

[0145] Having described the disclosure in detail, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims.

[0146] EXAMPLES

[0147] The following examples set forth methods in accordance with the disclosure. It is to be understood, however, that these examples are provided by way of illustration and nothing therein should be taken as a limitation upon the overall scope of the invention.

[0148] EXAMPLE

[0149] Modulating CAR-T Cells

[0150] I. Overview

[0151] The present Example describes new technology to load CAR-T cells with small-molecule drugs of interest to enhance CAR-T function while mitigating systemic toxicity. Glioblastoma (GBM) remains an incurable disease, with a median survival of 16-20 months. Maximal safe resection and adjuvant chemoradiation improve survival, but there is no cure. New therapies are desperately needed. The epidermal growth factor receptor (EGFR) is overexpressed in over 50% of GBM, making it an important therapeutic target. EGFR tyrosine kinase inhibitors are ineffective in GBM due to compensatory upregulation of other oncogenic receptors. The application of CAR- T cells to GBM raises a number of challenges including the immune-suppressive microenvironment. Transforming Growth Factor (TGFP) is highly enriched in the GBM TME and is highly immune-suppressive against T cells. TGFP inhibition improves CAR-T activity in various preclinical tumor models, but has also shown systemic toxicity in clinical trials, including one case of a patient’s death in a prostate cancer trial involving a CAR-T encoding a dominantnegative TGFP Receptor. A near complete biochemical PSA response was observed prior to macrophage histiocytosis and patient death. Toxicity has also been reported with bintrafusp alpha, a bifunctional fusion protein targeting TGFP and PD-L1. In clinical testing in recurrent GBM, 14% and 17% of patients had intracranial hemorrhage and gingival bleeding, respectively, thought to be related to the drug utilized. Finally, small molecule kinase inhibitors often have poor CNS penetration, further complicating systemic delivery in treating brain tumors. There is an urgent need to develop nanotechnology to augment CAR-T cell killing while mitigating systemic toxicity.

[0152] In this Example, we establish a nanoplatform technology to utilize CAR-T cells as a small- molecule drug-vehicle, dvCAR-T cells: a nanocarrier comprising of a cell-penetrating peptide called WTAS (PLKWPGKKKKGKPGKRKEQEKKKRRTR, SEQ ID NO: 11) as primary nanocarrier and a poly(b-amino ester) (PBAE) polymer as a secondary nanocarrier. Our preliminary data demonstrates that WTAS-PBAE significantly enhances PBAE penetration into T cells compared to PBAE alone (FIGS. 1-4). This is a substantial improvement over traditional PBAE nanocarriers’ T cell penetration capabilities, as it may result in reduced T cell toxicity allowing for sufficient drug loading. Recent work also demonstrated potent anti-tumor killing of intracranial delivered EGFR CAR-T cells in a murine brain tumor model, thus further highlighting the clinically proven intracranial route of CAR-T delivery.

[0153] The objective of the research is to improve CAR-T tumor cytolysis and mitigate toxicity by drug-loading. The data demonstrates that peptide (WTAS) labeled polymer poly (b-amino ester) (PBAE) can serve as a platform to establish dvCAR-T cells targeting GBM with enhanced efficacy compared to CAR-T cells alone, and reduced toxicity compared to systemically delivered Vactosertib + intracranial CAR-T. The work entailed an evaluation of (1) drug-loading WTAS- PBAE with a candidate drug TGF0 receptor Kinase I Vactosertib, (2) demonstrate CAR-T uptake without loss of T cell viability, (3) evaluate half-life of drug elution within dvCAR-T cells as compared to free-drug alone, (4) enhanced anti-tumor activity (and toxicity mitigation) in preclinical GBM models of dvCAR-T vs CAR-T administered concurrently with systemic Vactosertib.

[0154] Our preliminary data demonstrates that the nanocarrier-polymers can (1) penetrate T cells without loss in T cell viability, and (2) elute polymer conjugated targeted therapy (TGFb Receptor inhibitor Vactosertib) up to 21 days in vitro, and (3) enhance CAR-T mediated anti-tumor killing. The ultimate goal is to implement CAR-T therapy to GBM patients in a GMP compliant CAR-T production facility at University of Kansas.

[0155] Research Aims: Evaluate dosage, release, optimization and immunotoxicity of drug loaded T cells in vitro,' evaluate anti-tumor activity and toxicity of dvCAR-T ceils in vivo preclinical model, and evaluate anti -tumor activity of murine dvCAR-T cells in syngeneic orthotopic GBM model.

[0156] Thus, the work here demonstrates a novel and potentially transformative technology to load CAR-T cells with small-molecule drugs of interest to enhance CAR-T function while mitigating systemic toxicity. Because the drug-loaded nanocarrier (e.g., PBAE) is agnostic to the CAR-T target, this technology can be used during manufacturing across the adoptive T cell therapy landscape. Importantly, we seek to determine mechanistically how the cargo-loaded nanocarrier affects T cell function. This fundamental understanding will allow us to improve nanocarrier designs and potentially load multiple drugs simultaneously during CAR-T cell production, either for delivery into the TME or to further modify the function of the CAR-T cells themselves.

[0157] Small molecules have already been designed to target immune-suppressive pathways in the TME. However, these drugs are often limited by either systemic toxicity or lack of CNS penetration. Multiple strategies have been reported to avoid systemic toxicity such as coadministering small-molecule targeted therapy and CAR-T cells locally at site of the tumor. One example is to load hydrogels with small-molecule agents, such as STING agonists, and co-deliver CAR-T cells. However, this strategy is limited as hydrogels cannot be administered intravenously, such as in cases of multi-focal disease. Leveraging adoptively transferred T cells as drug-vectors (dvCARs) can potentially overcome these limitations. dvCARs can augment the quantity and variety of drug that can be delivered to tumors while mitigating the risk of systemic toxicity of the free drug. Furthermore, a dvCAR-T, once loaded with a drug and injected intracranially (bypassing the blood brain barrier issues), allows small-molecule drugs to now enter the CNS. Indeed, intracranial delivery of CAR-T cells via subcutaneous ommaya reservoir and rickham catheter into the intraventricular brain is now the standard across multiple pediatric and adult primary brain tumor trials (NCT05063682, NCT05474378, NCT04214392).

[0158] One engineering strategy is to label the T cell surface with synthetic nanoparticles such as an IL15 super-agonist. However, surface labeling of T cells has multiple limitations vs. intracellular cargo loading including potential disruption of T cell adhesion, migration, and signal transduction. CAR-T cell cytoplasmic loading of a small molecule has not been reported to date and would significantly advance the field.

[0159] There is an ongoing tremendous effort to genetically modify CAR-T cells to overcome TME. One common approach is the CRISPR-Cas9 deletion of the T cell immune-checkpoint PD- 1 receptor for adoptive T cell therapy. Loading CAR-T cells with small molecules is an attractive concept that can further “armor” CAR-T cells to overcome the barrier of the TME. To our knowledge, while this concept has been reviewed elsewhere, and reported in neutrophils and dendritic cells, it has not been implemented in T cells due to various obstacles. These obstacles include the toxicity of the polymer carrier PBAE in T cells and unknown variables of the minimum required T cell drug cargo half-life.

[0160] To begin with, T cells are resistant to common chemical transfection methods such as Lipofectamine® 2000, PolyFect™, and TurboFect™, resulting in poor T cell viability. Electroporation of T cells also results in poor T cell viability. Poly(b-amino ester) (PBAE) has more attractive properties for T cell penetration than cationic polymers. PBAE has been utilized mostly for non-viral gene-delivery. However, a small molecule drug-loaded version of PBAE for T cell loading has not previously been reported to the best of our knowledge.

[0161] The peptide Microtubule Associated Sequence (MTAS) was previously reported to improve DNA transfection by mimicking the microtubule mediated protein transport with conjugation to quantum dots and later used as a PBAE conjugate to enhance T cells as a gene delivery vehicle.

[0162] Described herein is a nanocarrier comprising two main components, the human cellpenetrating peptide called PFTAS as primary nanocarrier and PBAE as a secondary polymeric nanocarrier. WTAS was identified by screening MTAS mutants for improved cellular uptake, with a tryptophan mutation (W) showing the highest uptake in human cancer cell lines. The WTAS- PBAE conjugate demonstrates greater than 90% transfection of Green Fluorescent Protein in numerous cell lines. Here, PBAE is used to protect the WTAS peptide from early degradation while further facilitating the transport into cells. WTAS is a peptide that penetrates cell membranes within a few minutes after exposure, which makes it an ideal candidate to transport cargo into cells. The drug can be tethered to the polymer backbone using a variety of customizable and tailored linkages, including esterase cleavable and / or protease cleavable linkages. Esterase cleavable bonds are also protease cleavable:

[0163] Urea-type linkages R-NH-CO-NH-R are also esterase-cleavable, albeit slowly. Once taken up by the cells, the candidate drugs can then be released from PBAE by cytoplasmic human esterases or proteases. Cargo loaded dvCAR-T cells can only be an improvement if they are able to load sufficient drug to overcome the TME once the T cell has trafficked to the solid tumor site. Therefore, one important consideration is what length of time is sufficient for a drug loaded CAR- T to elicit an anti-tumor response prior to having the drug-cargo exhausted? Clinical data from CD 19 CAR-T cells in lymphoma demonstrates anti -tumor response is associated with cytokinerelease syndrome (CRS). Importantly, CRS typically occurs within the first 7 days of CAR-T infusion. More limited data from clinical trial experience of intracranial CAR-T delivery in primary adult and pediatric high grade brain tumors has shed light on the timing of treatment response. In an adult GBM clinical trial incorporating IL13Ra2 CAR-T, the MRI response of complete tumor regression was identified to 30 days after CAR-T intracranial-intraventricular (ICV) delivery. Importantly, this study demonstrated that at day 10 after ICV CAR-T infusion, cerebrospinal fluid analysis identified recruitment of host immune cells (MHC II antigen presenting cells and T cells) and cytokines. Strikingly, in murine syngeneic GBM models of the IL13Ra2 CAR-T, TME recruitment of host T cells and MHC II antigen presenting cells were required for CAR-T response and immunologic memory to tumor re-challenge.

[0164] Similarly, clinical trial results were reported for pediatric high grade brain tumor patients treated with ICV delivered GD2 CAR-T cells. One notable patient on this trial demonstrated MRI brain tumor shrinkage of 80% after 3 weeks and 90% tumor volume reduction within 30 days. This clinical and preclinical data supports the concept that a CAR-T mediated immune response requires between 10 to 30 days for tumor eradication after ICV CAR-T delivery. Together, this data supports our estimate that a dvCAR-T requires drug-cargo of 30 days to potentially augment an anti-tumor CAR-T response.

[0165] Given our preliminary data of highly penetrant (> 95%) WTAS-PBAE in T cells without loss of T cell viability (FIGs. 4 and 5), and up to at least 21 days of T cell resistance to TGF0 after Vactosertib conjugation (FIG. 6), we seek to evaluate this platform further for drug-loading CAR- T cells.

[0166] This work is highly innovative as we seek to introduce a nanoplatform technology that addresses two major barriers to clinical application of CAR-T cells in GBM: (1) the immune- suppressive tumor microenvironment. (2) Mitigating system toxicity: ICV delivered dvCARs has the potential to mitigate systemic toxicity and improve small molecule access across the blood brain barrier. Such a platform may allow multiple small-molecules to be simultaneously loaded into CAR-T cells while mitigating their well -characterized systemic toxicity, allowing for a cocktail of targeted therapy previously demonstrated to enhance CAR-T cells in preclinical models when introduced during ex vivo manufacturing (e.g., PI3K inhibitor, AKT inhibitors, or GSK-3b inhibitors).

[0167] Advantageously, the inhibitors are introduced during ex vivo manufacturing to avoid toxicity when given systemically to patients.

[0168] Materials and Methods. Evaluate kinetics, release, optimization, and immunotoxicity of drug loaded T cell.

[0169] Our central paradigm is that the WTAS-PBAE nanocarrier can serve as a platform for dvCAR-T cells. We selected the TGF0 receptor kinase inhibitor, Vactosertib, for drug conjugation, because it has previously been shown to enhance CAR-T killing in vitro, and has also been used in clinical trials in combination with chemo-immunotherapy (NCT04515979, NCT05588648).

[0170] 1. PBAE and peptide synthesis. PBAE synthesis followed known protocols, 1,4- butanediol diacrylate is reacted with 3 -amino- 1 -propanol to form the polymer backbone, which is then reacted with tris(2-aminoethyl)amine) to add the “end-caps” of PBAE. All reaction steps are verified by 'H-NMR analysis. Peptides are synthesized following solid phase peptide synthesis protocol, where the cycle goes through deprotection of Fmoc and activation of the carboxyl group to form a peptide bond, adding one amino acid at a time. The peptides synthesized are characterized by means ofUPLC-MS using aUPLC peptide CSH C18 column. The purity of WTAS was greater than 95%, for this reason no further purification was needed.

[0171] 2. PBAE-WTAS nanocarrier assembly. For the peptide and polymer assembly, the C- terminal end of the peptide WTAS (PLKWPGKKKKGKPGKRKEQEKKKRRTR, SEQ ID NO: 11) is linked to the amine-functional ends of the PBAE by means of EDC ( -ethyl-W-(3- dimethyl-aminopropyl)carbodiimide hydrochloride) and DMAP (4-dimethylaminopyridine) coupling, while Vactosertib is attached using CDI (carbonyl-di-imidazole) coupling.

[0172] The reaction time for all components to attach is 72 hours at room temperature. The nanocarrier is then purified by dialysis against water using a membrane tubing with a molecular weight cut-off of 3500 Da, where all unreacted components will be removed. The nanocarrier is then lyophilized for 2 days. Nanocarrier loaded with Vactosertib was characterized by zeta potential and TEM to determine charge and size (FIG. 2).

[0173] As shown in FIG. 2, PBAE NC loaded with Vactosertib exhibits a size range of 20 to 50 nm. At higher concentration (> 10 mg / ml it forms larger clusters and the range in size increases to 100-250 nm, as determined by Dynamic Light Scattering. The addition of WTAS increases the charge (zeta potential) of the PBAE compared to the polymer alone. This effect is not observed when the PBAE NC is loaded with Vactosertib. In general, nanoclusters possessing a zeta potential > +30mV are considered stable. Therefore, the concentration of attached WTAS is increased until a zeta potential of +30mV is reached.

[0174] 3. WTAS-PBAE vs PBAE: Toxicity in T cells. We first established the role of WTAS in improved PBAE in T cell uptake. To track the WTAS-PBAE nanocarrier (NC) by flow cytometry, we labeled it with a rhodamine B. Strikingly, WTAS-PBAE NC demonstrated statistically significant improvement in T cell uptake at various concentrations of PBAE (FIG. 3).

[0175] Next, we identified cytoplasmic loading of WTAS-PBAE NC (FIG. 4A) with mean fluorescence intensity (MFI) of 98.9% - 99.7% at 1-1 / 64 mg / ml range, respectively, by flow cytometry (FIG. 4B), without loss of T cell viability from 97.1% - 98.5% (FIG. 4C) (Guava Muse® Analyzer). This data demonstrates that WTAS significantly improves PBAE uptake in T cells and is non-toxic up to Img / ml loading.

[0176] 4. WTAS-PBAE conjugation with Vactosertib: Viability of T cells and Inhibition of phosphoSMAD. Next, we conjugated the TGF0 Receptor I kinase inhibitor Vactosertib to WTAS-PBAE through an esterase-cleavable urethane linkage. Our first question was to determine how WTAS-PBAE loading affects T cell proliferation after T cell stimulation with CD3-CD28 beads. Human CD4 / CD8 mixed T cells were plated at 3 million cells per well at day 0 and incubated in 10% FBS with CD3-CD28 T cell activation beads (TransAct Miltenyi). Groups were No Treatment (NT), Nanocarrier only (NC at @ 0.4 mg / ml), Vactosertib only (Vac), mixed NC+ Vac, and conjugated Vactosertib-NC-10, conjugated at 10%w / v Vactosertib and incubated with T cells at Img / ml (FIG. 5).

[0177] NC alone was at a lower dose (0.4mg / ml) compared to conjugated (Img / ml) based upon evaluating mean-fluorescence intensity of NC alone and Vactosertib-conjugated NC at same concentration to back calculate the correct amount of PBAE for NC alone that had the same MFI as conjugated (data not shown). 0.4mg / ml ofNC alone corresponds to Img / ml of Vactosertib-NC at same MFI.

[0178] This data demonstrates NC alone and Vactosertib alone decreases T cell proliferation. However, viability is maintained at > 90% in Vactosertib-NC conjugated at lOw / v Vactosertib (vacto-NC-10). Next, we determined if vacto-NC-10 is maintaining inhibition of downstream phospho- SMAD activation in T cells after TGFP stimulation. T cells were loaded overnight with NC only or vacto-NC-10 at Img / ml. We changed media the next day to remove excess vacto-NC-10 or NC and maintained the cells in culture. T cells were treated with TGFP at lOng / ml for 30 minutes immediately prior to lysis for immunoblot at 24 hrs, 7 days, and 21 days. Strikingly, only vacto-NC-10 treated T cells maintained the shutdown of phospho-SMAD Ser465 / 467 after TGFb stimulation at all time points (FIG. 6).

[0179] This data demonstrates the feasibility of WTAS-PBAE as a platform to drug load T cells with Vactosertib. We seek to further characterize and optimize vacto-NC-10 in T cells.

[0180] 5. Evaluate the kinetics of vacto-NC-10 in T cells at lmg / ml via evaluation of phospho-SMAD by immunoblotting.

[0181] Two brain tumor clinical trials utilizing IL13Ra2 or GD2 CAR-T cells delivered intracranially ICV) via ommaya reservoir and catheter into the brain ventricles have identified the anti-tumor response by MRI at a 20-30 day window after ICV CAR-T. Thus, our goal is to optimize dvCAR-T cells to maintain Vactosertib loaded elution of CAR-T cells at 30 days as defined by greater than 95% inhibition of TGFP by immunoblot densitometry. WTAS-PBAE nanocarrier will be conjugated to different amounts of vactosertib (10%, 20%, 30%, 40%, 50% w / v vacto) followed by evaluating the function in T cells a varying concentrations of vacto-NC in the T cells (1, 0.7, 0.4, and 0.1 mg / ml). T cell functions to be evaluated include in vitro T cell proliferation, duration of phosphor-SMAD inhibition in the presence of TGF-beta cytokine, CAR T killing, cytokine profile, and gene expression changes.

[0182] 6. Synthesis of PBAE / WTAS nanoformulations for the delivery of Vactosertib to CAR-T cells. According to Table 1, we conjugate vacto-NC at 5 different Vactosertib concentrations (10 - 50%) and evaluate each vacto-NC at varying loading concentrations on T cells (0.1 - 1 mg / ml). We synthesize PBAE, and link it to WTAS and Vactosertib, as described above and summarized in Table 1. A total of 20 different conditions will be prepared on human T cells (CD4+CD8) isolated from healthy male and female volunteers according to standard protocols. Importantly, after T cells are incubated overnight with vacto-NC, they are washed the next morning to remove any excess vacto-NC (or Vactosertib alone) in the media that has not penetrated the T cell. At day 7, 14, 21, and 30, T cells will be stimulated with TGFP at 10 ng / ml than lysed for immunoblotting. Upon conclusion of this work, we will have identified an optimal dose of vacto-NC that disrupts SMAD signaling while minimizing amount PBAE in T cells.

[0183] In general, drug loading ranges appear to be preferred at about 0.1 to about 0.35 mg / mL.

[0184] 7. Evaluate mechanism of immunotoxicology of WTAS-PBAE and conjugated with Nanocarrier.

[0185] Our preliminary data in FIG. 5 demonstrated diminished T cell proliferation with NC alone or Vacto-NC-10. However, the mechanism of reduced proliferation is unclear. We will evaluate how Vactosertib alone, NC alone, and conjugate vacto-NC-10 will affect T cell function.

[0186] (1 ) Intracellular complement activation in human T cells (ITA-26).

[0187] (2) Detection of nanoparticle-mediated total oxidative stress in T cells using CM-H2DC- FDA dye (ITA-31).

[0188] (3) Detection of mitochondrial oxidative stress in T cells using MitoSOX Red dye (ITA-32).

[0189] (4) Detection of changes in mitochondrial membrane potential in T cells using JC-1 dye (ITA-33).

[0190] (5) Immunophenotyping: Analysis of nanoparticle effects on the composition and activation status of human peripheral blood mononuclear cells (ITA-37.2).

[0191] 8. Characterization of PBAE / WTAS nanoformulations for the delivery of Vactosertib to CAR-T cells

[0192] To independently verify our novel delivery concept, the characterization of the nanocarriers that will be synthesized in Aim 1 will be performed. The following established Physical Characterization Protocols will permit us to gain a quantitative understanding of nanodelivery platform performance: PCC-1: Batch-mode dynamic light scattering will reveal the global size distribution ofNC vs. vacto-NC. PCC-7: Transmission electron microscopy and PCC- 15: High-resolution scanning electron microscopy will enable us to discern the association principles of nanosized PBAE clusters. PCC-18: Quantitation of active pharmaceutical ingredients in polymeric prodrug products will establish the real Vactosertib content in our nanoformulations. Finally, PCC-2: Zeta potential will ascertain the surface charge of the Vactosertib-PBAE / WTAS nanoformulations. According to our preliminary results, a positive surface charge will aid in the fast uptake by CAR-T cells.

[0193] Our goal is to optimize drug loading of CAR-T cells with Vactosertib to 30 days because the clinical trial data in ICV delivered CAR-T cells demonstrated radiographic response by day 30. We have tested for the extent of phospho-SMAD inhibition to about 18 days with vacto-NC T cells to date. However, this experiment was performed at only 10% w / v Vactosertib conjugation with T cells. Thus, we are confident that we can attain 30 days with optimization of Vactosertib conjugation greater than 10% w / v. However, if higher Vactosertib conjugation is toxic to T cells or the drug is unable to achieve greater than 90% inhibition, we will modify PBAE by introducing a carboxylic acid group prior to conjugation with Vactosertib by means of a tertiary amide bond (FIG. 7) to slow down the release kinetics.

[0194] FIG. 7 focuses on the attachment of Vactosertib to the PBAE backbone by means of a tertiary amide group. This tertiary amide bond will be cleaved significantly more slowly than a urethane group that was used to tether Vactosertib to PBAE above. The biochemical reason is that tertiary amides cannot be cleaved by esterases, but require cytosolic proteases, as for instance calpain. We envision exchanging up to 10% of terminal -OH groups of PBAE with -COOH. For reference, the copolymer repeat units are also shown in the figure. Previous research has demonstrated the feasibility of forming amide bond for prolonged doxorubicin delivery.

[0195] 9. Evaluate anti-tumor activity in vitro (tumor re-challenge assay), in vivo and toxicity of dvCAR-T (vacto-NC-10) cells.

[0196] We have characterized the IL13Ra2 CAR-T construct (IL13E13Y IgG4EQ CD4- Transmembrane 41BB-CD3z) that is currently in clinical trials for GBM(NCT04003649, NCT05168423) to evaluate CAR-T mediated killing of tumor cells in vivo and in vitro.

[0197] Results

[0198] As shown in the CAR-T Cell Activation Assay in FIG. 8a, vac-NC-10 does not disrupt IL13Ra2 CAR-T activation markers. FIG. 8a demonstrates similar activation of IL13Ra2 CAR-T cells after incubation with U87 cells compared to mock T cells .

[0199] The major endpoint #1 will be to identify how normal T cell cytolytic functions that are necessary for tumor killing (production of Perforin, Granzyme B, TNFa, IFNg, CD69, and CD 107a) will be affected by NC and vacto-NC-10. For CAR-T cell activation assays, 50,000 U87 cells will be plated per well of a 96 well plate and allowed to attach overnight. 200,000 CAR-T cells and untransduced Mock T cells will be added to the cells the following morning and allowed to incubate for 5 hours with Brefeldin A (BioLegend #420601). T cells will be collected in a V- Bottom 96 well plate. Cells will be washed three times before adding extracellular antibodies and allowed to incubate at 4°C for 30 minutes. After 2 washes, cells will be fixed with Fixation Buffer (BioLegend #420801) in the dark for 20 minutes at room temperature. Cells will be centrifuged at 350g for 5 minutes and washed with Cell Staining Buffer. Samples will be left under Cell Staining Buffer at 4°C overnight. The fixed cells will be suspended in Intracellular staining Perm Wash Buffer (BioLegend #421002) and centrifuged at 350g for 5 minutes twice. The cells will be then incubated with antibodies against Perforin (Biolegend #353312), GranzymeB (Biolegend #372208), TNFa (BioLegend #), IFNg (BioLegend #), CD69 (BioLegend #104528) and CD 107a (BioLegend #328634) in Perm Wash Buffer for 20 minutes in the dark at room temperature. Cells will be washed twice with Intracellular staining Perm Wash Buffer and centrifuged at 350g for 5 minutes. Cells will be resuspended in FSS and followed by flow cytometry. Data will be represented as mean±SD and analyzed by one-way ANOVA with multiple comparators. Strikingly, our preliminary data demonstrates no disruption of activation markers of IL13Ra2 CAR-T cells pre-treated with nanocarrier or vactosertib conjugated nanocarrier at lOmg / ml (FIG.

[0200] 8a)

[0201] 10. Evaluate anti-tumor activity of IL13Ra2 dvCAR-T (vacto-NC-10) cells in vitro tumor re-challenge assay.

[0202] This work is aimed at evaluating enhanced anti-tumor activity of dvCAR-T cells modified with vac-NC-10 in a tumor re-challenge assay, which best ascertains recursive CAR-T mediated killing. Our preliminary data in FIG. 8b, demonstrates CAR-T cell killing of U87 tumor cells is not impeded by vac-NC-10 (“Conj ”). As shown in FIG. 8b, vac-NC-10 does not disrupt IL13Ra2 CAR-T mediated killing of U87 cells at 72 hours (FIG. 8b). Similarly, FIG. 9 demonstrates vac- NC-10 dvCAR-T cells significantly improve exhaustion phenotype. Thus, we surmise the tumor rechallenge assay is best to evaluate exhaustion / suppression related phenotype related changes of vac-NC-10 dvCAR-T cells.

[0203] To test long term killing we propose a tumor re-challenge assay which repeatedly challenges CARs with tumor cells until the T cells become exhausted and cease to kill, usually checking on Day 3, 5, 7, 9, 11 or similar time splits. This assay is prepared similarly to a normal killing assay, with the difference being that it is plated in a non-attaching U bottom plate. This causes the U87 or other tumor cells to form into neurosphere tumor organoids and concentrating tumor and CAR-Together. Replicate plates are made for each day that the challenge will be checked. Tumors are added periodically to all remaining wells and plates and on separate days tumor count and viability, and T cell activation and exhaustion are all probed using flow cytometry as described in CAR-T killing assays and exhaustion assays below, in addition to statistical analysis detailed in below sections. Upon conclusion of this sub-aim, we will have characterized the role of vac-NC-10 in improvement of CAR-T mediated recursive killing of tumor cells in vitro.

[0204] IL13Ra2 CAR-T production methods: Peripheral blood samples from healthy human male and female patients < 45 years of age without prior cancer diagnosis will be collected in vacutainer tubes ± EDTA, followed by centrifugation at 4°C set at 1300g for 10 minutes, followed by collection of the buffy coat. Untouched human CD3 T cells will be isolated from the buffy coat using Dynabeads® utilizing the Untouched Human T Cells Kit (11344D; Invitrogen; Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. Briefly, RBC lysis buffer will be added to the buffy coat to lyse red blood cells and then magnetic beads will be added to the PBMCs. After washing and spinning down tagged cells, Depletion Dynabeads® will be added to the cells to negatively select the CD3 population. Finally, the supernatant will be centrifuged to collect untouched CD3 T cells.

[0205] The T cells will be cultured in gas-permeable tissue 24-well culture plates (80192M; Wilsonwolf, St Paul, MN) at a concentration of IxlO6cells / well in ImL of LymphoONE™ T-Cell Expansion Xeno-Free media (WK552S; Takara) supplemented with 10% FBS, 50 U / mL IL2 (200- 02; 117 Peprotech, Cranbury, NJ), IL15 (200-15; peprotech) and Dynabeads Human T-Activator CD3 / CD28 for T Cell Expansion and Activation (1116 ID; Invitrogen; Thermo Fisher Scientific, Waltham, MA) with a bead-to-cell ratio of 3:1. IL13Ra2 CAR-T lentivirus particles will be added on day 1 at multiplicity of infection (MOI) of 6. Media will be doubled on day 2 (2x cytokines will be added on this day only) and day 3, and 3mL added on day 4 for a total of 7mL. On day 5 and day 6 half of the media will be replaced with fresh media (lx cytokines will be added in each of these days). On day 7, Dynabeads will be removed using a magnet.

[0206] The cells will be allowed to grow for 2 more days. Cells will be collected on Day 10, followed by measuring CAR expression by flow cytometry, then concentrated using the Easy Sep Release Human CD 19 Positive Selection Kit and allowed to grow for 2 more days. CAR expression will be identified by flow cytometry using anti -human CD 19 antibody (BiolLegend, #363012). CAR-T cells will be treated with vacto-Nano-10, unconjugated nanocarrier, and Vactosertib. All assays will be plated for CAR-T mediated killing and CAR-T activation on day 7, 14, 21, and 30.

[0207] In vitro killing assay methods: Human Glioblastoma U87 cells (ATCC) or GBM39 (a low passage GBM Neurosphere line that is a generous gift of C. David James PhD, Northwestern University)) will be co-cultured with CAR-T cells and mock T cells at multiple effector cell-to- tumor cell ratios. After 72 hour co-culture, the supernatant will be collected in tubes and attached tumor cells will be detached using Tryspin for 5 minutes incubation while shaking at 37°C. All cells are transferred to tubes and were stained for CD3 and aLive / Dead viability dye as described above for evaluating killing by the CAR-T cells. CAR-T cells will be identified by anti-CD19. Flow cytometry will be acquired on Cytek Aurora and data analyzed using FlowJo software. Data has been represented as mean±SD and analyzed by one-way ANOVA with multiple comparators.

[0208] 11. Flow Cytometry (T Cell Exhaustion): The major endpoint will be to identify how T cell exhaustion markers (PD-1, TIM3, Lag3, 2B4) will be affected by the nanocarrier alone, or the Vactosertib-nanocarrier. Our preliminary data demonstrates NC alone and vacto-NC-10 treated T cells (NC Conj.) both significantly decrease exhaustion markers LAG-3 and TIM-3 at day 18 compared to untreated T cells (FIG. 9). There was no change in the PD-1 exhaustion marker at day 18 (data not shown). In brief, 3e6 T cells were plated at lx 106cells / mL of LymphoONE™ T- Cell Expansion Xeno-Free media supplemented with 10%FBS, 50 U / mL IL2 and IL 15 in a regular 24 Well Plate. Untransduced T Cells were treated with nanocarrier alone or nanocarrier conjugated with Vactosertib at 10%w / v and allowed to grow over 18 days. T Cells were collected, and exhaustion markers Tim-3 and Lag-3 were evaluated on these cells by Flow Cytometry. (A) Tim 3 and (B) Lag3 were significantly reduced in the Vactosertib conjugated nanocarrier treatment of T Cells as compared to nanocarrier only or no treatment. Data are represented as Mean +- SD, analyzed by Student's t-test and plotted using Prism software. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001. Upon conclusion of this sub-aim, we will have characterized exhaustion markers on human T cells and CAR-T cells by flow cytometery.

[0209] Treatment groups will be prepared with incubation of NC / Vactosertib combinations overnight, then washed after 12 hours. This will be followed by T Cell exhaustion flow cytometry. Live T cells will be suspended in FACS Stain Solution (FSS) (PBS w / o CaC12 & MgC12, .1%BSA (15260-037; Gibco), 0.5mM EDTA (351-027 721; Quality Biological, Gaithersburg, MD) and washed twice before adding antibodies or isotype control. Cells will be incubated with respective antibodies: Lag3 (BioLegend #369346) and Tim3 (BioLegend #345056) for 30 minutes in the dark at 4°C. Flow cytometry will be performed on BD LSRII (BD Biosciences) and cell viability determined using 4', 6-diamidino-2-phenylindole (DAPI, Sigma) (D9542; Sigma-Aldrich) or Ghost DyeTM Red 780 Viability Dye (13-0865-T100; Tonbo Biosciences; San Diego, CA). Data will be analyzed using FlowJo 137 software (vl 0.7.1 ; FlowJo; BD Biosciences). Data will be represented as Mean +- SD, analyzed by Student's t-test and plotted using Prism software. Upon conclusion of this sub-aim, we will have further characterized all exhaustion markers in T cells treated with NC and vac-NC-10.

[0210] 12. Gene Expression changes within T cells

[0211] It is unclear how NC alone inhibits immune-cell exhaustion markers. PBAE has not previously been known to have an immunomodulatory role, although it is being degraded by immune cells. We will evaluate bulk RNA sequencing of T cells only, NC treated, and vacto-NC groups at day 1, 10, 20 of culture in order to ascertain how Nanocarrier and Vacto-Nanocarrier affect T cell gene expression. Using normalized read counts we will be able to do principle component analysis, Peason’s correlation, and gene ontology mapping to determine which, if any of our transcriptional pathways (inflammation, exhaustion, activation) are elevated or suppressed.

[0212] 13. Evaluate anti-tumor activity in vivo and toxicity of IL13Ra2 dvCAR-T (vacto- NC-10).

[0213] As a proof of concept, we selected to incorporate a TGFP Receptor Kinase 1 inhibitor (Vactosertib) currently in clinical trials and previously demonstrated to rescue CAR-T killing after TGFP incubation. TGFP is a pleotropic cytokine and is highly enriched in GBM. TGFP suppresses CAR-T cells through engagement of the TGFP Receptor I and II expressed on T cells. Multiple mechanisms have been reported to interrupt this pathway including anti TGFP ligand sinks, small molecule inhibitors of TGFPRI or RII, or dominant negative TGFbPRII expressed in the T cell. Clinical trials of TGFpRII dominant negative CAR-T have been associated with multiple complete clinical responses after CAR-T in hematologic malignancies. However, TGFPRII dominant negative CAR-T are also associated with severe toxicity and reported patient death. We selected to evaluate the TGFPRI inhibitor, Vactosertib, because it has been tested in clinical trials in free drug form alone.

[0214] We have selected to do our toxicity evaluation of the vacto-NC- 10 CAR-T cells in an immunodeficient model as this can best characterize over-activation of CAR-T cells resulting in toxicity. Indeed, the aforementioned TGFPRII dominant negative CAR-T was evaluated in immunodeficient mouse model and demonstrated loss of mouse body weight after CAR-T infusion, although with potent tumor eradication.

[0215] Male and Female immunodeficient mice (NOD.Cg-PrkdcscldI12rgtm l W|1 / SzJ) will be procured from Jackson Labs (NSG) and on Day 0 GMB39 or U87 tumor cells expressing firefly luciferase (e.g., U87LUC) are implanted via intracranial injection to generate luciferase-expressing mouse glioblastoma models. Previous research has shown that these models are suitable for CAR T research and understanding mechanisms of action in immunotherapy of cancer. Once tumors are of sufficient burden as detected by bioluminescent imaging, the mice will be injected with experimental groups (20 mice per group) as follows:

[0216] Intracranial Injection Groups:

[0217] (1) Mock T cells

[0218] (2) Mock T cells + oral gavage vactosertib (free drug)

[0219] (3) dvCAR T (NC only)

[0220] (4) dvCAR T (NC + oral gavage vactosertib)

[0221] (5) dvCAR T (conjugated vacto-NC-10)

[0222] At moribund status, mice will be euthanized, and data plotted for survival. We expect Vacto-NC- 10 conjugated IL13Ra2 CAR-T treated mice (group 5) to have improved survival over dvCAR-T cells (NC only), or dvCAR-T (NC only + oral gavage Vactosertib). We selected 20 mice per group based upon prior data (not shown) that CAR-T alone eradicates tumors at 50% at day 50 with n=5 mice per group. At 20 mice per group we will be 80% powered to detect statistical differences.

[0223] The intracranial implantation of tumor cells will follow published protocols. Briefly, mice will be anesthetized by exposure to isoflurane. A burr hole will be drilled on the skull at 2mm lateral of the center line, 0.5mm anterior to the bregma. IxlO6U87FFLUC(or GBM39FFLUC) cells suspended in 3ml of PBS will be injected orthotopically in 1 ml increments at 2.5mm, 2mm, and 1.5mm deep from the dura. Engraftment will then be verified by bioluminescent imaging (IVIS Spectrum imager; PerkinElmer, Waltham, MA) using intraperitoneal injection of 200ml (lOOmM) D-Luciferin one day before CAR-T cell injection. The mice will be randomized into groups based on the BLI signal. IxlO6IL13Ra2 CAR-T cells or PBS will be injected intracranially through the same burr hole at 0.5ul per incremental depth (2.75mm, 2.5mm, 2.25mm, 1.75mm, 1.5mm, & 1.25mm) next day. Tumor growth will be monitored by utilizing the IVIS imager. Flux signals will be analyzed using Living ImageOsoftware (v4.5.5; PerkinElmer). At desired time points or at moribund status, mice will be euthanized and brain tissues will be processed for H&E histology. In order to evaluate Vactosertib toxicity, mice will also be weighed weekly as a surrogate of toxicity. Starting at day 14, these evaluations will be performed at least every other day. If we do not see enhanced anti -tumor activity of dvCAR (vacto-NC-10) T cells in vivo, we will evaluate CAR-T cells loaded with higher concentrations of Vactosertib (vacto-NC-20 or higher), or with modifications of release kinetics with tertiary amide bond introduction (FIG. 7).

[0224] We will also evaluate anti-tumor activity of murine dvCAR-T cells in syngeneic orthotopic intracranial GBM immune competent mouse model. These experiments seek to evaluate dvCAR- T in a syngeneic orthotopic intracranial GBM model, including changes in the tumor microenvironment after each treatment group. The syngeneic system will better represent an immune-suppressive TME as well as endogenous immune response after CAR-T treatment.

[0225] The murine CAR construct (mIL13BBz) was designed in a MSCV retroviral backbone (Addgene), containing the extracellular murine IL 13 and murine CD8 hinge, murine CD8 transmembrane domain, and intracellular murine 4- IBB costimulatory and murine CD3z signals. Following a T2A ribosomal skip, a truncated murine CD19 was inserted as a transduction marker. The resulting plasmid was transfected into PlatE cells using Fugene (Promega). After 48 hours, the supernatant is aliquoted and frozen until the time of transduction.

[0226] Murine T cells will be isolated from spleens of naive C57B1 / 6J mice with EasySep Mouse T cell Isolation Kit (STEMCELL Technologies) and stimulated with Dynabead Mouse T-Activator CD3 / CD28 beads (Gibco) at 1 : 1 ratio. T cells will be transduced on RetroNectin-coated plates (Takara Bio) using retrovirus-containing supernatant (described above). Cells will be next expanded for 4 days in RPMI 1640 (Lonza) supplemented with 10% FBS (Hyclone Laboratories), 55 mmol / L 2-mercaptoethanol (GIBCO), 50 U / mL recombinant IL2 (Novartis), and 10 ng / mL recombinant IL7 (Peptrotech). Before in vivo experiments, the beads will be magnetically separated from T cells. CAR-T expression will be determined by flow cytometry. Next, mice will undergo orthotopic intracranial implantation of the KR158B mouse GBM cell line, genetically modified to express murine IL13Ra2 tumor antigen. The KR158B cell line is a generous gift from the Tyler Jacks lab (MIT). KR158B was isolated from murine C57 / BL6 NF1 / Trp53 mutant mice, which developed spontaneous astrocytomas. KR158B will be transduced with firefly luciferase and murine IL13Ra2 (K-Luc-IL13Ra2+). This cell line will be maintained in DMEM (Gibco) supplemented with 10% FBS (Hyclone Laboratories), 25 mmol / L HEPES (Irvine Scientific), and 2 mmol / L L-glutamine (Lonza). Cell surface expression of mIL13Ra2 will be authenticated by flow cytometry.

[0227] Our previous data demonstrates that mIL13Ra2 CAR-T cells significantly improve overall survival, compared to mock T cell treatment in an orthotopic intracranial syngeneic K-Luc- mIL13Ra2 tumor line (not shown).

[0228] Once tumors are of sufficient burden as detected by bioluminescent imaging, the mice will be injected with experimental groups (20 mice per group) as follows: Intracranial Injection Groups:

[0229] (1) Mock T cells

[0230] (2) Mock T cells + oral gavage vactosertib (free drug)

[0231] (3) dvCAR T (NC only)

[0232] (4) dvCAR T (NC + oral gavage vactosertib)

[0233] (5) dvCAR T (conjugated vacto-NC-10)

[0234] At moribund status, mice will be euthanized, and data plotted for survival.

[0235] Oral gavage Vactosertib at 25 mg / kg / day will be done daily for 10 days based upon prior preclinical studies. The number of animals required is based on our experience with previous mouse cancer models. Our statistical parameters for statistical comparisons by means of an independent t-test are: Expected deviation between groups: 30%, alpha error: 5%, power: 80%. Using these parameters, and prior data of mouse survival with CAR-T alone after tumor implantation, the respective group sizes per experimental group were calculated at 20 mice per group. Experimental series will be repeated twice. We will use both male and female mice in our studies. An additional test will be repeated with 10 mice per group, which will all have mice euthanized 5 days after CAR-T injection. 5 mice from each group will be used for histology of mouse brain. 5 mice of each group will be used for tumor microenvironment tissue digestion and evaluation by flow cytometry: NK cells (NK1.1+, NK1.2+), T Cells (CD3+), TH cells (CD4+, CD25-), Tccells (CD8+), Tregcells (CD4+, CD25+), PMN (Ly6G+), dendritic cells (CDl lc+) Ml macrophages (CD64+, CD86+), and M2 macrophages (CD64+, CD163+). Total mice required 160 C57 / BL6.

[0236] FIG. 10 and FIG. 11 clearly demonstrate the enhanced CAR-T cells (vactosertib conjugated NC) with improved tumor killing and cytokine profde against GBM cells and the ability to throttle down the cytokine profile while maintaining CAR-T killing (loaded with paxilisib) without any reduction in tumor killing - thus potentially making a CAR-T cell with better toxicity profile. Indeed, toxicity is an important clinical issue on the FDA approved CAR-T constructs for lymphoma. Yescarta™ (CD19 CAR-T) is the construct used for comparison in FIG. 11, with clear demonstration of reduction in cytokines associated with the clinical phenomenon CRS, which can be life threatening, and is a limiting factor in CAR-T therapy. In similar studies, CD 19 CAR T Cells loaded with NC-Pax show delayed killing of tumor cells and able to eliminate tumor by 21 days.

[0237] 5000 Luciferase expressing Raji cells were treated with 10,000 CAR T Cells or untransduced (Mock) T Cells (E:T 2: 1). CAR T Cells were challenged once with 5000 tumor cells on Day 3. Killing was evaluated over 3 weeks by assessing luciferase signal and data represented as Mean ± S.D. (FIG. 12 and 13). CD19 CAR T Cells loaded with NC-Pax (0.25 mg / ml, 2.5 uM Pax dosages) show delayed killing of tumor cells and able to eliminate tumor by 21 days (FIG. 12). 5000 Luciferase expressing Raji cells were treated with 10,000 CAR T Cells or untransduced (Mock) T Cells (E:T 2: 1). CAR T Cells were challenged once with 5000 tumor cells on Day 3. Killing was evaluated over 3 weeks by assessing luciferase signal and data represented as Mean ± S.D. Similarly, at higher dosages of 0.35 mg / ml, PI3K-inhibitor loaded NC slows killing of T cells, and then after day 11, the killing is resumed as the paxalisib is eluting out of the cell, so its effect on the cell is lessening over time. (FIG. 13) Cells were co-cultured with luciferase-labeled Raji cells at E:T ratio of 2: 1. Luciferase activity was measured at indicated time points and CAR T cells were rechallenged with Raji cells every 4 days. Statistical testing by Two-way ANOVA p****<0.0001.

[0238] Both studies at the 0.25 and 0.35 mg / ml dosages used -COOH linkage (which other studies reported herein result in a faster release of the conjugated drug). These studies demonstrate that you can control the killing activity of the CAR T cells depending on the dose of Pax used.

[0239] EXAMPLE

[0240] A PI3K inhibitor and AKT inhibitors have been published, demonstrating enhanced CAR- T activity in preclinical tumor models when they are co-cultured during ex-vivo manufacturing. One such example, paxalisib, is a dual PI3K-mT0R inhibitor. It has demonstrated early evidence of clinical activity as a monotherapy in GBM patients, and our preliminary data above indicates its usefulness for modifying CAR-T cells.

[0241] Unfortunately, PI3K-Akt inhibitors are often poorly tolerated and exhibit systemic toxicity, such as hyperglycemia, and have required dose reduction in 23-64% of patients in trials due to toxicity. As a proof of concept, we loaded the NC with paxalisib, a PI3K-mT0R inhibitor. Paxalisib was selected because the PI3K class of inhibitors also enhances T cell memory phenotypes important for long term immune response, whilst also inhibiting the PI3K pathway common in cancer. Indeed, PI3K inhibitors are well-validated targets for cancer drugs with five FDA approved therapies (idelalisib, copanlisib, duvelisib, alpelisib, and umbralasib).

[0242] Importantly, mTOR-1 inhibitors (e.g. rapamycin) have previously demonstrated enhanced anti-tumor activity of CAR-T cells in murine tumor models. Therefore, a paxalisib loaded dvCAR- T cell is an alternative approach that can modulate and enhance CAR-T activity by increasing the memory T cell phenotype when delivered as a conjugate with the nanoparticle, while mitigating obvious systemic toxicity.

[0243] To demonstrate the strength of this approach, we conjugated WTAS-PBAE NC to paxalisib and treated human T cells. Ie6 CD3 human T cells were treated according to the groups and lysates collected 24 hours (14B) and 7 days (14C) post treatment. Western blot was run for pS6 (Ser235 / 236) with Actinin as loading control. As shown in the summary in FIG. 14A, loading of Pax-NC at 0.35mg / mL on T Cells inhibits S6 phosphorylation on Day 7.

[0244] We demonstrate in vitro that paxalisib conjugated NC preferential and persistent disruption of PI3K-mT0R downstream signaling phospho-S6 at day 7 (14C), as well as an enhanced retention of the memory phenotype (CCR7 - CD45RO double positve - Fig 15 A, B) at day 11 after T cells were incubated with paxalisib-conjugated NC, and the memory phenotype was retained higher in the cells treated with the paxalisib-conjugated NC as compared to the free paxalisib (or free nanocarrier or mock). At day 1, it can be seen that the free drug shuts inhibits PI3K-mT0R (14B) like the rest. However, by day 7 the free drug wears off. We conjugated PI3K inhibitor, Paxalisib to WTAS-PBAE NC via carboxyl group (PI3KINHIB-COOH)and evaluated extent of PI3K / mT0R pathway inhibition in T cells compared to free drug at luM and 2.5uM as well as varying unconjugated and conjugated doses. Of note luM paxalisib is the maximum tolerated plasma concentration in preclinical models, which is unable to maintain phospho-S6 inhibition at Day 7 immunoblot as compared to NC formulation at 0.35 mg / ml. Thus, not only is this approach able to use higher dosages of drug, but the patient is not directly exposed to the drug toxicity because it is contained within the CAR-T cells.

[0245] We also found T cell central memory phenotype markers (double positive CD45 RO / CCR7) are also highly elevated, inducing a central memory phenotype in treated cells. As shown in FIG. 15A, when the paxalisib-conjugated NCs (at either the 0.25 mg / ml or 0.35 mg / ml doses) are compared to various controls, a distinct upregulation of both markers is seen. The flow cytometry, raw data of the bar graph showing the memory phenotype is in FIG. 15B. Thus, the drug-conjugated NC induce central memory phenotype T cells (CCR7, CD45RO ++), and reduced cytokine activation in a CAR T Raji lymphoma model in vitro.

[0246] This data corroborates recently published data demonstrating mTOR inhibition enhances T cell central-cell memory (Tcm) phenotype. Thus, as an alternative approach to Vactosertib, we optimize paxalisib loaded dvCAR-T cells. Since hyperglycemia is a common side-effect of PI3K inhibitors, it can easily be quantified (and potentially mitigated) in mouse models treated with CAR-T loaded NC+ conjugated (conj.) paxalisib vs CAR-T plus free paxalisib.

[0247] EXAMPLE

[0248] Heterogenous Tumor Models

[0249] Across the spectrum of solid and hematologic malignanices therapeutics, tumor antigen heterogeneity remains a major barrier to cure. Early inroads to therapeutic cure have been attained in hematologic malignanices. Chimeric Antigen Receptor (CAR) T cells combine the cytolytic potency of a T cell with the tumor specificity of an antibody. CD 19 is uniformly expressed in B- cell lymphomas, and CD 19 CAR T cells are now FDA approved. Interestingly, two pivotal clinical trials leading to FDA approval did not demonstrate any association with CD 19 lymphoma expression and clinical response. However, patient tumor Fas expression is associated with overall survival after CAR T administration, demonstrating the central importance of this pathway in curative treatment. Endogenous anti-tumor T cells and CAR T cells release Fas-Ligand when engaged with tumor cell antigen, resulting in adjacent tumor killing, irrespective of target-antigen expression. This phenomenon is referred to as Bystander killing, and is especially important in solid tumors, such in brain tumors, which are often fatal and have even more heterogenous tumor antigen expression. Our central hypothesis is that harnessing potent polyfuncional cytokine activation of CAR T cells, including Fas-FasL mediated killing pathway will overcome tumor antigen heterogeneity, potentially resulting in therapeutic cure. Herein, we propose a drug- nanoparticle technology to leverage the bystander effect within CAR T cells to overcome tumorantigen heterogeneity in solid and liquid tumor models.

[0250] Tumor antigen heterogeneity remains a major barrier to immunotherapy cure across solid and hematologic malignancies. CAR T cell therapy has demonstrated early clinical success in CD19 lymphoma, and has taught scientists key lessons in long term response — highlighting the importance of Fas-FasL dependence killing of antigen-negative tumor cells. Antigen escape has been identified as the mechanism of disease relapse after CAR T cell therapy in 20-28% of patients with B cell lymphoma, and in 16-68% with B-ALL. Dual targeting of CD 19 and CD22 has been reported in two trials involving patients with R / R B-ALL. These trials included post-relapse biopsy sampling and reported loss of one or both antigens in 25-33% of patients with disease relapse. Another trial in which patients with R / R B cell malignancies received CD19 / CD22-targeted CAR T cells again identified CD19 loss in 5 / 10 (50%) patients with B-ALL and in 4 / 14 (29%) patients with B cell lymphomas. Similarly, recent clinical trial results of multi-antigen CAR T cells in GBM have failed. These data suggest that targeting more than one antigen does not circumvent the problem of antigen escape. However, lack of CAR T cytokine activation may explain these treatment failures.

[0251] In preliminary studies, I3A enhances antigen independent killing (Green tumor cells) by EGFR CAR T Cells in mixed populations of cells. 2500 EGFR positive (RFP-red) and 2500 EGFR Knock Out(CFSE-green) Tumor (MDA-MB-231) Cells were plated in a well with untransduced (Mock) or EGFR CAR T Cells with or without 150nM 13 A drug. 72 hours post co-culture, fluorescent images were taken and number of live RFP and GFP cells quantified and plotted. The results are shown in FIG. 16A and 16B. Data is represented as Mean ± S.D. and statistical analysis done using one-way ANOVA. The data shows that mock T cells demonstrate anti-tumor activity after 13 A incubation. FIG. 17 shows fluorescent images of cells plated with CAR 3: 1 and then with CAR in the presence of free 13 A. Red cells are EGFR+, Green cells are EGFR- cells, and blue are dying cells. This data demonstrates in vitro cytotoxicity assays against heterogenous EGFR expressing tumors demonstrate CAR T cytolysis of bystander EGFR negative tumor cells when treated with free drug PKCOAg

[0252] In addition, as shown in the preliminary data, CAR T cells loaded with conjugated NC-I3 A (NC-I3A-Conj) enhances the secretion of IL-2, TNF alpha, sFasL and Granzyme B over 9 days of co culture as compared to free I3A or unconjugated I3A (+NC+I3 A). The results are show in FIG. 18A-18D. Drug causes the Mock T cells to secret cytokines too (blue bar) - free drug and / or - COOH conjugated drug or mixed. 2000 T98 GBM tumor cells were treated with untransduced (Mock) or EGFR CAR T Cells at 2: 1 E:T including empty NC or I3A-NC at 0.25mg / mL and lOOnM 13 A according to the groups. Supernatant was collected 48 hours and 9 days post co-culture and evaluated for human cytokines in CD8-NK panel using Legendplex. Data is represented as Mean ± S.D. and statistical analysis done using two-way ANOVA.

[0253] As seen in the data loading CAR-T cells with I3A activates them and enhances proinflammatory cytokines. The drug-loaded nanocarrier improves IL2 secretion among other groups, which increase over time (e.g., Day 2 to 9) and then tires out as the drug is metabolized by the cells. Thus, dvCAR-T can be programmed for targeted activity in the vicinity of the tumor. As seen in the data with EGFR CAR T Cells, bystander cells in the vicinity of the tumor are killed as well by the dvCAR-T cells.

[0254] It is hypothesized that amide conjugation will work best for systemically delivered models (lymphoma delivered to the abdomen (intraperitoneal) and then CAR-T cells were also delivered intraperitoneally). Faster -COOH linkage likely works better for direct intratumoral injection / delivery.

[0255] CAR T cells will have enhanced polyfunctional cytokine release including FasL-mediated clearance of brain and lymphoma tumors with heterogenous antigen expression. We hypothesize toxicity will be mitigated, as maximal CAR T cell activation will be timed through differential elution kinetics of co-loaded T cell activator (PKC9Ag) and T cell cytokine suppressor (PI3KINHIB). Timing requirements of CAR T activation will be determined by disease model: 3-7 days for intratumoral brain tumor CAR T injection and 7-14 days for systemic intravenous CAR T delivery for metastatic model. It is hypothesized that amide conjugation will work best for systemically delivered models (lymphoma delivered to the abdomen and then CAR-T cells were delivered intraperitoneally). Faster -COOH linkage likely works better for direct intratumoral injection / delivery.

[0256] The summarized data as shown in FIG. 18A-D strongly supports our central hypothesis. We have demonstrated potent and delayed polyfunctional cytokine release with PKCOAg-NC loaded CAR T cells compared to free-drug or unconjugated evaluated at Day 9. Indeed, FasL secretion is 5-fold higher at Day 9 with this novel treatment compared to CAR T alone using carboxyl-conjugation to polymeric nanocarrier. Our preliminary data also demonstrates potent bystander killing of EGFR KO cells when co-cultured with EGFR+ cells and CAR T cells plus PKCOAg (FIG. 16 and 17 above). Similarly, in the above work, we have demonstrated potent central memory T phenotype and suppressed cytokine activation with PBK^^-NC loaded T cells. We have demonstrated the ability to modulate the release kinetics of the loaded drug. In particular, as shown in FIG. 19, the combination of two different drugs attached to the NC via two different tethers: a PKC agonist (13 A) and PI3K inhibitor (paxalisib) results in delayed and potent cytokine activation. In this work, the PKC agonist (13 A) is attached with the slower release amide, while the PI3K inhibitor (paxalisib) are attached via the faster release -COOH tether. Thus, the inhibitor is released first which gives the modified CAR-T cells time to find their target under the suppressive effect of the faster-eluting PI3K inhibitor (paxalisib). The PI3K inhibitor (paxalisib) results in initial suppression of cytokines involved in killing. However, over time the PI3K inhibitor (paxalisib) is eluted from the cell and the T cell is activated once it gets to its target as the slower-eluting PKC agonist (13 A) begins to elicit its effect on the T cells.

[0257] Single orthotopic intracranial tumoral injection PKCOAg alone or PKCOAg-NC does not demonstrate in vivo toxicity: Our platform technology is based currently on PKCOAg-NC loaded into T cells. Leaching of this potent drug outside of the intracranial administered CAR T may result in intracranial toxicity. Thus, in order to evaluate for any neurologic toxicity, we injected free drug PKCOAg and PKCOAg-NC, devoid of CAR T cells, orthotopically in immunocompetent brain tumor bearing mice. There was no evidence of toxicity, and an associated trend towards improved survival after single injection PKCOAg in the immunocompetent host and specifically that when the PKCOAg (13 A) is conjugated to the NC, toxicity is better than free drug or vehicle control (FIG. 20). We surmise the anti-tumor activity of this compound alone is due to polarization of immune-suppressive macrophages previously described in murine immunocompetent breast cancer models, which we have confirmed in human PBMC derived monocytes polarized into Ml and M2 macrophages (see examples below). Thus, establishing early evidence of safety and potential anti-tumor activity in this platform on the brain-tumor setting, which is the least hospitable organ-environment for unchecked immune-related toxicity.

[0258] Anti-tumor activity and bystander killing of PKCOAg-NC loaded EGFR CAR T cells in orthotopic heterogenous antigen expressed brain tumor models. We have previously demonstrated potent elimination of TNBC brain metastases in orthotopic brain tumor model with 100% EGFR expressing MDA-MB-231 lined 1 (FIG. 21).

[0259] Given our loaded CAR T cells exhibit potent Bystander killing in vitro, we next evaluated this effect in heterogenous orthotopically implanted tumor model (50% parental MDA-MB-231 EGFR 100% and 50% EGFR KO). Strikingly, only the EGFR CAR T (PKCOAg-NCCOOH) (FIG. 22) was able to demonstrate statistically survival benefit (Kaplan Meier p=0.005). This nanoparticle was synthesized at 10% w / v of PKCOAgCOOH to WTAS-PBAE polymer. In this Aim, we would like to further improve survival benefit by optimizing the nanocarrier drug-loaded cargo as well as nanocarrier composition with inclusion of CAR T cell memory phenotype enhancing PI3K inhibitors. PKCOAg-NCCOOH vs PKC0Ag-NC AMIDE and 10, 20, 30% w / v polymer load: We have demonstrated strong preliminary evidence of mouse survival benefit (p=0.005) with PKC0AgCOOH(lO% w / v) loaded into EGFR CAR T, ICT at 50 / 50 heterogenous tumor (FIG. 22). We would like to confirm this preliminary data and refine this nano-therapeutic by comparing EGFR CAR T alone, along with (1) PKC0AgCOOH (10, 20%w / v), and (2) PKC0AgAMIDE (20, 30%w / v).

[0260] The data show that PKC0AgAMIDE (20%w / v) outperforms PKC0AgCOOH (10%w / v) in CD19 CAR T cells delivered IP (not shown). PKC0Ag-NC loaded CD19 CAR-T cells control CD19 heterogenous Raji tumor w / o e / o toxicity. 1x6 Raji-Luc cells were injected IP. After confirmation of BLI engraftment, tumor only and CAR T groups were also injected IP and tumor burden was monitored weekly BLI imaging. Mouse weights were also measured weekly, with no statistical difference in weight. Nanoparticles were loaded into CAR T cells at 0.2 mg / ml, which is the optimal dose of T cell viability >90% after 10 days in vitro culture (data not shown). We will evaluate this formulation in orthotopic intracranial brain tumor model ofMDA-MB-231 and follow mice for survival. The fastest route to clinical implementation of this technology is to rescue a CAR T response from a study of patients that have failed CD 19 CAR Treatment. Rescuing a failed response with repeat treatment of CAR T cells with loaded nanoparticles strongly indicates the nanoparticles were responsible for the rescue. At Day 42, the mice treated with the PKC0AgCOOH CD 19 CAR T cells mostly did not survived. It is believed that this is due to the drug eluting too quickly from this nanocarrier construct with the -COOH linkage. In contrast, the mice treated with the PKC0AgAMIDE CD19 CAR-T cells have statistically significant reduced tumor burden at Day 42 compared to CAR T only group. (FIG. 23A-C).

[0261] EXAMPLE

[0262] In vivo delivery of drug-mRNA CAR-loaded Nanoparticles for anti-GBM CAR-Macrophages It is also theorized that I3A may be useful in modulating tumor associated macrophages

[0263] (M2) and specifically to modulate M2 macrophages to secrete Ml -type cytokines. As with the CAR-T cells, drugs can be conjugated to the nanocarriers and taken up by macrophages and have their activity modulated by the loaded drug. In this study, we aim to leverage our group’s expertise in CAR engineering to overcome the immunosuppressive tumor microenvironment (TME) towards optimizing cell-based immunotherapies for GBM and other brain cancers. We aim to utilize a proprietary, nanocarrier technology developed and validated for effective cellular engineering by our collaborator (Stefan Bossmann, PhD) to design a multi-targeting, nanomedicine comprising of an immune-activating drug and mRNA to polarize immunosuppressive tumor-associated macrophages (TAMs) towards an anti-tumor phenotype and additionally reprogram these macrophages into CAR-macrophages (CAR-M) capable of tumor-killing. Through this novel immunotherapy approach enabled by our state-of-the-art nanoengineering platform, we will further elucidate mechanisms of immunosuppression and treatment failure while developing a combinatorial immunotherapy with the potential to enhance conventional CAR-T cell therapy paradigms for brain cancer.

[0264] Investigation of mechanisms underlying treatment resistance and failure have uncovered local and systemic, tumor-induced immunosuppression as a key barrier to the efficacy of such therapies which precludes the successful responses seen in the application of this approach to other cancers. The TME and specifically TAMs have been implicated in playing a key role in immunosuppression and tumor progression with efforts underway to mitigate this effect. Furthermore, TAMs are enriched in the GBM TME and associated with poor clinical outcomes, making them an ideal target for modulation and engineering as therapeutic vehicles. Preclinical studies and clinical trials in systemic cancers have shown that peripheral monocytes and TAMs may be stably engineered ex vivo with viral and non-viral techniques to secrete immunomodulatory drugs, carry anti-tumor cargo, and to expressing immunomodulatory ligands. Additionally, various immunomodulatory therapeutics have been explored including combinatorial therapy with checkpoint inhibitors or immune-activating small molecules. These approaches are limited in the setting of brain cancer due to systemic toxicity from intravenous administration and poor brain / tumor penetration as is the case for conventional free drug therapeutics. Therefore, there is a need for the rational design of novel therapeutic strategies that can target and reprogram TAMs in situ for anti-tumor activity and immunomodulation. This would effectively harness the inherent recruitment properties of TAMs and overcome challenges of drug delivery.

[0265] Our groups have invented and validated a novel nanocarrier (I3A NC) platform utilizing poly( -amino ester) PBAE polymer, macrophage-targeting ligand CD206, and cell-penetrating peptide WTAS that can effectively deliver an immune-active small molecule I3A, capable of polarizing TAMs towards an anti-tumor phenotype, to Peripheral bone marrow-derived M2 protumor macrophages with minimal cell toxicity as proof-of-concept. Furthermore, we have shown that I3A treatment effectively polarizes M2 macrophages towards an anti-tumor phenotype. As shown in FIG. 24, 13 A loaded NC is efficiently taken up by human PBMC derived monocytes and macrophages resulting in potent repolarization of M2 into Ml -type macrophages (A) Successful dose-dependent uptake of nanocarrier(NC)-I3A by MO, Ml, and M2 macrophages. (B) Downregulation of M2 markers following NC-I3A treatment.

[0266] As shown in FIG. 25, (A) CD206-targeted 13 A nanocarrier treatment decreases phenotypic M2 marker CD1633 in THP-1 cells. (B-D) CD206-I3A NC treatment stimulates secretion of antitumor soluble factors and cytokines.

[0267] Though effective in preclinical studies, a limitation associated with single-agent, drugloading approaches remain including development of compensatory resistance mechanisms in the TME and tumor as well as durability and robustness of therapeutic effect. Consequently, our group has developed protocols for viral engineering of CAR-M targeting epidermal growth factor receptor (EGFR) which is highly expressed in GBM. CAR-M demonstrate tumor-killing in vitro similar to CAR-T, and importantly, this tumor-killing is potently enhanced with NC-I3A cotreatment suggesting a robust mechanism of action. Given the promising in vitro efficacy of multimodal I3A small molecule immune activator and EGFR CAR-M therapy, we propose to synthesize this nanocarrier system for in vivo co-delivery of both small molecule 13 A and EGFR CAR mRNA cargo which would represent a first-in-class, tunable nanoengineering technology.

[0268] B. NC-I3A EGFR CAR mRNA engineered CAR-M represents a novel, multi- mechanistic cellular therapy and tunable, platform therapy for diverse applications. This nanoengineered CAR-M system would be a first-in-class, proprietary cell therapy that overcomes drug delivery obstacles including the blood-brain barrier and off-target toxicity and utilizes a combinatorial small molecule-mRNA therapeutic. This system may be modified to deliver drugs or mRNA of interest with potential for multidisciplinary applications in neuro-oncology as well as other disease processes with immune dysfunction.

[0269] C. The WTAS-CD206-PBAE nanocarrier used to design the NC-I3A EGFR CAR mRNA is a novel, proprietary nanotherapeutic system that can be modified for target-specific and cell-specific cell engineering. This technology offers the potential for new therapeutic strategies to enhance the efficacy of conventional immunotherapy. This nanoparticle formulation can be used to deliver drugs and reprogram other cell-types in the tumor milieu and potentially in other disease contexts.

[0270] D. Preclinical studies and early clinical trials in systemic cancers have demonstrated promise in CAR-M therapy for malignant brain tumors in adults and children. As shown in FIG. 26 and 27A. Viral transduction of THP-1 (monocytic leukemia line) produces EGFR CAR macrophage with statistically significant anti -tumor activity against T98 Glioblastoma line when combined with I3A drug compound in vitro at 5 days. FIG. 26 demonstrates Day 5 result of this in vitro experiment, while FIG. 27 demonstrates killing at indicated time points from 0 hours to 108 hours. Furthermore, intracranial delivery of I3A compound in a murine orthotopic GBM line results in prolonged survival without evidence of toxicity.

[0271] Therapeutic strategies to mitigate tumor resistance mechanisms, namely immunosuppressive programs, are needed. The multimodal mechanism of action proposed in this work strives to increase the robustness of this approach and uncover important treatment considerations to inform future clinical trials.

[0272] Nanoparticle-based system to co-deliver an immune-activating small molecule I3A and EGFR-encoding CAR mRNA in vivo to reprogram the TME, particularly TAMs towards an antitumor phenotype and generate tumor-killing CAR-M. Through the following specific aims, we will develop and characterize a novel nanotherapeutic with the potential to shift current treatment paradigms and enhance the efficacy of cell-based immunotherapy for brain tumors.

[0273] The findings from this work will establish the groundwork for a novel immune-engineering technology with the potential for diverse applications in the field of neuro-oncology and cell-based immunotherapy. Furthermore, the candidate small molecule and mRNA cargo in this study serves as a proof-of-concept for other candidate-of-interest delivery for cell engineering and TME immunomodulation that is patient-specific and disease-specific. We believe that this work will produce readily translatable and patentable technologies that have the potential to address an urgent, unmet clinical need in the treatment of malignant brain tumors.

[0274] Conventional cell-based immunotherapy approaches such as ex vivo engineering of CAR- T cells are limited by immunosuppressive programs in the TME. In situ engineering of immunosuppressive cells in the TME such as TAMs is a new frontier with potential to overcome this limitation. A dual-cargo therapeutic confers the advantage of promoting anti-tumor response through disparate mechanisms of action and thereby also decreases the likelihood of treatment resistance; in this proposal, we propose a therapeutic capable of re-education of T AMs towards an anti -tumor phenotype as well as engineering of TAMs into CAR-M. Our central hypothesis is that our novel, proprietary, first-in-class nanocarrier platform can deliver a multimodal, therapeutic payload containing an immune-activating small molecule I3A and EGFR-encoding CAR mRNA in vivo to polarize the immunosuppressive TME and simultaneously engineer tumor-killing CAR- M.

[0275] EXPERIMENTAL DESIGN & CORE UTILIZATION

[0276] (2A, 2B) We will utilize protocols established and validated by our group to synthesize a novel EGFR CAR mRNA NC and I3A-EGFR CAR mRNA NC systems. We will characterize the biophysical properties of these novel NC and optimize them for drug delivery to macrophages to ensure minimal off-target toxicity similar to prior NC iterations. We will engage the Biomedical Engineering Core to develop high-throughput, microfluidic assays to assess the targeting efficiency of these NC systems (CD206 ligand-based TAM targeting). Subsequently, we will characterize the immunoactivity of the EGFR mRNA NC and its effect on TAM immunomodulation. We will conduct in vitro polarization assays to assess the ability of this NC system to polarize TAM into an anti-tumor phenotype; we will confirm this polarization using cytokine array and flow cytometry analysis. Using an immunocompetent, orthotopic murine model of GBM, we will assess the ability of the drug delivery system to recruit and polarize TAMs in vivo following intracranial administration. We will perform immunohistochemistry at specified time-points to gauge the magnitude of immune activation and recruitment.

[0277] (2C) W e will characterize the 13 A-EGFR CAR mRNA NC-generated CAR-M through cell surface marker and cytokine array studies. We will assess the efficacy of the engineered CAR-M in tumorkilling in vitro using co-culture killing assays. Finally, we will evaluate the in vivo efficacy of the 13 A-EGFR CAR mRNA-engineered CAR-M following intracranial administration of our NC drug delivery system. On histological analysis, we will quantify the efficiency of in situ TAM polarization as well as transformation to CAR-M. We will also characterize these immune changes leveraging the GeoMx DSP platform available through the Quantitative ‘OmiCs’ core to assess longitudinal evolution of the immune microenvironment at pre-specified timepoints. Additionally, isolated single-cell suspensions at these timepoints will undergo single-cell RNA sequencing to identify differentially-expressed gene profiles among treatment groups and across different timepoints. Survival studies will be performed to determine the efficacy of this therapeutic paradigm in decreasing tumor burden and prolonging survival in our in vivo model.

[0278] Novelty & Innovation: Nanocarrier system for co-delivery of small molecule and mRNA immunotherapeutic Platform technology for in situ, multi-modal activation and genetic engineering of the TME Simultaneous TAM polarization and CAR-M paradigm to overcome therapy resistance Utilization of GeoMx DSP platform and OmiCs Core (single-cell RNA sequencing) to interrogate spatiotemporal TME evolution and immune activation

[0279] EXAMPLE

[0280] Improved nanoscale Targeting Chimeras (NTC) for Drug Delivery to CAR-T cells and Tumor Associated Macrophages

[0281] Polypropylene imine) dendrimers (PPI Dendrimers) can be used to end-cap Poly(beta- amino ester (PBAE), replacing the formerly used poly(polyethylene imine) dendrimers. The polymer chain of PBAE can be then reacted with further PPI dendrimers via CDI (carbonyl-di- imidazole). The result is a delivery polymer that is virtually covered with PPI dendrimers. See FIG. 28

[0282] The primary amine groups permit the attachment of drugs, such as Ingenol Angelate (13 A) via esterase-cleavable urethane bonds. They permit the coupling of targeting peptides as well via amide bond formation EDC using (A-ethyl-jV’-(3-dimethyl-aminopropyl)carbodiimide hydrochloride) and DMAP (4-dimethylaminopyridine) coupling. For instance, for delivery in- vivo, a targeting sequence (e.g. for CD206 in tumor-associated macrophages, KFRKAFKRFFGsG SEQ ID NO:XX) and a don’t eat me sequence (for enhancing circulation time, kGNYTCEVTELSREGKTVIELKkG SEQ ID NO:XX) can be randomly attached to the primary amines.

[0283] Furthermore, the biophysical properties of the NTCs can be fine-tuned by attaching cholesterol or (designer) lipids or carbohydrates to the primary amine groups.

[0284] After cellular uptake via endocytosis, followed by rapid escape from early endosomes, the drug is release by a human esterase in the cytosol. To facilitate a slower release, the following chemical modifications can be achieved, as illusrated in FIG. 29. All of these reactions target the -CH2-OH in Ingenol Angelate, as an example, and can be used potentially with other drugs that are derived from natural products (Ingenol class: Ingenol, Ingenol-3-Angelate (Ingenol -mebutate), Ingenol-3-hexanoate, GSK445A, Bryostatin-1; Ingenol diterpenes: gnidimacrin, SJ23B; Diacylglycerol (DAG)-like Agonists: DAG lactone compounds: LMC03, LMC07; Phorbol ester compounds: PMA, Prostratin, and DPP).

[0285] A: With reference to the below reaction as well as the scheme in FIG. 29, conversion of the primary hydroxyl group to a primary amine group. This reaction targets the achiral -CH2-OH group, which is accessible to SN2 reactions (requiring backside attach of the nucleophile phthalimide, whereas at least some sterical hindrance exists for all other (chiral) hydroxyl groups. The Mitsunobu reaction is ideal to achieve the conversion of -OH to -NH2 in very high yields:

[0286] Ph3P Phthalimide Diethyl azodicarbonate (DEAD) b) N2H4 / H2O / Ethanol

[0287] B: The attachment of the drug by means of a urea group, which is more stable than a urethane will be achieved by means of CDI (carbonyl-di-imidazole) coupling. Urea bonds are chemically more stable than urethane bonds.

[0288] C: Chemically stable amide bond formation can be achieved by means of EDC (Wethyl- M -(3 -di methyl -ami nopropyl jcarbodii mi de hydrochloride) and DMAP (4- dimethylaminopyridine) coupling.

[0289] D: A Michael addition acceptor is attached via an amide forming reaction (C).

[0290] E: A Michael addition, also known as a 1,4-addition or conjugate addition, is performed between the acceptor, which is covalently tethered to the drug, and a primary amine of the dendrimer. Amine coupling leads to thermodynamically very stable connections between dendrimer and attached drugs.

[0291] F: The direct cross coupling of alcohols can be achieved using N-Iodosuccinimide as a Precatalyst.

[0292] G: This is then followed by the formation of an amide bond with the dendrimer. EXAMPLE

[0293] Further Experimentation

[0294] Ex vivo loading of CAR T cells with WTAS-PBAE-NC-drug:

[0295] WTAS-PBAE / Dendrimers / NTC may be loaded during ex vivo manufacturing with small molecules (PKC agonists and PI3K / mTOR inhibitors, alone or in combination ) to enhance CAR T cell killing, including killing of heterogenous tumors in vitro and in vivo. Furthermore, the combination of nanocarrier - PI3K / mT0R inhibitor (such as Paxalisib, Idelalisib, duvelisib, copanlisib) and PKC agonists bound to nanocarrier with differential elution kinetics, such as COOH for Paxalisib, and Amide bound for PKC agonists, will allow for delayed activation of CAR T cells in vivo.

[0296] Embodiment where CAR T cells refers to EGFR, IL13Ra2, HER2, CD 19, CD20, CD22, CSPG4, B7-H3, BCMA, GD2, Axl, Mesothelin, R0R2, TAG-72, PSMA, PSCA, Claudinl8.2, GPC-3, ROR1 or any variation / combination thereof.

[0297] Supporting data thus far demonstrates that in vitro PKC agonists conjugated WTAS-PBAE NC:

[0298] (1) Elicits potent cytokine relese of CAR T cell in vitro including FASL.

[0299] (2) Enhances EGFR CAR T killing of MDA-MD-231 cells in vitro, in mixed EGFR KO tumor cells in vitro.

[0300] (3) Enhances CD19 CAR T killing of Raji lymphoma cell in vitro

[0301] (4) Enhance control of tumor burden of NSG mice implanted with heterogenous Raji cells (50% CD19 KO) where parental Raji cells and CD19 KO both have firefly luciferase labeled to monitor tumor burden

[0302] (5) EGFR CAR T cells loaded with WTAS-PBAE-I3A NC enhances survival against heterogenous EGFR expressing (EGFRKO 50 / 50 MDA-MD-231) cells implanted into intracranial NSG to model breast cancer brain metastases. EGFR CAR T unloaded do not result in mouse survival. This is the highest level of data supporting this claim.

[0303] (6) EGFR CAR T cells loaded with combination of nanocarrier conjugated to PI3K inhibitor (Paxalisib) via -COOH “fast release”, and nanocarrier conjugated to PKC agonist (13 A) via -Amide “slow release” demonstrates delayed release of TNF-alpha and IL-2 cytokines.

[0304] Pending experiment to demonstrate importance of delayed CAR T activation in vivo mouse models: NSG immunodeficient mice will be injected intravenously with firefly luciferase labeled tumor cells such as MDA-MD-231. Mice group will seek to establish the importance of CAR T cells in mouse survival loaded with combination PKC agonists -Amide bound, and PI3K inhibitor - COOH bound.

[0305] Group 1 : tumor plus Mock T cells

[0306] Group 2: tumor plus CAR T cells

[0307] Group 3: tumor plus CAR T cells loaded with NC-PKC agonist (Amide bound)

[0308] Group 4: tumor plus CAR T cells loaded with NC-PKC agonist (Amide bound) and NC- PI3K-inhibitor (COOH bound)

[0309] FAS-dominant negative incorporation into CAR will allow for better performing PKC- agonists loaded CAR T cells, preventing activation induced cell death:

[0310] CAR T cells co-expressing Fas Receptor (also known as CD95) dominant negative will have enhanced anti-tumor activity and CAR T cell viability when loaded with PKC-agonist- WTAS-PBAE during ex vivo manufacturing.

[0311] -FAS Dominant negative construct First 1-191 Amino acids

[0312] -CAR T - FAS construct: CAR (EGFR, IL13Ra2, HER2, CD19, CD20, CD22, CSPG4, B7-H3, BCMA, GD2, Axl, Mesothelin, R0R2, TAG-72, PSMA, PSCA, Claudinl8.2, GPC-3, R0R1 or any variation / combination thereof) followed by skip sequence (T2A, or other skip sequence) followed by dominant negative Fas Receptor.

[0313] Planned Experiment: EGFR CAR T -T2A-FAS-Dominant negative plasmid lentivirus construct will be transduced into human T cells, followed by expansion. This construct will be compared to parental EGFR CAR T cells. Following production of both constructs, each will be loaded with PKC-agonist-bound to WTAS-PBAE Nanocarrier.

[0314] EGFR CAR T- T2A-FAS-Dominant negative will have enhanced in vitro viability compared to EGFR CAR T cells when both loaded with PKC agonist-NC.

[0315] EGFR CAR T- T2A-FAS-Dominant negative will have enhanced in vivo anti-tumor activity against tumor cell lines compared to EGFR CAR T cells when both loaded with PKC agonist-NC.

[0316] Planned Experiment: EGFR CAR T-T2A-FAS DN and EGFR CAR T parental will both be loaded with WTAS-PBAE nanocarrier conjugated with PKC agonist (Ingenol-3-angelate) or empty NC alone. NSG (NOD.Cg-Prkdcscid I12rgtml Wjl / SzJ) mice will be injected with MDA- MD-231 TNBC line (mixed parental MDA-MD-231 and EGFR Knockout) to model heterogeneous tumor expression. Tumor cells will be labeled with firefly luciferase for non- invasive tumor burden monitoring.

[0317] Mice will be treated in the following groups

[0318] 1) Mock T cells (WTAS-PBAE NC_

[0319] 2) Mock T cells plus WTAS-PBAE NC-I3 A)

[0320] 3) EGFR CAR T

[0321] 4) EGCAR T cells loaded with WTAS-PBAE NC-I3A

[0322] 5) EGFR CAR T-T2A-FAS DN

[0323] 6) EGFR CAR T-T2A-FAS DN loaded with WTAS-PBAE NC-I3A

[0324] Ex vivo loading of CAR Monocytes cells with NC-PKC agonist:

[0325] CAR Monocyte ex vivo loaded with CD206-WTAS-PBAE-conjugated to small molecules (PKC agonists / PI3K Inhibitors / TGF beta Receptor kinase inhibitors ) will have enhanced antitumor activity. FIG. 30 shows, for example, a CD206-targeting PBAE nanocarrier featuring targeting peptide for CD206 and an esterase cleavable linker for Ingenol-3-angelate (13 A).

[0326] Embodiment where CAR refers to any single chain variable fragment (ScFv) which targets any combination of: EGFR, IL13Ra2, HER2, CD19, CD20, CD22, CSPG4, B7-H3, BCMA, GD2, Axl, Mesothelin, ROR2, TAG-72, PSMA, PSCA, Claudinl8.2, GPC-3, ROR1.

[0327] Evidence thus far: in vitro: PKC agonists repolarized immune suppressive M2 macrophages.

[0328] PKC agonists induce potent loss of M2 marker (CD163), and enhance pro-inflammatory cytokines

[0329] THP-1 cell line transduced with Chimeric Antigen Receptor (CAR) targeting CAR demonstrate potent anti-tumor activity when treated with PKC agonists (13 A).

[0330] Planned experiments:

[0331] Planned Experiments: THP-1 monocytic leukemia cell line will be transduced with EGFR CAR lentivirus (THP-1 EGFR CAR), will then be transduced with CD206-WTAS-PBAE- I3A. T98G Glioblastoma cell lines will be labeled with firefly luciferase and implanted intracranially in NSG (NOD.Cg-Prkdcscid I12rgtmlWjl / SzJ) mice. Mice will be treated in the following groups

[0332] 1) Mock THP-1 cell

[0333] 2) Mock THP-1 cells loaded with CD206-PBAE NC-I3A

[0334] 3) EGFR CAR transduced THP-1

[0335] 4) EGFR CAR transduced THP-1 loaded with CD206 WTAS-PBAE NC-I3A

[0336] Tumor burden will be monitored by firefly luciferase. We expect to observe statistically significant survival with group 4 vs group 3 demonstrating potent ability of NC-PKC agonist to enhance antitumor activity of CAR-monocytes.

[0337] In vivo delivery of WTAS-PBAE-PKC agonist to engineer blood monocytes:

[0338] CD206-WTAS-PBAE-PKC agonist may be delivered intravenously to target immune suppressive Myeloid derived suppressor cells, in order to improve mouse survival in advanced tumor models as a stand-alone immunotherapy, or in combination with CAR T cell therapy.

[0339] Embodiment where CAR refers to any single chain variable fragment (ScFv) which targets any combination of: EGFR, IL13Ra2, HER2, CD 19, CD20, CD22, CSPG4, B7-H3, BCMA, GD2, Axl, Mesothelin, R0R2, TAG-72, PSMA, PSCA, Claudinl8.2, GPC-3, R0R1.

[0340] Evidence thus far: in vitro: PKC agonists repolarized immune suppressive M2 macrophages.

[0341] - PKC agonists induce potent loss of M2 marker (CD163), and enhance pro-inflammatory cytokines

[0342] THP-1 cell line transduced with Chimeric Antigen Receptor (CAR) targeting CAR demonstrate potent anti-tumor activity when treated with PKC agonists (13 A).

[0343] Planned experiments:

[0344] Planned Experiments: We will utilize a mouse brain tumor model (GL261 and KR158B) orthotopic intracranial implanted, labeled with firefly luciferase and treated with intravenous CD206-NC-I3A and evaluation of murine survival. We will also evaluate anti-tumor activity in combination with murine IL13Ra2 CAR T cells.

[0345] (1) Vehicle control

[0346] (2) CD206-WTAS-PBAE-PKC agonist

[0347] (3) Murine IL13Ra2 CAR T cells. (4) Murine IL13Ra2 CAR T cells plus CD206-WTAS-PBAE-PKC agonist

[0348] In vivo delivery of NC loaded with CAR Transgene and / or PKC agonist:

[0349] In these experiments, we would like to demonstrate the potency of combination CAR Transgene and PKC agonist after intravenous delivery to modify blood immune cells (such as T cells or monocytes) for anti-tumor efficacy.

[0350] Planned Experiment: Herein, we will employ CD206-WTAS-PBAE NC loaded with genetic material (mRNA or plasmid) encoding EGFR Chimeric Antigen Receptor as well as PKC agonist- I3A. We will employ a humanized mouse model. CD206 will be used to target blood monocytes. We predict only the combination will have potent anti-tumor activity in metastatic EGFR expressing MDA-MD-231 cancer model. Thus, mice will be implanted with MDA-MD- 231 human breast cancer cells labeled with firefly luciferase. Treatment groups are below:

[0351] 1) Tumor only

[0352] 2) CD206-WTAS-PBAE-EGFR-CAR

[0353] 3) CD206-WTAS-PBAE-PKC agonist

[0354] 4) Combination CD206-WTAS-PBAE-CAR-PKC agonist

[0355] Mice will be monitored for tumor burden by bioluminescent imaging as well as mouse survival. We predict the combination will result in enhanced survival of mice.

[0356] We also predict in vivo delivery of CAR transgene will have more significant tumor control compared to tumor only. ex vivo delivery of immune cells (T cells) with NC ( loaded with CAR Transgene and / or PKC agonist:

[0357] In these experiments, we would like to demonstrate the potency of combination CAR Transgene and PKC agonist after loading into immune cells (such as human T cells) prior to infusion in murine immunodeficient mouse model.

[0358] Planned Experiment: Herein, we will employ WTAS-PBAE NC loaded with genetic material (mRNA or plasmid) encoding EGFR Chimeric Antigen Receptor as well as PKC agonist- 13 A. We will employ an immunodeficient mouse model. We predict only the combination will have potent anti-tumor activity in metastatic EGFR expressing MDA-MD-231 cancer model. Thus, mice will be implanted with MDA-MD-231 human breast cancer cells labeled with firefly luciferase. Treatment groups are below:

[0359] 1) Tumor plus Mock T cells

[0360] 2) WTAS-PBAE-EGFR-CAR loaded into human T cells

[0361] 3) WTAS-PBAE-PKC agonist loaded into human T cells 4) Combination WTAS-PBAE-CAR-PKC agonist loaded into human T cells

[0362] Mice will be monitored for tumor burden by bioluminescent imaging as well as mouse survival.

Claims

CLAIMS:

1. A therapeutic composition comprising drug-loaded nanoparticles for modulating CAR-T cell or CAR-M cell activity, wherein the drug-loaded nanoparticles each comprise a cellpenetrating peptide (WTAS or rp-182) or a fragment thereof and a modified poly(P-amino ester) (PBAE) polymer associated or assembled with one or more cell modulating agents.

2. The composition of claim 1, wherein the drug-loaded nanoparticles each further comprise a detectable moiety.

3. The composition of claim 2, wherein the detectable moiety is a fluorescent dye.

4. The composition of claims 1-3, said composition comprising enhanced CAR-T cells or CAR-M cells comprising said drug-loaded nanoparticles.

5. The composition of claim 4, wherein said enhanced CAR-T cells or CAR-M cell s comprise the drug-loaded nanoparticles within the cytoplasm wherein the one or more cell modulating agents is released to thereby modulate the activity of the enhanced CAR-T cells or CAR-M cells.

6. A method of modulating CAR-T cell or CAR-M cell activity to prepare enhanced CAR-T cells or CAR-M cells, the method comprises incubating drug-loaded nanoparticles with T cells, macrophages, or monocytes for a sufficient period of time to allow the drug-loaded nanoparticles to be taken up by the T cells, macrophages, or monocytes ex vivo, wherein the drug- loaded nanoparticles each comprise a cell-penetrating peptide (WTAS or rp-182) or a fragment thereof and a modified poly(P-amino ester) (PBAE) polymer associated or assembled with one or more cell modulating agents.

7. The method of claim 6, wherein said drug-loaded nanoparticles are taken up within the cytoplasm of said T cells, macrophages, or monocytes, wherein the one or more cell modulating agents is released to thereby modulate the activity of the transfected T cells, macrophages, or monocytes.

8. The composition or method of any one of the preceding claims, where the one or more cell modulating agents is a small molecule drug.

9. The composition or method of any one of the preceding claims, where the one or more cell modulating agents is a biologic drug.

10. The composition or method of any one of the preceding claims, where the one or more cell modulating agents is a kinase-blocker.11 . The composition or method of any one of the preceding claims, where the one or more cell modulating agents is a TGFp receptor inhibitor.

12. The composition or method of any one of the preceding claims, where the one or more cell modulating agents is a DCLK1 (Doublecortin Like Kinase 1) inhibitor.

13. The composition or method of any one of the preceding claims, where the one or more cell modulating agents is an MK2 inhibitor.

14. The composition or method of any one of the preceding claims, where the one or more cell modulating agents is a lethality inducer against p53 deficient cells.

15. The composition or method of any one of the preceding claims, where the one or more cell modulating agents is an MIF inhibitor.

16. The composition or method of any one of the preceding claims, where the one or more cell modulating agents is a PKC agonist.

17. The composition or method of any one of the preceding claims, where the one or more cell modulating agents is Ingenol 3-angelate (I3A).

18. The composition or method of any one of the preceding claims, where the one or more cell modulating agents is a cell supplement / nutrient or energy resource, such as amino acid(s), fatty acid(s), and / or carboxylic acid(s).

19. The composition or method of any one of the preceding claims, wherein the drug- loaded nanoparticles further comprise a nucleic acid encoding for a chimeric antigen receptor.

20. A method of treating a subject diagnosed with cancer, the method comprising administering to a subject in need thereof an effective amount of enhanced CAR-T cells or CAR- M cells prepared according to any one of claims 6-19.

21. The method of claim 20, wherein said T cells, macrophages, or monocytes are autologous cells obtained from a biological sample from said subject.

22. The method of claim 20, wherein said T cells, macrophages, or monocytes are allogeneic cells obtained from a donor or cell line.

23. A method of treating a subject diagnosed with cancer, the method comprising administering to a subject in need thereof an effective amount of a composition according to any one of claims 1-5, wherein the drug-loaded nanoparticles further comprise a nucleic acid encoding for a chimeric antigen receptor for in vivo engineering of CAR-T cells or CAR-M cells in said subject.

24. A method of modulating CAR-T cell or CAR-M cell activity to prepare enhanced CAR-T cells or CAR-M cells, the method comprises contacting T cells, macrophages, or monocytes for a sufficient period of time with drug-loaded nanoparticles to allow the drug-loaded nanoparticles to be taken up by the T cells, macrophages, or monocytes to yield enhanced CAR-T cells or CAR-M cells, wherein the drug-loaded nanoparticles each comprise a cell -penetrating peptide (WTAS or rp-182) or a fragment thereof and a modified poly(P-amino ester) (PBAE) polymer associated or assembled with one or more cell modulating agents, wherein said one or more cell modulating agents comprise at least one cell modulating agent for inhibiting cytotoxic activity of said enhanced CAR-T cells or CAR-M cells and at least one cell modulating agent for activating cytotoxic activity of said enhanced CAR-T cells or CAR-M cells.

25. The method of claim 25, wherein said at least one cell modulating agent for inhibiting cytotoxic activity of said enhanced CAR-T cells or CAR-M cells is conjugated with said nanoparticle via a carboxyl linkage and wherein said at least one cell modulating agent for activating cytotoxic activity of said enhanced CAR-T cells or CAR-M cells is conjugated with said nanoparticle via an amide or ether linkage.

26. An enhanced CAR-T cell or CAR-M cell comprising drug-loaded nanoparticles in cytoplasm of the cell, each drug-loaded nanoparticle comprising a cell-penetrating peptide (WTAS or rp-182) or a fragment thereof and a modified poly(P-amino ester) (PBAE) polymer associated or assembled with one or more cell modulating agents, wherein the one or more cell modulating agents is released into the cytoplasm to thereby modulate the activity of the enhanced CAR-T cells or CAR-M cells.

27. The enhanced CAR-T cells or CAR-M cells of claim 26, where the one or more cell modulating agents is a small molecule drug.

28. The enhanced CAR-T cells or CAR-M cells of claim 26, where the one or more cell modulating agents is a biologic drug.

29. The enhanced CAR-T cells or CAR-M cells of claim 26, where the one or more cell modulating agents is a kinase-blocker.

30. The enhanced CAR-T cells or CAR-M cells of claim 26, where the one or more cell modulating agents is a TGFp receptor inhibitor.

31. The enhanced CAR-T cells or CAR-M cells of claim 26, where the one or more cell modulating agents is a DCLK1 (Doublecortin Like Kinase 1) inhibitor.

32. The enhanced CAR-T cells or CAR-M cells of claim 26, where the one or more cell modulating agents is an MK2 inhibitor.

33. The enhanced CAR-T cells or CAR-M cells of claim 26, where the one or more cell modulating agents is a lethality inducer against p53 deficient cells.

34. The enhanced CAR-T cells or CAR-M cells of claim 26, where the one or more cell modulating agents is an MIF inhibitor.

35. The enhanced CAR-T cells or CAR-M cells of claim 26, where the one or more cell modulating agents is a PKC agonist.

36. The enhanced CAR-T cell, CAR macrophage, or CAR monocyte of claim 26, where the one or more cell modulating agents is Ingenol 3-angelate (13 A).

37. The enhanced CAR-T cells or CAR-M cells of claim 26, where the one or more cell modulating agents is a cell supplement / nutrient or energy resource, such as amino acid(s), fatty acid(s), and / or carboxylic acid(s).

38. An enhanced CAR-T cells or CAR-M cells comprising a combination of two or more different cell modulating agents according to any one of claims 26-37, preferably wherein at least one cell modulating agent is conjugated with the nanoparticle nanocarrier via a fast-release linkage and wherein at least another of said cell modulating agents is conjugated with the nanoparticle nanocarrier via a slow-release linkage.

39. The enhanced CAR-T cells or CAR-M cells of any one of claims 26-38, wherein the drug-loaded nanoparticles further comprise a nucleic acid encoding for a chimeric antigen receptor.

40. Use of enhanced CAR-T cells or CAR-M cells of any one of claims 26-38 for treatment of a subject in need thereof.

41. The enhanced CAR-T cells or CAR-M cells of any one of claims 26-38 for use in treating cancer in a subject in need thereof, and preferably wherein the cancer is a brain cancer or a blood cancer.

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