Phosphoantigen polymeric compositions and methods for activating gamma delta t cells for cancer therapy

Abstract

The present disclosure relates to polymeric compositions and methods for activating gamma delta T cells, specifically Vγ9Vδ2 T cells, in cancer immunotherapy. These compositions include a cell-targeting moiety and a polymer incorporating phosphoantigens (pAgs) for targeted delivery to tumor cells. The method involves administering an effective amount of these compositions to a subject, allowing controlled release of pAgs in the tumor microenvironment. This approach overcomes limitations of systemic pAg administration and can be used alone or with adoptive transfer of Vγ9Vδ2 T cells or PBMCs. The treatment may involve repeated administrations and is applicable to various cancer types. This disclosure also covers methods for synthesizing these compositions and their use in cancer treatment applications.

Description

PATENT ATTORNEY DOCKET NO. JHU4800-1WO PHOSPHOANTIGEN POLYMERIC COMPOSITIONS AND METHODS FOR ACTIVATING GAMMA DELTA T CELLS FOR CANCER THERAPY CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 709,329, filed October 18, 2024. The disclosure of the prior applications is considered part of and is herein incorporated by reference in the disclosure of this application in its entirety. FIELD OF THE INVENTION

[0002] The present disclosure relates generally to compositions and methods for cancer immunotherapy and more specifically to the use of polymeric nanoparticle compositions incorporating phosphoantigens (pAgs) for targeted delivery to tumor cells. BACKGROUND INFORMATION

[0003] Cancer remains a leading cause of death worldwide, despite significant advances in treatment options. Traditional cancer therapies, such as chemotherapy and radiation, often come with severe side effects and may not be effective against all types of cancer. This has led to an increased focus on immunotherapy as a promising approach to cancer treatment.

[0004] One area of particular interest in cancer immunotherapy is the activation of gammadelta T cells, specifically the V 9V 2 subset. These cells have shown significant tumor-killingpotential and can recognize tumor cells in a major histocompatibility complex (MHC)- independent manner. This makes them attractive candidates for broad-spectrum cancer therapy.

[0005] V 9V 2 T cells are activated by small molecules called phosphoantigens (pAgs).While these cells have shown promise in preclinical studies, translating this potential into effective clinical treatments has been challenging. One major hurdle is the rapid clearance of pAgs from the body when administered systemically, which limits their ability to effectivelyactivate V 9V 2 T cells in the tumor microenvironment.

[0006] Adoptive cell therapies using ex vivo expanded V 9V 2 T cells have been exploredas a potential solution. However, these approaches are often prohibitively expensive and logistically complex, limiting their widespread applicability. Moreover, maintaining the 1 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO activation and tumor-targeting capabilities of these cells after reinfusion into patients remains a significant challenge.

[0007] There is a critical need for approaches that can effectively deliver pAgs to the tumormicroenvironment to activate V 9V 2 T cells in situ. Such a strategy could potentiallyovercome the limitations of both systemic pAg administration and adoptive cell therapies. Ideally, this approach would allow for targeted delivery of pAgs to tumor cells, minimize off-target effects, and provide sustained activation of V 9V 2 T cells.

[0008] The development of a targeted delivery system for pAgs could significantly enhancethe efficacy of V 9V 2 T cell-based cancer immunotherapies. This would potentially providea more accessible and effective treatment option for a wide range of cancer types, addressing an important unmet need in cancer therapy. SUMMARY

[0009] The present disclosure provides compositions and methods for activating gamma deltaT cells, specifically V 9V 2 T cells, for cancer immunotherapy. This approach utilizespolymeric compositions incorporating phosphoantigens (pAgs) for targeted delivery to tumor cells. The polymeric composition design allows for efficient encapsulation of pAgs and their controlled release in the tumor microenvironment, overcoming the limitations of systemic pAgadministration. These compositions can activate V 9V 2 T cells both in vitro and in vivo,harnessing their potent anti-tumor activity for treating various types of cancer.

[0010] In one embodiment, the present disclosure provides a polymeric composition including: a) a cell-targeting moiety; and b) a polymer composed of monomer units incorporating phosphoantigen (pAg) into the structure of the polymer.

[0011] In some aspects, the cell-targeting moiety is a tumor-targeting moiety.

[0012] In some aspects, the composition is in the form of a nano to microstructure.

[0013] In some aspects, the nano to microstructure is a nanoparticle.

[0014] In some aspects, the polymer is hydrophobic.

[0015] In some aspects, the polymer is hydrophilic.

[0016] In some aspects, the pAg is incorporated into the backbone of the hydrophobic polymer via hydrolytically degradable ester linkages.

[0017] In some aspects, the pAg is incorporated into the backbone or onto the side of the hydrophobic polymer via bio-degradable linkages. 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO

[0018] In some aspects, the pAg is incorporated into the backbone or onto the side of the hydrophilic polymer via bio-degradable linkages.

[0019] In some aspects, the bio-degradable linkages are ester linkages.

[0020] In some aspects, the tumor-targeting moiety is hyaluronic acid (HA) or cyclic internalizing RGD peptide (ciRGD).

[0021] In some aspects, the tumor-targeting moiety is hydrophilic.

[0022] In some aspects, the hydrophobic polymer includes poly(HPMA-pAg).

[0023] In some aspects, the phosphoantigen is a hydrophobically protected phosphoantigen methacrylate monomer (pAgMA).

[0024] In some aspects, the polymer further includes a biologically inert comonomer. In some aspects, the polymer further includes another small molecule pharmaceutical agent.

[0025] In some aspects, the nano to microstructure has a size range of about 200nm to 30 microns.

[0026] In some aspects, the nano to microstructure has a size range of about 80 nm to 30 microns.

[0027] In some aspects, the nano to microstructure has a size range of about 80 nm to 200 nm.

[0028] In another embodiment, the present disclosure provides a method of stimulating an immune response to a tumor including administering to a subject in need thereof an effective amount of the disclosed polymeric composition, wherein the polymeric composition activatesV 9V 2 T cells.

[0029] In some aspects, the disclosed method further includes administering V 9V 2 T cellsto the subject.

[0030] In some aspects, the V 9V 2 T cells are administered to the subject prior toadministering the polymeric composition, and wherein the polymeric composition activates ormaintains the activation of the administered V 9V 2 T cells.

[0031] In some aspects, the V 9V 2 T cells are autologous to the subject.

[0032] In some aspects, the present disclosure provides a method of preparing andadministering activated V 9V 2 T cells to a subject, the method including: a) activatingV 9V 2 T cells within the peripheral blood mononuclear cells (PBMCs) of the subject; b)isolating the activated V 9V 2 T cells from the remaining PBMCs; c) administering theisolated, activated V 9V 2 T cells to the subject; and d) subsequently administering to the3 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO subject an effective amount of the disclosed polymeric composition to maintain or enhanceactivation of the administered V 9V 2 T cells.

[0033] In some aspects, the activation of the V 9V 2 T cells includes exposing the PBMCsto the disclosed polymeric composition.

[0034] In some aspects, the polymeric composition is administered intratumorally, subcutaneously, or systemically.

[0035] In another embodiment, the present disclosure provides a method of treating tumor in a subject including administering to a subject with the tumor an effective amount of the disclosed polymeric composition.

[0036] In some aspects, the method is for the treatment of tumors such as melanoma, breast cancer, lung cancer, prostate cancer, colorectal cancer, pancreatic cancer, ovarian cancer, liver cancer, brain cancer, leukemia, lymphoma, bladder cancer, kidney cancer, thyroid cancer, esophageal cancer, stomach cancer, cervical cancer, endometrial cancer, head and neck cancer, sarcoma, multiple myeloma, gallbladder cancer, bile duct cancer, testicular cancer, skin cancer, and nasopharyngeal cancer.

[0037] In some aspects, the tumor is expressing butyrophilins 2A1 and 3A1 (BTN2A1, BTN3A1).

[0038] In some aspects, the method further includes administering V 9V 2 T cells or PBMCsto the subject.

[0039] In some aspects, the V 9V 2 T cells or PBMCs are autologous.

[0040] In another embodiment, the present disclosure provides a method of activating gammadelta T cells in vitro, including contacting V 9V 2 T cells with the disclosed polymericcomposition in the presence of V 9V 2 T cell activating cells, thereby activating the gammadelta T cells.

[0041] In some aspects, the V 9V 2 T cell activating cells are tumor cells.

[0042] In some aspects, the V 9V 2 T cell activating cells are antigen presenting cells.

[0043] In some aspects, the V 9V 2 T cells are isolated from PBMCs.

[0044] In some aspects, the method further includes measuring interferon gamma (IFN )production as an indicator of V 9V 2 T cell activation.

[0045] In some aspects, the V 9V 2 T cells are expanded from PBMCs prior to the contactingstep. 4 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO

[0046] In some aspects, the expansion of the V 9V 2 T cells includes activating the V 9V 2T cells with zoledronate; and maintaining the activated V 9V 2 T cells with human interleukin2 (hIL-2).

[0047] In another embodiment, the present disclosure provides a method of stimulating gamma delta T cells in vivo, including administering to a subject the disclosed polymeric composition.

[0048] In some aspects, the polymeric composition activates V 9V 2 T cells in the subject.

[0049] In some aspects, the method further includes administering V 9V 2 T cells or PBMCsto the subject.

[0050] In some aspects, the V 9V 2 T cells or PBMCs are autologous.

[0051] In some aspects, the method further includes measuring tumor size or survival rate asan indicator of V 9V 2 T cell activation and anti-tumor activity.

[0052] In another embodiment, the present disclosure provides a method of synthesizing the disclosed polymeric composition including a) synthesizing a phosphoantigen methacrylate monomer (pAgMA); b) incorporating the pAgMA into a hydrophobic polymer backbone via Reversible Addition-Fragmentation chain Transfer (RAFT) polymerization; and c) conjugating a hydrophilic tumor-targeting moiety to the hydrophobic polymer to form a block copolymer.

[0053] In some aspects, the hydrophilic tumor-targeting moiety is functionalized with a terminal azide for copper-free click chemistry conjugation to the hydrophobic polymer.

[0054] In another embodiment, the present disclosure provides a targeted, self-assembling nanoparticle for eliminating tumor cells via in situ activation of T cells, including a) a phosphoantigen (pAg) monomer suitable for Reversible Addition-Fragmentation chain Transfer (RAFT) polymerization; and b) a RAFT polymer including the pAg monomer and a tumor-targeting moiety.

[0055] In some aspects, the pAg monomer is a hydrophobically protected phosphoantigen methacrylate monomer (pAgMA).

[0056] In some aspects, the tumor-targeting moiety is hyaluronic acid (HA) or cyclic internalizing RGD peptide (ciRGD).

[0057] In some aspects, the RAFT polymer includes poly(HPMA-pAg).

[0058] In another embodiment, the present disclosure provides a method of analyzing targeting efficiency of a phosphoantigen (pAg) nanoparticle in vitro, including a) quantifying selective uptake of targeted pAg nanoparticles by tumor cells compared to untargeted control 5 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WOnanoparticles; and b) verifying activation of V 9V 2 T cells with a tumor-cytotoxic phenotypevia a co-culture assay with V 9V 2 T cells expanded and isolated from PBMCs.

[0059] In some aspects, the quantification of selective uptake includes a) incubating fluorescein-loaded pAg nanoparticles with tumor cells at varying doses, times, and temperatures; and b) measuring cellular uptake of fluorescent nanoparticles via flow cytometry.

[0060] In some aspects, the verification of activation of V 9V 2 T cells includes a) co-culturing pAg nanoparticle-treated tumor cells with V 9V 2 T cells; b) determining tumor cellviability; and / or c) quantifying interferon gamma (IFN ) production.

[0061] In some aspects, the tumor cells express butyrophilins 2A1 and / or 3A1 (BTN2A1, BTN3A1).

[0062] In some aspects, number of monomer units in the polymer ranges from 1-10000.

[0063] In some aspects, the monomer units of the polymer include the following structure:n is an integer ranging from 1 to 200; m is an integer ranging from 1 to 100; Y is CH2, NH or O; R1 is a sugar, polyethylene glycol (PEG), poly(nucleic acid), peptide, antibody, protein, positively charged synthetic polymer, or aliphatic linkage; R2 is an alkyl, poly(ethylene glycol), or ethyl ether R3 is an alkyl group, ester, trithiocarbonate, mixed thiocarbonate, dithio- oxycarbonate, dithio-amine carbonate; R4 is an alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, cycloalkane, amine, or alcohol; 6 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO R5 is an alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, cycloalkane, amine, or alcohol; R6 is a first protecting group; and R7 is a second protecting group.

[0064] In some aspects, the monomer units of the polymer include the following structure:, wherein: n is an integer ranging from 1 to 200; m is an integer ranging from 1 to 100; Y is CH2, NH or O; R1 is an alkyl, poly(ethylene glycol), or ethyl ether R2 is an alkyl group, ester, trithiocarbonate, mixed thiocarbonate, dithio- oxycarbonate, dithio-amine carbonate; R3 is an alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, cycloalkane, amine, or alcohol; R4 is an alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, cycloalkane, amine, or alcohol; R5 is a first protecting group; and R6 is a second protecting group. 7 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO

[0065] In some aspects, the monomer units of the polymer include the following structure:, wherein: n is an integer ranging from 1 to 1000, preferably 1 to 200; m is an integer ranging from 1 to 1000, preferably 1 to 200; Y is CH2, NH, OH, NH2, or O; Z is CH2, NH, OH, NH2, or O; R1 is a sugar, oligosaccharide, polyethylene glycol (PEG), poly(nucleic acid), peptide, antibody, protein, synthetic polymer, natural polymer, or aliphatic linkage; R2 is an alkyl or ethyl ether, heterocycle, cycloalkene, cycloalkyne, cycloalkane, conjugated heterocycles, or conjugated cylcloalkenes; R3 is an alkyl group, thioester trithiocarbonate, mixed thiocarbonate, dithio- oxycarbonate, dithio-amine carbonate; R4 is an alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, cycloalkane, amine, or alcohol; R5 is a protonR6 is an oxygen or first protecting group or; R7 is an oxygen or second protecting group; R8 is an oxygen or third protecting group; R9 is an oxygen or fourth protecting group; and R10 is an alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, and cycloalkane. 8 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO

[0066] In some aspects, the monomer units of the polymer include the following structure:n is an integer ranging from 1 to 200; m is an integer ranging from 1 to 100; Y is CH2, NH or O; R1 is a sugar, polyethylene glycol (PEG), poly(nucleic acid), peptide, antibody, protein, positively charged synthetic polymer, or aliphatic linkage; R2 is an alkyl, poly(ethylene glycol), or ethyl ether R3 is an alkyl group, ester, trithiocarbonate, mixed thiocarbonate, dithio- oxycarbonate, dithio-amine carbonate; R4 is an alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, cycloalkane, amine, or alcohol; R5 is a carbon, alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, cycloalkane, amine, or alcohol; R7 is an oxygen or protecting group; R8 is an oxygen or protecting group; and R9 is an oxygen or protecting group. 9 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO

[0067] In some aspects, the monomer units of the polymer include the following structure:, wherein: n is an integer ranging from 1 to 200; m is an integer ranging from 1 to 100; Y is CH2, NH or O; R1 is an alkyl, poly(ethylene glycol), or ethyl ether R2 is an alkyl group, ester, trithiocarbonate, mixed thiocarbonate, dithio- oxycarbonate, dithio-amine carbonate; R3 is an alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, cycloalkane, amine, or alcohol; R4 is a carbon, alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, cycloalkane, amine, or alcohol; R5 is a first protecting group; R6 is an oxygen or protecting group; and R7 is an oxygen or protecting group. 10 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO

[0068] In some aspects, the monomer units of the polymer include the following structure:, wherein: n is an integer ranging from 1 to 1000, preferably 1 to 200; m is an integer ranging from 1 to 1000, preferably 1 to 200; Y is CH2, NH, OH, NH2, or O; Z is CH2, NH, OH, NH2, or O; R1 is a sugar, oligosaccharide, polyethylene glycol (PEG), poly(nucleic acid), peptide, antibody, protein, synthetic polymer, natural polymer, or aliphatic linkage; R2 is an alkyl or ethyl ether, heterocycle, cycloalkene, cycloalkyne, cycloalkane, conjugated heterocycles, or conjugated cylcloalkenes; R3 is an alkyl group, thioester trithiocarbonate, mixed thiocarbonate, dithio- oxycarbonate, dithio-amine carbonate; R4 is an alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, cycloalkane, amine, or alcohol; R5 is a protonR6 is an oxygen or first protecting group orR7 is an oxygen or second protecting group; R8 is an oxygen or third protecting group; R9 is an oxygen or fourth protecting group; and R10 is an alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, and cycloalkane. 11 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO

[0069] In some aspects, the first protecting group and / or the second protecting group includes alcohols, methoxymethyl (MOM), or pivaloyloxymethyl (POM). BRIEF DESCRIPTION OF THE DRAWINGS

[0070] FIG. 1 illustrates a schematic representation of a self-assembled block copolymerphosphoantigen (pAg) delivery vehicle for V 9V 2 T cell programming and activation.

[0071] FIG. 2 illustrates a schematic representation of mechanism of action for V 9V 2 Tcell activation and tumor cell killing.

[0072] FIG. 3 illustrates an exemplary synthetic scheme for a hydrophobically protected phosphoantigen methacrylate monomer (pAgMA).

[0073] FIG. 4 illustrates an exemplary degradation of pAgMA over time in physiological conditions to pAg.

[0074] FIG. 5 illustrates a bar graph comparing relative viability of tumor cells after treatment with pAgMA or zoledronate. Viability normalized to untreated co-culture control. Statistical analysis was performed by One-way ANOVA. ****p<0.0001.

[0075] FIG. 6 illustrates an exemplary synthetic scheme for dibenzocyclooctyne (DBCO) containing reversible addition-fragmentation transfer (RAFT) agent (denoted as (5)).

[0076] FIG.7 illustrates an exemplary synthetic scheme for HA-poly(HPMA-pAg) (denoted as (8)).

[0077] FIG. 8 illustrates an exemplary synthetic scheme for RGD-poly(HPMA-pAg) (denoted as (10)).

[0078] FIG. 9 illustrates an exemplary synthetic scheme for PEG-poly(HPMA-pAg) (denoted as (12)).

[0079] FIG. 10 illustrates a representative transmission electron microscopy (TEM) image of HA-p(HPMA-pAg) particles.

[0080] FIG. 11 illustrates a graph comparing the uptake of FI-HA-pAg-NP at the indicated temperature and time as well as in the presence or absence of HA exposure measured by flow cytometry.

[0081] FIG. 12 illustrates a graph comparing the uptake of FI-RGD-pAG-NP and FI-PEG- pAG-NP at the indicated temperature measured by flow cytometry. Statistical analysis was performed by One-way ANOVA. *p<0.0001, comparison among the indicated four groups. 12 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO

[0082] FIG. 13 illustrates a bar graph comparing relative viability of tumor cells aftertreatment with therapeutic NPs, zoledronate, or pAgMA and co-culture with V 9V 2 T cellsisolated from peripheral blood mononuclear cells (PBMCs). Statistical analysis was performed by One-way ANOVA. ****p<0.0001, compared with untreated group.

[0083] FIG.14 illustrates a bar graph comparing the expression of IFN in cell culture mediaof tumor cells after treatment with RGD-decorated nanoparticles or phosphoantigen monomerand co-culture with V 9V 2 T cells. Statistical analysis was performed by One-way ANOVA.****p<0.0001, compared with untreated group.

[0084] FIGs.15A-15C illustrate relative viability of PC3, OVCAR3, and MeWo cells treatedwith RGD-P3NP or pAgMA after coculture with V 9V 2 T cells. FIG. 15A is a graphillustrating relative viability of PC3 cells treated with RGD-P3NP or pAgMA after 72hrcoculture with V 9V 2 T cells, normalized to equivalently treated mono-cultured cells. FIG.15B is a graph illustrating relative viability of OVCAR3 cells treated with RGD-P3NP orpAgMA after 72hr coculture with V 9V 2 T cells, normalized to equivalently treated mono-cultured cells. FIG. 15C is a graph illustrating relative viability of MeWo cells treated withRGD-P3NP or pAgMA after 72hr coculture with V 9V 2 T cells, normalized to equivalentlytreated mono-cultured cells. Ordinary one-way ANOVA with Dunnett's multiple comparisons test, comparisons to untreated control, * = p<0.0001. All other comparisons ns.

[0085] FIGs. 16A-16D illustrate surface expression of CD27, CD28.2, and CD107a ofV 9V 2 cells treated with RGD-P3NPs. FIG. 16A is a graph illustrating surface expressionof CD27 of V 9V 2 cells treated with RGD-P3NPs as determined via flow cytometry. FIG.16B is a graph illustrating surface expression of CD28.2 of V 9V 2 cells treated with RGD-P3NPs as determined via flow cytometry. FIG. 16C is a graph illustrating surface expressionof CD107a of V 9V 2 cells treated with RGD-P3NPs as determined via flow cytometry. FIG.16D is a graph illustrating levels of IFN in the supernatant media of cultures in (FIG. 15A)as measured by ELISA. Statistical comparisons to untreated control, ** = p<0.01; **** = p < 0.0001, all other comparisons ns. DETAILED DESCRIPTION

[0086] The present disclosure provides compositions and methods for treating cancer usingphosphoantigen (pAg) nanoparticles to activate V 9V 2 T cells, as an illustrative example.This approach utilizes polymeric nanoparticle compositions incorporating pAgs for targeted 13 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO delivery to tumor cells, offering a potentially more effective and versatile alternative to current cancer immunotherapies. The nanoparticle design allows for efficient encapsulation and controlled release of pAgs in the tumor microenvironment, overcoming limitations of systemicpAg administration. These compositions can activate V 9V 2 T cells both in vitro and in vivo,harnessing their potent anti-tumor activity for treating various types of cancer.

[0087] Before the present compositions and methods are described, it is to be understood that this invention is not limited to particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular aspects only, and is not intended to be limiting, since the scope of the present invention will be limited only in the appended claims.

[0088] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0089] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, it will be understood that modifications and variations are encompassed within the spirit and scope of the instant disclosure. The preferred methods and materials are now described.

[0090] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” include one or more methods, and / or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0091] As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0092] The present disclosure provides a polymeric composition including a cell-targeting moiety and a polymer composed of monomer units incorporating phosphoantigen (pAg) intothe structure of the polymer. This composition is designed to activate V 9V 2 T cells for cancerimmunotherapy. 14 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO

[0093] The term “cell-targeting moiety” refers to a specific molecular component or functional group within a larger compound (e.g., a drug, nanoparticle, or imaging agent) that is designed to recognize and bind to specific cells or cellular structures. These moieties are crucial in targeted therapies, diagnostics, and drug delivery systems because they help ensure that the therapeutic or diagnostic agent reaches the intended cells (e.g., cancer cells, immune cells, or cells expressing a particular receptor).

[0094] The term “phosphoantigen” “pAg” refers to a small molecule that contains a phosphate group and can activate some immune cells, particularly a subset of T cells knownas V 9V 2 T cells in humans. pAg molecules are important in immunology and cancer becausethey can stimulate immune responses without the need for classical antigen presentation via MHC molecules.

[0095] V 9V 2 T cells refers to a subset of T cells, which are part of the human immunesystem. T cells recognize antigens in a non-MHC-restricted manner, making them especiallyinteresting for immunotherapy and infectious disease. T cells express a V 9 and V 2 T-cellreceptor (TCR) chain and respond to pAgs. Examples of pAgs that V 9V 2 T cells responseto include but are not limited to HMBPP, IPP, DMAPP, aminobisphosphonates such as zoledronate, pamidronate, and risedronate, as well as synthetic analogs of HMBPP such as phosphonodiamidate prodrugs and fluorinated derivatives. Molecules on antigen-presenting cells such as BTN3A1 and BTN2A1 help present pAgs to the TCR.

[0096] The polymeric composition is typically in the form of a nano to microstructure, preferably a nanoparticle. The polymer is hydrophobic, with the pAg incorporated into the backbone of the hydrophobic polymer via hydrolytically degradable ester linkages. This design allows for controlled release of pAg in the tumor microenvironment.

[0097] The present disclosure describes a strategy to hydrophobically protect phosphoantigens (pAgs). This approach enhances the uptake and potency of pAgs compared to their native small molecule form. The disclosure includes a scaled-up synthesis process with an additional step that incorporates a methacrylate group directly onto the pAg. This process produces a hydrophobically protected pAg methacrylate monomer (pAgMA) that can be incorporated into a RAFT polymer. The pAgMA monomer is designed to degrade at its ester linkages under physiological conditions, releasing free pAg.

[0098] In some aspects, the cell-targeting moiety is preferably a tumor-targeting moiety. In some aspects, the tumor-targeting moiety includes hyaluronic acid (HA),cyclic internalizing 15 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO RGD peptide (ciRGD), or a combination thereof. These targeting moieties may be hydrophilic, creating a block copolymer structure that self-assembles into nanoparticles in aqueous environments.

[0099] The hydrophobic polymer typically includes poly(HPMA-pAg), where HPMA is N- (2-hydroxypropyl)methacrylamide. The phosphoantigen is preferably a hydrophobically protected phosphoantigen methacrylate monomer (pAgMA).

[0100] Reversible addition fragmentation chain-transfer polymerization (RAFT) is used to create polymers with high control of polymer length and low polydispersity. RAFT agent customization allows for click chemistry handles for post-polymerization modifications.

[0101] In some aspects, the composition is in the form of a nano to microstructure. In some aspects, the nano to microstructure is a nanoparticle. The nano to microstructure typically has a size range of about 80 nm to 30 microns, preferably about 200 nm to 30 microns, more preferably about 80 nm to 200 nm. In some aspects, the nano to microstructure has a size range of about 80 nm to 30 microns, about 100 nm to 30 microns, about 200 nm to 30 microns, about 400 nm to 10 microns, about 500 nm to 1 micron, or about 80 nm to 200 nm. In some aspects, the nano to microstructure has a size of about 80 nm, 100 nm, 150 nm, 200 nm, 300nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 micron, 2 microns, 4 microns, 5 microns, 10 microns, 15 microns, 20 microns, 25 microns, and 30 microns.

[0102] This size range allows for effective delivery to tumor cells while avoiding rapid clearance from the body.

[0103] In some aspects, the polymer is hydrophobic. In some aspects, the polymer is hydrophilic. In some aspects, the pAg is incorporated into the backbone of the hydrophobic polymer via hydrolytically degradable ester linkages.

[0104] In some aspects, the pAg is incorporated into the backbone or onto the side of the hydrophobic polymer via bio-degradable linkages. In some aspects, the pAg is incorporated into the backbone or onto the side of the hydrophilic polymer via bio-degradable linkages. In some aspects, the bio-degradable linkages are ester linkages.

[0105] In some aspects, the tumor-targeting moiety is hyaluronic acid (HA) or cyclic internalizing RGD peptide (ciRGD). In some aspects, the tumor-targeting moiety is hydrophilic. In some aspects, the hydrophobic polymer comprises poly(HPMA-pAg). In some aspects, the phosphoantigen is a hydrophobically protected phosphoantigen methacrylate monomer 16 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO (pAgMA). In some aspects, the polymer further includes a biologically inert comonomer. In some aspects, the polymer further includes another small molecule pharmaceutical agent.

[0106] The present disclosure provides a polymeric composition with a generalized structure including a cell-targeting moiety (X) and a polymer incorporating phosphoantigens (pAgs). The structure can be represented as X-(Z)n, where Z is a monomer including a pAg, and n is the number of repeated units in the polymer ranging from 1-10,000, preferably 25-2,000. The cell-targeting moiety (X) can include various polymeric structures, allowing for customization of the nanoparticle's targeting capabilities. This flexibility enables the composition to be adapted for different types of cancer cells or tissues. The number of repeated units (n) in the polymer can be adjusted to optimize the size and properties of the resulting nanoparticles. The preferred range of 25-2,000 units allows for fine-tuning of the composition’s characteristics to suit specific delivery needs.

[0107] In some aspects, the disclosure includes a modified pAg structure (Structure 1) that converts the pAg into a monomer unit compatible with RAFT polymerization. This modification is crucial for incorporating the pAg into the polymer backbone.

[0108] Structure 1:

[0109] In some aspects, a dibenzocyclooctyne (DBCO)-terminated RAFT polymer (Structure 2) incorporates Structure 1 and a biologically inert comonomer. This polymer serves as a hydrophobic core to the final construct. The structure can be represented as:

[0110] Structure 2:17 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO

[0111] In some aspects, a block copolymer (Structure 3) is composed of Structure 2 “clicked” to an azide-terminated, hydrophilic block with a biological targeting ligand. Structure 3 self- assembles into a nanoparticle formulation in the presence of water, providing a versatile platform for targeted delivery of pAgs.

[0112] Structure 3:

[0113] Wherein: n is an integer ranging from 0 to 200, preferably n is an integer ranging from 1 to 200.

[0114] m is an integer ranging from 1 to 100.

[0115] Y is CH2, NH or O.

[0116] R1 is a sugar, or polyethylene glycol (PEG), poly(nucleic acid), protein, antibody, peptide, or aliphatic linkage.

[0117] R2 is an alkyl or ethyl ether.

[0118] R3 is an alkyl group, thioester, trithiocarbonate, mixed thiocarbonate, dithio- oxycarbonate, dithio-amine carbonate..

[0119] R4 is an alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, cycloalkane, amine, or al-cohol.

[0120] R5 is an alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, cycloalkane, amine, or alcohol.

[0121] R6 is a first protecting group, such as alcohols, methoxymethyl (MOM), or pivaloyloxymethyl (POM).

[0122] R7 is a second protecting group, such as alcohols, methoxymethyl (MOM), or pivaloyloxymethyl (POM).

[0123] This polymeric composition offers several advantages for cancer immunotherapy. The incorporation of pAgs into the polymer structure allows for controlled release in the tumor microenvironment, potentially overcoming limitations of systemic pAg administration. The 18 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO self-assembly into nanoparticles provides a means for efficient delivery to tumor cells, while the targeting moiety enhances specificity. The composition can be used alone or in combinationwith adoptive transfer of V 9V 2 T cells or PBMCs. It has potential applications in treatingvarious types of cancers by activating V 9V 2 T cells in the tumor microenvironment.

[0124] In another embodiment, the present disclosure provides a method of stimulating an immune response to a tumor including administering to a subject in need thereof an effective amount of the polymeric composition described above. In some aspects, the polymericcomposition activates V 9V 2 T cells, which then target and kill tumor cells.

[0125] “Administering” refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and / or any other method of physical delivery described herein or known in the art. The terms “administration of” and or “administering” should be understood to mean providing a pharmaceutical composition in a therapeutically effective amount to the subject in need of treatment. Administration routes can be enteral, topical or parenteral. As such, administration routes include but are not limited to inhalation, otic, buccal, conjunctival, dental, endocervical, endosinusial, endotracheal, enteral, epidural, extra- amniotic, extracorporeal, hemodialysis, infiltration, interstitial, intraabdominal, intraamniotic, intraarterial, intraarticular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavernous, intracavitary, intracerebroventricular, intracisternal, intracorneal, intracoronal, intracoronary, intracorpous cavernaosum, intradermal, intradiscal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intrahippocampal, intraileal, intralesional, intraluminal, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular, intrathoracic, intratubular, intratumor, intratympanic, intrauterine, intravascular, intravenous, intravenous bolus, intravenous drip, intravesical, intravitreal, iontophoresis, irrigation, laryngeal, nasal, nasogastric, ophthalmic, oral, oropharyngeal, parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, retrobulbar, subarachnoid, subconjunctival, subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transplacental, transtracheal, transtympanic, ureteral, urethral, vaginal, infraorbital, intraparenchymal, intrathecal, intraventricular, stereotactic administration, or any combination thereof. 19 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO

[0126] The terms “therapeutically effective amount”, “effective dose,” “therapeutically effective dose”, “effective amount,” or the like refer to that amount of the subject compound that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician. Generally, the response is either amelioration of symptoms in a patient or a desired biological outcome (e.g., stimulating an immune response to a tumor). Such amount should be sufficient to treat a cancer. The effective amount can be determined as described herein.

[0127] The term “subject” as used herein refers to any individual or patient to which the subject methods are performed. Generally, the subject is human, although as will be appreciated by those in the art, the subject may be a non-human animal. Thus other animals, including vertebrate such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, chickens, and non- human primates (including monkeys, chimpanzees, orangutans and gorillas) are included within the definition of subject.

[0128] In some aspects, the method further includes administering V 9V 2 T cells to thesubject. These T cells may be administered prior to the polymeric composition, with thecomposition then activating or maintaining the activation of the administered V 9V 2 T cells.The V 9V 2 T cells are preferably autologous to the subject.

[0129] In one embodiment, the present disclosure also encompasses methods of preparingand administering activated V 9V 2 T cells to a subject. In some aspects, the method includes:a) activating V 9V 2 T cells within the peripheral blood mononuclear cells (PBMCs) of thesubject; b) isolating the activated V 9V 2 T cells from the remaining PBMCs; c) administeringthe isolated, activated V 9V 2 T cells to the subject; and d) subsequently administering to thesubject an effective amount of the polymeric composition described above to maintain orenhance activation of the administered V 9V 2 T cells.

[0130] In some aspects, activating the V 9V 2 T cells includes exposing the PBMCs to thepolymeric composition described above.

[0131] In vitro studies have demonstrated the efficacy of the pAg nanoparticles in activatingV 9V 2 T cells. Tumor cells treated with various nanoparticle formulations (PEG-pAg-NP,30%RGD-pAg-NP, 50%RGD-pAg-NP, HA-pAg-NP) and co-cultured with V 9V 2 T cellsshowed significant reduction in viability compared to untreated controls. The treatments alsoinduced high levels of IFN production, indicating effective V 9V 2 T cell activation.20 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO

[0132] The polymeric composition may be administered intratumorally, subcutaneously, or systemically, depending on the specific therapeutic needs and tumor characteristics of the subject.

[0133] In one embodiment, the present disclosure provides a method of treating tumor in a subject including administering to a subject with the tumor an effective amount of the polymeric composition described above.

[0134] As used herein, the terms “treatment” and “treating” refer to the reduction or amelioration of the progression, severity, and / or duration of a disease or disorder resulting from the administration of one or more therapies. Treating may be determined by assessing whether there has been a decrease, alleviation and / or mitigation of one or more symptoms associated with the underlying disorder such that an improvement is observed with the patient, despite that the patient may still be afflicted with the underlying disorder. The term “treating” includes both managing and ameliorating the disease.

[0135] The disclosure is applicable to a wide range of tumor types, including but not limited to melanoma, breast cancer, lung cancer, prostate cancer, colorectal cancer, pancreatic cancer, ovarian cancer, liver cancer, brain cancer, leukemia, lymphoma, bladder cancer, kidney cancer, thyroid cancer, esophageal cancer, stomach cancer, cervical cancer, endometrial cancer, head and neck cancer, sarcoma, multiple myeloma, gallbladder cancer, bile duct cancer, testicular cancer, skin cancer, and nasopharyngeal cancer. Exemplary types of cancers include but are not limited to Acute Lymphoblastic Leukemia, Adult; Acute Lymphoblastic Leukemia, Childhood; Acute Myeloid Leukemia, Adult; Adrenocortical Carcinoma; Adrenocortical Carcinoma, Glioblastoma, Childhood; AIDS-Related Lymphoma; AIDS-Related Malignancies; Anal Cancer; Astrocytoma, Childhood Cerebellar; Astrocytoma, Childhood Cerebral; Bile Duct Cancer, Extrahepatic; Bladder Cancer; Bladder Cancer, Childhood; Bone Cancer, Osteosarcoma / Malignant Fibrous Histiocytoma; Brain Stem Glioma, Childhood; Brain Tumor, Adult; Brain Tumor, Brain Stem Glioma, Childhood; Brain Tumor, Cerebellar Astrocytoma, Childhood; Brain Tumor, Cerebral Astrocytoma / Malignant Glioma, Childhood; Brain Tumor, Ependymoma, Childhood; Brain Tumor, Medulloblastoma, Childhood; Brain Tumor, Glioblastoma; Brain Tumor, Supratentorial Primitive Neuroectodermal Tumors, Childhood; Brain Tumor, Visual Pathway and Hypothalamic Glioma, Childhood; Brain Tumor, Childhood (Other); Breast Cancer; Breast Cancer and Pregnancy; Oral Squamous Cell Carcinoma, Breast Cancer, Childhood; Breast Cancer, Male; Bronchial Adenomas / Carcinoids, 21 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO Childhood: Carcinoid Tumor, Childhood; Carcinoid Tumor, Gastrointestinal; Carcinoma, Adrenocortical; Carcinoma, Islet Cell; Sarcoma; Carcinoma of Unknown Primary; Central Nervous System Lymphoma, Primary; Cerebellar Astrocytoma, Childhood; Cerebral Astrocytoma / Malignant Glioma, Childhood; Cervical Cancer; Childhood Cancers; Chronic Lymphocytic Leukemia; Chronic Myelogenous Leukemia; Osteosarcoma; Chronic Myeloproliferative Disorders; Clear Cell Sarcoma of Tendon Sheaths; Colon Cancer; Chondrosarcoma; Colorectal Cancer, Childhood; Fibrosarcoma; Chordoma; Cutaneous T-Cell Lymphoma; Endometrial Cancer; Ependymoma, Childhood; Epithelial Cancer, Ovarian; Esophageal Cancer; Esophageal Cancer, Childhood; Ewing's Family of Tumors; Extracranial Germ Cell Tumor, Childhood; Extragonadal Germ Cell Tumor; Extrahepatic Bile Duct Cancer; Eye Cancer, Intraocular Melanoma; Giant cell tumor of bone; Eye Cancer, Retinoblastoma; Gallbladder Cancer; Gastric (Stomach) Cancer; Gastric (Stomach) Cancer, Childhood; Gastrointestinal Carcinoid Tumor; Germ Cell Tumor, Extracranial, Childhood; Germ Cell Tumor, Extragonadal; Germ Cell Tumor, Ovarian; Gestational Trophoblastic Tumor; Glioma. Childhood Brain Stem; Glioma. Childhood Visual Pathway and Hypothalamic; Hairy Cell Leukemia; Osteoblastoma; Head and Neck Cancer; Hepatocellular (Liver) Cancer, Adult (Primary); Hepatocellular (Liver) Cancer, Childhood (Primary); Hodgkin's Lymphoma, Adult; Hodgkin's Lymphoma, Childhood; Hodgkin's Lymphoma During Pregnancy; Adamantinoma; Hypopharyngeal Cancer; Hypothalamic and Visual Pathway Glioma, Childhood; Intraocular Melanoma; Islet Cell Carcinoma (Endocrine Pancreas); Kaposi's Sarcoma; Kidney Cancer; Laryngeal Cancer; Laryngeal Cancer, Childhood; Leukemia, Acute Lymphoblastic, Adult; Leukemia, Acute Lymphoblastic, Childhood; Leukemia, Acute Myeloid, Adult; Leukemia, Acute Myeloid, Childhood; Leukemia, Chronic Lymphocytic; Leukemia, Chronic Myelogenous; Leukemia, Hairy Cell; Lip and Oral Cavity Cancer; Liver Cancer, Adult (Primary); Liver Cancer, Childhood (Primary); Lung Cancer, Non-Small Cell; Lung Cancer, Small Cell; Lymphoblastic Leukemia, Adult Acute; Lymphoblastic Leukemia, Childhood Acute; Lymphocytic Leukemia, Chronic; Lymphoma, AIDS— Related; Lymphoma, Central Nervous System (Primary); Lymphoma, Cutaneous T-Cell; Lymphoma, Hodgkin's, Adult; Lymphoma, Hodgkin's; Childhood; Lymphoma, Hodgkin's During Pregnancy; Lymphoma, Non-Hodgkin's, Adult; Lymphoma, Non-Hodgkin's, Childhood; Lymphoma, Non-Hodgkin's During Pregnancy; Lymphoma, Primary Central Nervous System; Macroglobulinemia, Waldenstrom's; Male Breast Cancer; Malignant Mesothelioma, Adult; Malignant 22 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO Mesothelioma, Childhood; Malignant Thymoma; Medulloblastoma, Childhood; Melanoma; Melanoma, Intraocular; Merkel Cell Carcinoma; Mesothelioma, Malignant; Metastatic Squamous Neck Cancer with Occult Primary; Multiple Endocrine Neoplasia Syndrome, Childhood; Multiple Myeloma / Plasma Cell Neoplasm; Mycosis Fungoides; Myelodysplasia Syndromes; Myelogenous Leukemia, Chronic; Myeloid Leukemia, Childhood Acute; Myeloma, Multiple; Myeloproliferative Disorders, Chronic; Nasal Cavity and Paranasal Sinus Cancer; Nasopharyngeal Cancer; Nasopharyngeal Cancer, Childhood; Neuroblastoma; Non- Hodgkin's Lymphoma, Adult; Non-Hodgkin's Lymphoma, Childhood; Non-Hodgkin's Lymphoma During Pregnancy; Non-Small Cell Lung Cancer; Oral Cancer, Childhood; Oral Cavity and Lip Cancer; Oropharyngeal Cancer; Osteosarcoma / Malignant Fibrous Histiocytoma of Bone; Ovarian Cancer, Childhood; Ovarian Epithelial Cancer; Ovarian Germ Cell Tumor; Ovarian Low Malignant Potential Tumor; Pancreatic Cancer; Pancreatic Cancer, Childhood', Pancreatic Cancer, Islet Cell; Paranasal Sinus and Nasal Cavity Cancer; Parathyroid Cancer; Penile Cancer; Pheochromocytoma; Pineal and Supratentorial Primitive Neuroectodermal Tumors, Childhood; Pituitary Tumor; Plasma Cell Neoplasm / Multiple Myeloma; Pleuropulmonary Blastoma; Pregnancy and Breast Cancer; Pregnancy and Hodgkin's Lymphoma; Pregnancy and Non-Hodgkin's Lymphoma; Primary Central Nervous System Lymphoma; Primary Liver Cancer, Adult; Primary Liver Cancer, Childhood; Prostate Cancer; Rectal Cancer; Renal Cell (Kidney) Cancer; Renal Cell Cancer, Childhood; Renal Pelvis and Ureter, Transitional Cell Cancer; Retinoblastoma; Rhabdomyosarcoma, Childhood; Salivary Gland Cancer; Salivary Gland Cancer, Childhood; Sarcoma, Ewing's Family of Tumors; Sarcoma, Kaposi's; Sarcoma (Osteosarcoma Malignant Fibrous Histiocytoma of Bone; Sarcoma, Rhabdomyosarcoma, Childhood; Sarcoma, Soft Tissue, Adult; Sarcoma, Soft Tissue, Childhood; Sezary Syndrome; Skin Cancer; Skin Cancer, Childhood; Skin Cancer (Melanoma); Skin Carcinoma, Merkel Cell; Small Cell Lung Cancer; Small Intestine Cancer; Soft Tissue Sarcoma, Adult; Soft Tissue Sarcoma, Childhood; Squamous Neck Cancer with Occult Primary, Metastatic; Stomach (Gastric) Cancer; Stomach (Gastric) Cancer, Childhood; Supratentorial Primitive Neuroectodermal Tumors, Childhood; T-Cell Lymphoma, Cutaneous; Testicular Cancer; Thymoma, Childhood; Thymoma, Malignant; Thyroid Cancer; Thyroid Cancer, Childhood; Transitional Cell Cancer of the Renal Pelvis and Ureter; Trophoblastic Tumor, Gestational; Unknown Primary Site, Cancer of, Childhood; Unusual Cancers of Childhood; Ureter and Renal Pelvis, Transitional Cell Cancer; Urethral Cancer; Uterine 23 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO Sarcoma; Vaginal Cancer; Visual Pathway and Hypothalamic Glioma, Childhood; Vulvar Cancer; Waldenstrom's Macro globulinemia; and Wilms' Tumor.

[0136] In some aspects, the tumor is expressing butyrophilins 2A1 and 3A1 (BTN2A1, BTN3A1).

[0137] In some aspects, the method further includes administering V 9V 2 T cells or PBMCsto the subject. In some aspects, the V 9V 2 T cells or PBMCs are autologous.

[0138] In vivo studies have been conducted using a flank tumor model in immunocompromised NSG mice. MeWo melanoma cells were used to establish tumors, with 2x106cells implanted subcutaneously. This model allows for assessment of tumor growth and response to treatment.

[0139] In one embodiment, the present disclosure provides a method of activating gammadelta T cells in vitro, including contacting V 9V 2 T cells with the polymeric compositiondescribed above in the presence of V 9V 2 T cell activating cells, thereby activating thegamma delta T cells. In some aspects, the V 9V 2 T cell activating cells are tumor cells. Insome aspects, the V 9V 2 T cell activating cells are antigen presenting cells. In some aspects,the V 9V 2 T cells are isolated from PBMCs.

[0140] The methods of the dislcosure may also involve measuring interferon gamma (IFN )production as an indicator of V 9V 2 T cell activation. In some aspects, IFN may be measuredin vitro, in vivo, or both in vitro and in vivo. In animal models, tumor size and survival rate canbe used as indicators of V 9V 2 T cell activation and anti-tumor activity.

[0141] In some aspects, the V 9V 2 T cells are expanded from PBMCs prior to the contactingstep. In some aspects, expanding the V 9V 2 T cells includes activating the V 9V 2 T cellswith zoledronate; and maintaining the activated V 9V 2 T cells with human interleukin 2 (hIL-2).

[0142] In one embodiment, the present disclosure provides a method of stimulating gamma delta T cells in vivo, including administering to a subject the polymeric composition described above.

[0143] In some aspects, the polymeric composition activates V 9V 2 T cells in the subject.

[0144] In some aspects, the method further includes administering V 9V 2 T cells or PBMCsto the subject. In some aspects, the V 9V 2 T cells or PBMCs are autologous. In some aspects,the method further includes measuring tumor size or survival rate as an indicator of V 9V 2 Tcell activation and anti-tumor activity. 24 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO

[0145] The disclosure also provides methods for synthesizing the polymeric composition. This typically involves synthesizing a phosphoantigen methacrylate monomer (pAgMA), incorporating the pAgMA into a hydrophobic polymer backbone via Reversible Addition- Fragmentation chain Transfer (RAFT) polymerization, and conjugating a hydrophilic tumor- targeting moiety to the hydrophobic polymer to form a block copolymer.

[0146] In one embodiment, the present disclosure provides a method of synthesizing the polymeric composition described above including: a) synthesizing a phosphoantigen methacrylate monomer (pAgMA); b) incorporating the pAgMA into a hydrophobic polymer backbone via Reversible Addition-Fragmentation chain Transfer (RAFT) polymerization; and c) conjugating a hydrophilic tumor-targeting moiety to the hydrophobic polymer to form a block copolymer.

[0147] In some aspects, the hydrophilic tumor-targeting moiety is functionalized with a terminal azide for copper-free click chemistry conjugation to the hydrophobic polymer.

[0148] In one embodiment, the present disclosure provides a targeted, self-assembling nanoparticle for eliminating tumor cells via in situ activation of T cells, including: a) a phosphoantigen (pAg) monomer suitable for Reversible Addition-Fragmentation chain Transfer (RAFT) polymerization; and b) a RAFT polymer comprising the pAg monomer and a tumor-targeting moiety.

[0149] In some aspects, the pAg monomer is a hydrophobically protected phosphoantigen methacrylate monomer (pAgMA). In some aspects, the tumor-targeting moiety is hyaluronic acid (HA) or cyclic internalizing RGD peptide (ciRGD). In some aspects, the RAFT polymer comprises poly(HPMA-pAg).

[0150] In one embodiment, the present disclosure provides a method of analyzing targeting efficiency of a phosphoantigen (pAg) nanoparticle in vitro, including: a) quantifying selective uptake of targeted pAg nanoparticles by tumor cells compared to untargeted controlnanoparticles; and b) verifying activation of V 9V 2 T cells with a tumor-cytotoxic phenotypevia a co-culture assay with V 9V 2 T cells expanded and isolated from PBMCs.

[0151] In some aspects, quantifying selective uptake includes: a) incubating fluorescein- loaded pAg nanoparticles with tumor cells at varying doses, times, and temperatures; and b) measuring cellular uptake of fluorescent nanoparticles via flow cytometry. 25 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO

[0152] In some aspects, verifying activation of V 9V 2 T cells includes: a) co-culturing pAgnanoparticle-treated tumor cells with V 9V 2 T cells; b) determining tumor cell viability;and / or c) quantifying interferon gamma (IFN ) production.

[0153] In some aspects, the tumor cells express butyrophilins 2A1 and / or 3A1 (BTN2A1, BTN3A1).

[0154] In some aspects, number of monomer units in the polymer ranges from 1-10000. In some aspects, the monomer units of the polymer comprise the following structure:integer ranging from 1 to 200; m is an integer ranging from 1 to 100; Y is NH or O; R1 is a sugar, polyethylene glycol (PEG), or aliphatic linkage; R2 is an alkyl or ethyl ether; R3 is an alkyl group, thirster, trithiocarbonate, mixed thiocarbonate, dithio-oxycarbonate, dithio-amine carbonate; R4 is an alkyl, allyl, or alcohol; R5 is an alkyl, allyl, or alcohol; R6 is a first protecting group; and R7 is a second protecting group.

[0155] In some aspects, the monomer units of the polymer include the following structure:, wherein: n is an integer ranging from 1 to 200; 26 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO m is an integer ranging from 1 to 100; Y is CH2, NH or O; R1 is an alkyl, poly(ethylene glycol), or ethyl ether R2 is an alkyl group, ester, trithiocarbonate, mixed thiocarbonate, dithio- oxycarbonate, dithio-amine carbonate; R3 is an alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, cycloalkane, amine, or alcohol; R4 is an alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, cycloalkane, amine, or alcohol; R5 is a first protecting group; and R6 is a second protecting group.

[0156] In some aspects, the monomer units of the polymer include the following structure:, wherein: n is an integer ranging from 1 to 1000, preferably 1 to 200; m is an integer ranging from 1 to 1000, preferably 1 to 200; Y is CH2, NH, OH, NH2, or O; Z is CH2, NH, OH, NH2, or O; R1 is a sugar, oligosaccharide, polyethylene glycol (PEG), poly(nucleic acid), peptide, antibody, protein, synthetic polymer, natural polymer, or aliphatic linkage; R2 is an alkyl or ethyl ether, heterocycle, cycloalkene, cycloalkyne, cycloalkane, conjugated heterocycles, or conjugated cylcloalkenes; R3 is an alkyl group, thioester trithiocarbonate, mixed thiocarbonate, dithio- oxycarbonate, dithio-amine carbonate; R4 is an alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, cycloalkane, amine, or alcohol; 27 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO R5 is a protonR6 is an oxygen or first protecting group or; R7 is an oxygen or second protecting group; R8 is an oxygen or third protecting group; R9 is an oxygen or fourth protecting group; and R10 is an alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, and cycloalkane.

[0157] In some aspects, the monomer units of the polymer include the following structure:n is an integer ranging from 1 to 200; m is an integer ranging from 1 to 100; Y is CH2, NH or O; R1 is a sugar, polyethylene glycol (PEG), poly(nucleic acid), peptide, antibody, protein, positively charged synthetic polymer, or aliphatic linkage; R2 is an alkyl, poly(ethylene glycol), or ethyl ether R3 is an alkyl group, ester, trithiocarbonate, mixed thiocarbonate, dithio- oxycarbonate, dithio-amine carbonate; R4 is an alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, cycloalkane, amine, or alcohol; R5 is a carbon, alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, cycloalkane, amine, or alcohol; 28 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO R7 is an oxygen or protecting group; R8 is an oxygen or protecting group; and R9 is an oxygen or protecting group.

[0158] In some aspects, the monomer units of the polymer include the following structure:, wherein: n is an integer ranging from 1 to 200; m is an integer ranging from 1 to 100; Y is CH2, NH or O; R1 is an alkyl, poly(ethylene glycol), or ethyl ether R2 is an alkyl group, ester, trithiocarbonate, mixed thiocarbonate, dithio- oxycarbonate, dithio-amine carbonate; R3 is an alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, cycloalkane, amine, or alcohol; R4 is a carbon, alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, cycloalkane, amine, or alcohol; R5 is a first protecting group; R6 is an oxygen or protecting group; and R7 is an oxygen or protecting group. 29 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO

[0159] In some aspects, the monomer units of the polymer include the following structure:, wherein: n is an integer ranging from 1 to 1000, preferably 1 to 200; m is an integer ranging from 1 to 1000, preferably 1 to 200; Y is CH2, NH, OH, NH2, or O; Z is CH2, NH, OH, NH2, or O; R1 is a sugar, oligosaccharide, polyethylene glycol (PEG), poly(nucleic acid), peptide, antibody, protein, synthetic polymer, natural polymer, or aliphatic linkage; R2 is an alkyl or ethyl ether, heterocycle, cycloalkene, cycloalkyne, cycloalkane, conjugated heterocycles, or conjugated cylcloalkenes; R3 is an alkyl group, thioester trithiocarbonate, mixed thiocarbonate, dithio- oxycarbonate, dithio-amine carbonate; R4 is an alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, cycloalkane, amine, or alcohol; R5 is a protonR6 is an oxygen or first protecting group orR7 is an oxygen or second protecting group; R8 is an oxygen or third protecting group; R9 is an oxygen or fourth protecting group; and R10 is an alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, and cycloalkane. 30 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO

[0160] In some aspects, the first protecting group and / or the second protecting group comprises alcohols, methoxymethyl (MOM), or pivaloyloxymethyl (POM).

[0161] The following examples are provided to further illustrate the aspects of the present disclosure but are not intended to limit the scope of the disclosure. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used. EXAMPLES

[0162] The following examples are provided to further illustrate aspects of the present disclosure but are not intended to limit the scope of the invention. EXAMPLE 1 Synthesis and Characterization of Phosphoantigen Nanoparticles

[0163] This example describes the synthesis and characterization of phosphoantigen (pAg)nanoparticles, demonstrating their potential for targeted delivery and activation of V 9V 2 Tcells.

[0164] A strategy to hydrophobically protect pAg was developed to increase uptake and potency compared to the native small molecule. This synthesis process was scaled up and modified to incorporate a methacrylate group directly onto the pAg (FIG. 3). This approach resulted in a hydrophobically protected pAg methacrylate monomer (pAgMA) suitable for incorporation into a RAFT polymer. The pAgMA monomer was designed to degrade at its ester linkages under physiological conditions, releasing free pAg (FIG. 4). The products of each synthetic step were verified via thin layer chromatography (TLC), nuclear magnetic resonance (NMR), and liquid chromatography mass spectrometry (LCMS). FIG.1 illustrates a schematic representation of the self-assembled block copolymer phosphoantigen (pAg) delivery vehiclefor V 9V 2 T cell programming and activation. FIG. 2 depicts the proposed mechanism ofaction for the pAg nanoparticle, showing how it interacts with tumor cells and activatesV 9V 2 T cells, leading to tumor cell killing.

[0165] Reversible addition fragmentation chain-transfer polymerization (RAFT) was used to create polymers with high control of polymer length and low polydispersity. RAFT agent customization allowed for click chemistry handles for post-polymerization modifications. To create an off-the-shelf delivery vehicle, tumor-targeting ligands based on commonly 31 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO overexpressed tumor surface markers were investigated: namely CD44 (hyaluronic acid, HA) and integrins (cyclic internalizing RGD peptide, ciRGD).

[0166] A DBCO RAFT agent (denoted as (5) in FIG. 6) and polymerization (FIG. 7) were developed to allow for a straightforward copper-free click addition between the hydrophilic targeting moiety (HA-N3) and DBCO-poly(HPMA-pAg), effectively creating a block copolymer HA-poly(HPMA-pAg) (denoted as (8) in FIG. 7). For conjugation of cysteine- terminated ciRGD, the DBCO RAFT agent was modified with azide-PEG10K-maleimide then conjugated the peptide prior to polymerization (denoted as (9) in FIG. 8). Using this RAFT agent for polymerization yielded RGD-poly(HPMA-pAg) (denoted as (10) in FIG.8).

[0167] RAFT agents were characterized after synthesis via NMR and, where appropriate, LCMS (for a compound denoted as (5) in FIGS. 7 and 8) or high-performance liquid chromatography (HPLC) (for compounds denoted as (7) and (10) in FIGS.7 and 8). Polymers (denoted as (6) in FIG.7, (10) in FIG.8, (12) in FIG.9) were characterized via gel permeation chromatography (GPC). Species with click handles (compounds denoted as (6) and (7) in FIG. 7) were incubated with appropriate click-dye (BroadPharm 647 (BP647)-N3 or BP647-DBCO) and characterized via HPLC.

[0168] After polymer synthesis, particles were formulated from compounds denoted as (6) in FIG. 7, (10) in FIG. 8, and (12) in FIG. 9 via self-assembly in phosphate-buffered saline (PBS), pH = 7.4, and characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM) imaging (FIG. 10). Particles ranged from 80-200nm. Resultant particles were lyophilized in PBS with 1wt% sucrose as an excipient. Table 1 summarizes the molecular weights of various polymers used in the disclosure: Table 1: Summary of polymer molecular weights32 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO Table 2 provides a summary of nanoparticle components and sizes: Table 2: Summary of nanoparticle components and sizes

[0169] These results demonstrate the successful synthesis and characterization of pAg nanoparticles with various targeting moieties. The range of sizes and compositions provides a versatile platform for targeted delivery of pAg to tumor cells, potentially enhancing theactivation of V 9V 2 T cells in the tumor microenvironment.EXAMPLE 2 In Vitro Uptake and Activation Studies

[0170] This example describes the assessment of selective uptake of targeted pAgnanoparticles by tumor cells and the demonstration of V 9V 2 T cell activation with a tumor-cytotoxic phenotype.

[0171] To demonstrate receptor-mediated NP uptake, fluorescein-loaded particles were formulated. HA particles from a compound denoted as (2) in FIG. 3, Fl-HA-pAg-NP, were incubated with tumor cells at varying doses (1-100 M pAgMA equivalent), times (2-6 hours), and temperatures (4°C or 37°C), and a competitive inhibition condition with pre-incubation of HA was also introduced at the 2h timepoint (FIG.11). Uptake of fluorescent NPs was analyzed 33 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO via flow cytometry. Cells incubated at 4°C exhibited reduced uptake compared to equivalent timepoints at 37°C. Additionally, cells exposed to HA competitive inhibition displayed a modest reduction in uptake.

[0172] RGD particles from mixed compounds denoted as (3) and (4) in FIG. 3, Fl-RGD- pAg-NP, and PEG particles from a compound denoted as (4) in FIG. 3 alone, Fl-PEG-pAg- NP, were incubated with tumor cells at varying doses (1-250 M pAgMA equivalent) for 6 hours at 4°C or 37°C (FIG. 12). Fl-RGD-pAg-NPs displayed superior uptake compared to untargeted Fl-PEG-pAg-NPs at 37°C, but not at 4°C. Taken together, these results demonstrate that RGD-pAg-NPs are taken up via an energy-dependent process and that RGD is essential to the enhanced uptake of pAg-NPs.

[0173] To assess anti-tumor activity of V 9V 2 T cells in the presence of pAg-basedtreatments, V 9V 2 T cells were expanded from PBMCs via activation with zoledronate andmaintenance with human interleukin 2 (hIL-2) for 14 days. MeWo tumor cells were cultured at a density of 10,000 cells / well and incubated with various NP formulations (PEG-pAg-NP, 30%RGD-pAg-NP, 50%RGD-pAg-NP, HA-pAg-NP) and positive small molecule controls (zoledronate (zol)), an FDA-approved pAg infusion therapeutic, and pAgMA, the hydrophobically modified pAg methacrylate monomer). Treatment doses were calculated byequivalent pAgMA dose. V 9V 2 T cells were then co-cultured with MeWo cells at a 5:1effector:target ratio for 72 hours.

[0174] After this time, media and suspension cells were removed, and viability of remaining adherent tumor cells were assessed via CellTiter-Glo® 2.0 assay and normalized to equivalent monoculture conditions. All conditions produced significantly more tumor cell cytotoxicitythan the untreated control (FIG. 13) and produced high levels of IFN (FIG. 14), indicatingthat pAgMA and all NP formulations could activate V 9V 2 T cells in anti-tumor phenotype.Notably, the zol treatment produced comparable cell killing to other treatments in coculture, but tumor cells treated with zol in monoculture also experienced off-target cytotoxicity, indicating that pAgMA on its own may have fewer off-target effects than zol.

[0175] Further experiments were conducted to optimize the effector:target ratio for future studies. The viability of untreated control, while higher than treated conditions, indicated thatunstimulated V 9V 2 T cells were producing adverse effects on tumor cells. As such, theeffector:target ratio was adjusted to specifically demonstrate the cytotoxicity produced byactivating V 9V 2 T cells with pAg. An effector:target ratio of 2:1 appeared to exhibit34 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO substantial cell killing in the presence of pAg treatment while maintaining near-total viability in the absence of pAgMA. This optimized ratio allows for a clearer demonstration of thespecific effects of pAg-activated V 9V 2 T cells on tumor cell viability.

[0176] These results demonstrate the successful uptake of targeted pAg nanoparticles bytumor cells and their ability to activate V 9V 2 T cells, leading to tumor cell cytotoxicity. Thedata suggest that the pAg nanoparticles may offer improved specificity compared to current pAg therapeutics like zoledronate. EXAMPLE 3 In Vivo Tumor Model and Biodistribution Studies

[0177] This example describes the generation of a flank tumor model in immunocompromised mice and the characterization of tumor retention and biodistribution of dye-encapsulating targeted nanoparticles.

[0178] For simplicity of tumor measurement and tracking, a flank tumor model was adopted. Based on an initial pilot in male and female NSG mice, and the general greater availability of male leukopak donors for PBMCs, a flank implantation scheme of 2×106MeWo cells suspended in incomplete RPMI medium was selected. Based on tumor growth rates, a timepoint of 21 days was selected for initial intratumoral injections and initiation of adoptive cell transfer.

[0179] To assess tumor retention and biodistribution of NPs in vivo, particles encapsulating 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine (DID') were formulated: DID-HA- pAg-NP, DID-30%RGD-pAg-NP, DID-50%RGD-pAg-NP, and DID-PEG-pAg-NP. Tumor- bearing mice were dosed with NPs (approx. 160nmol pAgMA equivalent) and imaged via IVIS. Retention of dye-loaded NPs was observed in all groups.

[0180] These results demonstrate the successful establishment of a tumor model suitable for testing the pAg nanoparticles. The biodistribution studies indicate that the nanoparticles are retained in the tumor, suggesting their potential for sustained delivery of pAg to the tumor microenvironment. 35 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO EXAMPLE 4 Assessment of therapeutic efficacy across multiple cancers

[0181] To assess anti-tumor activity of V 9V 2 T cells in the presence of pAg-basedtreatments, V 9V 2 T cells were expanded from human PBMCs via activation withzoledronate and maintenance with human interleukin 2 (hIL-2) for 14 days. PC3 (prostate cancer), OVCAR3 (ovarian cancer), and MeWo (melanoma) tumor cells were cultured at a density of 10,000 cells / well and incubated with polymeric phosphoantigen prodrug nanoparticles labeled with RGD (RGD-P3NP) or phosphoantigen monomer (pAgMA). Wells treated with RGD-P3NP were washed after 4 hours, and pAgMA was maintained at the dosingconcentration for the duration of the study. V 9V 2 T cells were then co-cultured with thetreated cancer cells at an effector:target ratio of 2:1 for 72 hours, after which time the suspended T cells were washed from the cancer cells and the viability of the cancer cells was evaluated with CellTiter-Glo® 2.0 Cell Viability Assay (Promega). The viability of co-cultured cancer cells for each treatment was normalized to equivalently treated cancer cells which did notundergo co-culture with V 9V 2 T cells.

[0182] Notably, dose-dependent tumor cell death occurred only in the presence of expandedV 9V 2 T cells (FIGs. 15A-15C), indicating that the cell killing is V 9V 2-mediated and notsimply due to toxicity of RGD-P3NP or pAgMA. Furthermore, this effect was observed in all three tumor cell lines of diverse organ origin, suggesting broad potential applications of this technology across multiple cancer types. EXAMPLE 5 Phenotypic profiling of RGD-P3NP-activated V 9V 2 T cells

[0183] Next the activation state of the V 9V 2 T cells achieved at the time of cell killing wasevaluated. V 9V 2 T cells were isolated from co-culture with PC3 prostate cancer cells treatedwith RGD-P3NPs and evaluated using flow cytometry. Elevated levels of CD27 (a marker for antigen experience in T cells), CD28 (a co-stimulatory T cell receptor upregulated upon pro- inflammatory activation), and CD107a (upregulated in T cells upon degranulation) were observed at the two highest doses of RGD-P3NP (FIGs.16A-16D), correlating with the same doses for which -mediated tumor cell killing were observed (FIGs. 15A-15C). Levels ofIFN in the cell culture media were also evaluated using enzyme-linked immunosorbent assay36 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO (ELISA) and an increase was observed in cytokine secretion at higher RGD-P3NP doses. Taken together, these data further suggest that tumor cell killing is a result of pro-inflammatoryV 9V 2 activation due to the presence of RGD-P3NP.

[0184] While preferred aspects of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the aspects of the disclosure described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered accordingly. 37 1624760838.1

Claims

PATENT ATTORNEY DOCKET NO. JHU4800-1WO WHAT IS CLAIMED IS:

1. A polymeric composition comprising: a) a cell-targeting moiety; and b) a polymer composed of monomer units incorporating phosphoantigen (pAg) into the structure of the polymer.

2. The polymeric composition of claim 1, wherein the cell-targeting moiety is a tumor- targeting moiety.

3. The polymeric composition of claim 1, wherein the composition is in the form of a nano to microstructure.

4. The polymeric composition of claim 3, wherein the nano to microstructure is a nanoparticle.

5. The polymeric composition of claim 4, wherein the polymer is hydrophobic.

6. The polymeric composition of claim 4, wherein the polymer is hydrophilic.

7. The polymeric composition of claim 5, wherein the pAg is incorporated into the backbone of the hydrophobic polymer via hydrolytically degradable ester linkages.

8. The polymeric composition of claim 5, wherein the pAg is incorporated into the backbone or onto the side of the hydrophobic polymer via bio-degradable linkages.

9. The polymeric composition of claim 6, wherein the pAg is incorporated into the backbone or onto the side of the hydrophilic polymer via bio-degradable linkages.

10. The polymeric composition of claims 8 or 9, wherein the bio-degradable linkages are ester linkages.

11. The polymeric composition of claim 2, wherein the tumor-targeting moiety is hyaluronic acid (HA) or cyclic internalizing RGD peptide (ciRGD).

12. The polymeric composition of claim 2, wherein the tumor-targeting moiety is hydrophilic.

13. The polymeric composition of claim 5, wherein the hydrophobic polymer comprises poly(HPMA-pAg).

14. The polymeric composition of claim 1, wherein the phosphoantigen is a hydrophobically protected phosphoantigen methacrylate monomer (pAgMA).

15. The polymeric composition of claim 1, wherein the polymer further includes a biologically inert comonomer. 38 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO 16. The polymeric composition of claim 1, wherein the polymer further includes another small molecule pharmaceutical agent.

17. The polymeric composition of claim 3, wherein the nano to microstructure has a size range of about 80 nm to 30 microns.

18. The polymeric composition of claim 3, wherein the nano to microstructure has a size range of about 200 nm to 30 microns.

19. The polymeric composition of claim 17, wherein the nano to microstructure has a size range of about 80 nm to 200 nm.

20. A method of stimulating an immune response to a tumor comprising: administering to a subject in need thereof an effective amount of the polymeric composition of claim 1,wherein the polymeric composition activates V 9V 2 T cells.

21. The method of claim 20, further comprising administering V 9V 2 T cells to thesubject.

22. The method of claim 21, wherein the V 9V 2 T cells are administered to the subjectprior to administering the polymeric composition, and wherein the polymeric compositionactivates or maintains the activation of the administered V 9V 2 T cells.

23. The method of claim 22, wherein the V 9V 2 T cells are autologous to the subject.

24. A method of preparing and administering activated V 9V 2 T cells to a subject, themethod comprising: a) activating V 9V 2 T cells within the peripheral blood mononuclear cells(PBMCs) of the subject; b) isolating the activated V 9V 2 T cells from the remaining PBMCs;c) administering the isolated, activated V 9V 2 T cells to the subject; andd) subsequently administering to the subject an effective amount of the polymeric composition of claim 1 to maintain or enhance activation of the administered V9V 2 T cells.

25. The method of claim 24, wherein activating the V 9V 2 T cells comprises exposingthe PBMCs to the polymeric composition of claim 1.

26. The method of claim 20 or 24, wherein the polymeric composition is administered intratumorally, subcutaneously, or systemically.

27. A method of treating tumor in a subject comprising: administering to a subject with the tumor an effective amount of the polymeric composition of claim 1. 39 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO 28. The method of claim 27, wherein the tumor is selected from the group consisting of melanoma, breast cancer, lung cancer, prostate cancer, colorectal cancer, pancreatic cancer, ovarian cancer, liver cancer, brain cancer, leukemia, lymphoma, bladder cancer, kidney cancer, thyroid cancer, esophageal cancer, stomach cancer, cervical cancer, endometrial cancer, head and neck cancer, sarcoma, multiple myeloma, gallbladder cancer, bile duct cancer, testicular cancer, skin cancer, and nasopharyngeal cancer.

29. The method of claim 27, wherein the tumor is expressing butyrophilins 2A1 and 3A1 (BTN2A1, BTN3A1).

30. The method of claim 27, further comprising administering V 9V 2 T cells or PBMCsto the subject.

31. The method of claim 30, wherein the V 9V 2 T cells or PBMCs are autologous.

32. A method of activating gamma delta T cells in vitro, comprising contacting V 9V 2 Tcells with the polymeric composition of claim 1 in the presence of V 9V 2 T cell activatingcells, thereby activating the gamma delta T cells.

33. The method of claim 32, wherein the V 9V 2 T cell activating cells are tumor cells.

34. The method of claim 32, wherein the V 9V 2 T cell activating cells are antigenpresenting cells.

35. The method of claim 32, wherein the V 9V 2 T cells are isolated from PBMCs.

36. The method of claim 32, further comprising measuring interferon gamma (IFN )production as an indicator of V 9V 2 T cell activation.

37. The method of claim 32, wherein the V 9V 2 T cells are expanded from PBMCsprior to the contacting step.

38. The method of claim 37, wherein expanding the V 9V 2 T cells comprises activatingthe V 9V 2 T cells with zoledronate; and maintaining the activated V 9V 2 T cells withhuman interleukin 2 (hIL-2).

39. A method of stimulating gamma delta T cells in vivo, comprising administering to a subject the polymeric composition of claim 1.

40. The method of claim 39, wherein the polymeric composition activates V 9V 2 T cellsin the subject.

41. The method of claim 39, further comprising administering V 9V 2 T cells or PBMCsto the subject.

42. The method of claim 41, wherein the V 9V 2 T cells or PBMCs are autologous.40 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO 43. The method of claim 39, further comprising measuring tumor size or survival rate asan indicator of V 9V 2 T cell activation and anti-tumor activity.

44. A method of synthesizing the polymeric composition of claim 1 comprising: a) synthesizing a phosphoantigen methacrylate monomer (pAgMA); b) incorporating the pAgMA into a hydrophobic polymer backbone via Reversible Addition-Fragmentation chain Transfer (RAFT) polymerization; and c) conjugating a hydrophilic tumor-targeting moiety to the hydrophobic polymer to form a block copolymer.

45. The method of claim 44, wherein the hydrophilic tumor-targeting moiety is functionalized with a terminal azide for copper-free click chemistry conjugation to the hydrophobic polymer.

46. A targeted, self-assembling nanoparticle for eliminating tumor cells via in situ activation of T cells, comprising: a) a phosphoantigen (pAg) monomer suitable for Reversible Addition-Fragmentation chain Transfer (RAFT) polymerization; and b) a RAFT polymer comprising the pAg monomer and a tumor-targeting moiety.

47. The nanoparticle of claim 46, wherein the pAg monomer is a hydrophobically protected phosphoantigen methacrylate monomer (pAgMA).

48. The nanoparticle of claim 46, wherein the tumor-targeting moiety is hyaluronic acid (HA) or cyclic internalizing RGD peptide (ciRGD).

49. The nanoparticle of claim 46, wherein the RAFT polymer comprises poly(HPMA- pAg).

50. A method of analyzing targeting efficiency of a phosphoantigen (pAg) nanoparticle in vitro, comprising: a) quantifying selective uptake of targeted pAg nanoparticles by tumor cells compared to untargeted control nanoparticles; and b) verifying activation of V 9V 2 T cells with a tumor-cytotoxic phenotype via a co-culture assay with V 9V 2 T cells expanded and isolated from PBMCs.

51. The method of claim 50, wherein quantifying selective uptake comprises: a) incubating fluorescein-loaded pAg nanoparticles with tumor cells at varying doses, times, and temperatures; and b) measuring cellular uptake of fluorescent nanoparticles via flow cytometry. 41 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO52. The method of claim 50, wherein verifying activation of V 9V 2 T cells comprises:a) co-culturing pAg nanoparticle-treated tumor cells with V 9V 2 T cells;b) determining tumor cell viability; and / or c) quantifying interferon gamma (IFN ) production.

53. The method of claim 50, wherein the tumor cells express butyrophilins 2A1 and / or 3A1 (BTN2A1, BTN3A1).

54. The polymeric composition of claim 1, wherein number of monomer units in the polymer ranges from 1-10000.

55. The polymeric composition of claim 1, wherein the monomer units of the polymer comprise the following structure:, wherein: n is an integer ranging from 1 to 200; m is an integer ranging from 1 to 100; Y is CH2, NH or O; R1 is a sugar, polyethylene glycol (PEG), poly(nucleic acid), peptide, antibody, protein, positively charged synthetic polymer, or aliphatic linkage; R2 is an alkyl, poly(ethylene glycol), or ethyl ether; R3 is an alkyl group, ester, thirster, trithiocarbonate, mixed thiocarbonate, dithio- oxycarbonate, dithio-amine carbonate; R4 is an alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, cycloalkane, amine, or alcohol; R5 is an alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, cycloalkane, amine, or alcohol; 42 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO R6 is a first protecting group; and R7 is a second protecting group.

56. The polymeric composition of claim 1, wherein the monomer units of the polymer include the following structure:, wherein: n is an integer ranging from 1 to 200; m is an integer ranging from 1 to 100; Y is CH2, NH or O; R1 is an alkyl, poly(ethylene glycol), or ethyl ether R2 is an alkyl group, ester, trithiocarbonate, mixed thiocarbonate, dithio- oxycarbonate, dithio-amine carbonate; R3 is an alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, cycloalkane, amine, or alcohol; R4 is an alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, cycloalkane, amine, or alcohol; R5 is a first protecting group; and R6 is a second protecting group. 43 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO 57. The polymeric composition of claim 1, wherein the monomer units of the polymer include the following structure:, wherein: n is an integer ranging from 1 to 1000, preferably 1 to 200; m is an integer ranging from 1 to 1000, preferably 1 to 200; Y is CH2, NH, OH, NH2, or O; Z is CH2, NH, OH, NH2, or O; R1 is a sugar, oligosaccharide, polyethylene glycol (PEG), poly(nucleic acid), peptide, antibody, protein, synthetic polymer, natural polymer, or aliphatic linkage; R2 is an alkyl or ethyl ether, heterocycle, cycloalkene, cycloalkyne, cycloalkane, conjugated heterocycles, or conjugated cylcloalkenes; R3 is an alkyl group, thioester trithiocarbonate, mixed thiocarbonate, dithio- oxycarbonate, dithio-amine carbonate; R4 is an alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, cycloalkane, amine, or alcohol; R5 is a protonR6 is an oxygen or first protecting group or ; R7 is an oxygen or second protecting group; R8 is an oxygen or third protecting group; R9 is an oxygen or fourth protecting group; and R10 is an alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, and cycloalkane. 44 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO 58. The polymeric composition of claim 1, wherein the monomer units of the polymer include the following structure:, wherein: n is an integer ranging from 1 to 200; m is an integer ranging from 1 to 100; Y is CH2, NH or O; R1 is a sugar, polyethylene glycol (PEG), poly(nucleic acid), peptide, antibody, protein, positively charged synthetic polymer, or aliphatic linkage; R2 is an alkyl, poly(ethylene glycol), or ethyl ether R3 is an alkyl group, ester, trithiocarbonate, mixed thiocarbonate, dithio- oxycarbonate, dithio-amine carbonate; R4 is an alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, cycloalkane, amine, or alcohol; R5 is a carbon, alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, cycloalkane, amine, or alcohol; R7 is an oxygen or protecting group; R8 is an oxygen or protecting group; and R9 is an oxygen or protecting group.

59. The polymeric composition of claim 1, wherein the monomer units of the polymer include the following structure: 45 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO, wherein: n is an integer ranging from 1 to 200; m is an integer ranging from 1 to 100; Y is CH2, NH or O; R1 is an alkyl, poly(ethylene glycol), or ethyl ether R2 is an alkyl group, ester, trithiocarbonate, mixed thiocarbonate, dithio- oxycarbonate, dithio-amine carbonate; R3 is an alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, cycloalkane, amine, or alcohol; R4 is a carbon, alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, cycloalkane, amine, or alcohol; R5 is a first protecting group; R6 is an oxygen or protecting group; and R7 is an oxygen or protecting group.

60. The polymeric composition of claim 1, wherein the monomer units of the polymer include the following structure: 46 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO, wherein: n is an integer ranging from 1 to 1000, preferably 1 to 200; m is an integer ranging from 1 to 1000, preferably 1 to 200; Y is CH2, NH, OH, NH2, or O; Z is CH2, NH, OH, NH2, or O; R1 is a sugar, oligosaccharide, polyethylene glycol (PEG), poly(nucleic acid), peptide, antibody, protein, synthetic polymer, natural polymer, or aliphatic linkage; R2 is an alkyl or ethyl ether, heterocycle, cycloalkene, cycloalkyne, cycloalkane, conjugated heterocycles, or conjugated cylcloalkenes; R3 is an alkyl group, thioester trithiocarbonate, mixed thiocarbonate, dithio- oxycarbonate, dithio-amine carbonate; R4 is an alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, cycloalkane, amine, or alcohol; R5 is a protonR6 is an oxygen or first protecting group or; R7 is an oxygen or second protecting group; R8 is an oxygen or third protecting group; R9 is an oxygen or fourth protecting group; and R10 is an alkyl, allyl, polyethylene, heterocycle, cycloalkene, cycloalkyne, and cycloalkane. 47 1624760838.1PATENT ATTORNEY DOCKET NO. JHU4800-1WO 61. The polymeric composition of claim 55, wherein the first protecting group and / or the second protecting group comprises alcohols, methoxymethyl (MOM), or pivaloyloxymethyl (POM). 48 1624760838.1

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