Brain targeted nanoconjugates and uses thereof

Abstract

The disclosure relates to nanoconjugates and uses thereof. Specifically, the disclosure provides nanoconjugates comprising a biodegradable polymeric backbone conjugated to a blood-brain barrier penetrating peptide and an immunotherapeutic agent, and uses thereof for treating cancers of the central nervous system.

Description

[0001] BRAIN TARGETED NANOCONJUGATES AND USES THEREOF STATEMENT REGARDING RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Application No.

[0003] 63 / 753,566, filed February 4, 2025, the entire contents of which are incorporated herein by reference for all purposes.

[0004] SEQUENCE LISTING

[0005] The text of the computer readable sequence listing filed herewith, titled “STDU2-43902-601_SQL.xml”, created February 4, 2026, having a file size of 24,632 bytes, is hereby incorporated by reference in its entirety.

[0006] FIELD

[0007] The disclosure relates to nanoconjugates and uses thereof. Specifically, the disclosure provides nanoconjugates comprising a biodegradable polymeric backbone conjugated to an peptide that facilitates crossing of the blood-brain barrier and an immunotherapeutic agent, and uses thereof for treating cancers of the central nervous system.

[0008] BACKGROUND

[0009] Glioblastoma Multiforme (GBM) remains a significant clinical challenge among central nervous system (CNS) tumors, with a dismal mean survival rate of less than 8 months, a statistic that has remained largely unchanged for decades. The specialized anatomical features of the brain, notably the blood-brain barrier (BBB), pose major challenges for effective therapeutic interventions, limiting the potential reach of modern advancements in immunotherapy to impact these types of tumors. As such, what are needed are effective treatments for glioblastoma multiforme and other central nervous system cancers.

[0010] SUMMARY

[0011] In some aspects, provided herein are nanoconjugates (also referred to throughout as “nanoparticles”). In some embodiments, provided here are nanoconjugates comprising a biodegradable polymeric backbone with an immune checkpoint inhibitor conjugated to the biodegradable polymeric backbone and a peptide that facilitates crossing of the blood-brainbarrier (e.g. an angiopep peptide) conjugated to the biodegradable polymeric backbone. A peptide that facilitates crossing of the blood-brain barrier is also referred to herein as a “bloodbrain barrier penetrating peptide” or a “BBB penetrating peptide”. The nanoconjugates are shown herein to cross the blood brain barrier and deliver the immune checkpoint inhibitor locally, e.g. at the site of a central nervous system tumor.

[0012] In some embodiments, the biodegradable polymeric backbone comprises a plurality of monomeric units, and each monomeric unit comprises a pendant -COOH group. In some embodiments, the biodegradable polymeric backbone comprises polymalic acid (PMLA).

[0013] In some embodiments, the immune checkpoint inhibitor comprises a peptide. For example, in some embodiments the immune checkpoint inhibitor comprises a peptide that antagonizes signaling at a receptor selected from CTLA4, PD-1, VISTA, BTLA, TIM-3, KIR, LAG-3, TIGIT, CD-96, and SIRPa. In some embodiments, the immune checkpoint inhibitor is a peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the immune checkpoint inhibitor comprises NP-12. In some embodiments, the immune checkpoint inhibitor comprises a peptide selected from: NP-12, TPP-1 (SGQYASYHCWCWRDPGRSGGSK, SEQ ID NO: 3), CLP002 (WHRSYYTWNLNT, SEQ ID NO: 4), C8 (CKWYRPSEC, SEQ ID NO: 5), P-F4 (FSGTVTTAGLLF, SEQ ID NO: 6), OPBP-1 (GQSEHHMRVYSE, SEQ ID NO: 7), PD-LlPep-1 (CLQKTPKQC, SEQ ID NO: 8), PDL1-Pep2 (CVRARTR, SEQ ID NO: 9), Pep-39 (AMSDHHWTQRDK, SEQ ID NO: 10), nABP284 (SRLKEIANSPTQFWRMVARNTLGNGAKQSLNIEHARL, SEQ ID NO: 11), C25 (CVPMTYRAC. SEQ ID NO: 12), ERY2-4 (CAWGQAILEGELAWLEGGGGGAGQLADLKRQLAWWKQAC, SEQ ID NO: 13), p344 (ARHPSWYRPFEGCG, SE ID NO: 14). PPA (NYSKPTDRQYHF, SEQ ID NO: 15), HVEM(14-39) (ESCPKCSPGYRVKEACGELTGTVCEP (SEQ ID NO: 16), P16 (cyc(EIDTVLTPTGWVAKRYS) (SEQ ID NO: 17), LC4 (WGHSHFSHWKGR, SEQ ID NO: 18), TBP-3 (GGYTFFIWHRLNP, SEQ ID NO: 19), D4-2 (RYSAVYSIHPSW. SEQ ID NO: 20), and PPL-C (SVSVSHFQKVWVVGGGSK, SEQ ID NO: 27). In some embodiments, the peptide comprises a terminal -NH2 group (e.g. at the C-terminus). In some embodiments, the immune checkpoint inhibitor peptide is conjugated to the biodegradable polymeric backbone via an amide bond.In some embodiments, about 0.1 % to about 5% of monomeric units within the biodegradable polymeric backbone are conjugated to the immune checkpoint inhibitor. In some embodiments, about 0.1% to about 5% of monomeric units within the biodegradable polymeric backbone are conjugated to NP-12.

[0014] In some embodiments, the peptide that facilitates crossing of the blood-brain barrier (e.g. the BBB penetrating peptide) facilitates crossing of the blood-brain barrier by interaction with LRP1. In some embodiments, the blood-brain barrier penetrating peptide comprises an angiopep peptide. In some embodiments, the angiopep peptide is angiopep-2 (“AP-2”). Angiopep-2 is a synthetic peptide having the sequence TFFYGGSRGKRNNFKTEEY (SEQ ID NO: 21). In some embodiments, the peptide that facilitates crossing of the blood-brain barrier comprises L57 (TWPKHFDKHTFYSILKLGKH, SEQ ID NO: 23), D1 (QSHYRHISPAQVC, SEQ ID NO: 24), D3 (RPRTRLHTHRNRC, SEQ ID NO: 25), or ACI-89 (PSHYRHISPAQKC, SEQ ID NO: 26).

[0015] In some embodiments, the BBB penetrating peptide (e.g. AP-2, L57, DI, D3, or ACI-89) is conjugated to the biodegradable polymeric backbone via a polyethylene glycol (PEG)-containing spacer. In some embodiments, the PEG-containing spacer has a molecular weight of about 1000 Da to about 5000 Da. In some embodiments, 0.1% to about 5% of monomeric units within the biodegradable polymeric backbone are conjugated to the BBB penetrating peptide (e.g. AP-2, L57, DI, D3, or ACI-89). In some embodiments, 0.1% to about 5% of monomeric units within the biodegradable polymeric backbone are conjugated to AP-2.

[0016] In some embodiments, the nanoconjugate further comprises a trileucine moiety (LLL) conjugated to the biodegradable polymeric backbone. In some embodiments, the trileucine moiety is conjugated to the biodegradable polymeric backbone via an amide bond. In some embodiments, about 10% to about 30% of the monomeric units within the biodegradable polymeric backbone are conjugated to the trileucine moiety.

[0017] In some embodiments, the nanoconjugate comprises an immune checkpoint inhibitor peptide and a BBB penetrating peptide (e.g. AP-2, L57, D1, D3, or ACI-89) conjugated to the biodegradable polymeric backbone (e.g. PMLA). In some embodiments, the nanoconjugate comprises an immune checkpoint inhibitor peptide comprising the amino acid sequence of SEQID NO: 1 and a BBB penetrating peptide (e.g. AP-2, L57, DI, D3, or ACI-89) conjugated to the biodegradable polymeric backbone (e.g. PMLA backbone). In some embodiments, the nanoconjugate comprises an immune checkpoint inhibitor peptide comprising NP-12, TPP-1, CLP002, C8, P-F4, OPBP-1, PD-LlPep-1, PDL1-Pep2, Pep-39, nABP284, C25, ERY2-4, p344, PPA, HVEM(14-39), P16, LC4, TBP-3, D4-2, or PPL-C and a BBB penetrating peptide (e.g. AP-2, L57, D1, D3, or ACI-89) conjugated to the biodegradable polymeric backbone (e.g.

[0018] PMLA). In some embodiments, the nanoconjugate comprises NP-12 and AP-2 conjugated to a PMLA backbone. In some embodiments, about 0.1% to about 5% of monomeric units within the biodegradable polymeric backbone are conjugated to the immune checkpoint inhibitor peptide and about 0.1% to about 5% of the monomeric units within the biodegradable polymeric backbone are conjugated to the BBB penetrating peptide (e.g. AP-2, L57, D1, D3, or ACI-89). In some embodiments, about 0.1% to about 5% of monomeric units within the biodegradable polymeric backbone are conjugated to the NP-12 and about 0.1% to about 5% of the monomeric units within the biodegradable polymeric backbone are conjugated to AP-2.

[0019] In some embodiments, the nanoconjugate comprises an immune checkpoint inhibitor peptide, a BBB penetrating peptide, and a trileucine moiety (LLL) conjugated to the biodegradable polymeric backbone (e.g. PMLA). In some embodiments, the nanoconjugate comprises an immune checkpoint inhibitor peptide comprising the amino acid sequence of SEQ ID NO: 1, a BBB penetrating peptide (e.g. AP-2, L57, DI, D3, or ACI-89), and a trileucine moiety conjugated to the biodegradable polymeric backbone (e.g. PMLA backbone). In some embodiments, the nanoconjugate comprises an immune checkpoint inhibitor peptide comprising NP-12, TPP-1, CLP002, C8, P-F4, OPBP-1. PD-LlPep-1, PDL1-Pep2, Pep-39, nABP284, C25, ERY2-4, p344. PPA, HVEM( 14-39), P16, LC4, TBP-3, D4-2, or PPL-C, a BBB penetrating peptide (e.g. AP-2, L57, DI, D3, or ACI-89), and a trileucine moiety conjugated to the biodegradable polymeric backbone (e.g. PMLA). In some embodiments, the nanoconjugate comprises NP-12, an angiopep peptide (e.g. AP-2), and a trileucine moiety conjugated to the biodegradable polymeric backbone (e.g. PMLA). In some embodiments, the nanoconjugate comprises NP-12, AP-2. and a trileucine moiety conjugated to a PMLA backbone. In some embodiments, about 0.1% to about 5% of monomeric units within the biodegradable polymeric backbone are conjugated to the immune checkpoint inhibitor peptide, about 0.1% to about 5% of the monomeric units within the biodegradable polymeric backbone are conjugated to the BBBpenetrating peptide (e.g. AP-2, L57, D1, D3, or ACI-89) and about 10% to about 30% of monomeric units within the biodegradable polymeric backbone are conjugated to the trileucine moiety. In some embodiments, about 0.1% to about 5% of monomeric units within the biodegradable polymeric backbone are conjugated to NP-12, about 0.1% to about 5% of the monomeric units within the biodegradable polymeric backbone are conjugated to AP-2, and about 10% to about 30% of monomeric units within the biodegradable polymeric backbone are conjugated to the trileucine moiety.

[0020] In some embodiments, the nanoconjugate further comprises a fluorophore conjugated to the biodegradable polymeric backbone.

[0021] The nanoconjugates herein find use in methods of treating a central nervous system cancer in a subject. In some aspects, provided herein are methods of treating a central nervous system cancer in a subject comprising providing to the subject a nanoconjugate described herein. In some embodiments, the central nervous system cancer is a brain cancer. In some embodiments the brain cancer is glioblastoma multiforme. In some embodiments, the subject is a mammal. For example, in some embodiments the subject is a human.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1A is a schematic showing an overview of the nanoconjugates provided herein and the putative mechanism of action thereof after crossing the blood brain barrier. FIG. IB and FIG. 1C shows a suitable synthesis method for an exemplary nanoconjugate herein. The nanoconjugate comprises a PMLA backbone conjugated to the immunotherapeutic peptide P-12 and cysteine-modified angiopep-2.

[0023] FIG. 2A is a schematic showing an exemplary method for production of polymalic acid (PMLA). FIGs. 2B-2C show characterization of the nanoconjugate using 1H NMR (FIG. 2B) and SEC-HPLC (FIG. 2C) for structure and molecular weight elucidation.

[0024] FIG. 3A is a schematic representation of a BBB-transwell model demonstrating nanoparticle receptor-mediated transcytosis and PDL1 receptor- mediated interaction with GBM spheroids wherein human brain microvascular endothelial cells (HBMECs) are incorporated on a fibronectin layer to mimic the blood-brain barrier (BBB). The model illustrates the successfulcrossing of AP-2 / P- 12 / RhB NCs through the BBB and their subsequent interaction with glioblastoma multiforme (GBM) tumor spheroids in a separate compartment. FIG. 3B shows cellular uptake of nanoconjugates in a BBB transwell model, assessed by confocal laser scanning microscopy (CLSM) analysis of AP-2 / P-12 / RhB NCs uptake by HBMECs (seeded as a monolayer on fibronectin-coated transwells) mimicking the BBB and incubated with 0.1 mg / mL of AP-2 / P-12 / RhB NCs for 2 h. FIG. 3C shows results from CLSM analysis demonstrating cellular uptake of nanoconjugates in a complete transwell model with the constructed BBB and GBM tumor spheroid. Tumor spheroids were stained with DAPI and green Cell Tracker for cell membrane. Free rhodamine and rhodamine-labeled AP-2 / P-12 nanoparticles were then incorporated. Free rhodamine did not have significant staining on the periphery. In contrast, successful nanoparticle binding to the tumor spheroid by AP-2 / P-12 conjugates was observed as indicated by rhodamine staining colocalized with Cell Tracker staining (overlay). Results are quantified in FIG. 3D.

[0025] FIG. 4A is a schematic showing an exemplary experimental method for evaluating T-cell activation and proliferation. FIG. 4B shows the effect of a-PDl antibody (“a-PD-1”), free P-12 peptide (“peptide”), and AP-2 / P-12 nanoconjugate (“conjugate”) on cell proliferation and expression of cytokines. Data are mean ± SEM. ** p < 0.01, *** p < 0.001, **** p < 0.0001 (One way ANOVA). The results suggest a synergistic effect of the AP-2 / P-12 NCs in promoting T cell activation and proliferation, which directly enhance antitumor immune responses.

[0026] FIGs. 5A-5B show that AP-2 / P-12 nanoconjugates (NCs) enhance T cell-mediated cytotoxicity against glioblastoma multiforme (GBM) spheroids and inhibit their growth. FIG.

[0027] 5A shows a visualization of T cell-mediated cytotoxicity: fluorescence microscopy analysis demonstrating the cytotoxic effect of T cells on GBM spheroids in the presence of P-12 peptide and AP-2 / P-12 NCs. Live cells are visualized using calcein AM (green fluorescence) while dead cells are identified by ethidium bromide (EtBr) staining (red fluorescence) This dualfluorescence assay allows for the simultaneous quantification of live and dead cells within the spheroids. This provides a direct measure of T cell-mediated cytotoxicity. Results are quantified in the bar graphs to the right. FIG. 5B shows temporal analysis of 3D tumor spheroid growth: quantitative assessment of GBM spheroid size over 7 days when cocultured with T cells in the presence of P-12 peptide alone or AP-2 / P-12 NCs using brightfield microscopy. A reducedgrowth rate of spheroid size in the AP-2 / P-12 NC treatment group over the 4 days can be observed. Data are presented as mean ± standard error of the mean (SEM). Statistical significance was determined using one-way ANOVA followed by posthoc tests, with ** p < 0.01, *** p < 0.001, and **** p < 0.0001, indicating significance levels between treatment groups.

[0028] FIG. 6A shows in vivo biodistribution and FIG. 6B shows ex vivo organ biodistribution of AP-2 / P-12 nanoparticles 2 hours after intravenous administration. FIG. 6C shows in vivo biodistribution and FIG.6D shows ex vivo organ biodistribution of AP-2 / P-12 nanoparticles 6 hours after intravenous administration. A sustained presence of AP-2 / P-12 nanoparticles is shown in the brain.

[0029] FIG. 7 shows organ toxicity observed by histopathological analysis. No necrosis / toxicity was observed for P-12 nanoconjugates or AP-2 / P-12 nanoconjugates.

[0030] FIG. 8 shows a Kaplan-Meier Survival plot in MBM model, showing improved survival in the AP-2 / P-12 nanoconjugate compared to Free P-12, mouse PD-L1 antibody (ATZ) and P-12 NCs (p<0.05).

[0031] DEFINITIONS

[0032] To facilitate an understanding of the present technology, a number of terms and phrases are defined below. Additional definitions are set forth throughout the detailed description.

[0033] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; in the event, however, of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0034] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element, e.g., a plurality of elements.As used herein, the modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (for example, it includes at least the degree of error associated with the measurement of the particular quantity). The modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4.” The term “about” may refer to ±10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9 - 1.1. Other meanings of “about” may be apparent from the context, such as rounding off; for example, “about 1” may also mean from 0.5 to 1.4.

[0035] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0036] For the recitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0037] As used herein, the terms “treat”, “treatment”, and “treating” refer to administration of an agent or therapy to a subject for the purpose of achieving a desired clinical result. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of a condition, disorder, or disease stabilized (i.e.. not worsening) state of condition, disorder, or disease; delay in onset or slowing of condition, disorder, or disease progression; amelioration of the condition, disorder, or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder, or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.The term “subject” or “patient” are used interchangeably herein and broadly refers to any living organism, and more specifically to an animal including human and non-human animals. In some embodiments, the subject is a mammal. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. In some embodiments, the subject is a human. The subject may be an adult subject (e.g. an adult human of 18 years of age or older). The subject may be a juvenile (e.g. pediatric) subject. The term “juvenile” or “pediatric” when used in reference to a human subject refers to a subject 17 years of younger.

[0038] DETAILED DESCRIPTION

[0039] This disclosure provides nanoconjugates (also referred to throughout as “nanoparticles”) and uses thereof for treating cancer. In some aspects, provide herein is a nanoconjugate comprising a biodegradable polymeric backbone, an immune checkpoint inhibitor conjugated to the biodegradable polymeric backbone, and a BBB penetrating peptide conjugated to the biodegradable polymeric backbone.

[0040] A “biodegradable” polymeric backbone, refers to a polymer that degrades, decomposes, or breaks down within a suitable timeframe in a biologically relevant environment, including in a cell or in a subject, as a result of natural processes. In some embodiments, a “biodegradable” polymeric backbone refers to a polymer that degrades within a suitable timeframe within a cell or a subject after administration to the cell or subject. For example, a “biodegradable” polymer may degrade within 6 months after contact with the cell or subject, within 5 months, within 4 months, within 3 months, within 2 months, within 1 month, within 2 weeks, or within 1 week. In some embodiments, the biodegradable polymeric backbone comprises a natural biodegradable polymer. In some embodiments, the biodegradable polymeric backbone comprises a plurality of monomeric units, and each monomeric unit comprises a pendant -COOH group. The -COOH groups provide convenient reactive handles for conjugation of the immune checkpoint inhibitor and the angiopep peptide, and optional additional moieties, to the polymeric backbone. In someembodiments, the biodegradable polymeric backbone comprises polymalic acid (PMLA). In some embodiments, the biodegradable polymeric backbone, prior to conjugation of the immune checkpoint inhibitor and angiopep peptide, has a molecular weight in a range of about 10-1000 kDa, or about 50-100 kDa.

[0041] In some embodiments, nanoconjugate (i.e., nanoparticle) comprises an immune checkpoint inhibitor conjugated to the biodegradable polymeric backbone. In some embodiments, the immune checkpoint inhibitor comprises a peptide. In some embodiments, the immune checkpoint inhibitor comprises a peptide that antagonizes signaling at one or more immune checkpoint receptors selected from CTLA4, PD-1, VISTA, BTLA, TIM-3, KIR, LAG-3, TIGIT, CD-96, and SIRPa. The immune checkpoint inhibitor may antagonize signaling by binding to the receptor itself thus preventing or reducing binding of a ligand to the receptor (e.g. a direct antagonist, such as a competitive antagonist or a non-competitive antagonist), by binding to the ligand for the receptor (thereby preventing the ligand from binding to the receptor), or by other means (e.g. allosteric modulation).

[0042] In some embodiments, the immune checkpoint inhibitor comprises a peptide comprising the sequence SNTSESF (SEQ ID NO: 1). In some embodiments, the immune checkpoint inhibitor comprises NP-12, referred to interchangeably herein as “P-12”. NP-12 is described in Mol Cancer Ther 2019 Jun;18(6):1081-1091, the entire contents of which are incorporated herein by reference for all purposes. NP-12 or P-12 are used interchangeably herein and refer to a branched peptide comprising the sequence SNTSESFKFRVTQLAPKAQIKE (SEQ ID NO: 2), wherein the lysine residue at position 8 is linked to the amino acid sequence SNTSESF (SEQ ID NO: 1). This linkage forms an amide bridge between the lys-8 and Phe-7 in a branch chain. In some embodiments, the C-terminus of NP-12 is terminated with NH2.

[0043] In some embodiments, the immune checkpoint inhibitor comprises TPP-1 (SGQYASYHCWCWRDPGRSGGSK, SEQ ID NO: 3), CLP002 (WHRSYYTWNLNT, SEQ ID NO: 4), C8 (CKWYRPSEC, SEQ ID NO: 5), P-F4 (FSGTVTTAGLLF, SEQ ID NO: 6), OPBP-1 (GQSEHHMRVYSE, SEQ ID NO: 7), PD-LlPep-1 (CLQKTPKQC, SEQ ID NO: 8), PDL1-Pep2 (CVRARTR, SEQ ID NO: 9), Pep-39 (AMSDHHWTQRDK, SEQ ID NO: 10), nABP284 (SRLKEIANSPTQFWRMVARNTLGNGAKQSLNIEHARL, SEQ ID NO: 11), C25 ( CVPMTYRAC, SEQ ID NO: 12), ERY2-4(CAWGQAILEGELAWLEGGGGGAGQLADLKRQLAWWKQAC. SEQ ID NO: 13), p344 (ARHPSWYRPFEGCG, SE ID NO: 14), PPA (NYSKPTDRQYHF, SEQ ID NO: 15), HVEM(14-39) (ESCPKCSPGYRVKEACGELTGTVCEP (SEQ ID NO: 16), P16 (cyc(EIDTVLTPTGWVAKRYS) (SEQ ID NO: 17), LC4 (WGHSHFSHWKGR, SEQ ID NO: 18), TBP-3 (GGYTFHWHRLNP, SEQ ID NO: 19), D4-2 (RYSAVYSIHPSW, SEQ ID NO: 20), or PPL-C (SVSVSHFQKVWVVGGGSK, SEQ ID NO: 27).

[0044] In some embodiments, the immune checkpoint inhibitor comprises a peptide having or modified to have a terminal -NH₂ group. In some embodiments, the terminal -NH₂ group facilitates conjugation of the immune checkpoint inhibitor peptide to the biodegradable polymeric backbone (e.g. to the PMLA backbone).

[0045] In some embodiments, the immune checkpoint inhibitor is conjugated to the biodegradable polymeric backbone by coupling the immune checkpoint inhibitor to a free - COOH group on the polymeric backbone, using conjugation chemistries known to those skilled in the art. For example, PMLA includes pendant -COOH groups attached to the polymeric backbone. These can be converted to activated esters, such as succinimidyl esters, p-nitrophenyl esters, tetrafluorophenyl esters, or the like. The activation reaction can be performed using an appropriate coupling agent, such as a carbodiimide coupling agent (e.g., dicyclohexylcarbodiimide (DCC) or l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Such a reaction generates an activated PMLA product. From there, conjugation to the polymeric backbone can be conducted by straightforward coupling reaction with the immune checkpoint inhibitor containing a complementary reactive group, such as an -NH₂ group (e.g., the free N- terminal -NH₂ group of the peptide) forms an amide bond.

[0046] In some embodiments, about 0.1% to about 5% of monomeric units within the biodegradable polymeric backbone are conjugated to the immune checkpoint inhibitor. For example, in some embodiments, about 0.1%, about 0.5%, about 1%, about 2%, about 3%, about 4%, or about 5% of monomeric units within the biodegradable polymeric backbone are conjugated to the immune checkpoint inhibitor. Loading of the immune checkpoint inhibitor can be controlled, for example, by adjusting the reaction stoichiometry and / or reaction time.

[0047] In some embodiments, the nanoconjugate (z.e., nanoparticle) comprises a peptide that facilitates crossing of the blood-brain barrier. In some embodiments, the peptide facilitatescrossing of the blood-brain barrier by interaction with LRP1. In some embodiments, the peptide that facilitates crossing of the blood-brain barrier comprises an angiopep peptide conjugated to the biodegradable polymeric backbone. The angiopep family of peptides refers to a family of peptides derived from the Kunitz domain of human aprotinin. The angiopep family of peptides are able to cross the blood-brain barrier and are thus useful for delivery of agents to the central nervous system. In some embodiments, the angiopep peptide is angiopep-2 (“AP-2”).

[0048] Angiopep-2 is a synthetic peptide having the sequence TFFYGGSRGKRNNFKTEEY (SEQ ID NO: 21). AP-2 is thought to cross the blood brain barrier through interaction with LRP-1 receptors. In some embodiments, the peptide that facilitates crossing of the blood-brain barrier comprises L57 (TWPKHFDKHTFYSILKLGKH, SEQ ID NO: 23), D1 (QSHYRHISPAQVC, SEQ ID NO: 24), D3 (RPRTRLHTHRNRC. SEQ ID NO: 25), or ACI-89 (PSHYRHISPAQKC, SEQ ID NO: 26).

[0049] In some embodiments, the BBB penetrating peptide (e.g. AP-2, L57, DI, D3, or ACI-89) is conjugated to the biodegradable polymeric backbone by using a similar coupling reaction as that used to conjugate the immune checkpoint inhibitor, or using different conjugation chemistry. For example, in some embodiments, after the PMLA backbone is conjugated to the immune checkpoint inhibitor, additional active ester moieties can be functionalized with a moiety containing an orthogonal reactive group, such as a thiol, for example by coupling mercaptoethylamine (MEA) to the polymeric backbone. The -NH₂ group of the MEA forms an amide bond to the polymeric backbone by reaction with the active ester (e.g., succinimidyl ester), leaving the free thiol group available for conjugating the angiopep peptide. For example, in some embodiments, the angiopep peptide is first functionalized with a maleimide moiety, which is then coupled to the free thiol group conjugated to the biodegradable polymeric backbone via a Michael addition reaction. In some embodiments, the BBB penetrating peptide (e.g. AP-2, L57, DI, D3, or ACI-89) is further functionalized with a polyethylene glycol (PEG) moiety to provide a spacer between the angiopep peptide and the polymeric backbone. For example, in some embodiments, a precursor compound is prepared by coupling the BBB penetrating peptide to a polyethylene glycol (PEG) moiety, and the precursor compound is subsequently conjugated to the biodegradable polymeric backbone. In some embodiments, the PEG moiety has a molecular weight of about 1000 Da to about 5000 Da, or about 2000 Da to about 4000 Da. Preparation of such an BBB penetrating peptide precursor can be effected, for example, by coupling the BBBpenetrating peptide (e.g. AP-2 peptide) including a C-terminal cysteine, (e.g.

[0050] TFFYGGSRGKRNNFKTEEYC-NH₂ (SEQ ID NO: 22)), to maleimide-PEG-maleimide (e.g., Mal-PEG₃₄₀₀-Mal). The remaining free maleimide moiety is then available to react with the free thiol group conjugated to the polymeric backbone.

[0051] In some embodiments, about 0.1% to about 5% of monomeric units within the biodegradable polymeric backbone are conjugated to the BBB penetrating peptide (e.g. AP-2, L57, DI, D3, or ACI-89). For example, in some embodiments, about 0.1%, about 0.5%, about 1%, about 2%, about 3%, about 4%, or about 5% of monomeric units within the biodegradable polymeric backbone are conjugated to the BBB penetrating peptide (e.g. AP-2, L57, DI, D3, or ACI-89). Loading of the angiopep peptide can be controlled, for example, by adjusting the reaction stoichiometry and / or reaction time.

[0052] In some embodiments, the nanoconjugate comprises a PMLA backbone conjugated to: (1 ) the immune checkpoint inhibitor peptide NP-12, and (2) an angiopep peptide (e.g. AP-2). In some embodiments, the PMLA backbone is conjugated to NP-12 and AP-2. In some embodiments, about 0.1% to about 5% of monomeric units within the PMLA backbone are conjugated to the immune checkpoint inhibitor peptide and about 0.1%, about 0.5%, about 1%, about 2%, about 3%, about 4%, or about 5% of monomeric units within the PMLA backbone are conjugated to the angiopep peptide (e.g. AP-2). In some embodiments, additional groups or moieties can be conjugated to the biodegradable polymeric backbone. For example, in some embodiments, the nanoconjugate further comprises an additional peptide conjugated to the biodegradable polymeric backbone, such as a trileucine moiety (LLL). Such moieties display pH-responsive lipophilicity and promote endosomal escape of PMLA bound agents once they are internalized and part of the intracellular endosomal pathway. In some embodiments inclusion of a trileucine moiety improves blood brain barrier permeability. In some embodiments, the trileucine moiety is conjugated to the biodegradable polymeric backbone via an amide bond. For example, an activated PMLA compound such as those described above (e.g., one in which the pendant -COOH groups have been converted to active esters) can be conjugated to the N- terminal -NH₂ group of a trileucine peptide to form an amide bond. In some embodiments, about 10% to about 30% of monomeric units within the biodegradable polymeric backbone are conjugated to the trileucine moiety. For example, in some embodiments, about 10%, about 15%,about 20%, about 25%, or about 30% of monomeric units within the biodegradable polymeric backbone are conjugated to the trileucine moiety.

[0053] In some embodiments, the nanoconjugate further comprises a fluorophore conjugated to the biodegradable polymeric backbone, such as rhodamine, fluorescein, or the like. In some embodiments, about 0.1% to about 2% of monomeric units (e.g., about 1%) within the polymeric backbone are conjugated to the fluorophore. Fluorophore conjugation can facilitate visualization of the nanoconjugates via fluorescence imaging. The fluorophore can be conjugated using conjugation chemistries similar to those disclosed above. For example, many fluorophores are commercially available with pendant reactive moieties, such as maleimides, which can be attached to free thiol groups that had previously been conjugated to the polymeric backbone as described above.

[0054] In some aspects, the nanoconjugates provided herein find use in methods of treating central nervous system cancers. The term “central nervous system cancer” or “cancer of the central nervous system” indicates any cancer within the central nervous system (e.g. brain or spinal cord). The cancer may originate in the central nervous system or may have metastasized to the central nervous system. In some embodiments, the cancer is a glioblastoma, meningioma, pituitary adenoma, astrocytoma, schwannoma, oligodendroglioma, ependymoma, or medulloblastoma. In some embodiments, the cancer is a cancer that has metastasized to the brain. In some embodiments, the cancer is a cancer that has metastasized to the spinal cord.

[0055] In some embodiments, provided herein is a method of treating a central nervous system cancer in a subject, comprising providing to the subject a nanoconjugate provided herein.

[0056] Without wishing to be bound by theory, the blood-brain barrier penetrating peptide (e.g. angiopep-2) in the nanoconjugates herein is thought to interact with LRP1 receptors that typically maintain blood brain barrier integrity. This interaction with LRP1 reduces blood brain barrier integrity and function, enabling the nanoconjugate to cross the blood brain barrier and enter the central nervous system such that the immune checkpoint inhibitor exerts local effects at the site of a tumor. As such, the nanoconjugates provide herein represent a viable treatment for central nervous system cancers that are typically difficult to treat due to the relative impermeability of the blood brain barrier that otherwise prevents therapeutic agents from reaching the desired site of action (e.g. the tumor environment).In some embodiments, the method of treating a central nervous system cancer comprises providing to the subject a nanoconjugate provided herein. The nanoconjugate may be formulated into a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers. Reference to provide to the subject a nanoconjugate is inclusive of providing to the subject a composition (e.g. a pharmaceutical composition) comprising the nanoconjugate. The phrase “pharmaceutically acceptable” refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a subject (e.g., a mammal, a human). Preferably, as used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans. Pharmaceutically acceptable carriers, including buffers, are well known in the art, and may comprise phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; amino acids; hydrophobic polymers; monosaccharides; disaccharides; and other carbohydrates; metal complexes; and / or non-ionic surfactants. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.

[0057] The effective amount and / or dosage of the nanoconjugate to treat a cancer in a subject depends on the particular cancer being treated, the severity of the cancer, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. It will be appreciated that appropriate dosages can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the treatments of the present disclosure. In some embodiments, the effective amount alleviates, relieves, ameliorates, improves, reduces the symptoms, or delays the progression of the cancer in the subject. In some embodiments, the subject is a human. The amount and route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side -effects to the patient.Administration can be in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. In some embodiments, the nanoconjugate is administered to the subject continuously or intermittently over the course of a suitable dosing window. For example, the dosing window may be 10 minutes to 6 hours, 20 minutes to 5 hours, 30 minutes to 4 hours, or about 1 to 3 hours. In some embodiments, the nanoconjugate is provided to the subject once per day. In some embodiments, the nanoconjugate is provided to the subject multiple times per day. In some embodiments, the nanoconjugate is provided to the subject every other day, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every 8 days, every 9 days, every 10 days, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, annually, etc. The nanoconjugate may be administered until a desired reduction of symptoms is achieved.

[0058] Other therapies, as included in the above methods, may be used in combination with the nanoconjugate. For example, other anti-cancer therapies or other medications used to alleviate one or more symptoms of cancer (e.g. pain medications, anti-nauseam medications, etc.) in combination with the nanoconjugates described herein. Administered “in combination,” as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject's affliction with the disorder, e.g.. the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery.” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins.

[0059] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject has or is suspected of having a central nervous system cancer. In some embodiments, the subject is an adult. In some embodiments, the subject is a pediatric subject (e.g. less than 18 years of age).

[0060] EXAMPLESEXAMPLE 1

[0061] This example demonstrates the development and use of an innovative, actively- targeted immunotherapeutic nanoconjugate (P-12 / AP-2 / NCs). designed to serve as an immunotherapeutic agent capable of traversing the BBB via LRP-1 receptor-mediated transcytosis. P-12 / AP-2 / NC exerts its immune-modulating effects by inhibiting the PD-1 / PD-L1 axis through a small molecule PD-L1 antagonist peptide (P-12). P-12 / AP-2 / NCs is synthesized from completely biodegradable, functionalized high molecular weight β-polymalic acid polymer, conjugated with P-12 (also referred to herein are “NP-12”) and angiopep-2 (AP-2) to yield P-12 / AP-2 / NCs. Evaluation of nanoconjugates for BBB permeability using an in vitro 3D tumor-BBB static model demonstrated successful crossing of the BBB and internalization by brain 3D tumor environments. In addition, the nanoconjugate mediated successful tumor cell killing in a U87 GBM spheroid model. AP-2 / P-12 is selectively internalized in T cells, increasing inflammatory cytokine secretion and T cell proliferation. Notably, in an in-vivo murine brain environment, rhodamine-labeled AP-2 / P-12 displayed significantly increased accumulation in the brain and prolonged bioavailability over unconjugated peptides. Provided herein is a novel, programmable nanoconjugate platform, capable of penetrating the with BBB for directed delivery and significant immune environment modulation potential, offering promise for treating challenging brain diseases like glioblastoma multiforme, and beyond.

[0062] Results:

[0063] Synthesis and Characterization of AP-2 / P-12 functionalized Nanoconjugates (AP-2 / P-12 NCs) The AP-2 / P-12 immunotherapeutic peptide-functionalized NCs were synthesized. FIG. IB and FIG. 1C shows an exemplary synthesis scheme for the nanoconjugate. The process shown in FIG. 1C is understood to be performed after forming the P-12 / thiolated PMLA in FIG. IB. FIG. 2A is a schematic illustrating an exemplary method for isolation, purification, and characterization of PMLA. Initially, the biocompatible poly (β-L-malic acid polymer was extracted from sp. Physarum polycephalum and purified to yield pure polymer with molecular weight of 50-100 kDa. Due to its biocompatibility and ease in tailorable nature, the PMLA possessing free COOH groups were utilized as a polymer backbone for the immune-nanoconjugate (FIG. 1B, FIG. 2A). The purified biosynthesized PMLA polymer was reacted with DCC / NHS to activate the free COOH sites of the polymer, followed by reaction with trileucine (LLL), P-12 (PD-L1 blocker) or P-12-FITC, and mercaptoethylamine (MEA) to obtain preconjugate P-12 / thiolated PMLA. Thereafter, the preconjugate with free thiol (SH) group was reacted with compound maleimide-PEG-AP-2 peptide (LRP1 peptide) and PDP to obtain AP- 2 / P-12 NCs. The fluorescent labelled AP-2 / P-12 NCs were prepared by reacting Rhodamine Red™-C2-maleimide with AP-2 / P-12 NCs to obtain AP-2 / P-12 / RhB NCs (FIG. IB, FIG. 1C). During the synthesis process, the efficiency of AP-2 and P-12 peptide conjugation was determined using size exclusion and high-pressure liquid chromatography, resulting in purity of >95 % (FIG. 2B). The obtained AP-2 / P-12 NCs were characterized using Zetasizer Nano ZS90 (Malvern Instruments. Malvern, UK) exhibiting an average particle size of less than 100 nm, and a polydispersity index of < 0.3 (Table 1).

[0064] Table 1. Hydrodynamic diameter, polydispersity index (PDI) and pol tentials.

[0065] Nanoconjugate I Hydrodynamic Diameter Polydispersity Index potentials (nm) (PDI)

[0066] PMLA -4.440 3.140 0.264

[0067] PMLA / LLL -35.666 45.2 0.270 PMLA / LLL / Rh -5.970 3.934 0.254 PMLA / LLL / AP2 / Rh -7.133 94.57 0.230 PMLA / LLL / P-12 / Rh -8.160 81.396 0.250 PMLA / LLL / AP2 / / P- -8.363 39.490 0.227

[0068] 12 / Rh

[0069] P / LLL / AP2 / FITC- -6.613 61.41 0.212

[0070] P12 / Rh

[0071]

[0072] AP-2 / P-12 NCs Successfully Crosses the BBB-on Chip-GBM model in vitro

[0073] To investigate the efficacy of the AP-2 / P-12 nanoconjugate in crossing the BBB, human 3D GBM tumor spheroids composed of U87, PDM 140 and LN229 cells were constructed. A model BBB was synthesized using human brain endothelial cells (HBMEC) coated on afibronectin layer (extracellular matrix) to serve as a barrier to block entry of particles into the tumor spheroid (Fig. 3a). This model referred to herein as a BBB on-chip-GBM model. After the construction of the BBB model, the transepithelial / trans-endothelial electrical resistance (TEER) was measured to verify barrier integrity. The developed HBMEC BBB on-chip model was validated using immunocytochemistry (ICC) for their characteristic protein marker such as zona occludens-1 (ZO-1) (red color) for protein located on a cytoplasmic membrane surface of intercellular tight junctions, VE-cadherin (green color) for the protein localized at the intercellular boundaries of endothelial cells, GLUT-1 (red color) protein for facilitative glucose transporter, and CD31 (green color) for platelet endothelial cell adhesion molecule- 1 (PEC AM -1) protein. After the construction of the BBB model, the transepithelial / trans-endothelial electrical resistance (TEER) was measured to verify barrier integrity. Next, the ability of nanoconjugates to first target and cross the model BBB was investigated. Cells comprising the BBB model were stained with DAPI nuclei staining, where rhodamine red colocalization by immunocytochemistry (IHC) indicated successful nanoparticle uptake (Fig. 3b). Using free rhodamine as a control, there was no binding or colocalization to BBB cells. When the rhodamine-labeled nanoparticle without AP-2 was incorporated, a modest rhodamine signal on microscopy was observed. With AP-2 conjugation (e.g. in the AP-2 / P-12 NC), there was significant increase in signal, indicating successful targeting of BBB endothelial cells. Next, nanoconjugate crossing was investigated in a complete transwell model with the constructed BBB and GBM tumor spheroid (Fig. 3c, Fig. 3d). Tumor spheroids were stained with DAPI and green Cell Tracker for cell membrane. Free rhodamine and rhodamine-labeled nanoparticles with or without AP-2 were then incorporated. Successful nanoparticle binding to the tumor spheroid in AP-2 / P-12 nanoparticles was observed, as indicated by a positive rhodamine red signal on the spheroid appreciated by microscopy (Fig. 3c). The efficacy of the nanoparticle crossing the BBB and binding to tumor spheroid in a murine model was also investigated, with similar findings observed. Taken together, these results indicate that AP-2 conjugation facilitates nanoparticle crossing of the BBB.

[0074] P-12 induces robust expansion and function of proinflammatory T cells

[0075] After characterizing the kinetics of P-12, the effect of the particle in the tumor-immune microenvironment (TiME) of GBM was evaluated. First, the T-cell activating properties of P-12compared to a-PDl antibody, free P-12 peptide, and AP-2 / P-12 nanoconjugates was assessed. Mouse splenocytes were activated with anti-CD3a and anti-CD28 antibodies, followed by recombinant PD-L1 or PD-L2 (Figure 4a). Splenocytes were then treated with anti-mouse PD-1 as a positive control, free P-12 peptide, AP-2 / P-12 nanoparticles, or AP-2 nanoparticles with no P-12 as a negative control. Cytokine levels were measured by ELISA and intracellular staining was assessed by FACs. Results are shown in Fig. 4b. T cell populations were analyzed with CD4, CD8, Ki67 and FOX-P3 (regulatory T cell, Treg). Both the CD4 and CD8 T-cell populations were reduced in the presence of recombinant PD-L1. Treatment with AP-2 / P-12 nanoconjugate (“conjugate”) significantly expanded the CD8+ and CD4+ T cell subsets compared with free P-12 (“peptide”) and free anti-PDl antibody (a-PD-1). In addition, there was no significant difference in Treg expansion between all groups (Fig. 4b). ELISA-based assays were also used to investigate the effects of therapies on proinflammatory cytokine production of TNF-a, IL-2, and IFN-y. Notably, inhibiting the PD-1 / PD-L1 axis using any treatment modality led to an increase in the panel of proinflammatory cytokines compared with control. The AP-2 / P-12 nanoconjugate treatment produced robust proinflammatory cytokine secretion by T-cells compared with P-12 or anti-PD-1 antibodies of TNF-a, IL-2, and IFN-y (Fig. 4b).

[0076] P-12 activated T cells mediate cytotoxicity of 3D GBM spheroids:

[0077] After verifying the in-vitro activation of T cells utilizing P-12, the ability of the nanoconjugate to activate lymphocytes and other immune mileu to mediate tumor cell cytotoxicity was evaluated. Human brain cancer spheroids were generated from three different (LN-229, PDM-140, and U-87) cancer cell lines co-cultured with peripheral blood immune cells from healthy donors (HD- PBMCs). The growth of different cancer spheroids was monitored using the Maestro Z platform and the immune cell-mediated killing of cells was compared between different samples of treated spheroid cultures. The cytotoxic effect of samples-treated immune cells on spheroids was evaluated by measuring the ratios of live and dead cells in the spheroids. This coculture system was treated with control, P-12 peptide alone, or the AP-2 / P-12 nanoconjugate. Without any therapy, the intrinsic tumor immune microenvironment of the spheroid poses a significant challenge to the immune activation of T cells. The untreated spheroids in the wells were viable while maintaining their round shape. Treatment AP-2 / P-12 provided highly effective tumor cell killing compared with P-12 alone across all spheroid models(Fig. 5 A). The tumor spheroid size was also monitored following treatment, with AP-2 / P-12 nanoconjugates displaying the greatest reduction in tumor volume at day 2 and day 4 following treatment (Fig. 5B).

[0078] In vivo P-12 cellular uptake and biodistribution in C57Balb / C mice and toxicity data analysis The uptake of the AP-2-targeted nanoconjugate (AP-2 / P-12 NC) and nanoparticles lacking AP-2 into the brain capillary endothelial cells to differentiate between active and passive uptake mechanisms was investigated through confocal laser scanning microscopy. Furthermore, different in vitro co-incubation experiments were performed with competing ligands of the respective receptor. For studies concerning the cellular uptake and intracellular distribution of the nanoconjugates, confocal laser scanning microscopy (CLSM) was used. The bEnd3 cells were incubated with the unspecific nanoconjugates (P-12 nanoparticles) or specific AP-2-modified nanoparticles (AP-2 / P-12 nanoparticles) for 4 h at 37°C. In the case of the incubation with the specific AP-2-modified nanoconjugates, a clear intracellular uptake and accumulation could be observed in contrast to the unspecific control nanoconjugates (Figure 3B). After establishing successful BBB crossing of the nanoparticle in vitro and in vivo through brain parenchyma, an in vivo investigation on the trafficking of P-12 in a living organism was investigated using C57Balb / C mice. FITC-labeled dye was conjugated to P-12 and IVIS was used to track the dispersion of P-12 in the murine model over time. Mice were administered saline, P-12 peptide, or P-12 conjugated to the nanoparticle with or without the LRP1 targeting moiety AP-2. Biodistribution was evaluated in vivo by live imaging, and ex vivo by isolating selective tissues and evaluating fluorescence therein. The in vivo trafficking of nanoconjugates was determined using an In vivo imaging system (IVIS) in healthy C57BL / 6 using FITC dye as a fluorescent probe (FIG. 6A-6D). The mice were injected with saline, and free FITC-P-12 peptide showed no significant signal intensity at 2h and 6 h. On the contrary, upon administration of AP-12 / FITC-P-12 NCs, an intense signal was observed throughout the body in 2 and 6 h. See FIG.

[0079] 6A and FIG. 6C. To better delineate the organ distribution of the nanoconjugates, the animals were euthanized at 2 and 6 h, and ex vivo signals of majorly excised organs (brain, kidney, liver, spleen, heart, and lungs) were determined (FIG. 6B for 2 hour time point, FIG. 6D for 6 hour time point). Meanwhile, saline and free P-12 peptide-injected animals showed no significantsignal in any organ. Upon administration of AP-12 / P-12 NCs, an intense signal was seen in the brain at 2 and 6 h compared to the P-12 NCs and free P-12-pep group. The signal was also observed in other organs, including the kidney, liver, spleen, and lungs, at both 2 and 6 h, indicating the beneficial properties of AP-2 / P-12 NCs in BBB targeting. AP-12 / P-12 NCs penetrate into the brain more than P-12 nanoconjugate without AP2. Both nanoconjugates also distribute into multiple viscera and promote longer drug bioavailability than free P-12 peptides. In vivo survival study

[0080] AP-2 / P-12 NCs were evaluated in a preclinical model of melanoma brain metastases (MBM). The study employed established Immune checkpoint blockade (ICB)-resistant MBM models having characterized immunogenicity and response to PD-L1 directed therapy, including Yale University Mouse Melanoma (YUMM1.7) lines derived from GEMM parent tumors that carried activating BrafV600E mutations and inactivating PTEN- / -Cdkn2- / - mutations. C57BL / 6 female mice, aged 8-10 weeks were used. YUMM-GFP-Luc cells (1,000) were stereotactically injected in a total volume of 2 p into the right striatum. Tumor growth was tracked by bioluminescent in vivo imaging system (IVIS) imaging every three days through postimplantation day 21. Survival experiments were conducted in triplicate, with n = 5 mice per group for survival analysis. Therapy was initiated once tumors were established and compared systemic anti-PD-1 therapy with AP-2 / P-12 NCs. Five treatment groups were evaluated: (1) untreated control, (2) standard InVivoMAb anti-mouse PD-L1, (3) free P-12 peptide, (4) P-12 NCs, and (5) AP-2 / P-12 NCs. Tumor growth was monitored by bioluminescence imaging (BLI), and endpoints included survival, brain tumor burden, and metastatic volume. Mice were euthanized at humane endpoints, which included signs of CNS disturbances, hunched posture, lethargy, significant weight loss, and inability to ambulate. Results are shown in FIG. 8. As shown, AP-2 / P-12 NCs significantly improved survival.

[0081] Discussion and Conclusion

[0082] GBM is a highly aggressive cancer that is severely limited in therapeutic options due to its sequestration within the BBB, which prevents passage of 90% of all small-molecule drugs, along with the profoundly immunosuppressive environment of the brain. Utilizing targetedtherapy that can navigate past this barrier and access the TiME is of paramount importance. Provided herein is nanoparticle-based therapy that can cross the BBB and deliver immunotherapy to the GBM TiME. Specifically, the nanoconjugate AP-2 / P-12 is shown herein to cross the BBB and modulate the immune cell milieu within the GBM TiME. The AP-2 peptide component of P-12 / NCs allows the nanoconjugates to bind to LRP1 and cross the BBB by transcytosis, and then mediate endocytosis in the tumor cell. P-12 (also referred to herein as “NP-12”), an a-PD-Ll molecule conjugated to the nanoparticle, inhibits immune checkpoints and restore cytotoxic activity. AP-2 / P-12 is able to cross an in vitro BBB model, target both human and murine GBM tumor spheroids, and demonstrates successful biodistribution into murine brains while posing minimal toxicity in off-target sites. In addition, AP-2 / P-12 had the most profound activation of the immune cell milieu compared to immune peptide molecules alone without nanoconjugate targeting. These findings demonstrate that this conjugate can successfully infiltrate the BBB to reach the GBM tumor and activate players of the immune system to mediate tumor cell killing. Other suitable immune checkpoint inhibitor peptides may be used in lieu of P-12 the targeted nanoconjugates herein to exert similar local anti-tumor effects upon nanoconjugate crossing of the BBB driven by the angiopep peptide (e.g. AP-2).

[0083] One of the traditionally limiting factors of using PMLA is its difficulty in synthesis. Creating usable PMLA conjugates takes multiple purification steps and the usage of different solvents and apparatuses make reproducibility challenging. Herein, this barrier was overcome by generating an optimized PMLA nanocarrier generation protocol to successfully synthesize the polymer in large volumes for further investigation. The AP-2 / P-12 conjugate has favorable intrinsic chemical properties that allow it to infiltrate past the BBB and deliver therapies of interest to the TiME, making it a powerful potential anticancer therapeutic with the feasibility to scale the synthesis protocol for wide use in treating brain tumors. In conclusion, the AP-2 / P-12 nanoconjugate herein overcomes significant obstacles in GBM treatment and has therapeutic potential extending beyond GBM to all difficult-to-treat brain tumors or other inflammatory brain diseases. Provided herein is a scalable nano-conjugate production method, capable of being individually tailored and modified for target-specific delivery for future personalized drug therapies including using immune-peptides over current standard antibody-based

[0084] immunotherapies.Materials:

[0085] Highly purified poly β-L-malic acid (PMLA) with a molecular mass of 33,800 g / mol (as determined by SEC-HPLC using polystyrene sulfonate standards) was prepared from the culture broth of Physarum polycephalum M3CVII, following previously reported methods [1]. The cysteine-modified Angiopep-2 peptide (AP-2) (sequence: TFFYGGSRGKRNNFKTEEYC-NH2 (SEQ ID NO: 22, which contains an additional cystine and an amidated terminus) was obtained from NovoPro Bioscience Inc. (Minhang, Shanghai, China). Trileucine (LLL) was sourced from Bachem (Torrance, CA, USA). Mal-PEG3400-Mal and mPEG5000-NH2 were purchased from Laysan Bio. Rhodamine Red C2 maleimide (rh) was ordered from Thermo Fisher Scientific (Canoga Park, CA, USA). PD-10 columns were acquired from GE Healthcare (Chicago, IL, USA). Additionally, the following chemicals were obtained from Sigma Aldrich (St. Louis, MO, USA): N, N'-Dicyclohexylcarbodiimide (DCC), N-Hydroxysuccinimide (NHS), tri-fluoroacetic acid (TFA), cysteamine (2-mercaptoethylamine, MEA), dithiothreitol (DTT), deuterated acetone, dimethylformamide (DMF), 3-(2-pyridyldithio)propionic acid (PDP), triethylamine (NEt3), and 5,5'-disulfanediylbis(2-nitrobenzoic acid) (DTNB).

[0086] Synthesis of PMLA Bio-polymer:

[0087] The PMLA was produced by culturing single cells slime mold (Physarum polycephalum) and obtained crude was purified using previously described method to yield poly (P-L-malic acid; 67,000 g / mol (Ref: 10.3791 / 50668) polymers. Physarum polycephalum seed culture was grown from spherules on agar plate; the plasmodia was transferred to 500 mL culture medium and incubated at 25 °C under dark conditions. After that, the 500 mL containing plasmodia was transferred to a 10 L bioreactor containing CaCO3 basal medium (80 g CaCO3 in 8 L basal medium) and processed at 25 °C. with an airflow of 10 L / min and 150 rpm stirring for 75 h until the broth gets acidified to pH 4.8 The end of production of PMLA content was measured with a hydroxamate / Fe(III) assay. The broth was cooled to 17 °C and the cells were allowed to settle down, followed by cooling the suspension to 4 °C, and the pH was adjusted to 7.5 using 2 M NaOH. The obtained supernatant was pumped through a DEAE-cellulose column (equilibrated with 20 mM Tris-HCl pH 7.5 at 4°C), washed with a 0.3 M NaCl buffer, and the crude PMLA mixture was eluted with 0.7 M NaCl. A solution of 0.1 M CaC12 was added to PMLA crude to yield PMLA-calcium complex, followed by precipitation with 80% ice-cold ethanol and size-fractionated over Sephadex G25 into 80-300 kDa, 50-80 kDa, and 10-50 kDa PMLA-calcium using purified water. Subsequently, the fractions were acidified through an activated Amberlite IR 120H+ column and the flow-through (PMLA-COOH) was collected, snap-frozen in liquid nitrogen, and lyophilized to yield dried PMLA powder and stored at -20 °C until further use. The material was dissolved in dry acetone, filtered, dried, lyophilized, and stored at -20 °C to keep the polymer under anhydrous condition under storage.

[0088] Synthesis of P-12 peptide nanoconjugates (P-12 / NCs) (PMLA / LLL / P-12 / RhB) conjugate:

[0089] The P-12 NCs were synthesized using carbodiimide coupling. Briefly, 320 mg of PMLA was dissolved in 3 mL of DMF. Subsequently, a mixture of 320 mg of N-hydroxysuccinimide and 592 mg of N, N'-dicyclohexylcarbodiimide (DCC) in 3 mL of dimethylformamide (DMF) was added dropwise to activate the PMLA, followed by stirring for 2 hours at room temperature. Afterward, a DMF solution of 394.40 mg of tri-leucine, 2.15 mg of P-12 peptide or FITC-P-12 and 101 pL of trifluoroacetic acid was added in portions for 1 h, along with triethylamine (NEt3) in specified increments totaling 213.12 pL in 1.6 mL of DMF. The reaction was stirred for 12 h, followed by the addition 56 mg of DL-dithiothreitol (DTT) in 400 pL of DMF, mercaptoethylamine hydrochloride (MEA. HC1) (31.36 mg) in 86.4 pL of DMF, and NEt3 (in a 1:1 ratio with MEA). The reaction mixture was allowed to stir for 3 h to yield thiol pendant polymer PMLA / LLL / (20%) / P-12 or FITC-P-12 (0.22%) / MEA(10%). The polymer were purified using a PD-10 column and subsequently lyophilized.

[0090] Synthesis of Angiopep-2-PEG3400-maleimide (AP-2) pre-conjugate:

[0091] The Angiopep-2-PEG3400-maleimide (AP-2) pre-conjugate was prepared by dissolving Maleimide-PEG3400-Maleimide (molecular weight 3400 g / mol; 7.4 mg, 2.2 pmol, 1.05 equiv.) in 500 pL phosphate buffer (100 mM, pH 6.3) supplemented with 2 mM of EDTA.

[0092] Subsequently, 5 mg of cysteine-modified Angiopep-2 peptide (H-TFFYGGSRGKRNNFKTEEYC-OH) (cys-AP-2) was dissolved in 500 pL of phosphate buffer (pH 6.3). Thereafter, the cys-AP-2 was added to the mixture dropwise at 4 °C. The reaction was stirred for 1 h under nitrogen conditions, followed by purification and freeze-drying. The resulting product was reconstituted in phosphate buffer (10 mg / mL, pH 6.3) for the synthesis of PMLA conjugates.Synthesis of AP-2 / P-12 NCs (P / LLL(20%) / P-12(l%) / AP-2(l%) / RhB(l%) conjugate):

[0093] The final AP-2 / P-12 NCs were synthesized by dissolving 30 mg of PMLA / LLL / (20%) / P-12 or FITC-P-12 (1%) MEA(5%) (130 g / mol, 7.5 nmol) in 3 mL of degassed PBS (150 mM, pH 6.3) in a glass vial. Thereafter, 1% peptide loading, 14.24 mg of AP-2-PEG3400-maleimide (5802.7 g / mol, pmol) was dissolved in 1.3 mL of PBS (150 mM, pH 6.3) and added dropwise to the reaction mixture, allowed to stir for 1.5 h under ice-cold (4 °C) nitrogen conditions.

[0094] Rhodamine Red™ C2 Maleimide (680.79 g / mol, 0.153 pmol) dissolved in DMF from a 2.5 mg / mL solution in DMF (332.8 pL) was added in the dark conditions and stirred for 1.5 h under at 4 C. Subsequently, 120 pL of PDP (10 mg / mL solution in DMF) was introduced to cap the free thiol groups and the solution was stirred for an additional hour. The crude reaction mixture was then purified using a PD-10 column with PBS as the solvent. The amount of rhodamine conjugated to the PMLA backbone was analyzed using fluorescence and HPLC analysis, followed by freeze-dried and subjected to lyophilization. The samples were stored at -20 °C until further use. The polymer conjugates were purified using a PD-10 column and subsequently lyophilized.

[0095] Preconjugates and Nanoconjugate Characterization:

[0096] 1H NMR Characterization: The polymers, pre-conjugates and nanoconjugates were initially characterized using NMR spectroscopy (Bruker Avance-III HD 600 MHz) in DMSO-d6as a solvent and analyzed using Mestrenova software.

[0097] Polymer and Peptide Quantification: The synthesis of nanoconjugates was monitored through size-exclusion chromatography (SEC-HPLC) using the Agilent instrument with a Hitachi L-2455 detector and EZChrome Software. The SEC-HPLC column was Polysep 4000, at Iml / min flow rate and PBS (pH 7.4, 150 mM). Components and intermediates in purified PMLA and synthesized nanoconjugates validated using SEC-HPLC. The SEC-HPLC elution profiles of PMLA and nanoconjugates are shown at wavelength 220 nm in Fig. 2B Purified PMLA molar mass measured by SEC-HPLC in phosphate-buffered saline (pH 7.3) using polystyrene sulfonate as molecular mass standards. Reverse-phase HPLC was used to quantify the P-12 peptide innanoconjugates. The samples were monitored at 220 nm with a flow rate of 1 mL / min, and PBS (pH 7.4) was used as the eluent at 25 °C.

[0098] Particle size, Polydispersity index and Zeta Potential Characterization: For particle size, polydispersity index (PDI) and zeta potential analysis of nanoconjugates, samples were prepared at a concentration of 2 mg / mL in PBS saline and filtered using a 0.2 pm pore membrane. The 1.5 mL of sample solution was measured by the Malvern Nano-ZS zeta sizer instrument.

[0099] Thiol quantification Assay:

[0100] To prepare the experimental solutions, 2 mg of poly(malic acid) (PMLA) in a 40% polymer and 10% monoethylamine (MEA) composition were dissolved in 0.4 mL of ethanol to achieve a final concentration of 5 mg / mL. Concurrently, 5 mg of Ellman's Reagent (DTNB: 5,5-dithio-bis(2-nitrobenzoic acid)) was dissolved in 0.5 mL of ethanol, yielding a 10 mg / mL solution. Following this, a 50 pL aliquot of the DTNB solution was combinatorially mixed with 50 pL of the prepared PMLA / LLL / MEA solution and 900 pL of phosphate-buffered saline (PBS) adjusted to pH 8.5. The resultant mixture was gently stirred for 30 minutes at ambient temperature to facilitate the reaction. Absorbance measurements were conducted at 412 nm using a spectrophotometer to quantify the formation of the thioketone derivative. For calibration, a reference solution was prepared by diluting 50 pL of DTNB in 950 pL of PBS (pH 8.5). The absorbance readings obtained at 412 nm were subsequently converted to concentration based on the molar absorptivity coefficient of 14,150 M-1cm-1[1], The loading efficiency of MEA onto the PMLA / LLL / MEA matrix was determined and expressed as a percentage (4.6-5.4 %), reflecting the total malic acid content incorporated within the PMLA polymer.

[0101] 3D Tumor Spheroid Formation:

[0102] The method utilized for creating humanized 3D-brain tumor spheroids involved using 3D Petri Dishes® technology (MicroTissues Inc, Sigma-Aldrich, St. Louis, MO, USA), Briefly, sterile 2 % agarose in 0.9% NaCl was used to create 12-series agarose micro-molds equilibrated with DMEM containing 10% PBS and 0.1% Penicillin-Streptomycin mixture. The U87MG, PDM140, LN229, and GL261 cell line suspension was then seeded into the micro-molds at a density of around 50 xl03 cells per micro-mold. After settling for 5 min at 4°C additional 1 mLof media was added to the wells. Spheroid formation within the 3D Petri Dish® was observed every day using an inverted microscope for cell culture examination.

[0103] Spheroid Live / Dead assay:

[0104] Following staining with a live / dead viability / cytotoxicity kit, confocal fluorescence images were captured using an ECHO microscope. The staining kit used Calcein AM to stain live cells with green fluorescence and ethidium homodimer- 1 to label dead cells with red fluorescence as per manufacturer protocol. The process involved adding Calcein AM (2 pM) and Ethidium Homodimer (4 pM) staining solution to the spheroids post-drug exposure and incubating the dye solution for 10 min at 37°C shielded from light, followed by twice washing with sterile PBS. Live / dead cell visualization was achieved using a fluorescent ECHO microscope with the appropriate Calcein AM and ethidium homodimer- 1 fluorescence filters.

[0105] In vitro Cellular uptake of Nanoconjugates in BBB-transwell-3D spheroids system:

[0106] Immortalized human cerebral microvascular endothelial cells (HBMEC) were cultured in Transwell plates to create an in vitro blood-brain barrier (BBB) model. 50x103 cells were seeded on the apical side of 12-well polystyrene transwell plates with 0.4 pm pores and 1.12 cm2 inserts (Coming™ 3402, Cat. 07-200-157). The cells were cultured in differentiation medium (EBM-2 supplemented with 5% FBS, 1% penicillin / streptomycin, 1.4 pM hydrocortisone, 5 pg / mL ascorbic acid, 1% lipid concentrate, 10 mM HEPES buffer, and 1 ng / mL bFGF) for 3 days at 37 C with 5% CO2 and saturated humidity (500 pL in apical compartment, 1 mL in basolateral compartment). Subsequently, the medium was switched to growth medium (EBM-2 supplemented with 5% FBS, 1% penicillin / streptomycin, 1.4 pM hydrocortisone, 5 pg / mL ascorbic acid, 1% lipid concentrate, 10 mM HEPES buffer, and 10 mM LiCl) for 6-7 days, with medium changes occurring every 3rd day. Trans-endothelial electrical resistance (TEER), a measure of tight junction integrity, was assessed daily using an epithelial voltammeter (EVOM2) from World Precision Instruments (FL, USA). In vitro, a blood-brain barrier (BBB) model was created by culturing human brain endothelial cells (HBMEC) 50 xlO 3 cells on the top of fibronectin (2 pg / cm2) layer as an extracellular matrix on collagen-coated inserts, 3.0 pm, 12 mm, 12 well plates for 4th day. Every day, the cellular layers were observed to conform to amonolayer and subsequently, the transepithelial / trans-endothelial electrical resistance (TEER) was quantified to confirm the integrity of the barrier in the BBB model. The Tight junction proteins ZO-1, Occludins, claudins, and VEGF were evaluated on the BBB layers at day 7.

[0107] To determine the in vitro blood-brain barrier (BBB) permeability and 3-D tumor Spheroids of nanoconjugates, following 7th day and 4th day growing culture of HBMEC cells and 3-D tumor spheroids growth on BBB-3D spheroid model, cell medium was removed from each upper and down layer well, and cell growth medium were replaced with fresh medium without FBS. The Free Rhodamine, Rhodamine-labeled nanoconjugates P-12 / MEA / LLL / Rhodamine and P-12 / MEA / LLL / Rhodamine / PEG-AP-2 were added to the apical compartment at a concentration equivalent to 0.30X (46.7 pg / mL for 4 hours dye analyzed on cellular uptake of nanoconjugate in HBMEC cells and green-labeled 3D Spheroids using confocal fluorescence.

[0108] Efficacy of nanoconjugates on T cell proliferation inhibiting PDL1:

[0109] To determine in vitro efficacy of peptide nanoconjugates, the proliferation of CD4 and CD8 lymphocytes and Cytokines was analyzed. The P-12 peptides at 10 mmol / L and an equivalent concentration of P-12 / NCs conjugate stock were initially prepared in sterile PBS with a pH of 7.2. These solutions were further diluted with cell medium in the assay media. Mouse splenocytes were cultured in cells culture media (RPMI with 10% FBS + 2mM L-glutamine + 0.5 mM of 2-mercaptoethanol + 2 mM Nonessential Amino acid (NEM) solution + 10,000 units / mL penicillin and 10,000 mg / mL streptomycin). Following the washing steps, the first fresh splenocytes were stimulated with specific anti-CD3a and anti-CD28 antibodies (1 pg / mL each) and incubated for 72 hours at 37°C with 5% CO2. On the 4th day, the splenocytes at a concentration of 10 x 106cells / mL were treated with 1 mmol / L carboxyfluorescein succinimidyl ester (CFSE) for 30 min at 37°C in a pre-warmed 1 mL solution of PBS with 0.1% BSA solution. After this, 4 times of cold growth media were added to the cells and incubated on ice for 5 min to remove any remaining free CFSE dye. The CFSE-labeled splenocytes underwent triple washing in cold PBS to replace with T cell culture media. CFSE-labeled splenocytes at a concentration of 1 x 105cells / well were exposed to a solution containing recombinant PD-L1 or PD-L2 (10 nmol / L each) and simultaneously treated with P-12 peptide, InVivoMAb anti-mouse PD-1 (CD279) (positive control), P / P-12 / AP-2 NCs and P / AP-2 NCs (negative control) in a 96-well plate incubated for 72 hours. After, the incubation period the supernatants were collected for cytokines IL-2, TNF-a, and IFN-y levels were measured at 450 nm by using an ELISA assay kit, and on the other end treated cells were harvested by centrifugation at 400 x g for 5 min at 4°C and underwent three washes with PBS and further resuspended in cold FACS buffer. The cells were treated with fluorescently labeled antibodies targeting CD4+ and CD8+ T cells. They were then incubated in a dark condition for 30 min at 4°C. Following incubation, the cells underwent three washes with FACS buffer. Next, the cell suspension was treated with fixation / permeabilization buffer to enable intracellular staining with anti-Ki67 and FoxP3 antibodies. After labeling, the cells were washed and resuspended in FACS buffer. The analysis was carried out using FACS Calibur (make and model), with a minimum of 50,000 total events recorded on the live gate scatter plot. The cells positive for each marker were identified and the percentage of Ki67-positive cells was determined for interpretation.

[0110] PBMCs Isolation and T Cell Isolation, Activation, and Culturing

[0111] To verify if free P-12 peptide and peptide nanoconjugates exert a functional effect on human T cells and 3D Spheroid co-culture in Trans well, formulation performance was assessed on freshly isolated human peripheral blood mononuclear cells (PBMC). Briefly, human PBMCs were isolated from healthy donor’ s blood by density gradient centrifugation using Ficoll-Paque PLUS. In brief, blood was diluted at 1:1 with phosphate-buffered saline (PBS), layered on Ficoll-Paque and centrifuged at 400 x g for 30 min. To remove RBC, the PBMC was diluted with RBC lysis buffer for 2 min at 4°C and washed twice with PBS re-suspended in phosphate-buffered saline. Isolated PMBCs were immediately enriched by PMA for 24 hours. For stimulating and activating T cells, isolated cells were cultured in 6 well-plates in RPMI-1640 medium with 10% fetal bovine serum (FBS), 100 lU / mL penicillin, and 100 pg / mL streptomycin. Then PBMC inoculated with human-specific anti-CD3a and anti-CD28 antibodies (1 pg / mL each) incubated for 72 hours at 37°C with 5% CO2 supplemented with 50 TU / mL of recombinant human IL-2 (Novus Biologicals, Littleton, CO, USA).

[0112] Investigation of the nanoconjugates interactions with T cells and 3D Spheroids coculture:

[0113] Rhodamine-labeled peptide nanoconjugates cellular uptake on the CD3 / CD28 activated CFSE labeled PBMC. PBMC were plated in a 24-well culture plate and then treated with 1 mL1% bovine serum albumin (BSA) for 30 min at 37 °C. Next, the cells were incubated with 300 nM P-12 peptide nanoconjugates at 37°C for 1 hour under dark conditions. After incubation, cells were washed twice with PBS and resuspended in PMBC media. Finally, PBMC was collected, washed twice with PBS, and the uptake of rhodamine-labeled nanoconjugates was detected on CD3- / CD28- and CD3+ / CD28+ treated cells surface by confocal fluorescence microscopy (OLYMPUS IX53, Tokyo, Japan). For the detection of rhodamine (red) and CFSE (green) fluorescence, the excitation wavelength is 490-495 nm and the emission wavelength is 520-530 nm. For the detection of red fluorescence, the excitation wavelength is 549 nm and the emission wavelength is 565 nm. The binding interaction of PBMC with 3-D tumor spheroid were performed by co-culturing the CFSE labeled PMBC with Hoechst (blue) labeled GBM spheroid in the presence and absence of nonfluorescent nanoconjugate. The images were captured under a fluorescence microscope and assessed using ImageJ software and the Fiji plug-in by analyzing the fluorescence channels (green for PBMC cells, Blue for GBM spheroids).

[0114] Live / Dead assay analysis in T cells-3D spheroid Co-culture:

[0115] In this assay, activated PBMC were co-cultured with GBM spheroids in the presence of P-12 peptide. P-12 / AP-2 / NCs, and control conditions for 96 hours. After the incubation period, the spheroids were washed thrice with cold PBS and then stained with Calcein AM (2 pM) and Ethidium Homodimer (4 M) staining solution. The spheroids were incubated in the dye solution for 10 min at 37 °C shielded from light, followed by two washes with sterile PBS. Live / dead cell visualization was performed using a fluorescent ECHO microscope with Calcein AM and ethidium homodimer- 1 fluorescence filters. The viability of GBM spheroids was assessed using ImageJ software and the Fiji plug-in by analyzing the fluorescence channels (green for live cells, red for dead cells) from raw images taken with fluorescence microscopy.

[0116] In-vivo and ex-vivo Biodistribution:

[0117] For the biodistribution studies in healthy animals, the experiment involved dividing the animals into three groups. One group received saline as a control, while the other group received nanoconjugates at a dose of 10 mg / kg via tail vein injection (IV administration). Following the drug administration, the animals were imaged at 2 hours, 6 hours, and 12 hours intervals for invivo imaging using the IVIS Imaging system. For ex vivo tissue biodistribution analysis, animals were sacrificed at 2 hours and 6 hours after administration. Various organs such as the brain, kidney, liver, spleen, heart, and lungs were then isolated from each group. The excised organs were promptly rinsed in ice-cold normal saline to remove blood, dried, and subsequently imaged using the IVIS Imaging system. This process allowed for the assessment of how the nanoconjugates were distributed among different organs over time.

[0118] Animal safety and toxicity study:

[0119] In this study, animals were divided into two dose levels of approximately 10 mg / kg and 20 mg / kg equivalent amount of P-12 peptides, administered with nanoconjugates composed of different formulations: P / LLL (40%) / AP-2(2%) / rh(1%), P / LLL(40%) / P-12(0.22%) / rh(1%), and P / LLL(40%) / AP-2(2%) / P-12(0.23%) / rh(1%) with a total of 21 animals. Each animal received a single intravenous dose. Mice were randomly assigned to receive either the nanoconjugates groups or a control group intravenously. Body weights were monitored twice weekly, and clinical observations were conducted at least twice daily during dosing and once daily thereafter. Mortality checks were performed twice daily with at least 6 hours between each check. On day 15, blood samples were obtained via transcardiac aspiration for hematological evaluation, and vital organs (brain, heart, kidney, lung, liver, and spleen) were collected for histopathological analysis immediately after euthanasia.

[0120] Statistical Analysis:

[0121] Data were analyzed using GraphPad software. Results are expressed as mean + / - s.e.m. or s.d. Data were analyzed by t-test or ANOVA and results are considered significant at p<0.05.

Claims

CLAIMS1. A nanoconjugate comprising:a) a biodegradable polymeric backbone;b) an immune checkpoint inhibitor conjugated to the biodegradable polymeric backbone; andc) a blood-brain barrier penetrating peptide conjugated to the biodegradable polymeric backbone.

2. The nanoconjugate of claim 1, wherein the biodegradable polymeric backbone comprises a plurality of monomeric units, and each monomeric unit comprises a pendant -COOH group.

3. The nanoconjugate of claim 1 or claim 2, wherein the biodegradable polymeric backbone comprises polymalic acid (PMLA).

4. The nanoconjugate of any one of the preceding claims, wherein the immune checkpoint inhibitor comprises a peptide.

5. The nanoconjugate of any one of the preceding claims, wherein the immune checkpoint inhibitor comprises a peptide that antagonizes signaling at a receptor selected from CTLA4, PD-1, VISTA, BTLA, TIM-3, KIR, LAG-3, TIG IT, CD-96, and SIRPa.

6. The nanoconjugate of any one of the preceding claims, wherein the immune checkpoint inhibitor comprises a peptide comprising the amino acid sequence of SEQ ID NO: 1, or comprises a peptide selected from: NP-12, TPP-1 (SGQYASYHCWCWRDPGRSGGSK, SEQ ID NO: 3), CLP002 (WHRSYYTWNLNT. SEQ ID NO: 4), C8 (CKWYRPSEC, SEQ ID NO: 5), P-F4 (FSGTVTTAGLLF, SEQ ID NO: 6), OPBP-1 (GQSEHHMRVYSE, SEQ ID NO: 7), PD-LlPep-1 (CLQKTPKQC, SEQ ID NO: 8), PDL1-Pep2 (CVRARTR, SEQ ID NO: 9), Pep-39 (AMSDHHWTQRDK, SEQ ID NO: 10), nABP284(SRLKEIANSPTQFWRMVARNTLGNGAKQSLNTEHARL, SEQ ID NO: 11). C25 (CVPMTYRAC, SEQ ID NO: 12), ERY2-4 (CAWGQAILEGELAWLEGGGGGAGQLADLKRQLAWWKQAC, SEQ ID NO: 13), p344 (ARHPSWYRPFEGCG. SE ID NO: 14), PPA (NYSKPTDRQYHF, SEQ ID NO: 15), HVEM(14-39) (ESCPKCSPGYRVKEACGELTGTVCEP (SEQ ID NO: 16), P16 (cyc(EIDTVLTPTGWVAKRYS) (SEQ ID NO: 17), LC4 (WGHSHFSHWKGR, SEQ ID NO: 18), TBP-3 (GGYTFHWHRLNP, SEQ ID NO: 19). D4-2 (RYSAVYSIHPSW, SEQ ID NO: 20), and PPL-C (SVSVSHFQKVWVVGGGSK, SEQ ID NO: 27),7. The nanoconjugate of any one of claims 4-6, wherein the immune checkpoint inhibitor peptide comprises a terminal -NH₂ group.

8. The nanoconjugate of any one of claims 4-7, wherein the immune checkpoint inhibitor peptide is conjugated to the biodegradable polymeric backbone via an amide bond.

9. The nanoconjugate of any one of the preceding claims, wherein about 0,1% to about 5% of monomeric units within the polymeric backbone are conjugated to the immune checkpoint inhibitor.

10. The nanoconjugate of any one of the preceding claims, wherein the blood-brain barrier penetrating peptide comprises an angiopep family peptide, L57, DI, D3, or ACI-89.

11. The nanoconjugate of claim 10, wherein the angiopep family peptide is angiopep-2 (AP-2).

12. The nanoconjugate of any one of the preceding claims, wherein the blood-brain barrier penetrating peptide is conjugated to the biodegradable polymeric backbone via a polyethylene glycol (PEG)-containing spacer.

13. The nanoconjugate of any one of the preceding claims, wherein the PEG-containing spacer has a molecular weight of about 1000 Da to about 5000 Da.

14. The nanoconjugate of any one of the preceding claims, wherein about 0.1% to about 5% of monomeric units within the polymeric backbone are conjugated to the angiopep peptide.

15. The nanoconjugate of any one of the preceding claims, further comprising a trileucine moiety conjugated to the polymeric backbone.

16. The nanoconjugate of claim 15, wherein the trileucine moiety is conjugated to the polymeric backbone via an amide bond.

17. The nanoconjugate of claim 15 or claim 16. wherein about 10% to about 30% of the monomeric units within the polymeric backbone are conjugated to the trileucine moiety.

18. The nanoconjugate of any one of the preceding claims, further comprising a fluorophore conjugated to the polymeric backbone.

19. A method of treating a central nervous system cancer comprising providing to the subject the nanoconjugate of any of the preceding claims.

20. The method of claim 19, wherein the central nervous system cancer is a brain cancer.

21. The method of claim 20, wherein the brain cancer is glioblastoma multiforme.

22. The method of any one of claims 19-21, wherein the subject is a mammal.

23. Use of the nanoconjugate of any one of claims 1-18 in a method of treating a central nervous system cancer in a subject.

24. Use of claim 23, wherein the central nervous system cancer is a brain cancer.

25. Use of claim 24, wherein the brain cancer is glioblastoma multiforme.

26. Use of any one of claims 23-25, wherein the subject is a mammal.

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