WO2024167958A1

WO2024167958A1 - Antigen-capturing nanoparticles and formulations for immunotherapy

Info

Publication number: WO2024167958A1
Application number: PCT/US2024/014664
Authority: WO
Inventors: Hong Ren; Sonke Svenson; Dapeng Qian; Andrew Wang
Original assignee: Archimmune Therapeutics, Inc.
Priority date: 2023-02-06
Filing date: 2024-02-06
Publication date: 2024-08-15

Abstract

The present invention provides novel antigen-capturing nano-particles (ACNPs) and pharmaceutical formulations containing such ACNPs. Also provided herein are methods for preparing the ACNP formulations, and methods for the treatment of a disease (such as cancer) in a subject with the ACPNs or a pharmaceutical formulation containing the ACPNs.

Description

Antigen-Capturing Nanoparticles and Formulations for Immunotherapy Cross-Reference to Related Application [1] This application claims the priority of US patent application No. 63/443,555, filed on February 6, 2023, the contents of which are incorporated herein by reference in their entirety. Background of the Invention [2] Immunotherapy is a type of cancer treatment that uses the power of a person’s own immune system to prevent, control, and eliminate cancer. Immunotherapy comes in a variety of forms and holds tremendous promise for improving cancer treatment. Recent development of immune checkpoints inhibitors, such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell death protein 1 (PD-1), and programmed cell death ligand 1 (PD-L1), has shown to be safe and effective. To date, these immune checkpoints inhibitors have been used in Hodgkin’s lymphoma, non-small cell lung cancer, melanoma, renal cell cancer, bladder transitional cell carcinoma and squamous cell. However, research estimates that only 10-20% of patients obtain long-lasting benefits from these treatments (See, Ignacio Morales-Orue et al, Reports of practical oncology and radiotherapy 24 (2019) 86-91). Such findings have led to high interest in developing strategies to further improve cancer immunotherapy. [3] Today, the growing consensus is that combining radiotherapy (RT) with immunotherapy provides an opportunity to boost the “abscopal effect”, a phenomenon of tumor regression where distant tumors outside of a radiation field shrink in response to treatment that was not directed toward them. R. H. Mole coined the word first “abscopal” (‘ab’ - away from; ‘scopus’ - target) in 1953 to describe radiation effects “at a distance from the irradiated volume but within the same organism” when he observed this phenomenon in 2 cases of lung and esophageal cancer (See, Mole R., Br J Radiol.1953; 26(305): 234-241; Demaria S, Formenti SC., Br J Radiol.2020; 93(1109): 20200042). The mechanism of the synergy of radiotherapy with immunotherapy is thought to be due to radiotherapy-induced cell death leading to improved antigen exposure to the antigen presenting cells. Radiotherapy-induced cell death induces the release of many tumor antigens and enables the development of an antigenic “cascade” (expansion of T cell clones that are reactive against a variety of tumor antigens) rather than an immune response against a few antigens. Radiotherapy can also induce the release of immune modulating molecules that can further enhance the effects of immunotherapy (See, Nikhila ReddySultanpuram, et al, Acta Biomaterialia, 2022(153): 299-307). However, published data evaluating abscopal effect using radiotherapy together with immunotherapy remain limited. Considering this, other methods to enhance the abscopal effect by exposure of immune cells to cancer-specific antigens after RT are in high demand. [4] In this invention, Applicant provides novel antigen-capturing nanoparticles (“ACNP”) and formulations containing such nanoparticles. They significantly improve the synergy between radiotherapy and immunotherapy, thereby treating cancer more effectively. Brief Summary of the Invention [5] One aspect of the present invention is directed to novel antigen-capturing nanoparticles (“ACNPs”) and formulations containing such ACNPs. The formulations can be used in treating cancer or inducing an immune response. [6] In some embodiments, the present invention provides a nanoparticle comprising a core, wherein the core comprises Polymer-X-J in which X is PEG', linker, PEG’-Linker, or absent; and J is a reactive group or absent; and when J is a reactive group, a portion or all of the J is optionally covalently bound to PEP-PEG", in which the PEP is a protease sensitive protein sequence, and the Polymer comprises poly(lactic-co-glycolic acid) (“PLGA”). [7] In some embodiments, the ratio of lactic acid to glycolic acid (“LA:GA”) in the Polymer is in a range of 10: 90 to 90: 10 (weight/weight). [8] In some embodiments, the ratio of the lactic acid to glycolic acid (“LA:GA”) in the Polymer is in a range of 25:75, 50:50 or 75: 25 (weight/weight). [9] In some embodiments, the preferred ratio of the lactic acid to glycolic acid (“LA:GA”) in the polymer is about 75:25 (weight/weight). A higher molecular weight and a higher lactic acid to glycolic acid ratio contribute to increased hydrophobicity in the ACNP core, resulting in enhanced stability due to reduced susceptibility to hydrolysis. [10] In some embodiments, the molecular weight (MW) of the Polymer is in a range of 10- 100KDa, 30-70 KDa, or 40-65KDa; preferably, in a range of in a range of 55-65KDa, 42-62KDa or 45-55KDa. In some embodiments, molecular weight (MW) of the X moiety can be further optimized. [11] In some embodiments, the nanoparticle is free from any antigen. [12] In some embodiments, J is maleimide (“Mal”). [13] In some other embodiments, J is absent, or J and X are both absent. The nanoparticle core can provide a functional group (such as with maleimide) or a hydrophobic surface (such as with unmodified PLGA) to capture proteins via covalent or hydrophobic-hydrophobic interactions. [14] In some embodiments, the core of the nanoparticle comprises PLGA-X-Maleimide, PLGA or PLGA-X. [15] In some embodiments, the nanoparticle comprises a mixture of PLGA-X-J and PLGA-X at a mass ratio of 0.05: 1 to 1:0.05, wherein X is PEG', linker, PEG’-Linker, or absent, and a portion or all of J is optionally covalently bound to PEP-PEG" in which PEP is a protease sensitive protein sequence. [16] In some embodiments, the nanoparticle comprises a mixture of PLGA-X-Maleimide and PLGA at a mass ratio ranging from 0.05:1 to 1:0.05, wherein maleimide is optionally covalently bound to PEP-PEG" in which PEP is a protease sensitive protein sequence. In some preferred embodiments, the mass ratio of PLGA-X-Maleimide to PLGA is 1:1. [17] In some embodiments, when J is a reactive group, a portion or all of J is optionally covalently bound to PEP-PEG", thereby, the nanoparticle described herein comprises a PEG corona. The PEG corona may block premature and nonspecific protein adsorption before radiotherapy and accumulation in the tumor. The PEG corona may also prevent phagocytosis before radiotherapy and accumulation in the tumor. [18] In some embodiments, PEP, the protease sensitive sequence, is capable of being cleaved by a protease (such as Caspase 3, Cathepsin B and MM2) to remove the PEG corona (PEG’’). In some preferred embodiments, PEP, the protease sensitive sequence, is selected from the group consisting of Gly-Phe-Leu-Gly, Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln, and Glu-Val-Asp-Gly. [19] In some embodiments, the nanoparticle further comprises an adjuvant. [20] In some embodiments, the adjuvant is selected from a small molecule, a double-stranded RNA molecule, and a single-stranded DNA molecule. [21] Examples of the small molecule adjuvant include, but not limited to, imiquimod, resiquimod, and gardiquimod. [22] Examples of the double-stranded RNA molecule include, but not limited to, poly(inosinic- cytidylic acid) (“Poly IC” or “Poly (I:C)”), poly IC, Riboxxol RGI®50, poly IC mixed with the stabilizers, poly ICLC, complexes between poly IC and poly(ethylene imine) (“PEI”), and PEI. [23] Examples of the single-stranded DNA molecule include but not limited to CpG oligodeoxynucleotides (CpG ODN). [24] In some embodiments, the adjuvant is poly IC, PEI or poly IC/PEI. [25] In some embodiments, the nanoparticle is optionally lyophilized by a process comprising freeze-drying or spray drying. And such a process requires the presence of a lyoprotectant. [26] In some embodiments, the lyoprotectant is a sugar molecule or a polymer. [27] Examples of lyoprotectant include, but not limited to, HEPES buffered saline (“HBS”), mannose, sucrose, trehalose, mannitol, poly(ethylene glycol) (“PEG”), poly(ethyleneimines), PEI, Poly(vinyl alcohol) (“PVA”), or a mixture thereof. [28] In some embodiments, the lyoprotectant is HEPES buffered saline (“HBS”), sucrose, PVA or a mixture thereof. [29] Another aspect of the present invention provides pharmaceutical formulations each comprising the nanoparticle as described above and a pharmaceutically acceptable carrier or excipient. [30] In some embodiments, the pharmaceutical formulation further comprises PVA associated with the nanoparticle. Preferably, the mass ratio of PVA to the core of the nanoparticle is 20-70% or 30-50%. [31] In some embodiments, the pharmaceutical formulation further comprises a buffer with pH range of 7-8. preferably, the buffer is HEPES buffer at pH7.4 or HEPES buffered saline (HBS) at pH 7.4. [32] Yet still another aspect of this invention provides a method for enhancing effectiveness of cancer treatment or inducing an immune response in a subject in need thereof, comprising administering to the subject a nanoparticle or a pharmaceutical composition as described above. [33] In some embodiments, the composition is administrated after a previous treatment of the subject. In some embodiments, the previous treatment is radiation. [34] In some embodiments, the composition is used in combination with a second therapeutic agent. [35] In some embodiments, the second therapeutic agent is an immune checkpoint inhibitor. Example of the immune checkpoint inhibitor, without limitation, is PD-1 antibody. [36] Cancers that can be treated (including reduction in the likelihood of recurrence) by the methods of the present teachings include, but not limited to, brain cancer, non-small cell lung cancer, small cell lung cancer, esophageal cancer, gastric cancer, pancreatic cancer, colorectal cancer, renal cell carcinoma, bladder cancer, prostate cancer, breast cancer, non-Hodgkins lymphoma, Hodgkin's lymphoma, anal cancer, head and neck cancer, or melanoma. Brief Descriptions of the Drawings [37] Fig. 1 is a schematic depiction of utilizing ACNP for improving cancer immunotherapy. Following radiotherapy, ACNPs bind to tumor antigens and enhance their presentation to dendritic cells. The enhanced antigen-presentation and immune activation is synergistic with αPD-1 treatment. [38] Figs. 2b and 2c show an average tumor growth curve and survival curve of antigen- capturing nanoparticles (ACNPs), respectively, in combination with anti-PD1 on mice inoculated with B16F10 melanoma cancer cells. Abscopal effect is based on tumor growth inhibition by comparing tumor growth curves between ACNP treatment group and control group. [39] Fig.3a and Fig.3b show size stability over time during storage at 4-8°C for PLGA-PEG-Mal and PLGA ACNP formulation candidates, respectively. Preferred particle size is 50-200 nm, more preferred size is 70-150 nm. [40] Fig. 4 shows the stability of the maleimide surface in PLGA-PEG-Mal at 7.00 ppm compared to the internal standard maleic acid with a peak at 6.20 ppm. The ratio between the peak areas allows quantification of the maleimide surface. The result shows that introducing the maleimide active cap to an ACNP does not accelerate its degradation in vitro versus an uncoupled standard. [41] Fig.5 shows association scenarios between ACNP and adjuvant (e.g., PLGA-PEG-Mal and poly IC). [42] Fig.6 shows three processes for associating ACNP (e.g., PLGA) with adjuvant (e.g., poly IC). [43] Fig.7 shows tumor model, treatment schedule and tumor growth delay in mice inoculated with CT26 carcinoma cells. Fig.7a depicts tumor model and treatment schedule. Figs.7b and 7c show tumor growth delay on left (primary) and right (secondary) flank of mice, respectively. [44] Fig.8 shows a flow chart of TFF run on PLGA ACNP in Example 26. [45] Fig.9 shows an NMR spectrum of TFF collection in Example 26 [46] Fig.10 shows an NMR spectrum of TFF wash in Example 26. [47] Fig.11 shows standard curves of PLGA, PLGA-PEG-Mal and the mixture of PLGA and PLGA- PEG-Mal solutions. All the three curves fit well in polynomial trendlines. [48] Figs.12a and 12b show good stability of PLGA ACNP and PLGA-PEG-Mal ACNP at tested concentrations, respectively. [49] Fig.13 shows exemplary images of different ACNPs. [50] Fig.14a depicts ACNP in vivo efficacy study design in CT26 bi-lateral tumor models and treatment schedule. Figs.14b and 14c show the significant abscopal tumor growth inhibition (TGI) effect of co-injection of AT1011 and polyIC-40µg, in comparison to base line treatment (i.e., RT+anti-PD1). Fig. 14b shows individual tumor growth of primary and secondary tumor and overall tumor growth (mean tumor volume and SEM). Fig.14c shows two-way ANOVA analysis of Group 3, 4, 5 in comparison with base line treatment (Group 2). [51] Fig.15 shows improved survival of treated animals by co-injection of AT1011 and poly IC, in comparison to base line treatment (i.e., RT+anti-PD1). [52] Fig.16 shows mean body weight change percentages with SEM. [53] Fig.17a depicts ACNP in vivo efficacy study design in CT26 bi-lateral tumor models and treatment schedule. Fig. 17b and 17c are intratumoral injection of AT1011 or co-injection of AT1011+AT1014, which shows significant abscopal tumor growth inhibition (TGI) effect in comparison to base line treatment RT+anti-PD1 at both dose levels. Fig. 17b shows individual tumor growth of primary and secondary tumor and overall tumor growth (mean tumor volume and SEM). Fig.17c shows two-way ANOVA analysis of Group 3, 4, 5, 6 in comparison with base line treatment RT+anti-PD1(Group 2). [54] Fig.18 shows improved survival of treated animals of co-injection of AT1011 and polyIC in comparison to base line treatment RT+anti-PD1. [55] Fig.19 shows mean body weight change percentages with SEM. [56] Fig.20 shows CT26-WT and EMT6 tumor growth in previously cured mice and tumor naive mice. Fig.20a shows overall tumor growth of 4 study groups (mean tumor volume and SEM). Fig. 20b shows individual tumor growth in each study group. [57] Fig.21 shows the changes of body weight in the integrated safety study. [58] Fig.22 schematically depicts the treatment timelines for in vivo cancer immunotherapy experiments in Example 47. [59] Fig. 23 shows ACNPs improved immunotherapy and the abscopal effect in CT26 WT xenografts. (A) Growth curves of irradiated (primary) and unirradiated (secondary) tumors in individual mice treated with immunotherapy and AT1019 at various dose levels; (B) Average tumor-growth curves of unirradiated (secondary) tumors in the mice treated in (A); (C) Survival curves of the mice in various groups. Data represent mean ± s.e.m. Differences in survival were determined for each group by the Kaplan–Meier method and the overall p value was calculated by the log-rank test. *p < 0.05, vs group 2 (RT+anti-PD-1). [60] Fig.24 shows body weight changes over time for various groups. [61] Fig.25 shows tumor growth curves. Detailed Description of the Invention [62] The presently disclosed subject matter will now be described more fully hereinafter. It is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed, and modifications and other embodiments are intended to be included within the scope of the appended claims. [63] The present invention provides novel antigen-capturing nanoparticles (“ACNP”) and compositions comprising such nanoparticles, which can enhance cancer immunotherapy and induce the abscopal effect. In another aspect, the present invention provides methods of treating cancer or inducing immune responses by administering the ACNP or composition as described to a subject in need thereof. ACNP, compositions and methods of the present invention find use in, among other things, clinical (e.g., therapeutic and preventative medicine) and research applications. [64] The ACNP comprises a polymer core that in embodiments have surface modifications that allow for binding of antigens. In an embodiment, the nanoparticles are formulated using poly (lactic-co-glycolic acid) (“PLGA”), a biocompatible and biodegradable polymer. In another embodiment, the nanoparticle's surface was modified to enable binding of tumor derived protein antigens (TDPAs) by a variety of mechanisms. Without being bound by theory, it is believed that unmodified PLGA nanoparticles bind to proteins principally through non-covalent interactions (weak ionic/coulombic/hydrogen bonding), and ACNPs coated with maleimide bind to proteins predominantly by reaction with thiol groups to create thioether bonds. In addition, both ACNP species are capable of hydrophobic interactions with antigen proteins. I. Definitions [65] To facilitate an understanding of the present invention, a number of terms and phrases are defined below. Unless otherwise stated, the following terms used in the specification and claims have the meanings discussed below: [66] As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. [67] As used herein, the term “about” in front of a number or numerical range means that another number, even not identical to the number following “about” or outside the range following “about,” is within the scope of this invention as long as it is not a significant deviation from the number or range following “about” and results in significantly similar effect as the number or a number in the range that follows the work “about.” [68] As used herein, the term “formulation” and “composition” are interchangeable. [69] As used herein, the term “abscopal effect” or “abscopal response” refers to an oncologic phenomenon in which localized treatment of a tumor affects not only on the treated tumor, but also tumors outside the scope of the localized treatment. [70] As used herein, the phrase “potentiating the abscopal effect” refers to an increase in the abscopal effect of 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%, following administration of antigen-capturing nanoparticles, relative to the abscopal effect seen in the absence of the antigen-capturing nanoparticles. [71] As used herein, the term “liposome” refers to an artificial microscopic vesicle consisting of an aqueous core enclosed in one or more phospholipid layers, used to convey vaccines, drugs, enzymes, or other substances to target cells or organs. [72] As used herein, the term “non-covalent” refers to the interactions between two or more species wherein the interactions are, for example, hydrogen bonds, Coulombic interactions, ionic bonds, van der Waals forces, and/or hydrophobic interactions. [73] The term “covalently bound” or “covalently linked” refers to a chemical bond formed by sharing of one or more pairs of electrons. [74] As used herein, the term “ionic bond” refers to the formation of ions by transfer of one or more electrons from one atom to another, thus generating two oppositely charged ions. [75] As used herein, the term “contacting” refers to reagents in close proximity so that a reaction may occur. [76] As used herein, the term “linker” refers to a chemical moiety comprising a chain of atoms that covalently attaches the core of a nanoparticle to other chemical moieties. [77] As used herein, the term “antibody” is used in the broadest sense and covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multi-specific antibodies (e.g., bispecific antibodies), and antibody fragments, including chimera thereof, nanobodies, and the like, so long as they exhibit the desired biological activity (Miller et al (2003) Jour. of Immunology 170:4854- 4861). Antibodies may be murine, human, humanized, chimeric, or derived from other species. An antibody is a protein generated by the immune system that is capable of recognizing and binding to a specific antigen. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). A target antigen generally has numerous binding sites, also called epitopes, recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody. An antibody includes a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that immunespecifically binds an antigen of a target of interest or part thereof, such targets including, but not limited to, cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulin disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. The immunoglobulins can be derived from any species. In some embodiments, the immunoglobulin is of human, murine, or rabbit origin. [78] As used herein, the term “checkpoint inhibitor” or “immune checkpoint inhibitor” is any molecule that directly or indirectly inhibits, partially or completely, an immune checkpoint pathway. Without wishing to be bound by any particular theory, it is generally thought that immune checkpoint pathways function to turn on or off aspects of the immune system, particularly T cells. Following activation of a T cell, a number of inhibitory receptors can be upregulated and present on the surface of the T cell in order to suppress the immune response at the appropriate time. In the case of persistent immune stimulation, such as with chronic viral infection, for example, immune checkpoint pathways can suppress the immune response and lead to immune exhaustion. Aspects of the disclosure are related to the observation that inhibiting such immune checkpoint pathways and administering synthetic nanocarrier compositions comprising antigens and immunostimulators, can result in the generation of enhanced immune responses to the antigen and/or a reduction in immunosuppressive immune responses against the antigen. Examples of immune checkpoint pathways include, without limitation, PD-1/PD-L1, CTLA4/B7-1, TIM-3, LAG3, By-He, H4, HAVCR2, ID01, CD276 and VTCN1. In the instance of the PD-1/PD-L1 immune checkpoint pathway, an inhibitor may bind to PD-1 or to PD-L1 and prevent interaction between the receptor and ligand. Therefore, the inhibitor may be an anti-PD-1 antibody or anti-PD-L1 antibody. Similarly, in the instance of the CTLA4/B7-1 immune checkpoint pathway, an inhibitor may bind to CTLA4 or to B7-1 and prevent interaction between the receptor and ligand. Non-limiting examples of immune checkpoint inhibitors include fully human monoclonal antibodies, such as BMS-936558/MDX-1106, BMS-936559/MDX-1105, ipilimumab/Yervoy, and tremelimumab; humanized antibodies, such as CT-011 and MK-3475; and fusion proteins, such as AMP-224. [79] As used herein, the terms “capture” or “captured” refers to the binding of a nanoparticle to an antigen. [80] As used herein, the term “lipid” refers to a member of a group of organic compounds that has lipophilic or amphipathic properties, including, but not limited to, fats, fatty oils, essential oils, waxes, steroids, sterols, phospholipids, glycolipids, sulpholipids, aminolipids, chromolipids (lipochromes), and fatty acids. The term “lipid” encompasses both naturally occurring and synthetically produced lipids. “Lipophilic” refers to those organic compounds that dissolve in fats, oils, lipids, and non-polar solvents, such as organic solvents. Lipophilic compounds are sparingly soluble or insoluble in water. Thus, lipophilic compounds are hydrophobic. Amphipathic lipids, also referred to herein as “amphiphilic lipids” refer to a lipid molecule having both hydrophilic and hydrophobic characteristics. The hydrophobic group of an amphipathic lipid, as described in more detail immediately herein below, can be a long chain hydrocarbon group. The hydrophilic group of an amphipathic lipid can include a charged group, e.g., an anionic or a cationic group, or a polar, uncharged group. Amphipathic lipids can have multiple hydrophobic groups, multiple hydrophilic groups, and combinations thereof. Because of the presence of both a hydrophobic group and a hydrophilic group, amphipathic lipids can be soluble in water, and to some extent, in organic solvents. [81] As used herein, the term “hydrophobic” refers to a physical property of a molecule that is repelled from a mass of water and can be referred to as “nonpolar,” or “apolar,” all of which are terms that can be used interchangeably with “hydrophobic.” Hydrophobicity can be conferred by the inclusion of apolar groups that include, but are not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic or heterocyclic group(s). [82] As used herein, the term “hydrophilic” refers to a physical property of a molecule that is capable of hydrogen bonding with a water molecule and is soluble in water and other polar solvents. The terms “hydrophilic” and “polar” can be used interchangeably. Hydrophilic characteristics derive from the presence of polar or charged groups, such as carbohydrates, phosphate, carboxylic, sulfato, amino, sulfhydryl, nitro, hydroxy and other like groups. [83] Examples of amphipathic compounds include, but are not limited to, phospholipids, aminolipids and sphingolipids. Representative examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoy-loleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoyl-phosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoyl phosphatidic acid, and dilinoleoylphosphati-dylcholine. Other compounds lacking in phosphorus, such as sphingolipid, glycosphingolipid families, diacylglycerols and β-acyloxy acids, also are within the group designated as amphipathic lipids. [84] As used herein, the term “cationic lipid” encompasses any of a number of lipid species that carry a net positive charge at physiological pH, which can be determined using any method known to one of skill in the art. These include, but are not limited to, N-methyl-N-(2-(argin- oylamino)ethyl)-N,N-Di octadecyl ammonium chloride or distearoyl arginyl ammonium chloride (DSAA), N,N-dimyristoyl-N-methyl-N-2 [Nʹ(N6-guanidino-L-lysinyl)] aminoethyl ammonium chloride (DMGLA), N,N-dimyris-toyl-N-methyl-N-2[N2-guanidino-L-lysinyl]aminoethyl ammonium chloride, N,N-dimyristoyl-N-methyl-N-2 [Nʹ(N2,N6-di-guanidino-L-lysinyl)] aminoethyl ammonium chloride, and N,N-di-stearoyl-N-methyl-N-2[Nʹ (N6-guanidino-L-lysinyl)] aminoethyl ammonium chloride (DS-GLA). Other non-limiting examples of cationic lipids that can be present in the liposome or lipid bilayer of the presently disclosed delivery system complexes include N,N-dioleyl-N, N-dimethylammonium chloride (DODAC); N-(2,3-dioleoyloxy)propyl)- N,N,N-trimethylammonium chloride (DOTAP); N-(2,3-dioleyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTMA) or other N (N,N-1-dialkoxy)-alkyl-N,N,N-trisubstituted ammonium surfactants; N,N-distearyl-N,N-dimethylammoniumbromide (DDAB); 3-(N(Nʹ,Nʹ dimethylaminoethane)carbamoyl) cholesterol (DC-Chol) and N-(1,2-dimyristyloxyprop-3-yl)-N,N- dimethyl-N-hydroxyethyl ammonium bromide (DMRIE); 1,3-dioleoyl-3-trimethylammonium- propane,N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethyl- lammoniumtrifluoro acetate (DOSPA); GAP-DLRIE; DMDHP; 3-β[4N(1N,N-diguanidinospermidine) carbamoyl] cholesterol (BGSC); 3-β[N,N-diguanidinoethyl-aminoethane)-carbamoyl] cholesterol (BGTC); N,N1,N2,N3 Tetra-methyltetrapalmityl-spermine (cellfectin); N-t-butyl-Nʹ-tetradecyl-3- tetradecyl-aminopropion-amidine (CLONfectin); dimethyldioctadecyl ammonium bromide (DDAB); 1,3-dioleoyloxy-2-(6-carbox-yspermy1)-propyl amide (DOSPER); 4-(2,3-bis-palmitoy- loxy-propy1)-1-methy1-1H-imidazole (DPIM) N,N,Nʹ,Nʹ-tet-ramethyl-N,Nʹ-bis(2-hydroxyethyl)- 2,3-dioleoyloxy-1,4-butanediammonium iodide) (Tfx-50); 1,2 dioleoyl-3-(4ʹ-trimethylammonio) butanol-sn-glycerol (DOBT) or cholesteryl (4ʹ trimethylammonia) butanoate (ChOTB) where the trimethylammonium group is connected via a butanol spacer arm to either the double chain (for DOTB) or choles-teryl group (for ChOTB); DL-1,2-dioleoyl-3-dimethylami-nopropy1-β- hydroxyethylammonium (DORI) or DL-1,2-O-dioleoyl-3-dimethylaminopropy1-β- hydroxyethylammonium (DORIE); 1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC); cholesteryl hemisuccinate ester (ChOSC); lipopolyamines such as dioctadecylamidoglycylspermine (DOGS) and dipalmitoyl phosphatidylethanolamylspermine (DPPES); cholestery1-3-β-carboxyl-amido-ethylenetrimethylammoniumiodide; 1- dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl carboxylate iodide; cholesteryl-3- β-carboxyamidoethyleneamine; cholesteryl-3-β-oxysuccinamido-ethylenetrimethylammonium iodide; 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-3-β-oxysuccinateiodide; 2-(2-trimethylammonio)-ethylmethylaminoethyl-cholesteryl-3-β-oxysuccinate iodide; and 3-β- N-(polyethyleneimine)-carbamoylcholesterol. [85] As used herein, the term “target” refers to a biological system of interest including unicellular or pluricellular living organisms or any portion thereof, and include in vitro or in vivo biological systems or any portion thereof. [86] As used herein, the term “polymer” refers to a large molecule composed of repeating structural units typically connected by covalent chemical bonds. A suitable polymer may be linear and/or branched, and can take the form of a homopolymer or a co-polymer. If a co-polymer is used, the co-polymer may be a random co-polymer or a branched co-polymer. Exemplary polymers comprise water-dispersible and in particular water-soluble polymers. For example, suitable polymers include, but are not limited to polysaccharides, polyesters, polyamides, polyethers, polycarbonates, polyacrylates, polyethyleneimines and derivatives thereof. A derivative of a polymer may be either commercially available or it can be prepared as described herein. For therapeutic and/or pharmaceutical uses and applications, the polymer should have a low toxicity profile and in particular that are not toxic or cytotoxic. In other words, the polymer should be biocompatible. For example, maleimide derivatized PLGA is a preferable polymer material. [87] As used herein, the term “nanoparticle” refers to particles of any shape having at least one dimension that is less than about 1000 nm. In some embodiments, nanoparticles have at least one dimension in the range of about 1 nm to about 1000 nm, including any integer value between 1 nm and 1000 nm (including about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, and 1000). In certain embodiments, the nanoparticles have at least one dimension that is about 150 nm. Particle size can be determined using any method known in the art, including, but not limited to, sedimentation field flow fractionation, photon correlation spectroscopy, disk centrifugation, and dynamic light scattering. [88] As used herein, the term “alarmin” refers to any molecule released from a damaged or diseased cell that stimulates an immune response. Non-limiting examples of alarmins are heat- shock proteins, interleukin-la, HMGB1, and nucleosomes. [89] The term “antigen” as used herein refers broadly to any antigen to which an individual can generate an immune response. “Antigen” as used herein refers broadly to molecules that contains at least one antigenic determinant to which the immune response may be directed. The immune response may be cell mediated or humoral or both. As is well known in the art, an antigen may be protein in nature, carbohydrate in nature, lipid in nature, or nucleic acid in nature, or combinations of these biomolecules. An antigen may include non-natural molecules such as polymers and the like. Antigens include self-antigens and foreign antigens such as antigens produced by another animal or antigens from an infectious agent. Infectious agent antigens may be bacterial, viral, fungal, protozoan, and the like. [90] The term “tumor antigen” as used herein refers to a protein which is present on tumor cells, and on normal cells during fetal life (onco-fetal antigen), after birth in selected organs, or on many normal cells, but at much lower concentration than on tumor cells. A variety of tumor antigens have been described. Non-limiting examples of tumor antigens are mucin such as MUC1 or the HER2 (neu) antigen. [91] As used herein, the phrase “antigen presenting cell” or “APC,” has its art understood meaning referring to cells which process and present antigens to T-cells. Non-limiting examples of antigen cells include dendritic cells, macrophages and certain activated epithelial cells. [92] As used herein, the term “Cathepsin” or “Cathepsin family” refers to the family of proteases distinguished by their structure, catalytic mechanism, and which proteins they cleave. The Cathepsin family includes Cathepsin A, B, C, D, E, F, G, H, K, L1, L2, O, S, W, and Z. [93] As used herein, the term “Cathepsin cleavable peptide” refers to a peptide which is cleaved by a member of the Cathepsin family of enzymes. [94] As used herein, the term “MMP2” refers to the protein known as matrix metalloproteinase 2. It is an enzyme that in humans is encoded by the MMP2 gene. [95] As used herein, the term “Caspase” or “Caspase family” refers to a family of cysteine aspartic proteases. The Caspase family includes Caspase 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 14. [96] By “therapeutically effective amount” or “dose” is meant the concentration of a delivery system or a bioactive compound comprised therein that is sufficient to elicit the desired therapeutic effect. Non-limiting examples of a therapeutically effective amount includes range between 50 μg to 1 g, 100 μg to 500 mg, 200 μg to 250 mg, 300 μg to 100 mg, 400 μg to 50 mg, or 500 μg to 1 mg. [97] As used herein, the term “effective amount” refers to an amount sufficient to effect beneficial or desired clinical or biochemical results. An effective amount can be administered one or more times. [98] As used herein, the phrase “at least partially necrotizing” refers to a group of cancer cells or tumor within which at least some of the cancer cells are dying and releasing antigens. “At least partially necrotizing” means the tumor has partially responded to therapy. This also includes tumors in the process of releasing antigens. [99] As used herein, the term “immune cell” refers to cells of the immune system that are involved in protecting the body. Non-limiting examples of immune cells are myeloid cells, lymphoid cells, dendritic cells, T-cells, B-cells, and natural killer cells. [100] As used herein, the term “adjuvant” refers to a substance or mixture that enhances the immune response to an antigen. In some embodiments, the adjuvant refers to an additional compound added to the nanoparticle. Non-limiting examples of adjuvants include (1) small molecules such as imiquimod or resiquimod; (2) double-stranded (ds) RNA molecules such as poly(inosinic-cytidylic acid) (“poly (I:C)” or “poly IC”), and its derivatives and formulations, i.e., Riboxxol RGI®50; poly IC mixed with the stabilizers carboxymethylcellulose and poly(lysine), poly ICLC (Hiltonol); and complexes between poly IC and poly(ethylene imine), PEI, such as BO-112 or [poly IC-(in vivo-jet PEI)]; and (3) single-stranded DNA molecules such as CpG oligodeoxynucleotides (CpG ODN), which contain a cytosine triphosphate deoxynucleotide ("C") followed by a guanine triphosphate deoxynucleotide ("G"). The "p" refers to the phosphodiester link between consecutive nucleotides. Unmethylated CpG motifs act as immunostimulants. [101] As used herein, the term “cleavage peptide” refers to a peptide that is capable of being cleaved by an enzyme. Non-limiting examples of enzymes that may cleave the peptide are Cathepsin, Cathepsin B, Caspase 3, and MMP2. [102] As used herein, the term “reactive group” is defined as a group that will bind to an antigen. The reactive group may bind to the antigen using a covalent bond or a non-covalent interaction, such as hydrophobic-hydrophobic or ionic interactions. Non-limiting examples of a reactive group include NH₂, maleimide COOH, —CHO, —NHS, —SH, -epoxy, -azide, -alkyne, —NHNH₂, — Si(OCH₂CH₃)₃, orthopyridyl disulfide, nitrophenyl carbonate, carbonyl imidazole, tosylate, mesylate, acrylate, and vinylsulfone. [103] As used herein, the term PEG, PEG’, PEG’’ or poly(ethylene glycol) is used broadly to encompass any polyethylene glycol molecule, without regard to size or to modification at an end of the PEG. [104] As used herein, the term “PEG corona” refers to a PEG polymer which encapsulates at least a portion of the surface of the nanoparticle. The PEG corona may be designated as PEGʺ and has a molecular weight range of between 100 to 10,000 Da, 200 to 5,000 Da, 300 to 1,000 Da, or 400 to 700 Da. [105] As used herein, the term “surface” refers to the outside part or uppermost layer. [106] As used herein, the term “protease sensitive” refers to a protein sequence that may be cleaved by an enzyme. Non-limiting examples of protein sequences which may be protease sensitive include Gly-Phe-Leu-Gly, Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln, and Glu-Val-Asp-Gly. Non- limiting enzymes that may cleave such a protein sequence are Cathepsins, Caspases, and MMP2. [107] As used herein, the phrase “wherein at least one” refers to at least 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the available J groups are bound to the PEP-PEG”. [108] As used herein, the terms “buffer” or “buffering agents” refer to materials, that when added to a solution, cause the solution to resist changes in pH. The term “solution” refers to an aqueous or non-aqueous mixture. [109] As used herein, the term “a composition for inducing an immune response” refers to a composition that, once administered to a subject [e.g., once, twice, three times or more (e.g., separated by weeks, months or years)], stimulates, generates and/or elicits an immune response in the subject (e.g., resulting in total or partial immunity to and/or clearance of an immunogen (e.g., tumor) and/or prevents growth and/or metastasis of an immunogen (e.g., tumor) in a subject). An immune response may be an innate (e.g., a non-specific) immune response or a learned (e.g., acquired) immune response. [110] As used herein, the term “lyoprotectant” to refers to a molecule that prevents or reduces chemical and/or physical instability of a substance upon freeze-drying or spray-drying process and subsequent storage. Exemplary lyoprotectants include, but are not limited to, sugar molecules such as mannose, sucrose, trehalose, and mannitol as well as polymers such as poly(vinyl alcohols) (“PVA”), poly(ethylene glycol) (“PEG”), and poly(ethyleneimines) (“PEI”), to name a few examples, are being utilized to preserve the structural integrity of nanoparticular systems such as ACNPs during solvent removal by lyophilization, freeze-drying, or spray-drying and facilitate the redispersion of those nanoparticular systems such as ACNPs into aqueous solvents and buffers. [111] As used herein, the term “pharmaceutically acceptable carrier” includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds also can be incorporated into the compositions. [112] As used herein, the term “therapeutic activity,” when referring to a bioactive compound, is intended to mean that the molecule is able to elicit a desired pharmacological or physiological effect when administered to a subject in need thereof. [113] As used herein, the term “treatment” or “prevention” refers to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a particular infection or disease or sign or symptom thereof and/or may be therapeutic in terms of a partial or complete cure of an infection or disease and/or adverse effect attributable to the infection or the disease. Accordingly, the method “prevents” (i.e., delays or inhibits) and/or “reduces” (i.e., decreases, slows, or ameliorates) the detrimental effects of a disease or disorder in the subject receiving the compositions of the invention. The subject may be any animal, including a mammal, such as a human, and including, but by no means limited to, domestic animals, such as feline or canine subjects, farm animals, such as, but not limited to, bovine, equine, caprine, ovine, and porcine subjects, wild animals (whether in the wild or in a zoological garden), research animals, such as mice, rats, rabbits, goats, sheep, pigs, dogs, cats, etc., avian species, such as chickens, turkeys, songbirds, etc., i.e., for veterinary medical use. II. ACNP Formulations [114] As one of ordinary skill in the art would appreciate, a presently disclosed pharmaceutical composition is formulated to be compatible with its intended route of administration. Solutions or suspensions used for parenteral (e.g., intravenous), intramuscular, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates; and agents for the adjustment of tonicity, such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. [115] Pharmaceutical compositions suitable for injectable use typically include sterile aqueous solutions or dispersions such as those described elsewhere herein and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). The composition should be sterile and should be fluid to the extent that easy syringeability exists. In some embodiments, the pharmaceutical compositions are stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. In general, the relevant carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In some embodiments, isotonic agents, for example, sugar, polyalcohol, such as mannitol or sorbitol, or sodium chloride are included in the formulation. Prolonged absorption of the injectable formulation can be brought about by including in the formulation an agent that delays absorption, for example, aluminum monostearate and gelatin. [116] Sterile injectable solutions can be prepared by filter sterilization as described elsewhere herein. In certain embodiments, solutions for injection are free of endotoxin. Generally, dispersions are prepared by incorporating the delivery system complexes into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In those embodiments in which sterile powders are used for the preparation of sterile injectable solutions, the solutions can be prepared by vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. [117] Oral compositions generally include an inert diluent or an edible carrier. Oral compositions can be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The oral compositions can include a sweetening agent, such as sucrose or saccharin; or a flavoring agent, such as peppermint, methyl salicylate, or orange flavoring. [118] For administration by inhalation, the presently disclosed compositions can be delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Liquid aerosols, dry powders, and the like, also can be used. [119] Systemic administration of the presently disclosed compositions also can be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. [120] It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical or cosmetic carrier. The specification for the dosage unit forms of the invention is dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of individuals. [121] The disease or unwanted condition to be treated can encompass any type of condition or disease that can be treated therapeutically. In some embodiments, the disease or unwanted condition that is to be treated is a cancer. The term “cancer” encompasses any type of unregulated cellular growth and includes all forms of cancer. In some embodiments, the cancer to be treated is a metastatic cancer. Examples of cancer to be treated herein include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cell carcinoma, brain cancer, esophageal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, non-hodgkins lymphoma, hodgkin's lymphoma, as well as head and neck cancer. [122] In particular, the cancer may be resistant to known therapies. Methods to detect the inhibition of cancer growth or progression are known in the art and include, but are not limited to, measuring the size of the primary tumor to detect a reduction in its size, delayed appearance of secondary tumors, slowed development of secondary tumors, decreased occurrence of secondary tumors, and slowed or decreased severity of secondary effects of disease. METHODS Down-selection and identification of PLGA-PEG-Mal and PLGA ACNP formulation candidates [123] In some embodiments, poly(lactide-co-glycolide)-block-poly(ethylene glycol)-maleimide (PLGA-PEG-Mal) and PLGA ACNP formulation candidates were evaluated. Selection criteria include: (1) different molecular weight/weight ratios of the PLGA and PEG polymers, (2) different lactic acid-to-glycolic acid (LA:GA) ratios, and (3) surface compositions, i.e., maleimide modification for conjugation of thiol-containing antigens to the carbon-carbon double bond; unmodified PLGA surface for electrostatic interaction between the negatively charged PLGA surface and positively charged antigen proteins. In addition, both maleimide modified surface and unmodified PLGA surface are capable of hydrophobic interactions with antigen proteins. [124] Three physical selection filters were applied during down-selection: (1) particle sizes, polydispersity index (PDI) and zeta potential (ZP) of the ACNP formulations as measured by Dynamic Light Scattering (DLS); (2) particle stability over eight weeks at 4-8^oC; and (3) batch-to- batch reproducibility and re-dispersibility after lyophilization of the ACNP formulations. [125] Figs. 3a and 3b illustrate the size changes over storage time with examples, without claiming to be exclusive of other ACNP-forming compounds. [126] An additional filter was applied to PLGA-PEG-Mal formulations to confirm the stability of the maleic acid carbon-carbon double bond. The intact presence of the maleimide unit was confirmed using (i) proton (¹H)-NMR spectroscopy with malic acid of known concentration as the internal standard; and (ii) a maleic acid-specific fluorophore kit, such as the Amplite® Colorimetric Maleimide Quantitation Kit. The ¹H-NMR quantification uses the peak area ratio of the maleimide component of PLGA-PEG-Mal and the internal standard as exemplarily shown in Fig. 4. The fluorophore kit relies on the release of a spectroscopically active component after reaction between the carbon-carbon double bond and the 4,4'-dithiodipyridine (DTDP) compound of the kit. [127] Fig. 4 shows the stability of the maleimide surface in PLGA-PEG-Mal at 7.00 ppm compared to the internal standard maleic acid with a peak at 6.20 ppm. The ratio between the peak areas allows quantification of the maleimide surface. The results showed that introducing maleimide active group to ACNP surface does not accelerate its degradation in vitro. Adjuvant addition to improve the antigen-capturing ability of ACNP [128] In some embodiments, the combination of commercially available adjuvants with the ACNP compounds was evaluated. Examples for commercially available adjuvants include but are not limited to: (1) small molecules, such as imiquimod or resiquimod; (2) double-stranded (ds) RNA molecules, such as poly(inosinic-cytidylic acid), poly IC, and its derivatives and formulations, i.e., Riboxxol RGI®50; poly IC mixed with the stabilizers carboxymethylcellulose and poly(lysine), poly ICLC (Hiltonol); and complexes between poly IC and poly(ethylene imine), PEI, such as BO- 112 or [poly IC-(in vivo-jet PEI)]; and (3) single-stranded DNA molecules, such as CpG oligodeoxynucleotides (CpG ODN), which contain a cytosine triphosphate deoxynucleotide ("C") followed by a guanine triphosphate deoxynucleotide ("G"). The "p" refers to the phosphodiester link between consecutive nucleotides. Unmethylated CpG motifs act as immunostimulants. [129] Examples for the combination between ACNPs and adjuvants include: (1) Mixing of ACNP with free adjuvants, for example free poly IC. Free poly IC can be dissolved in water or physiological buffer solutions such as phosphate-buffered saline (PBS), or phosphate buffer solution (PB) prior to mixing with the ACNP formulation, or poly IC can be added to the reconstitution solvent of lyophilized ACNP formulations. The advantage of this approach is easy variability of ACNP-to-adjuvant ratios. (2) Association of adjuvant, i.e., poly IC, with one ACNP population, then mixing with another ACNP population, for example association of poly IC with PLGA ACNP, then mixing with PLGA-PEG-Mal. Advantages of this approach are (i) easy variability of the two ACNP population ratios, and therefore, adjuvant presence, and (ii) different, potentially complementary association between the ACNP populations with negatively charged and maleimide surfaces and antigen proteins. Alternatively, commercially available poly IC complex formulations such as poly ICLC (Hiltonol) or poly IC/PEI can be utilized in this approach. More alternatively, ACNPs based on other nanoparticle structures such as liposomes can be utilized. Liposomes have an aqueous inner phase that can dissolve hydrophilic adjuvants such as poly IC. (3) Encapsulation of adjuvant, i.e., poly IC, into one ACNP population, for example either PLGA ACNP or PLGA-PEG-Mal. The advantage of this approach is that ACNP and adjuvant are at the same time in the same location during in vivo application. The disadvantage is limited ability to change the ACNP-to-adjuvant ratio. [130] Fig.5 shows different ACNP and adjuvant association scenarios. [131] Examples of each scenario have been prepared and evaluated, including in in vivo studies using a mouse model with CT26 cells, a N-nitroso-N-methylurethane-(NNMU) induced, undifferentiated colon carcinoma cell line, which was cloned to generate the cell line designated CT26 (ATCC CRL-2638). [132] Several methods to achieve the association/encapsulation between ACNPs and adjuvants are available, as exemplarily demonstrated for poly IC, and illustrated in Fig.6. (1) Nanoprecipitation from an organic solvent containing the ACNP components and poly IC into an antisolvent such as water; optionally, poly IC can be complexed with a cationic lipid or polymer to improve the solubility in the organic solvent; (2) Nanoprecipitation from an organic solvent containing the ACNP components into an antisolvent such as water containing the water-soluble poly IC; and (3) Water-in-oil-in-water (W/O/W) double emulsion, where the poly IC is dissolved into the internal water phase and the ACNP components are dissolved into the internal oil phase, and both internal phases are stabilized for dispersion into the bulk water phase. Lyoprotectant selection to preserve ACNP structural integrity during lyophilization/freeze- drying and facilitate redispersion of the lyophilized ACNP solids [133] A wide selection of water-soluble compounds including sugar molecules such as mannose, sucrose, trehalose, and mannitol as well as polymers such as poly(vinyl alcohols), PVA, poly(ethylene glycol), PEG, and poly(ethyleneimines), PEI, to name a few examples, are being utilized to preserve the structural integrity of nanoparticular systems such as ACNPs during solvent removal by lyophilization, freeze-drying, or spray-drying and facilitate the redispersion of those nanoparticular systems such as ACNPs into aqueous solvents and buffers. [134] PLGA-PEG-Mal and PLGA ACNP have been lyophilized in the presence of sucrose and PVA as cytoprotectants. It has been demonstrated in in vivo studies in a mouse model using the CT26 colon carcinoma cell line that the sucrose/PVA composition can have an important effect on the tumor growth delay as exemplarily shown in Fig.7. Briefly, CT26 cells were implanted into the left (primary) and right (secondary) flank of mice. The primary tumors underwent radiotherapy and intratumoral PLGA-PEG-Mal treatments. In addition, mice received intraperitoneal injections with an anti-PD1 immunotherapy. Different sucrose-to-PVA ratios were used during the lyophilization of PLGA-PEG-Mal, resulting in different tumor volume changes. In some embodiments, a lyoprotectant of 5-10% sucrose is preferred. EXAMPLES [135] The following examples are offered by way of illustration and not by way of limitation. General Experimental Methods Reagents AI052: Poly(lactide-co-glycolide)-b-poly(ethylene glycol)-maleimide (PLGA-PEG-MAL); LA:GA=75:25; M_W: ˜63,400 Da AP059: Poly(D,L lactide-co-glycolide), acid-terminated (PLGA ACNP); LA:GA=50:50; M_W: 45,000-55,000 Da AI020: Poly(lactide-co-glycolide)-b-poly(ethylene glycol)-maleimide (PLGA-PEG-MAL); LA:GA=50:50; M_W: ˜25,000 Da AP091: Poly(D,L lactide-co-glycolide), acid-terminated (PLGA ACNP); LA:GA=75:25; M_W: 15,000-25,000 Da AP230: Poly(D,L lactide-co-glycolide), acid-terminated (PLGA ACNP); LA:GA=75:25; M_W: 55,000-65,000 Da AT1011 = PLGA-PEG-MAL with optimized MW and LA:GA ratio AT1012 = AI052 with 20mg/ml sucrose + 4mg/ml PVA as lyoprotection AT1013 = AI052 with 20mg/ml sucrose + 16mg/ml PVA as lyoprotection AT1014 = PLGA ACNP with optimized MW and LA:GA ratio AT1015 = AP040: Poly(D,L lactide-co-glycolide), acid-terminated (PLGA ACNP); LA:GA=50:50; MW: 15,000-25,000 Da AT1001.10 = AI020 AT1016: PLGA-PEG-Mal: PLGA ACNP =1:1, HMW AT1017: PLGA-PEG-Mal: PLGA ACNP =1:1, LMW AT1018: PLGA-PEG-Mal: PLGA ACNP =1:3, LMW AT1019: mixture of AT1011 (PLGA-PEG-Mal) and AT1014 (PLGA ACNP) at a mass ratio of 1:1. [136] The cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP) was obtained from Avanti Polar Lipids (Alabaster, Ala., USA). Soybean lecithin consisting of 90- 95% phosphatidylcholine was obtained from MP Biomedicals (Solon, Ohio, USA). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise noted. [137] Collagenase/Hyaluronidase and Bovine Pancreas DNase I-PBS solution were obtained from Stemcell Technologies. LIVE/DEAD® Fixable Yellow Dead Cell Stain Kit and ACK lysis buffer were obtained from Life Technology. Recombinant Murine IL-2 was obtained from PeproTech. anti-PD-1 (clone: RMP1-14) was from BioXcell. Animal Model Mice [138] 6- to 8-week-old female C57BL/6 mice were used. Sample sizes were calculated based on our preliminary data. We calculated an effect size of 1.821. The nonparametric analog of this effect size can be stated in terms of p₁=Pr (X<Y), or an observation in Group X is less than an observation in Group Y, when H₁ is true. The null hypothesis being tested is p₁=0.5. For effect size 1.821, p₁=0.099. A sample size of at least 8 in each group will have 80% power to detect a probability of 0.099 that an observation in Group X is less than an observation in Group Y, using a Wilcoxon (Mann-Whitney) rank-sum test, with a 0.05 two-sided significance level. Mice were assigned to treatment groups based on cage numbers. The groups were not blinded. Cell line [139] The cell line was acquired from ATCC, where the cell line was authenticated using morphology, karyotyping, and PCR based approaches and tested for mycoplasma. Example 1: Preparation of PLGA-PEG-MAL Formulations by Drip Method ^ 8.0 mg/mL PLGA-PEG-MAL (60kDA-3.4kDA, LA:GA ratio 75:25) stock solution: o AI052 PLGA-PEG-MAL (60kDA-3.4kDA, LA:GA ratio 75:25) 187.5 mg o Acetonitrile (organic solvent) 23.4 mL o Endotoxin-free water (antisolvent) 70.0 mL PLGA-PEG-Mal Formulation Preparation: [140] 187.5 mg of PLGA-PEG-MAL (60kDA-3.4kDA, LA:GA ratio 75:25, PolySciTech catalog# AI052) were dissolved at room temperature in 23.4 mL of acetonitrile (ACN) by sonication to give a clear solution. This organic solution was then added dropwise within 2-3 minutes under stirring into 70 mL of endotoxin-free water (mixing ratio of organic solvent-to-water 1:3) through a syringe (21G needle, needle tip above the solution). Next, the organic-aqueous mixture was stirred at room temperature under mild vacuum (evaporation arm) for about 2.5 h to remove the organic solvent, measured by the fill-height reduction (volume) of the aqueous phase. The remaining aqueous nanoparticle dispersion was filtered through an Amicon Ultra-15 Centrifugal Filter Unit (100 kDa cutoff, MilliporeSigma, centrifuge speed at 1500x g) for about 15 min and the filtrate discarded. The nanoparticle retentate above the filter was redissolved twice in water and filtered again by centrifugation to remove free polymer from the nanoparticle dispersion. The filter was washed with endotoxin-free water, and the retentate and wash water were combined (7.0 mL total).150 µl of this aqueous dispersion were used to measure particle size, polydispersity index (PDI), and zeta potential (ZP) of the PLGA-PEG-Mal formulation by Dynamic Light Scattering (DLS, Litesizer 500 from Anton Paar). Optional, a fraction of the aqueous dispersion was lyophilized to dryness to measure the PLGA-PEG-Mal yield gravimetrically, giving recovery yields of 80-85%. PLGA-PEG-Mal Lyophilization and Reconstitution: o PLGA-PEG-Mal, based on 80% recovery (187.5 mgx0.8) 150.0 mg o PVA (MW 6,000 Da, 80% hydrolyzed) stock solution in water 40.0 mg/mL o Sucrose stock solution in water 40.0 mg/mL [141] 150.0 mg PLGA-PEG-Mal in 7.0 mL from above formulation were mixed with 1.875 mL (75.0 mg) of the sucrose stock solution and 0.75 mL (30.0 mg) of the PVA stock solution, divided into five 15-mL tubes (30.0 mg PLGA-PEG-Mal per tube), and lyophilized overnight. Each tube was finally reconstituted with 0.75 mL of Dulbecco's phosphate-buffered saline 1x (DPBS) for use in in vivo studies. Final concentration of each reconstituted formulation: 40.0 mg/mL PLGA-PEG- Mal, 20.0 mg/mL sucrose, and 8.0 mg/mL PVA. Example 2: Preparation of High Molecular Weight PLGA ACNP Formulations by Drip Method ^ 8.0 mg/mL PLGA (45-55kDa, LA:GA ratio 50:50) stock solution: o AP059 PLGA (45-55kDa, LA:GA ratio 50:50) 10.0 mg o Acetonitrile (organic solvent) 2.5 mL o Endotoxin-free water (antisolvent) 7.5 mL PLGA ACNP Formulation Preparation: [142] 10.0 mg of PLGA (45-55kDA, acid, LA:GA ratio 75:25, PolySciTech catalog# AP059) were dissolved at room temperature in 2.5 mL of acetonitrile (ACN) by sonication to give a clear solution. This organic solution was then added dropwise within 2-3 minutes under stirring into 7.5 mL of endotoxin-free water (mixing ratio of organic solvent-to-water 1:3) through a syringe (20G needle, needle tip above the solution). Next, the organic-aqueous mixture was stirred at room temperature under mild vacuum (evaporation arm) for about 2.5 h to remove the organic solvent, measured by the fill-height reduction (volume) of the aqueous phase. The remaining aqueous nanoparticle dispersion was filtered through an Amicon Ultra-15 Centrifugal Filter Unit (100 kDa cutoff, MilliporeSigma, centrifuge speed at 800x g) for about 15 min and the filtrate discarded. The nanoparticle retentate above the filter was redissolved twice in water containing 0.5% PVA (by weight) and filtered again by centrifugation to remove free polymer from the nanoparticle dispersion. The filter was washed with endotoxin-free water, and the retentate and wash water were combined. 150 µl of this aqueous dispersion were used to measure particle size, polydispersity index (PDI), and zeta potential (ZP) of the PLGA ACNP formulation by Dynamic Light Scattering (DLS). Optional, a fraction of the aqueous dispersion was lyophilized to dryness to measure the PLGA-PEG-Mal yield gravimetrically, giving recovery yields of 80-85%. PLGA ACNP Lyophilization and Reconstitution: o PLGA-PEG-Mal, based on 80% recovery (187.5 mgx0.8) 8.0 mg o PVA (MW 6,000 Da, 80% hydrolyzed) stock solution in water 40.0 mg/mL o Sucrose stock solution in water 40.0 mg/mL [143] 8.0 mg PLGA ACNP in 2.0 mL from above formulation were mixed with 0.1 mL (4.0 mg) of the sucrose stock solution and 0.04 mL (1.6 mg) of the PVA stock solution, divided into two 10- mL tubes (4.0 mg PLGA ACNP per tube), and lyophilized overnight. Each tube was finally reconstituted with 0.15 mL of Dulbecco's phosphate-buffered saline 1x (DPBS) for use in in vivo studies. Final concentration of each reconstituted formulation: 40.0 mg/mL PLGA-PEG-Mal, 20.0 mg/mL sucrose, and 8.0 mg/mL PVA. Example 3: Preparation of High Molecular Weight PLGA-PEG-MAL Formulations by Micromixing Method ^ 8.0 mg/mL PLGA-PEG-MAL (60kDA-3.4kDA, LA:GA ratio 75:25) stock solution in acetonitrile o AI052 PLGA-PEG-MAL (60kDA-3.4kDA, LA:GA ratio 75:25) o Degassed HPLC grade, endotoxin-free water (antisolvent) o Dolomite Micromixer with 100-µm microchannel chip PLGA-PEG-Mal Formulation Preparation [144] The PLGA-PEG-Mal stock solution in acetonitrile was prepared with sonication as described in Example 1. The stock solution was then filtered through a 0.20 um nylon filter to remove residues that could block the microchannel chip. Syringes A and B of the Dolomite micromixer were filled with 5.0 mL each, the PLGA-PEG-Mal stock solution and degassed, endotoxin-free water. Both syringes were set for a flow rate of 5.0 mL/min, and a total volume of 10.0 mL PLGA-PEG-Mal formulation was collected in 20.0-mL glass vials containing a magnetic stirring bar to keep the formulation agitated.150 µl samples were taken from each vial, and the particle size and polydispersity were measured by Dynamic Light Scattering (DLS). Target particle size was 50-70 nm by number detection. Formulation vials meeting the target size were further diluted under stirring with 10.0 mL of endotoxin-free water to bring the final organic solvent-to- water ratio to 1:3. The organic-aqueous mixture was stirred at room temperature under mild vacuum (evaporation arm) for about 2.5 h to remove the organic solvent, measured by the fill- height reduction (volume) of the aqueous phase. The remaining aqueous nanoparticle dispersion was filtered through an Amicon Ultra-15 Centrifugal Filter Unit (100 kDa cutoff, MilliporeSigma, centrifuge speed at 1000x g and 4^oC) for about 25 min and the filtrate discarded. The nanoparticle retentate above the filter was redissolved twice in water and filtered again by centrifugation to remove free polymer from the nanoparticle dispersion. The filter was washed with endotoxin- free water, and the retentate and wash water were combined. PLGA-PEG-Mal Lyophilization and Reconstitution: [145] The same protocol as described in Example 1 was applied to lyophilize and reconstitute the PLGA-PEG-Mal formulations, resulting in a final concentration of 40.0 mg/mL PLGA-PEG-Mal, 20.0 mg/mL sucrose, and 8.0 mg/mL PVA. Example 4: Preparation of Low Molecular Weight PLGA-PEG-MAL Formulations by Micromixing Method ^ 8.0 mg/mL PLGA-PEG-MAL (20kDA-5.0kDA, LA:GA ratio 50:50) stock solution in acetonitrile o AI020 PLGA-PEG-MAL (20kDA-5.0kDA, LA:GA ratio 50:50) o Degassed HPLC grade, endotoxin-free water (antisolvent) o Dolomite Micromixer with 100-µm microchannel chip PLGA-PEG-Mal Formulation Preparation: [146] For the preparation of low molecular weight (LMW) PLGA-PEG-Mal stock solution, 315.0 mg PLGA-PEG-MAL (20kDA-5.0kDA, LA:GA ratio 50:50) were dissolved under sonication in 39.4 mL ACN to make 8.0 mg/mL solution. The protocols for PLGA-PEG-Mal formulation and lyophilization followed the protocols disclosed in Example 3. Example 5: Preparation of Low Molecular Weight PLGA ACNP Formulations by Micromixing Method ^ 8.0 mg/mL PLGA (15-25kDa, LA:GA ratio 75:25) stock solution in acetonitrile o AP091 PLGA (15-25kDa, LA:GA ratio 75:25) o Degassed HPLC grade, endotoxin-free water (antisolvent) o PVA (MW 6,000 Da, 80% hydrolyzed) o Dolomite Micromixer with 100-µm microchannel chip PLGA ACNP Formulation Preparation: [147] For the preparation of low molecular weight (LMW) PLGA ACNP stock solution, 320.0 mg PLGA (15-25kDa, LA:GA ratio 75:25) were dissolved under sonication in 40.0 mL acetonitrile ACN to make 8.0 mg/mL solution. The solution was filtered through a 0.20 µm nylon filter to remove residues that could block the microchannel chip. Syringes A and B of the Dolomite micromixer were filled with 5.0 mL each, the PLGA stock solution and degassed, endotoxin-free water. Both syringes were set for a flow rate of 5.0 mL/min, and a total volume of 10.0 mL PLGA ACNP formulation was collected in 20.0 mL glass vials containing a magnetic stirring bar set to spin at 300 rpm to keep the formulation agitated. 150 µl samples were taken from each vial, and the particle size and polydispersity were measured by Dynamic Light Scattering (DLS). Target particle size was 50-80 nm by number detection. Formulation vials meeting the target size were further diluted under stirring with 10.0 mL of endotoxin-free water to bring the final organic solvent-to- water ratio to 1:3. The organic-aqueous mixture was stirred at room temperature under mild vacuum (evaporation arm) for about 2.5 h to remove the organic solvent, measured by the fill- height reduction (volume) of the aqueous phase. The remaining aqueous nanoparticle dispersion was filtered through an Amicon Ultra-15 Centrifugal Filter Unit (100 kDa cutoff, MilliporeSigma, centrifuge speed at 1000x g and 4^oC) for about 30 min and the filtrate discarded. The nanoparticle retentate above the filter was redissolved twice in water containing 0.5 mg/L PVA and filtered again by centrifugation to remove free polymer from the nanoparticle dispersion. The filter was washed with endotoxin-free water, and the retentate and wash water were combined. PLGA ACNP Lyophilization and Reconstitution: [148] The same protocol as described in Example 2 was applied to lyophilize and reconstitute the PLGA ACNP formulations, resulting in a final concentration of 40.0 mg/mL PLGA ACNP, 20.0 mg/mL sucrose, and 8.0 mg/mL PVA. Example 6: Preparation of High Molecular Weight PLGA ACNP Formulations by Micromixing Method ^ 8.0 mg/mL PLGA (55-65kDa, LA:GA ratio 75:25) stock solution in acetonitrile o AP230 PLGA (55-65kDa, LA:GA ratio 75:25) o Degassed HPLC grade, endotoxin-free water (antisolvent) o PVA (MW 6,000 Da, 80% hydrolyzed) o Dolomite Micromixer with 100-µm microchannel chip PLGA ACNP Formulation Preparation [149] The preparation, lyophilization, and reconstitution of high molecular weight (HMW) PLGA ACNP formulations using PLGA (55-65kDa, LA:GA ratio 75:25) followed the protocol disclosed in Example 5. Example 7: Stability of PLGA ACNP and PLGA-PEG-Mal at 4-8 ^oC [150] PLGA ACNP and PLGA-PEG-Mal of different compositions were prepared following the method disclosed in Examples 1 and 2. The vials containing the different ACNP formulations were stored in the refrigerator at 4-8^oC for up to eight weeks. At several time points, T= 0, 1, 3, 7 days and 2, 4 and 8 weeks, 150 µL of each vial was removed, diluted with DI water as needed, and the size, polydispersity index (DPI) and zeta potential (ZP) were measured using the Litesizer^TM 500 from Anton Paar USA Inc. Examples of size/DPI and ZP of freshly prepared PLGA ACNP and PLGA- PEG-Mal formulations in water, as well as after storage for 8 weeks are shown in Tables 1 and 2, and Figs. 3a and 3b. All changes are within the experimental error, demonstrating storage stability. Data in Table 1-2 and Figs.3a and 3b show good nanoparticle size stability over storage at 4-8 Celsius. Table 1. Size/PDI and zeta potential changes for PLGA ACNP formulations after storage at 4-8 ^oC over 8 weeks PLGA Freshly made 4°C for 8 weeks )

AP040 15-25 50:50 Acid 107.2/0.18 -13.6±0.2 96.3/0.15 -13.8±0.3 AP 1 2 E 1 12 2 1 1 1 3 5 4 3 5 2 3 7

ticles improve the abscopal effect and cancer immunotherapy. Nature Nanotech, 12, 877–882 (2017). https://doi.org/10.1038/nnano.2017.113) Table 2. Size/PDI and zeta potential changes for PLGA-PEG-Mal formulations after storage at 4-8^oC over 8 weeks Freshly made 4°C for 8 weeks Vendor PLGA-PEG-Mal ) ² 4 7 2

Example 8: Stability of the Maleimide Surface in PLGA-PEG-Mal Formulations [151] The intact presence of the maleimide unit was confirmed using proton ¹H-NMR spectroscopy with malic acid of known concentration as the internal standard. The ¹H-NMR quantification uses the peak area ratio of the maleimide component of PLGA-PEG-Mal and the internal standard. For sample preparation, 10.0 mg of PLGA-PEG-Mal was freeze-dried to remove the aqueous solvent. The residual solid was then dissolved in 0.5 mL of deuterated dimethyl sulfoxide (DMSO-d6). For the maleic acid internal standard, 3.0 mg of maleic acid were dissolved in 3.0 mL DMSO-d6 to make a 1.0 mg/mL stock solution. To further dilute the solution, 0.6 mL of the stock were added to 0.9 mL DMSO-d6, resulting in a 0.4 mg/mL stock solution. Finally, 0.1 mL of this solution was added to the 0.5 mL of the PLGA-PEG-Mal solution in DMSO-d6 prepared before. The combined solutions were transferred into a 5 mm x 7 inch NMR tube for ¹H-NMR measurement. [152] Fig. 4 shows the stability of the maleimide during storage for at least 8 weeks by comparing the peak area for the maleimide component of PLGA-PEG-Mal to the maleic acid internal standard using ¹H-NMR measurements. Example 9: Encapsulation of Adjuvant poly IC into PLGA ACNP by Nanoprecipitation o PLGA (15-25kDa, LA:GA ratio 75:25), AP091 o Acetonitrile (solvent) o PVA (MW 6,000 Da, 80% hydrolyzed) o HMW poly IC (InvivoGen; dissolved in endotoxin-free water to prepare a 5.0 mg/mL stock solution; stored at -20 ^C) PLGA ACNP/poly IC Formulation Preparation: [153] HMW poly IC stock solution (5.0 mg/mL) from the freezer was heated to 70^oC to prepare a homogeneous, clear solution. In parallel, PLGA ACNP was dissolved in acetonitrile at room temperature under sonication to prepare a 10.0 mg/mL stock solution. Then 7.35 mL of endotoxin-free water were mixed at room temperature with 0.15 mL of the poly IC stock solution under stirring for 1 min. To this solution, 2.5 mL of the PLGA ACNP stock solution in acetonitrile was added dropwise through a syringe (21G needle), resulting in an organic solvent-to-water ratio of 1:3. Next, the organic-aqueous mixture was stirred at room temperature under mild vacuum (evaporation arm) for about 2.5 h to remove the organic solvent, measured by the fill- height reduction (volume) of the aqueous phase. The remaining aqueous nanoparticle dispersion was filtered through an Amicon Ultra-15 Centrifugal Filter Unit (100 kDa cutoff, MilliporeSigma, centrifuge speed at 1,500x g at 4 ^C) for about 60 min and the filtrate discarded. The nanoparticle retentate above the filter was redissolved twice in water containing 0.5 mg/L PVA and filtered again by centrifugation to remove free polymer from the nanoparticle dispersion. The filter was washed with endotoxin-free water, and the retentate and wash water were combined.150 µl of this aqueous dispersion were used to measure particle size, polydispersity index (PDI), and zeta potential (ZP) of the PLGA ACNP formulation by Dynamic Light Scattering (DLS). Quantification of poly IC association with PLGA ACNP by Nanodrop UV-Vis Detection: [154] The poly IC concentration associated with PLGA ACNP was measured using the Thermo Scientific Nanodrop 2000 Spectrophotometer. For sample preparation, 0.2 mL of the PLGA ACNP were freeze-dried, and the residue dissolved in 0.4 mL dichloromethane (DCM) to disintegrate the ACNP and free encapsulated poly IC. Then 0.4 mL water were added, and the mixture agitated for 1h to extract the poly IC into the aqueous phase. The aqueous and organic phases were separated by centrifugation (10,000x g for 15 min), and 0.3 mL of the top aqueous phase were removed by pipette. The concentration of poly IC was measured by UV-Vis at a wavelength of 260 nm. Three independent measurements were conducted to measure the average concentration as C1, each using 2.0 µL solution. The extraction using 0.4 mL water was repeated three times, giving the concentrations C2, C3, and C4. The total amount of poly IC (W) released from the ACNP in the 0.2 mL suspension was calculated by: W (µg) = C1 (µg/mL) x 0.3 (mL) + C2 (µg/mL) x 0.4 (mL) + C3 (µg/mL) x 0.4 (mL) + C4 (µg/mL) x 0.5 (mL) [155] Finally, the association efficiency was calculated by: W x 10/total amount of poly IC used for encapsulation. [156] Examples of PLGA ACNP size/PDI (polydispersity index), and Zeta Potential are shown in Table 4. Example 10: Encapsulation of Adjuvant poly IC into PLGA ACNP by Water/Oil/Water Double Emulsion Reagents o PLGA (15-25kDa, LA:GA ratio 75:25), AP091 o Ethyl acetate (solvent, oil phase) o PVA (MW 6,000 Da, 80% hydrolyzed) o HMW poly IC (InvivoGen; dissolved in endotoxin-free water to prepare a 5.0 mg/mL stock solution; stored at -20 ^C) PLGA ACNP/poly IC Formulation Preparation: [157] HMW poly IC stock solution (5.0 mg/mL) from the freezer was heated to 70^oC to prepare a homogeneous, clear solution. In parallel, PLGA ACNP was dissolved in ethyl acetate at room temperature to prepare a 25.0 mg/mL stock solution.1.0 mL of the PLGA ACNP stock solution in ethyl acetate was cooled in an ice water bath, followed by addition of 0.1 mL of the aqueous poly IC stock solution, and briefly (~5 seconds) mixed by vortex mixer. The mixture was then agitated by ultrasonication under ice water cooling (probe sonicator in pulse mode with 5 seconds on/5 seconds off to avoid heating of the mixture, with total sonication time of 30s and 25% amplitude) to prepare a crude, opaque water/oil emulsion. The crude emulsion with then added into 2.0 mL of a 20.0 mg/mL PVA solution (pre-cooled in an ice water bath), mixed by vortexing (~5 seconds), and ultrasonicated in an ice water bath using the same settings as before. Finally, the emulsion was transferred into a 5-mL syringe (21G needle) and added dropwise into 7.0 mL of a 20.0 mg/mL PVA solution under magnetic stirring at 600 rpm to complete the water/oil/water double emulsion. The emulsion was stirred at room temperature under mild vacuum (evaporation arm) overnight to remove excess organic solvent. Purification was carried out by filtration through an Amicon Ultra-15 Centrifugal Filter Unit (100 kDa cutoff, MilliporeSigma, centrifuge speed at 1,500x g at 4 ^C) for about 60 min and the filtrate discarded. The nanoparticle retentate above the filter was redissolved three times in water, filtered again by centrifugation, and concentrated to a final volume of 1.0 mL. The filter was washed with endotoxin-free water, and the retentate and wash water were combined.150 µl of this double emulsion were used to measure particle size, polydispersity index (PDI), and zeta potential (ZP) by Dynamic Light Scattering (DLS). Quantification of poly IC in the W/O/W double emulsion by Nanodrop UV-Vis Detection: [158] The poly IC concentration of the double emulsion was measured using the Thermo Scientific Nanodrop 2000 Spectrophotometer. For sample preparation, 0.2 mL of the double emulsion was freeze-dried, and the residue dissolved in 0.4 mL dichloromethane (DCM) to disintegrate the ACNP and free encapsulated poly IC. Then 0.4 mL water were added, and the mixture agitated for 1h to extract the poly IC into the aqueous phase. The aqueous and organic phases were separated by centrifugation (10,000x g for 15 min), and 0.3 mL of the top aqueous phase were removed by pipette. The concentration of poly IC was measured by UV-Vis at a wavelength of 260 nm. Three independent measurements were conducted to measure the average concentration as C1, each using 2.0 µL solution. The extraction using 0.4 mL water was repeated three times, giving the concentrations C2, C3, and C4. The total amount of poly IC (W) released from the ACNP in the 0.2 mL suspension was calculated by: W (µg) = C1 (µg/mL) x 0.3 (mL) + C2 (µg/mL) x 0.4 (mL) + C3 (µg/mL) x 0.4 (mL) + C4 (µg/mL) x 0.5 (mL) [159] Finally, the association efficiency was calculated by: W x 10/ total amount of poly IC used for encapsulation [160] Table 4: Examples of ACNP size/DPI, ZP are shown for PLGA ACNP and poly IC made by nanoprecipitation and W/O/W double emulsion methods. Results of Table 4 indicate that the particle size of ACNP is significantly impacted by the relative amount of poly IC encapsulated. Large particle size is associated with higher amounts of poly IC. Table 4 Nanoparticles P St ti l IC ) 4 0 6 8 1

Example 11: Encapsulation of Adjuvant CpG ODN into PLGA ACNP by Nanoprecipitation o PLGA (15-25kDa, LA:GA ratio 75:25), AP091 o Acetonitrile (solvent) o PVA (MW 6,000 Da, 80% hydrolyzed) o CpG ODN 1585 VacciGrade (InvivoGen; a class A CpG ODN specific for mouse TLR9) PLGA ACNP/CpG ODN 1585 Formulation Preparation: [161] The formulation preparation and quantification of CpG ODN association with PLGA ACNP by Nanodrop UV-Vis detection followed the protocol disclosed in Example 9. Examples of ACNP physical parameters and association efficiencies are shown in Table 5. [162] Table 5: Examples of ACNP size/PDI, ZP, and adjuvant association efficiency are shown for PLGA ACNP and CpG ODN made by the nanoprecipitation method. Association efficiency measurements were done using the Thermo Scientific Nanodrop 2000 Spectrophotometer. Table 5 P^{rocess PLGA} CpG CpG/PLGA % (_mg) (mg) _(wt/wt) ^{Size (nm)/PDI ZP (mv)}

Example 12: Preparation of PLGA-PEG-MAL Formulations by Drip Method with Different Ratios of Sucrose and Poly(vinyl alcohol) as Lyoprotectants ^ 8.0 mg/mL PLGA-PEG-MAL (60kDA-3.4kDA, LA:GA ratio 75:25) stock solution in acetonitrile o AI052 PLGA-PEG-MAL (60kDA-3.4kDA, LA:GA ratio 75:25) o Endotoxin-free water (antisolvent) o PVA (MW 6,000 Da, 80% hydrolyzed) stock solution in water 40.0 mg/mL o Sucrose stock solution in water 40.0 mg/mL PLGA-PEG-Mal Formulation Preparation: [163] The preparation, lyophilization, and reconstitution of high molecular weight (HMW) PLGA-PEG-Mal formulations using PLGA-PEG-MAL (60kDA-3.4kDA, LA:GA ratio 75:25) followed the protocol disclosed in Example 1. The final lyoprotectant mixtures used for the formulation preparations were sucrose (20.0 mg/mL) combined with poly(vinyl alcohol), PVA at 4.0, 8.0, and 16.0 mg/mL. The physical parameters of the PLGA-PEG-Mal formulations as measured by Dynamic Light Scattering (DLS) before lyophilization and after reconstitution in PBS solution are shown in Table 6. Generally, reconstituted PLGA-PEG-Mal is slightly larger than freshly prepared PLGA-PEG-Mal. Reconstitution in the presence of 8.0 and 16.0 mg/mL PVA resulted in comparable and reproducible (8.0 mg/mL) particle sizes, polydispersity indices, and zeta potentials; however, redispersion in the presence of 4.0 mg/mL PVA resulted in larger particle sizes. [164] Table 6: PLGA-PEG-Mal particle sizes, polydispersity indices (PDI), and zeta potentials (ZP) of formulations containing different sucrose-to-PVA ratios before and after reconstitution. The reduced ZP after redispersion is indicative of the presence of the lyoprotectants. Table 6 Compound Lyoprotectant Mixture Size (nm) / PDI ZP (mV) before l o hilization 1207±205 / 019 -85±03

Example 13: Preparation of PLGA-PEG-MAL ACNP by handheld drip addition method Materials • 8 mg/mL PLGA-PEG-MAL (60kDA-3.4kDA, LA:GA ratio 75:25) • AI052 PLGA-PEG-MAL (60kDA-3.4kDA, LA:GA ratio 75:25) 150 mg • Acetonitrile (organic solvent) 18.75 mL • Endotoxin-free water (antisolvent) 56.25 mL PLGA-PEG-MAL ACNP Formulation Preparation: [165] For each 1x preparation, 37.5 mg of PLGA-PEG-MAL (60kDA-3.4kDA, LA:GA ratio 75:25, PolySciTech catalog# AI052) were dissolved at room temperature in 4.69 mL of acetonitrile (ACN) by sonication to give a clear solution at 8 mg/mL. This organic solution was then added dropwise under stirring into 14 mL of endotoxin-free water (mixing ratio of organic solvent to water 1:3) through a syringe (21G needle, needle tip above the solution) as one 1x scale. Four 1x scale were prepared. Next, the organic-aqueous mixtures were stirred at room temperature under mild vacuum (evaporation arm) for about 2.5 h to remove the organic solvent, measured by the fill- height reduction (volume) of the aqueous phase. Then these dispersions were filtered through Amicon Ultra-15 Centrifugal Filter Units (100 kDa cutoff, MilliporeSigma, 1000xg) for about 20 min and the filtrate discarded. The nanoparticle retentate above the filter was resuspended twice in endotoxin free water and filtered again by centrifugation to remove free polymer from the nanoparticle dispersion. The filter was washed with endotoxin-free water, and the retentate and wash water were combined (about 8mL total for four 1x scale).150 µL of aqueous dispersion was used to measure particle size, PDI, and ZP of the PLGA-PEG-Mal ACNP formulation DLS. Example 14: Preparation of PLGA ACNP by handheld drip addition method Materials • 8 mg/mL PLGA (55-65kDA, LA:GA ratio 75:25) • AP230 PLGA (55-65kDA, LA:GA ratio 75:25) 160 mg • Acetonitrile (organic solvent) 20 mL • Endotoxin-free water (antisolvent) 60 mL PLGA ACNP Formulation Preparation: [166] 160 mg of PLGA (55-65kDA, LA:GA ratio 75:25, PolySciTech catalog# AP230) were dissolved at room temperature in 20 mL of acetonitrile by sonication to give a clear solution at 8 mg/mL.4.69mL of this organic solution was then added dropwise under stirring into 14.07 mL of endotoxin-free water (mixing ratio of organic solvent-to-water 1:3) through a syringe (21G needle, needle tip above the solution) as one 1x scale. Four 1x scale were prepared. Next, the organic-aqueous mixtures were stirred at room temperature under mild vacuum (evaporation arm) for about 3 h to remove the organic solvent, measured by the fill-height reduction (volume) of the aqueous phase. Then 0.175 mL of 40 mg/mL PVA solution were added into each remaining 1x aqueous nanoparticle dispersion. Each 1x dispersion was filtered through Amicon Ultra-15 Centrifugal Filter Units (100 kDa cutoff, MilliporeSigma, centrifuge speed at 1000x g) for about 45 min and the filtrate discarded. The nanoparticle retentate above the filter was resuspended twice in 0.5 mg/mL PVA solution and filtered again by centrifugation to remove free polymer from the nanoparticle dispersion. The filter was washed with endotoxin-free water, and the retentate and wash water were combined (8 mL total for these four 1x scale).150 µL of aqueous dispersion was used to measure particle size, PDI, and ZP of the PLGA ACNP formulation DLS. Example 15: Preparation of PLGA ACNP by handheld drip addition method using different PLGA starting material [167] A series of PLGA ACNP were prepared by handheld drip addition method using different PLGA starting material having different molecular weight of PLGA and LA:GA ratio or terminal group (acid vs ester). The particle size, PDI and zeta potential data are summarized in Table 7. Table 7 PLGA Mn LA:GA Endcap Size (nm)/PDI ZP (mV) 55-65 kD* 75:25 Acid 116.3/0.13 -

15-25 kD 50:50 Acid 107.2/0.18 -13.6±0.2 15-25 kD 50:50 Ester 137.5/0.12 -6.0±0.2 35-45 kD 50:50 Acid 103.5/0.15 -8.2±0.4 35-45 kD 50:50 Ester 105.2/0.17 -11.9±0.5 45-55 kD 50:50 Acid 105.7/0.14 -18.6±2.0 15-25 kD 75:25 Acid 106.9/0.16 -19.5±1.2 15-25 kD 75:25 Ester 133.1/0.11 -25.1±0.3 35-45 kD 75:25 Acid 116.9/0.13 -21.8±0.7 35-45 kD 75:25 Ester 117.8/0.06 -23.6±1.1 Example 16: Preparation of PLGA-PEG-Mal ACNP by handheld drip addition method using different PLGA-PEG-Mal starting material [168] A series of PLGA ACNP were prepared by handheld drip addition method using different PLGA-PEG-Mal starting material having different molecular weight of PLGA and LA:GA ratio. The particle size, PDI and zeta potential data are summarized in Table 8 below. Table 8 Vendor # PLGA-PEG-Mal Mn (PLGA/PEG) LA:GA Size (nm)/PDI ZP (mV)

AI020* 20k/5k 50:50 82.6/0.17 -10.6±0.9 AI1 2 k 2k 1 221

p p y g g ent PLGA-PEG-Mal starting material [169] A series of PLGA ACNP were prepared by microfluidic mixing method using different PLGA-PEG-Mal starting material having different molecular weight of PLGA or LA:GA. The particle size, PDI are summarized in Table 9 below. Table 9 PLGA-PEG-Mal Mn (PLGA/PEG) LA:GA Size (nm) PDI 4 3 2 8 3

Example 18: Preparation of PLGA ACNP by impingement jets mixing method [170] PLGA ACNP were prepared by the impingement jets mixing (IJM) method under various conditions using the IJM-skid from Knauer. PLGA from Ashland (DLG 7505 A, batch# 0002594415) was used as the starting material. A jet of PLGA solution in acetonitrile was directly impinged with a water jet inside the impinging jets mixer. This mixture was then immediately diluted into a 25 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer solution, pH 7.4, containing 0.625 mg/mL Polyvinyl Alcohol (PVA), resulting in a final solution concentration of 20 mM HEPES with 0.5 mg/mL PVA. The mixing conditions and results are summarized in Table 10. Table 10 S^{N PLGA Concentration (m /mL) Flow (mL/min) Size (nm) PDI}

1 5:5 111 0.08 6 2 20:20 136 0.06

5 10:10 130 0.09 6 20:20 131 0.1 Example 19: Preparation of PLGA-PEG-Mal ACNP by impingement jets mixing method [171] PLGA-PEG-Mal ACNP were prepared by the impingement jets mixing (IJM) method under various conditions using the IJM-skid from Knauer. PLGA-PEG-Mal (48k/3.4k, LA:GA 75:25) was used as the starting material. A jet of PLGA-PEG-Mal solution in acetonitrile was directly impinged with a water jet inside the IJM. This mixture was then immediately diluted into a 25 mM HEPES buffer solution, pH 7.4, containing 0.625 mg/mL PVA, resulting in a final concentration of 20 mM HEPES with 0.5 mg/mL PVA. The mixing conditions and results are summarized in Table 11. Table 11 S^{N PLGA-PEG-Mal Concentration (mg/mL) Flow (mL/min)} (_{organic : water)} ^{Size (nm) PDI} 4 3 3 3 4

Example 20. Preparation of PLGA ACNP (AT1014) by microfluidic mixing followed by immediate dilution [172] PLGA ACNP dispersion was prepared by the same method described in Example 27 and 28 except that the product out of the microfluidic mixer was immediately diluted into a 25 mM HEPES buffer solution, pH 7.4, containing 0.625 mg/mL PVA, resulting in a final concentration of 20 mM HEPES with 0.5 mg/mL PVA. The PLGA ACNP dispersion was first filtered through 0.45 µm PES filter, then concentrated on a flat sheet TFF 10 times, buffer exchanged with 4 times volume of 20 mM HEPES pH 7.4 first, followed 5 volume of 25 mM HEPES buffer solution, pH 7.4, containing 140 mM NaCl. The purified product was then filtered through 0.2 µm PES filter for storage. Example 21: Preparation of PLGA-PEG-Mal ACNP (AT1011) by microfluidic mixing followed by immediate dilution [173] PLGA-PEG-Mal ACNP dispersion was prepared by the same method described in Example 31 except that the product out of the microfluidic mixer was immediately diluted into a 25 mM HEPES buffer solution, pH 7.4, containing 0.625 mg/mL PVA, resulting in a final solution concentration of 20 mM HEPES with 0.5 mg/mL PVA. The PLGA-PEG-Mal ACNP dispersion was first filtered through 0.45 µm PES filter, then concentrated on a flat sheet TFF 10 times, buffer exchanged with 4 times volume of 20 mM HEPES pH 7.4 first, followed 5 volume of 25 mM HEPES buffer solution, pH 7.4, containing 140 mM NaCl. The purified product was then filtered through 0.2 µm PES filter for storage. Example 22: Preparation of PLGA ACNP using PolySciTech AP230 as starting material [174] AP230 was dissolved in acetonitrile (ACN) at 4 mg/mL and followed by filtration through 0.2 µM nylon filter. Nanoparticles was made by using Dolomite System with 100 µm chip with the flow rate at 5 mL/min for both water and the above PLGA solution. The nanoparticles were collected in 20 mL glass vials with 10 mL endotoxin free water. Put these vials under snorkel to evaporate ACN with mild stirring. When ACN was completely evaporated (about 2.5 to 3h), the dispersion was transferred into an Amicon Ultra-15 centrifugal filter units (100 kDa cutoff, MilliporeSigma, centrifuge speed at 1000 g, 4 ^oC) for about 30 min. Two washes were performed with 0.5 mg/mL PVA (MW 6,000, 80% hydrolyzed) and centrifuged at 1000 g at 4 ^oC for about 30 min. Finally, the PLGA nanoparticle dispersion was concentrated into small volume and transferred into a Falcon tube. The Amicon tubes were washed with small amount of endotoxin free water, and the washed solutions were combined into the Falcon tube and make its final concentration to about 40 mg/mL (by use the yield of 80%) with PBS (AT1014) and stored at 4 ^oC in fridge. The size and zeta potential of the nanoparticles were measured by DLS. The concentration of PLGA and PVA in nanoparticle dispersion was determined by lyophilization of the obtained PLGA nanoparticle dispersion, followed by dissolving in DMSO-d6 with maleic acid of known concentration for NMR (maleic acid as the internal standard). Example 23: Preparation of PLGA-PEG-Mal ACNP using PolySciTech AI052 as starting material [175] AI052 from PolySciTech was dissolved in acetonitrile at 6-8 mg/mL, filtered, then made into nanoparticles by Dolomite and purified with Amicon Ultra-15 centrifugal filter units in the similar way to above PLGA nanoparticles described in Example 22, except no PVA was used during the washing procedure. Therefore, the collected PLGA-PEG-MAL nanoparticles from the Amicon tubes contained no PVA. The concentration of PLGA-PEG-MAL nanoparticles was measured by gravimetrical method from the lyophilized samples. The yield of PLGA-PEG-MAL nanoparticles was in the range of 75-85%. Then it was formulated with PVA and PBS to 40 mg/mL (AT1011). Example 24: Preparation of PLGA ACNP using Ashland PLGA (GMP grade) as starting material [176] Ashland PLGA was dissolved in acetonitrile at 4 mg/mL, filtered, then made into nanoparticles by Dolomite system and purified with Amicon Ultra-15 centrifugal filter units with the same method described in Example 22 except with a different flow rate (6mL/min Ashland PLGA solution and 4 mL/min water). Example 25: Preparation of PLGA-PEG-Mal ACNP using SEQENS PLGA-PEG-Mal as starting material [177] SEQENS PLGA-PEG-Mal was dissolved in acetonitrile at 4 mg/mL, filtered, then made into nanoparticles by Dolomite micromixer and purified with Amicon Ultra-15 centrifugal filter units with the same method described in Example 22. Example 26: Preparation of PLGA ACNP (AT1014) by microfluidic mixing and tangential flow filtration (TFF) purification using PolySciTech AP230 at 0.8g scale Materials ^ AP230 PLGA (55-65kDa, LA:GA ratio 75:25) (Lot#171121YSK-A) 4 mg/mL PLGA ^ Acetonitrile (Fisher) ^ Endotoxin free water (EMD Millipore) ^ Dolomite Micromixer with 100-µm microchannel chip ^ 0.2 µm Nylon syringe filters, sterile, 25 mm (Fisher) ^ 0.45 µm PES filter (Fisher) ^ PVA (MW 6,000 Da, 80% hydrolyzed) (Polysciences) ^ DPBS, 1x (Corning) TFF Set-up ^ Repligen KrosFlo® KR2i TFF System ^ Repligen 500 kDa MWCO hollow-fiber filter (Part# D06-E500-05-N) ^ PharmaPure or C-Flex tubing #16 PLGA ACNP (AT1014) dispersion preparation [178] The PLGA stock solution in acetonitrile was prepared by dissolving 800 mg PLGA (AP230) in 200 mL acetonitrile using sonication for 2-5 minutes and vortex to get a clear solution. The stock solution was then filtered through a 0.2 µm nylon filter to remove residues that might block the microchannel chip. Syringes A and B of the Dolomite micromixer were filled with each of the PLGA stock solution and endotoxin-free water. Both syringes were set for a flow rate of 5.0 mL/min, and a total volume of 100.0 mL ACNP formulation was collected into a 250-mL glass bottle that contained 100 mL of 0.5 mg/mL PVA in 1x DPBS buffer and a magnetic stirring bar to keep the formulation agitated.30 µL samples were taken from the 200 mL solution in the bottle, and the particle size and polydispersity were measured by Dynamic Light Scattering (DLS). Target particle size was ~100 nm by zeta-avg. Repeated above steps three times to collect a total of 800 mL dispersion, and then 1200 mL of 0.5 mg/mL PVA in 1x DPBS buffer was added to make the final dispersion volume of ~2 L with acetonitrile about 10%. This dispersion was then filtered by 0.45 µm PES filter. PLGA ACNP purification by TFF processing [179] The hollow fiber TFF system was set up and connected with #16 tubing. The filter was sanitized using 0.2 M NaOH (pH 13) for 30-60 minutes with the flow rate of 125 mL/min if necessary and was rinsed with endotoxin free water twice afterwards. The PLGA ACNP dispersion (~2 L) was then applied to the TFF filter with the settings of TMP 5 psi and flow rate 125 mL/min. Two cycles of concentration, diafiltration and concentration were carried out as illustrated in the flow chart (Fig.8). Total volume of ~ 50 mL concentrated ACNP was collected at the end of the TFF process. The filter was then recirculated with about 50 mL 0.5 mg/mL PVA in 1x DPBS buffer as the TFF wash. Analysis DLS using Litesizer 500 from Anton Paar Table 12 particle size (nm) and PDI for the dispersion Particle size (nm) Hydrodynamic By number PDI 6 3 1 ¹H-NMR using 40

0MHz Bruker magnet by custom NMR services. [180] 10 mg of lyophilized PLGA ACNP nanoparticles was dissolved in 0.5 mL DMSO-d6. PBS was removed by filtration. Fig.9 shows NMR spectrum of TFF collection. [181] 10 mg of lyophilized PLGA ACNP nanoparticles was dissolved in 0.5 mL DMSO-d6. PBS was removed by filtration. Fig.10 shows NMR spectrum of TFF wash. Sample Integrated proton G A A A

[182] Based on NMR, the estimated yield of PLGA (TFF collection and wash combined) was about 78%. Example 27: Preparation of PLGA ACNP (AT1014) by microfluidic mixing and tangential flow filtration (TFF) purification using Ashland PLGA at 1.8 g scale Materials ^ PLGA from Ashland (batch# 0002594415) 4 mg/mL PLGA ^ Acetonitrile (Fisher) ^ Endotoxin free water (EMD Millipore) ^ Dolomite Micromixer with 100-µm microchannel chip ^ 0.2µm Nylon syringe filters, sterile, 25mm (Fisher) ^ 0.45 µm PES filter (Fisher) ^ 1M HEPES buffer, sterile (Teknova) ^ PVA (Polyvinyl alcohol 4-88) (EMD Millipore) TFF Set-up ^ Repligen KrosFlo® KR2i TFF System ^ Repligen 500 kDa MWCO hollow-fiber filter (Part# D06-E500-05-N) ^ PharmaPure or C-Flex tubing #16 PLGA ACNP dispersion preparation [183] The PLGA stock solution in acetonitrile was prepared by dissolving 1.8 g PLGA in 450 mL acetonitrile using sonication for 2-5 minutes and vortex to get a clear solution. The stock solution was then filtered through a 0.2 µm nylon filter to remove residues that might block the microchannel chip. Syringes A and B of the Dolomite micromixer were filled with each of the PLGA stock solution for A and endotoxin-free water for B. Syringe A was set for a flow rate of 6.0 mL/min and syringe B was set for a flow rate of 4.0 mL/min. A total volume of 200.0 mL PLGA ACNP dispersion was collected into a 450-mL glass bottle that contained 200 mL of 2.5 mg/mL PVA in 100 mM HEPES buffer and a magnetic stirring bar to keep the formulation agitated.30 µL samples were taken from the 400 mL solution in the bottle for the particle size and polydispersity measurement by dynamic light scattering (DLS). Target particle size was ~100 nm (Z-average). Repeated above steps twice to collect a total of 1.2 L dispersion in 2.5 mg/mL PVA, 100 mM HEPES buffer. The last collection was made by collecting the rest 150 mL ACNP into a 450-mL glass bottle that contained 150 mL of 2.5 mg/mL PVA in 100 mM HEPES buffer and a magnetic stirring bar to keep the formulation agitated. The four ACNP dispersions were then combined to make a total volume of 1.5 L and were filtered by 0.45 µm PES filter. Approximate 2.7 L of endotoxin free water was added to the filtered dispersion to make the final volume of about 4.2 L of ACNP in 0.5 mg/mL PVA, 20 mM HEPES with acetonitrile at 10.7%. Purification of PLGA ACNP by TFF [184] The TFF hollow fiber filter system was set up and connected with #16 tubing. The system was sanitized by recirculating 0.2 N NaOH for 30 - 60 minutes at flow rate of 125 mL/min followed by flushing with endotoxin free water twice. The PLGA ACNP dispersion (4.14 kg) was then applied to the TFF filter with the settings of TMP 9 psi and flow rate 200 mL/min for the first concentration cycle. The second cycle of concentration, diafiltration and concentration was carried out with the settings of TMP 5 psi and flow rate 125 mL/min. Total volume of 42 mL concentrated ACNP was collected at the end of the TFF process. Analysis DLS using Litesizer 500 from Anton Paar Table 13. Particle size (nm) and PDI for the dispersion Particle size (nm) H d d i B b PDI

PLGA ACNP concentration and yield calculation [185] PLGA ACNP concentration determined by HPLC was 38.2 mg/mL. With a volume of ~40 mL, the total amount after TFF was 1.53 g. The calculated yield was 85%. Example 28: Preparation of PLGA ACNP (AT1014) by microfluidic mixing and flat sheet TFF purification using Ashland PLGA at 1.8 g scale Materials ^ PLGA from Ashland (batch# 0002594415) 4 mg/mL PLGA ^ Acetonitrile (Fisher) ^ Endotoxin free water (EMD Millipore) ^ Dolomite Micromixer with 100-µm microchannel chip ^ 0.2µm Nylon syringe filters, sterile, 25mm (Fisher) ^ 0.45 µm PES filter (Fisher) ^ 1M HEPES buffer, sterile (Teknova) ^ PVA (Polyvinyl alcohol 4-88) (EMD Millipore) Flat Sheet Cassette TFF Set-up ^ Repligen KrosFlo® KR2i TFF System ^ Repligen 100 kDa MWCO flat sheet cassette filter (Part# XP300M01L) ^ Repligen cassette filter plate insert (Part# TFPLS-SA08) ^ Repligen manual clamp holder for cassettes (Part#TSLDI-2BMC) ^ PharmaPure or C-Flex tubing #16 PLGA ACNP dispersion preparation [186] PLGA ACNP dispersion was prepared by the same method described in Example 27. Purification of PLGA ACNP by flat sheet TFF [187] The flat sheet TFF system was set up and connected with #16 tubing. The system was sanitized by recirculating 0.5 M NaOH for 30 - 60 minutes at flow rate of 200 mL/min followed by flushing with endotoxin free water twice. The PLGA ACNP dispersion (4.1 kg) was then applied to the TFF filter with the settings of TMP 9 psi and flow rate 200 mL/min for the first concentration cycle. The second cycle of concentration, diafiltration and concentration was carried out with the settings of TMP 5 psi and flow rate 125 mL/min. Approximately 50 mL concentrated PLGA ACNP dispersion was collected at the end of the TFF process. Analysis DLS using Litesizer 500 from Anton Paar Table 14. Particle size (nm) and PDI for dispersion Particle size (nm) I 8 7 9

PLGA collection -200mL-4 106.5 ± 0.4 70.3 ± 7.9 0.19 PLGA collection -100mL-5 108.9 ± 1.0 75.0 ± 4.8 0.16 6 PLGA ACNP c

[188] PLGA ACNP concentration determined by HPLC was 29.7 mg/mL. With a volume of ~50 mL, the total amount after TFF was 1.49 g. The calculated yield was 83%. Example 29: Preparation of PLGA-PEG-Mal ACNP using PolySciTech AI052 as starting material and hollow fiber TFF for purification at 0.9 g scale Materials ^ AI052 PLGA-PEG-MAL (60kDA-3.4kDA, LA:GA ratio 75:25) (Lot#230505RAI-A) 6 mg/mL in acetonitrile ^ Acetonitrile (Fisher) ^ Endotoxin free water (EMD Millipore) ^ Dolomite Micromixer with 100 µm microchannel chip ^ 0.2 µm Nylon syringe filters, sterile, 25 mm (Fisher) ^ 0.45 µm PES filter (Fisher) ^ PVA (MW 6,000 Da, 80% hydrolyzed) (Polysciences) ^ DPBS, 1x (Corning) TFF Set-up ^ Repligen KrosFlo® KR2i TFF System ^ Repligen 500 kDa MWCO hollow-fiber filter (Part# D06-E500-05-N) ^ PharmaPure or C-Flex tubing #16 PLGA-PEG-Mal dispersion preparation [189] The PLGA-PEG-Mal stock solution in acetonitrile was prepared by dissolving 1.2 g PLGA- PEG-MAL in 200 mL acetonitrile using sonication for 2-5 minutes and vortex to get a clear solution. The stock solution was then filtered through a 0.2 µm nylon filter to remove residues that might block the microchannel chip. Syringes A and B of the Dolomite micromixer were filled with each of the PLGA-PEG-Mal stock solution and endotoxin-free water. Both syringes were set for a flow rate of 6.0 mL/min, and a total volume of 100.0 mL ACNP formulation was collected into a 250-mL glass bottle that contained 100 mL of 0.5 mg/mL PVA in 1x DPBS buffer and a magnetic stirring bar to keep the formulation agitated.30 µL samples were taken from the 200 mL solution in the bottle, and the particle size and polydispersity were measured by DLS. Repeated the above steps twice to collect a total of 600 mL dispersion, and then 900 mL of 0.5 mg/mL PVA in 1x DPBS buffer was added to make the final dispersion volume of ~1.5 L with about 10% acetonitrile. This dispersion was then filtered by 0.45 µm PES filter. Purification of PLGA-PEG-Mal ACNP by hollow fiber TFF [190] The hollow fiber TFF system was set up and connected with #16 tubing. The system was sanitized by recirculating 0.2 M NaOH for 30 - 60 minutes at flow rate of 125 mL/min following by flushing with endotoxin free water twice. The ACNP dispersion (~1.5 L) was then applied to the TFF filter with the settings of TMP 5 psi and flow rate 125 mL/min. Two cycles of concentration, diafiltration and concentration were carried out. Approximately 50 mL concentrated ACNP was collected at end of concentration. The filter was then recirculated with about 65 mL 0.5 mg/mL PVA in 1x DPBS buffer and the product was collected as the TFF wash. Analysis DLS using Litesizer 500 from Anton Paar Table 15. Particle size (nm) and PDI for dispersion Particle size (nm)

Example 30: Preparation of PLGA-PEG-Mal ACNP (AT1011) by microfluidic mixing and hollow fiber TFF using SEQENS PLGA-PEG-Mal at 1.8 g scale Materials ^ PLGA-PEG-Mal from SEQENS (batch# 2585-149) 6 mg/mL in acetonitrile ^ Acetonitrile (Fisher) ^ Endotoxin free water (EMD Millipore) ^ Dolomite Micromixer with 100-µm microchannel chip ^ 0.2µm Nylon syringe filters, sterile, 25mm (Fisher) ^ 0.45 µm PES filter (Fisher) ^ 1M HEPES buffer, sterile (Teknova) ^ PVA (Polyvinyl alcohol 4-88) (EMD Millipore) TFF Set-up ^ Repligen KrosFlo® KR2i TFF System ^ Repligen 500 kDa MWCO hollow-fiber filter (Part# D06-E500-05-N) ^ PharmaPure or C-Flex tubing #16 PLGA-PEG-Mal dispersion preparation: [191] The PLGA-PEG-Mal stock solution in acetonitrile was prepared by dissolving 1.8 g PLGA- PEG-MAL in 300 mL acetonitrile using sonication for 2-5 minutes and vortex to get a clear solution. The stock solution was then filtered through a 0.2 µm nylon filter to remove residues that might block the microchannel chip. Syringes A and B of the Dolomite micromixer were filled with each of the PLGA-PEG-Mal stock solution and endotoxin-free water. Both syringes were set for a flow rate of 6.0 mL/min, and a total volume of 200 mL ACNP dispersion was collected into a 450-mL glass bottle that contained 200 mL of 2.5 mg/mL PVA in 100 mM HEPES buffer and a magnetic stirring bar to keep the dispersion agitated.30 µL samples were taken from the bottle, and the particle size and polydispersity were measured by DLS. Repeated above steps twice to collect a total of 1.2 L dispersion in 2.5 mg/mL PVA, 100 mM HEPES buffer. This dispersion was then filtered by 0.45 µm PES filter. 1.8 L of endotoxin free water was added to the filtered dispersion to make the final volume of ~3 L of ACNP in 0.5 mg/mL PVA, 20 mM HEPES with acetonitrile at 10%. Purification of PLGA-PEG-Mal ACNP by hollow fiber TFF [192] The hollow fiber TFF system was set up and connected with #16 tubing. The system was sanitized by recirculating 0.2 M NaOH for 30-60 minutes with the flow rate of 125 mL/min followed by flushing with endotoxin free water twice. The ACNP dispersion ( ~3 L )was then applied to the TFF filter with the settings of TMP 9 psi and flow rate 200 mL/min for the first concentration cycle. The second cycle of concentration, diafiltration and concentration was carried out with the settings of TMP 5 psi and flow rate 125 mL/min.42 mL concentrated ACNP was collected at the end of TFF process. Analysis DLS using Litesizer 500 from Anton Paar Table 16. Particle size (nm) and PDI for dispersion Particle size (nm) Hydrodynamic By number PDI PLGA

-PEG-Mal ACNP concentration and yield calculation [193] PLGA-PEG-Mal ACNP concentration determined by HPLC was 29.7 mg/mL. With a volume of ~40 mL, the total amount after TFF was 1.25 g. The calculated yield was 69%. Example 31: Preparation of PLGA-PEG-Mal ACNP (AT1011) by microfluidic mixing and flat sheet TFF using SEQENS PLGA-PEG-Mal at 1.8 g scale Materials ^ PLGA-PEG-Mal from SEQENS (batch# 2585-149) 6 mg/mL in acetonitrile ^ Acetonitrile (Fisher) ^ Endotoxin free water (EMD Millipore) ^ Dolomite Micromixer with 100-µm microchannel chip ^ 0.2µm Nylon syringe filters, sterile, 25mm (Fisher) ^ 0.45 µm PES filter (Fisher) ^ 1M HEPES buffer, sterile (Teknova) ^ PVA (Polyvinyl alcohol 4-88) (EMD Millipore) Flat Sheet Cassette TFF Set-up ^ Repligen KrosFlo® KR2i TFF System ^ Repligen 100 kDa MWCO flat sheet cassette filter (Part# XP300M01L) ^ Repligen cassette filter plate insert (Part# TFPLS-SA08) ^ Repligen manual clamp holder for cassettes (Part#TSLDI-2BMC) ^ PharmaPure or C-Flex tubing #16 PLGA-PEG-Mal ACNP dispersion preparation [194] PLGA-PEG-Mal ACNP dispersion was prepared by the same method as described in Example 30. Purification of PLGA-PEG-Mal ACNP by flat sheet TFF [195] The flat sheet cassette TFF system was set up and connected with #16 tubing. The system was sanitized by recirculating 0.5 M NaOH for 30 - 60 minutes at flow rate of 200 mL/min followed by flushing with endotoxin free water twice. The ACNP dispersion (~2.9 kg) was then applied to the TFF filter with the settings of TMP 9 psi and flow rate 200 mL/min for the first concentration cycle. The second cycle of concentration, diafiltration and concentration was carried out with the settings of TMP 5 psi and flow rate 125 mL/min. About 50 mL concentrated ACNP was collected at the end of TFF process. Analysis DLS using Litesizer 500 from Anton Paar Table 17. Particle size (nm) and PDI for dispersion Particle size (nm)

PLGA-PEG-Mal ACNP concentration and yield calculation [196] PLGA-PEG-Mal ACNP concentration determined by HPLC was 25.2 mg/mL. With a volume of ~50 mL, the total amount after TFF was 1.26 g. The calculated yield was 70%. Example 32: Determination of PLGA and PVA concentration in ACNP by NMR [197] Concentration of PLGA ACNP containing PVA cannot be determined directly by gravimetrical method after lyophilization. NMR was one of the methods that can be used to measure PLGA and PVA concentrations. 0.2-0.3mL of PLGA nanoparticles (purified but not formulated) was lyophilized to remove water, then dissolved in 0.5 mL DMSO-d6 with 0.1 mL 0.4 mg/mL maleic acid (MA) in DMSO-d6 added for NMR analysis. The known concentration of maleic acid (proton chemical shift at 6.2 ppm) was used as internal standard to quantify PLGA (protons from LA at 5.2 ppm, and from GA at 4.9 ppm) and PVA at 3.8 ppm. For example, 0.225 mL of purified PLGA ACNP was lyophilized and dissolved in 0.5 mL DMSO-d6 with 0.1mL MA in DMSO- d6 for proton NMR. The NMR analysis results were listed in Table 18. Table 18 PLGA amount by NMR (mg) PVA amount by NMR (mg) Calculated by LA Calculated by GA Average [19

8] The PLGA nanoparticle concentration was calculated as 52.4 mg/mL, with PVA at 10.2 mg/mL. Example 33: Quantification of PLGA, PLGA-PEG-Mal in ACNPs by HPLC 1. Materials and Methods 1.1. HPLC method [199] The HPLC system was bought from Shimadzu (Maryland, USA). It consists of 5-channel degasser (DGU-405), two pumps (LC-40D), system controller (CBM-40), column oven (CTO-40C) and evaporative light scattering detector (ELSD-LT III). A ZORBAX RR Eclipse Plus C18 column (95 Å, 2.1 x 50 mm, 3.5 µm) was used as analytical column. Chromatograms were recorded and analyzed via LabSolutions software. Nitrogen was used as carrier gas for ELSD-detection. [200] Gradient elution was applied to elute all components of ACNPs in a short time maintaining baseline separation. Mobile phase A consisted of water and 0.1% TFA. Mobile phase B consisted of acetonitrile and 0.1% TFA. The gradient profile is depicted in Table 19. The gradients change linearly between the timepoints. Duration of one run was 6 min. Injection volume was 10 µL; column oven was heated to 40 ˚C and flow rate was set to 0.5 mL/min. Table 19. Gradient profile for HPLC measurement. Time (min) Mobile A (%） Mobile B (%) 0 55 45 1.2. Prepar

[201] Stock solutions of PLGA and PLGA-PEG-Mal were prepared in acetonitrile with concentrations of 1 mg/mL, respectively. The working standard solutions were prepared as needed by appropriate dilution of the concentrated stock solutions in acetonitrile. Standard solutions containing 50, 100, 150, 200 and 250 µg/mL of PLGA and PLGA-PEG-Mal were prepared freshly before each measurement. In addition, the standard solutions of the mixture of PLGA and PLGA-PEG-Mal were prepared by mixing the same concentrations of PLGA and PLGA-PEG-Mal solutions at 50, 100, 150, 200 and 250 µg/mL. 1.3. Sample Preparation [202] The formulated ACNPs were first diluted 10 times in DI water. After that, the solution was further diluted 20 times in acetonitrile. The diluted solution was then filtered using a 0.22 µm PVDF filter to remove any precipitations. The filtrate solution was collected and measured by HPLC-LESD. 2. Results 2.1. Standard Curves of Polymer Solutions [203] Three standard curves of PLGA, PLGA-PEG-Mal and the mixture of PLGA and PLGA-PEG- Mal were prepared. As shown in Figure 16, all the three curves fit well in polynomial trendlines. In addition, PLGA solutions had stronger responses than PLGA-PEG-Mal solutions at the same concentrations. [204] Fig.11 shows standard curves of PLGA, PLGA-PEG-Mal and the mixture of PLGA and PLGA- PEG-Mal solutions. 2.2. Application for ACNP Measurement [205] ACNPs were diluted in acetonitrile and then filtered using PVDF filter to remove the interferences (salts from PBS and PVA) from the tested samples. The concentration before and after filtration didn’t show any change, indicating that the filtration process would not affect the HPLC measurement of PLGA and PLGA-PEG-Mal. ACNP samples were analyzed by this HPLC-ELSD method. Example 34 Formulation of ACNPs in 20 mM HEPES, 10% sucrose, pH 7.4 Concentrations of ACNPs in 20 mM HEPES pH 7.4 from Examples 28 and 31 were determined by the HPLC methods from Example 33. The ACNPs were diluted to the specified concentrations by adding calculated amount of 50% sucrose, 1 M HEPES pH 7.4 and water for injection to obtain a final formulation in 20 mM HEPES, 10% sucrose at pH 7.4. The final product was then sterile filtered through 0.2 µm PES filter into the storage container. Example 35: Formulation of mixed ACNPs [206] Different ACNPs from Examples above, such as PLGA ACNP and PLGA-PEG-Mal ACNP, can be mixed at specific ratios as an ACNP cocktail. For example, AT1019 is a 1:1 mass mixture of PLGA ACNP (AT1014) and PLGA-PEG-Mal ACNP (AT1011). AT1019 can be further adjusted to the target concentration, sterile filtered and fill-finished in the vials for future use. Example 36: Quantification of PVA by colorimetric method [207] The PVA concentration in ACNPs were determined by colorimetric method. The ACNP dispersion was diluted to appropriate concentration with water. For example, the formulated ACNPs at about 20 mg/mL can be diluted 50 times for test. PVA solutions made from PVA 4-88 (31kD, from MilliporeSigma) ranging from 25 to 500 µg/mL were prepared as standards.0.75 mL boric acid and 0.15mL of KI/I₂ solution were added to 0.2 mL of PVA standard solutions or test sample dilutions, mixed at room temperature for 30 minutes. The reaction mixtures were pipetted into 96-well plate to measure the absorbance at 630 nm. The PVA concentration of the tested samples were calculated based on the standard curve made from the PVA standards. Two batches of PLGA nanoparticles formulated at 20 mg/mL were measured to have PVA concentration at 6.9 and 6.4 mg/mL respectively. One batch of PLGA-PEG-MAL nanoparticles formulated 20 mg/mL were measured to have PVA concentration at 8.1 mg/mL. Example 37: Quantification of maleimide content in ACNP [208] The maleimide content of PLGA-PEG-MAL can also be analyzed by colorimetric method (modified Ellman’s assay, Ellman GL (1959). "Tissue sulfhydryl groups". Arch. Biochem. Biophys. 82 (1): 70–7.) by using N-ethylmaleimide (NEM) as standard. Briefly, the tested PLGA-PEG-MAL and standard NEM were dissolved in acetonitrile. To 0.9 mL of PLGA-PEG-MAL or standard NEM in acetonitrile was added 90 µL of water, 10 µL of 5 mM fresh cysteine-HCl aqueous solution, 2 µL of 1 M HEPES pH 7.0, and 10 µL of 0.1 M NaOH to react for 30 min, followed by the addition of 10 µL of 20 mM DTNB (Ellman's reagent) for 10 minutes. The absorbance of the mixture was measured at 412 nm. [209] For example, the maleimide content from one batch of PLGA-PEG-MAL was calculated as 40% using the above mentioned maleimide quantification method. In a repeated experiment, the maleimide content was quantified as 41%. Example 38: Stability Measurement of PLGA ACNP at -80 ^oC and 4 ^oC [210] PLGA ACNP were prepared following the method disclosed in Example 34. The vials were stored at -80 ^oC and 4 ^oC for up to 70 days (10 weeks). At time points of T= 0, 5 days and 2, 10 weeks, 30 µL sample from each vial was removed, diluted with 1 mL DI water, and the particle size and polydispersity index (PDI) were measured by DLS using the Litesizer 500 from Anton Paar. Results are shown in Table 20 below. [211] Control samples: in 20 mM HEPES buffer; Samples A in 20 mM HEPES buffer with 10% sucrose. Samples at -80 ^oC were thawed to room temperature before the measurements. Table 20. Size and PDI changes for PLGA ACNP formulations after storage at -80 ^oC and 4 ^oC over 10 weeks. The particles of ACNPs are stable under all tested conditions. P^{LGA Stora e Freshly made 5 days 2 weeks 10 weeks} I

Control- 0 4 ^oC 106.1±0.6 0.18 106.2±2.1 0.15 103.8±1.4 0.18 103.3±1.8 0.17 6 7 7 8 ng

storage sta ty. Example 39 Stability of PLGA ACNP at accelerated conditions: room temperature with light exposure [212] PLGA ACNP were prepared following the method disclosed in Example 34. The vials were stored at room temperature with light exposure for up to 70 days (10 weeks). At time points of T= 0, 1, 2, 8, 15, 30, and 70 days, 30 µL sample was removed, diluted with 1 mL DI water for the measurement of size and polydispersity index (PDI) by DLS using the Litesizer 500 from Anton Paar. Results are shown in Table 21, indicating nanoparticle ACNPs are stable at room temperature. Table 21 Day Z-Avg Size (nm) N-Avg Size (nm) PDI 4 8 8 5 5 6 6 6

Example 40: Stability Measurement of ACNPs at different concentrations [213] ACNPs from Example 34 were at 20 mg/mL. They were further diluted into 2 mg/mL and 0.2 mg/mL with 10%sucrose and 20 mM HEPES for stability evaluation. These 0.2, 2, and 20 mg/mL ACNPs were stored at -80 ^oC, and taken out at different time points to warm to room temperature for particle size measurement by DLS. Good stability was observed for both PLGA and PLGA-PEG-Mal ACNPs at all concentrations as shown in Figs.12a and 12b. Example 41: Morphology of ACNP by cryo electron microscope imaging [214] The morphology of ACNPs were characterized by cryo electron microscope (cryo-EM) imaging. Quantifoil R 1.2/1.3 grids (copper, 300 mesh) were glow discharged for 60 s using a Ted Pella EasiGlow device. Grids were frozen using a Vitrobot Mark IV system operating at 22 °C and 100% relative humidity. Undiluted samples were extensively vortexed and a 5 μL droplet was placed onto the glow-discharged surface of a grid. The grid was blotted for 4s using a blot force of +2, allowed a 0.5 s “drain time” and plunged into liquid-nitrogen-cooled liquid ethane. Frozen grids were stored in liquid nitrogen until they were clipped and loaded into a Thermo Fisher Talos Arctica electron microscope. The clipping, loading and storage within the Arctica until imaging occurred were all performed in liquid nitrogen or at liquid nitrogen temperatures. Minimal dose images of the frozen grids were acquired using the Thermo Fisher EPU software. All imaging was performed with the Arctica operating at 200 kV. Images that show entire holes were acquired at a nominal magnification of 36,000x using a Falcon 3 direct electron detecting camera operating in counting mode. The dose rate was at the upper extreme of the camera’s tolerance of 1 electron per pixel per second. Total exposure time was ~20 seconds and the individual frames (1 second exposures) were aligned, summed and saved. Targeted defocus while imaging was set to -5, -4 and -3 microns defocus, and actual defocus was not determined for each image. Fig.13 shows exemplary images of different ACNPs. All ACNPs are spherical solid nanoparticles. Example 42: Evaluation of the in vivo abscopal anti-tumor effects of AT1011 with or without poly IC in the treatment of bi-lateral subcutaneous syngeneic colorectal cancer CT26.WT tumors in female Balb/c mice [215] The objective of this study was to evaluate the abscopal anti-tumor effect of AT1011 with or without poly IC in the treatment of bilateral subcutaneous syngeneic CT26.WT-Dual Flank colorectal cancer tumors in female BALB/c mice. [216] Compared to baseline treatment of RT+anti-mPD1 (Group 2), i.t. injection of poly IC (Group 3) and ACNP AT1011 (Group 4) into primary tumor induced abscopal TGI in the secondary tumor at 27% and 15%, respectively. Co-injection of poly IC-40µg and poly IC-80µg with AT1011 (Groups 5 and 6) induced synergistic TGI at 49% and 43%, respectively. The TGI induced by AT1011+polyIC-40µg (Group 5) is significantly increased compared to baseline treatment (p=0.022*). This treatment combination also significantly improved the survival of treated mice with a median survival time of 32.5 days, compared with 23.5 days of baseline treatment group (Group 2). [217] No treatment-related body weight loss (>20%) or animal death was observed during the study, suggesting that AT1011 alone or in combination with poly IC are well tolerated when administered intratumorally. Materials and methods 1. Animal and model 1.1 Animal information Species Mus musculus St i B lb/ 1.2 Ho

using condition Cage Type Polysulfone IVC cage (325mm × 210mm × 180mm)

Humidity 40 - 70% Light Cycle 12 hours light and 12 hours dark , C

Model ID Cancer Type Medium Inoculation Method CT 26 WT Colorectal cancer RPMI-1640 +10%FBS 5 x 10⁵/mouse in 0.1 ml of PBS 2.

[218] The CT26 WT tumor cells were maintained in vitro with RPMI1640 medium supplemented with 10% fetal bovine serum at 37°C in an atmosphere of 5% CO₂ in air. The cells in exponential growth phase were harvested and quantitated by cell counter before tumor inoculation. A number of 120 Balb/c mice (7-8 weeks) were inoculated subcutaneously in both the right and left lower flank with CT26 WT tumor cells (5x 10⁵) in 0.1 mL of PBS on day 0 and day 3, respectively. The left tumor was the primary tumor and the right tumor was the secondary tumor. [219] Day 9~10 after the first inoculation, 48 mice with left tumor size in the range of 80- 120mm³ and the right tumor size in the range of 30-40mm³ are enrolled into the study. Mice are randomized into 6 study groups (Table 21) based on the size of the right tumors using the “Matched distribution” method (StudyDirector^TM software, version 3.1.399.19) with an average right tumor size of ~36mm³. 3. Test article preparation [220] Anti-mPD1 was purchased from BioXcell and used for i.p. injection at 100 µl/mouse, assuming body weight of each babl/c mouse to be 20g (equivalent to 5mg/kg). [221] Poly IC (polyinosine-polycytidylic acid, or Poly(I:C) ((HMW) VacciGrade) was purchased from Invivogen (Cat. Vac-pic). Poly IC was prepared at 1.6mg/ml in PBS according to the manufacturer’s protocol. [222] ACNP AT1011 was prepared using nanoprecipitation (refer to NP generation session). Lyophilized AT1011 was reconstituted in PBS or poly IC solution as shown in Table 21 right before intratumoral injections. In each group treated with ACNPs, each dose was prepared fresh with the following protocol: a) Reagent A should be kept at 4°C once arrived at the study site. Reagent B should be kept at -20 °C. Each reagent A tube will be used for 3 doses, and each reagent B will be used for single dose. b) Reconstitute Reagent B 30 minutes prior to each dose: Take out 1 vial of reagent B and equilibrate at room temperature for 5min, then add 0.66 mL Reagent A onto the inside wall of the vial to give the final concentration of test articles of 36 mg/mL. Then allow the vials to sit for 3-5 mins to ensure proper wetting of the lyophilized cake. Vortex the vial by using a vortex mixer (Fisherbrand Analog Vortex Mixer or similar model) for 10 seconds to ensure complete disintegration and dissolution of the cake. Store the rest of Reagent A back to the refrigerator for next doses. c) Slight foaming might be present upon reconstitution. Allow the vials to stand undisturbed for approximately 5 mins. d) Inspect visually for particulates and discoloration. The final solutions should be free of visible particulates. Test articles will be opalescent. e) Use the reconstituted articles immediately within 1 hour and discard the unused portion. Table 21. Intratumoral injection (i.t.) reagents preparation table for the study Reagent A Reagent B Group ID Treatment se ul r .

AT1011 (MAL ACNP)_dose 2 AT1011(MAL ACNP)_dose 3

[223] The treatment was initiated on the same day of randomization (day 0) per study design. The treatment design is illustrated in Fig.14a and treatment groups are summarized in Table 22. Fig.14a shows ACNP in vivo efficacy study design in CT26 bi-lateral tumor models. Table 22. Treatment design M^ic Dosing Dosing Dosing G_r ^e _{Tr tm nt D L l} S l ti n V l m ROA Fr n &

Radiation l_{eft tumor} ^{8 Gy - - - Day 0/1/2}

dy (5mg/kg) on days 0 and 3, radiation therapy (RT, 8Gy) on the left side (primary) tumor on days 0, 1 and 2; and 50µl of intratumoral (i.t., 50µl) injection of PBS or test articles (AT1011, poly IC or their combinations) in the left side tumor on days 2, 3 and 4. Irradiation on primary tumor was conducted with a small animal image guided radiation therapy system, X-RAD SMART precision X-Ray, which is a focal irradiation system that mimics clinical computerized tomography (CT) and RT, provides accurate delivery of radiation treatment with minimal impact on surrounding tissue. Treatments were conducted in the following order: i.p. injection, radiation therapy and i.t. injection. On day 0 when both i.p. and RT are conducted, mice are rested for 2 hours before administration of RT. The right side (secondary) tumors are left untreated. 5. Observation and Data Collection [225] After tumor cells inoculation, the animals were checked daily for morbidity and mortality. During routine monitoring, the animals were checked for any effects of tumor growth and treatments on behavior such as mobility, food and water consumption, body weight gain/loss (Body weights were measured three times per week after randomization), eye/hair matting and any other abnormalities. Mortality and observed clinical signs were recorded for individual animals in detail. [226] Tumor volumes of both left and right tumors were measured three times per week after randomization in two dimensions using a caliper, and the volume was expressed in mm³ using the formula: V = (L x W x W)/2, where V is tumor volume, L is tumor length (the longest tumor dimension) and W is tumor width (the longest tumor dimension perpendicular to L). Dosing as well as tumor and body weight measurements were conducted in a Laminar Flow Cabinet. The body weights and tumor volumes were reported by using StudyDirector^TM software (version 3.1.399.19). The study was terminated on day 53 after randomization. 6. Humane endpoints and toxicity [227] The individual mouse was euthanized if its tumor volume exceeds 3000 mm³ or if they lose over 20% of their body weight relative to the weight on the first day of treatment. [228] Any mouse exhibiting an ulcerated or necrotic tumor was separated immediately and singly housed and monitored daily before the animal was euthanized or until tumor regression was completed. The mouse was euthanized rapidly if tumor ulcerates, and the ulceration diameter was greater than 5 mm, or pus or necrosis observed, or if tumor burden, including metastasis, compromises animal’s normal physiologic performances, e.g., orientation, access to food or water, etc. [229] Animals were weighed twice weekly for the first 30 days and once a week for the remainder of the study. The mice were observed frequently for overt signs of any adverse side effects such as lethargy, death or infections. Acceptable toxicity is defined as a group mean body- weight loss of less than 15% during the study and not more than one treatment-related (TR) death among ten treated animals. A death is classified as TR if attributable to treatment side effects as evidenced by clinical signs and/or necropsy or if due to unknown causes during the dosing period or within 10 days of the last dose. A death is classified as an NTR if there is no evidence that death was related to treatment side effects. 7. Data analysis [230] Treatment outcome was determined from percent tumor growth inhibition (TGI). TGI is defined as the percent decrease in average tumor volume of treated versus control group and calculated from the following equation: TGI= (1- ^{^^^^ ୈୟ^ ௫ ି ^^^^ ୈୟ^ ^} ^_{^^^୦ ୈୟ^ ௫ ି ^^^^୦ ୈୟ^ ^} ) x 100% [231] Where TVtx DayX=Average tumor volume of treated group on Day X; TVtx Day= average tumor volume of treated group on Day 0; TVveh DayX=Average tumor volume of vehicle group on Day X; TVveh Day= Average tumor volume of vehicle group on Day 0. [232] TGI of each treatment group was assessed on Day 13, the last day before the first mouse in control group (G1) is euthanized due to tumor burden. [233] Graphpad Prism 9.4.1 software was used for graphical and statistical analysis in this study. Tumor growth and mouse survival data, including individual tumor growth on both left and right tumors, overall tumor growth, survival curves, median survival time were generated. [234] Statistical differences in the average tumor growth curves were determined by two-way ANOVA using the variables of time and mean tumor volume with Bonferroni correction with Graphpad Prism 9.4.1. [235] The differences in survival in each group were determined using the Kaplan–Meier method and the overall P value was calculated by the log-rank test using GraphPad Prism 5.0 (P value: *P < 0.05, **P < 0.01, ***P < 0.005) or GraphPad Prism 9.4.1 (P value: *P < 0.05, **P < 0.01, ***P < 0.005). Results [236] 6 groups of animals were treated in accordance with the protocol summarized in Table 21. The study was concluded on Day 53. Table 23 summarizes the treatment responses for each group. TGI on day 13 was assessed as described in “Materials and methods”. Compare to baseline treatment of RT+anti-mPD1 (Group 2), i.t. injection of both poly IC (Group 3) and ACNP AT1011 (Group 4) into primary tumor induced abscopal TGI in the secondary tumor. Combination of poly IC-40µg and poly IC-80µg with AT1011 (Groups 5 and 6) induced synergistic TGI at 49% and 43%, respectively. The TGI induced by AT1011+polyIC-40µg (Group 5) is significantly increased compared to baseline treatment (p=0.022) based on the two-way ANOVA analysis using the variables of time and mean tumor volume with Bonferroni correction with Graphpad Prism 9.4.1. This treatment combination also significantly improved the survival of treated mice with a median survival time of 32.5 days, compared with 23.5 days of baseline treatment group (Group 2). Table 23. Response summary of study groups in the bi-lateral CT26 tumor model efficacy study n al

w/ RT+PD1 (D13) 8

CT26 tumor model [237] As shown in Table 23 and Fig. 14b, individual tumor growth curve for primary tumor (treated) and secondary tumor (untreated) showed that although RT+anti-PD1 treatment did not eradicate the primary tumor, when compared to baseline treatment of RT+anti-mPD1 (Group 2), i.t. injection of both poly IC (Group 3) and ACNP AT1011 (Group 4) into primary tumor induced abscopal TGI in the secondary tumor. As shown in Table 23, co-injection of poly IC-40µg and poly IC-80µg with AT1011 (Groups 5 and 6) induced synergistic TGI at 49% and 43%, respectively. In addition, 2 out of 8 animals treated with co-injection of poly IC-40µg and AT1011 (Group 5) are cured (tumor free) at the end of study on Day 53. The TGI induced by AT1011+polyIC-40µg (Group 5) is significantly increased compared to baseline treatment (p=0.022) (Fig. 14c) based on the two-way ANOVA analysis using the variables of time and mean tumor volume with Bonferroni correction with Graphpad Prism 9.4.1. [238] Figs. 14b and 14c: Co-injection of AT1011 and polyIC-40µg showed significant abscopal TGI effect when compared to base line treatment RT+ant-PD1. [239] Fig. 14b shows individual tumor growth of primary and secondary tumor and overall tumor growth (mean tumor volume and SEM). [240] Fig. 14c shows two-way ANOVA analysis of Group 3, 4, 5 in comparison with base line treatment (Group 2). Effect of AT1011 and poly IC co-injection on the survival of treated animals. [241] Survival time of all 6 study groups is analyzed using Graphpad Prism 9.4.1. as shown in Fig. 15 with medium survival time (MS) calculated and indicated for each group. Co-injection of AT1011 and poly IC (Groups 5 and 6) improved the survival of treated mice compared with baseline treatment (Group 2) as shown in the Kaplan Meier plots. Among the two co-injection treated groups, AT1011+poly IC-80µg (Group 5) has a median survival time of 32.5 days, compared with 23.5 days of baseline treatment group (Group 2). Statistical analysis showed that survival of this group is significantly improved from the untreated group (p=0.0019**) [242] Fig. 15: Co-injection of AT1011 and poly IC improved survival of treated animals in comparison to base line treatment RT+anti-PD1. 9. Tolerability and Mortality [243] Although some mice showed slight body weight loss (BWL) several days after radiation (Fig.16), they recovered within 2 weeks. Two mice in Group 6 were found dead on day 1 and 2 after randomization therefore are not included in the study. Since other mice in the study are not showing death or lethargy during the study, we consider the death of these mice not treatment related. These data suggest that the treatment of RT+anti-PD1+ACNP is well tolerated. [244] Fig.16 shows mean body weight change percentages with SEM. Example 43: Evaluation of the in vivo abscopal anti-tumor effects of AT1011 (PLGA-PEG-MAL), AT1014 (PLGA ACNP) and their combination in the treatment of bi-lateral subcutaneous syngeneic colorectal cancer CT26.WT tumors in female Balb/c mice [245] The objective of this study was to evaluate the abscopal anti-tumor effect of AT1011(PLGA-PEG-MAL) and the combination of 2 ACNPs: AT1011(PLGA-PEG-MAL) and AT1014 (PLGA ACNP) at 2mg/dose and 1mg/dose in the treatment of bilateral subcutaneous syngeneic CT26. WT Flank colorectal cancer tumors in female BALB/c mice. [246] Compared to baseline treatment of RT+anti-mPD1 (Group 2), i.t. injection of AT1011 or AT1011+AT1014 at both dose levels showed significant abscopal TGI. Tumor free mouse frequencies in AT1011_2mg, AT1011+AT1014_2mg and AT1011+AT1014_1mg treated groups are 3/8, 2/7 and 1/8, respectively. Interestingly, AT1011+AT1014_1mg treated animals showed a significant improvement of survival compared with the baseline treated group, as indicated by the survival curve statistical analysis (p=0.0016*) and extended medium survival time. [247] No treatment-related body weight loss (>20%) or animal death was observed during the study, suggesting that AT1011 or AT1011+AT1014 at both doses are well tolerated when administered intratumorally and combined with anti-PD1 systemic administration and primary tumor focused radiation therapy. Materials and methods 1. Animal and model 1.1 Animal information Species Mus musculus Strain Balb/c 1.

Cage Type Polysulfone IVC cage (325mm × 210mm × 180mm) r,

Cage identification label: project no., group no., number of Cage Labelling animals, gender, strain, supplier, date of receipt, treatment, c. 1

[248] The CT26 WT tumor cells were maintained in vitro with RPMI1640 medium supplemented with 10% fetal bovine serum at 37ºC in an atmosphere of 5% CO₂ in air. The cells in exponential growth phase were harvested and quantitated by cell counter before tumor inoculation.1207-8 weeks Balb/c mice were inoculated subcutaneously in both the right and left lower flank with CT26 WT tumor cells (5x 10⁵) in 0.1 ml of PBS on day 0 and day, respectively. The left tumor was the primary tumor and the right tumor was the secondary tumor. [249] Day 9~10 after the first inoculation, 48 mice with left tumor size in the range of 80- 120mm³ and the right tumor size in the range of 30-40mm³ are enrolled into the study. Mice are randomized into 6 study groups (Table 24) based on the size of the right tumors using the “Matched distribution” method (StudyDirector^TM software, version 3.1.399.19) with an average right tumor size of ~36mm³. 3. Test article preparation [250] Anti-mPD1 was purchased from BioXcell and used for i.p. injection at 100ul/mouse, assuming body weight of each babl/c mouse to be 20g (equivalent to 5mg/kg). [251] ACNPs AT1011 (PLGA-PEG-Mal) and AT1014 (PLGA ACNP) were prepared separately using nanoprecipitation (refer to NP generation session). Lyophilized AT1011 or AT1011+AT1014 were reconstituted in PBS as shown in Table 24 right before intratumoral injections. In each group treated with ACNPs, each dose was prepared fresh with the following protocol: a) Reagent A should be kept at 4°C once arrived at the study site. Reagent B should be kept at -20°C. Each reagent A tube will be used for 3 doses, and each reagent B will be used for single dose. b) Reconstitute Reagent B 30 minutes prior to each dose: Take out 1 vial of reagent B and equilibrate at room temperature for 5min, then add 0.66 mL Reagent A onto the inside wall of the vial to give the final concentration of test articles of 36 mg/mL. Then allow the vials to sit for 3-5 mins to ensure proper wetting of the lyophilized cake. Vortex the vial by using a vortex mixer (Fisherbrand Analog Vortex Mixer or similar model) for 10 seconds to ensure complete disintegration and dissolution of the cake. Store the rest of Reagent A back to the refrigerator for next doses. c) Slight foaming might be present upon reconstitution. Allow the vials to stand undisturbed for approximately 5 mins. d) Inspect visually for particulates and discoloration. The final solutions should be free of visible particulates. Test articles will be opalescent. e) Use the reconstituted articles immediately within 1 hour and discard the unused portion. Table 24. Intratumoral injection (i.t.) reagents preparation table for the study Group Reagent A Reagent B I_D ^Treatment e l t.

4. Mouse treatment [252] The treatment was initiated on the same day of randomization (day 0) per study design. The treatment design is illustrated in Fig.17a and treatment groups are summarized in Table 25. [253] Fig.17a ACNP in vivo efficacy study design in CT26 bi-lateral tumor models Table 25. Treatment design Dose Dosing Dosing Volu Dosing G_roup ^Mice _Treatment me Solution ^ROA Frequency &

Radiation l_{eft tumor} ^{8 Gy - - - Day 0/1/2}

ody (5mg/kg) on days 0 and 3, radiation therapy (RT, 8Gy) on the left side (primary) tumor on days 0, 1 and 2; and 50µl of intratumoral (i.t., 50µl) injection of PBS or ACNPs: Mal-ACNP (AT1011) or combination of two ACNPs, Mal-ACNP (AT1011) + PLGA-ACNP (AT1014), at 18mg/dose or 0.9mg/dose, in the left side (primary) tumor on days 2, 3 and 4. Irradiation on primary tumor was conducted with a small animal image guided radiation therapy system, X-RAD SMART precision X-Ray, which is a focal irradiation system that mimics clinical computerized tomography (CT) and RT, provides accurate delivery of radiation treatment with minimal impact on surrounding tissue. Treatments were conducted in the following order: i.p. injection, radiation therapy and i.t. injection. On day 0 when both i.p. and RT are conducted, mice are rested for 2 hours before administration of RT. The right side (secondary) tumors are left untreated. 5. Observation and Data Collection [255] After tumor cells inoculation, the animals were checked daily for morbidity and mortality. During routine monitoring, the animals were checked for any effects of tumor growth and treatments on behavior such as mobility, food and water consumption, body weight gain/loss (Body weights were measured three times per week after randomization), eye/hair matting and any other abnormalities. Mortality and observed clinical signs were recorded for individual animals in detail. [256] Tumor volumes of both left and right tumors were measured three times per week after randomization in two dimensions using a caliper, and the volume was expressed in mm³ using the formula: “V = (L x W x W)/2, where V was tumor volume, L was tumor length (the longest tumor dimension) and W was tumor width (the longest tumor dimension perpendicular to L). Dosing as well as tumor and body weight measurements were conducted in a Laminar Flow Cabinet. The body weights and tumor volumes were reported by using StudyDirector^TM software (version 3.1.399.19). The study was terminated on day 53 after randomization. 6. Humane endpoints and toxicity [257] The individual mouse was euthanized if its tumor volume exceeds 3000 mm³ or if they lose over 20% of their body weight relative to the weight on the first day of treatment. [258] Any mouse exhibiting an ulcerated or necrotic tumor was separated immediately and singly housed and monitored daily before the animal was euthanized or until tumor regression was completed. The mouse was euthanized rapidly if tumor ulcerates, and the ulceration diameter was greater than 5 mm, or pus or necrosis observed, or if tumor burden, including metastasis, compromises animal’s normal physiologic performances, e.g., orientation, access to food or water, etc. [259] Animals were weighed twice weekly for the first 30 days and once a week for the remainder of the study. The mice were observed frequently for overt signs of any adverse side effects such as lethargy, death or infections. Acceptable toxicity is defined as a group mean body- weight loss of less than 15% during the study and not more than one treatment-related (TR) death among ten treated animals. A death is classified as TR if attributable to treatment side effects as evidenced by clinical signs and/or necropsy or if due to unknown causes during the dosing period or within 10 days of the last dose. A death is classified as an NTR if there is no evidence that death was related to treatment side effects. 7. Data analysis [260] Treatment outcome was determined from percent tumor growth inhibition (TGI). TGI is defined as the percent decrease in average tumor volume of treated versus control group and calculated from the following equation: TGI= (1- ^{^^^^ ୈୟ^ ௫ ି ^^^^ ୈୟ^ ^} ^_{^^^୦ ୈୟ^ ௫ ି ^^^^୦ ୈୟ^ ^} ) x 100% [261] Where TVtx

on Day X; TVtx Day= Averag tumor volume of treated group on Day 0; TVveh DayX=Average tumor volume of vehicle group on Day X; TVveh Day= Averag tumor volume of vehicle group on Day 0. [262] TGI of each treatment group was assessed on Day 13, the last day before the first mouse in control group (G1) is euthanized due to tumor burden. [263] Graphpad Prism 9.4.1 software was used for graphical and statistical analysis in this study. Tumor growth and mouse survival data, including individual tumor growth on both left and right tumors, overall tumor growth, survival curves, median survival time were generated. [264] Statistical differences in the average tumor growth curves were determined by two-way ANOVA using the variables of time and mean tumor volume with Bonferroni correction with Graphpad Prism 9.4.1. [265] The differences in survival in each group were determined using the Kaplan–Meier method and the overall P value was calculated by the log-rank test using GraphPad Prism 5.0 (P value: *P < 0.05, **P < 0.01, ***P < 0.005) or GraphPad Prism 9.4.1 (P value: *P < 0.05, **P < 0.01, ***P < 0.005). Results [266] 6 groups of animals were treated in accordance with the protocol summarized in Table 24. The study was concluded on Day 70. Table 26 summarizes the treatment responses for each group. TGI on day 13 was assessed as described in “Materials and methods”. Compared to baseline treatment of RT+anti-mPD1 (Group 2), i.t. injection of AT1011_1.8mg, AT1011_0.9mg, AT1011_0.9mg+AT1014_0.9mg and AT1011_0.45mg+AT1014_0.45mg into primary tumor induced significant abscopal TGI in the secondary tumor with TGI of 56, 46, 47 and 56%, respectively. Two-way ANOVA analysis using the variables of time and mean tumor volume with Bonferroni correction with Graphpad Prism 9.4.1. showed significant TGI increase compared to baseline treatment with p<0.05. The frequencies of tumor free animals at the end of the study (Day70) are 3/8, 2/7 and 1/8 in AT1011_1.8mg, AT1011_0.9+AT1014_0.9mg and AT1011_0.45+AT1014_0.45mg, respectively. ACNP intratumorally treatment resulted in improved survival as shown by the extended medium survival time compared with base line treatment (Group 2). Among all treated groups, AT1011_0.45+AT1014_0.45mg treatment showed most significant survival improvement with a p value of 0.016, compared to the baseline treatment (Group 2). TABLE 26. Response of different study groups in the bi-lateral CT26 tumor model efficacy study Abscopal Abscopal # of Median l

w/ RT+PD1 (D19) 8.

h in bi-lateral CT26 tumor model. [267] As shown in Table 26 and Fig. 17b, individual tumor growth curve for primary tumor (treated) and secondary tumor (untreated) showed that while RT+anti-PD1 treatment eradicated most of the primary tumors, no abscopal cure was found in Group 2 and Group 4. However, 3/8, 2/7 and 1/8 abscopal cure were found in Group 3., Group 5 and Group 6, respectively. When compared to baseline treatment of RT+anti-mPD1 (Group 2), i.t. injection of AT1011_1.8mg, AT1011_0.9mg, AT1011_0.9mg+AT1014_0.9mg and AT1011_0.45mg +AT1014_0.45mg into primary tumor induced significant abscopal TGI in the secondary tumor with TGI of 56, 46, 47 and 56%, respectively. Two-way ANOVA analysis using the variables of time and mean tumor volume with Bonferroni correction with Graphpad Prism 9.4.1. showed significant TGI increase of all ACNP treated groups compared to baseline treatment with p<0.05 (Fig.17c). [268] Figs. 17b and 17c: Intratumoral injection of AT1011 or co-injection of AT1011+AT1014 showed significant abscopal TGI effect when compared to base line treatment RT+ant-PD1 at both dose levels. [269] Fig. 17b shows individual tumor growth of primary and secondary tumor and overall tumor growth (mean tumor volume and SEM). [270] Fig.17c shows two-way ANOVA analysis of Group 3, 4, 5, 6 in comparison with base line treatment RT+anti-PD1(Group 2). 9. Effect of AT1011 and AT1011+AT1014 co-injection on the survival of treated animals [271] Survival time of all 6 study groups were analyzed using Graphpad Prism 9.4.1. as shown in Fig.18 with medium survival time (MS) calculated and indicated for each group. Intratumoral injection of AT1011_1.8mg, AT1011_0.9mg+AT1014_0.9mg and AT1011_0.45mg+AT1014_0.45mg resulted in extended median survival time from 31.5 days of the baseline treatment group to 39.5, 40 and 51.5 days, respectively, with the changes in Group 5 being significant (p=0.016*). [272] Fig. 18: Co-injection of AT1011 and poly IC improved survival of treated animals in comparison to base line treatment RT+anti-PD1 10. Tolerability and Mortality [273] Although some mice showed slight body weight loss (BWL) several days after radiation (Fig.19), they recovered within 2 weeks. One mouse in Group 5 was found dead on day 6 after randomization therefore are not included in the study. Since other mice in the study are not showing death or lethargy during the study, we consider the death of this mouse not treatment related. These data suggest that the treatment of RT+anti-PD1 (i.p.) +ACNP (i.t.) are well tolerated. [274] Fig.19 shows mean body weight change percentages with SEM. Example 44: Extension Tumor rechallenge of mice which showed complete response to ACNP intratumoral treatments when combining with RT+anti-PD1 [275] The objective of this study was to evaluate the tumor specific immunity inducing potential of AT1011 and AT1011+AT1014 in mice that were cured with these reagents when combined with RT+anti-PD1. ACNP cured mice were resistant to CT26.WT cell rechallenge while susceptible to EMT6 rechallenge. The data indicated that AT1011_1.8mg, AT1011_0.9mg+AT1014_0.9mg and AT1011_0.45mg+AT1014_0.45mg, when combined with RT+anti-PD1 were able to induce tumor specific immunity which protected them from rechange of the same tumor cells. Materials and methods 1. Animal and model 1.1 Animal information Species Mus musculus

No. of Animals Assigned to 48 Study 1.2 Ho

Cage Type Polysulfone IVC cage (325mm × 210mm × 180mm) Environment Autoclaved tissue paper, cardboard cylinder, chew stick, club

2. Tumor implantation and randomization [276] The CT26 WT tumor cells were maintained in vitro with RPMI1640 medium supplemented with 10% fetal bovine serum at 37°C in an atmosphere of 5% CO₂ in air. The cells in exponential growth phase were harvested and quantitated by cell counter before tumor inoculation. [277] The EMT6 tumor cells were maintained in vitro with DMEM medium supplemented with 10% fetal bovine serum at 37ºC in an atmosphere of 5% CO₂ in air. The cells in exponential growth phase were harvested and quantitated by cell counter before tumor inoculation. [278] 6 tumor free mice which were tumor free 70 days after the treatment described in study #2, and 8 age matched tumor naïve balb/c mice (18-20 wks) were inoculated subcutaneously in the right front flank region with CT26 WT tumor cells (5x 10⁵) and EMT6 tumor cells (5x 10⁵) in 0.1 ml of PBS for tumor development. Animals were randomized into 4 study groups on the day of inoculation as shown in Table 27. Table 27. Study groups in CT26-WT and EMT6 rechallenge study Group Mouse ID Previous treatment in Study #2 Rechallenge T T T T T T T

3. Observation and Data Collection [279] After tumor cells inoculation, the animals were checked daily for morbidity and mortality. During routine monitoring, the animals were checked for any effects of tumor growth and treatments on behavior such as mobility, food and water consumption, body weight gain/loss (Body weights were measured three times per week after randomization), eye/hair matting and any other abnormalities. Mortality and observed clinical signs were recorded for individual animals in detail. [280] Tumor volumes of both left and right tumors were measured three times per week after randomization in two dimensions using a caliper, and the volume was expressed in mm3 using the formula: V = (L x W x W)/2, where V is tumor volume, L was tumor length (the longest tumor dimension) and W is tumor width (the longest tumor dimension perpendicular to L). Dosing as well as tumor and body weight measurements were conducted in a Laminar Flow Cabinet. The body weights and tumor volumes were reported by using StudyDirectorTM software (version 3.1.399.19). 4. Humane endpoints and toxicity [281] The individual mouse was euthanized if its tumor volume exceeds 3000 mm³ or if they lose over 20% of their body weight relative to the weight on the first day of treatment. [282] Any mouse exhibiting an ulcerated or necrotic tumor was separated immediately and singly housed and monitored daily before the animal was euthanized or until tumor regression was completed. The mouse was euthanized rapidly if tumor ulcerates, and the ulceration diameter was greater than 5 mm, or pus or necrosis observed, or if tumor burden, including metastasis, compromises animal’s normal physiologic performances, e.g., orientation, access to food or water, etc. Results [283] As shown in Fig.20a and Fig.20b, all three cured mice re-challenged with CT26-WT cells (Group 1) were able to resist same cell line (CT26.WT) re-challenge while mice from the same treatment groups are still susceptible to the rechallenge of a different cell line (EMT6 rechallenge, Group 2). All previously tumor naïve mice are susceptible to rechallenge of both cell lines (Group 3 and Group 4). These data indicated that AT1011_1.8mg, AT1011_0.9mg+AT1014_0.9mg and AT1011_0.45mg+AT1014_0.45mg, when combined with RT+anti-PD1 were able to induce tumor specific immunity which protected them from same tumor re-challenge. [284] Fig.20 shows CT26-WT and EMT6 tumor growth in previously cured mice and tumor naive mice. [285] Fig.20a shows overall tumor growth of 4 study groups (mean tumor volume and SEM). [286] Fig.20b shows individual tumor growth in each study group. Example 45 Exploratory toxicity study of ACNPs in Sprague Dawley rats [287] The purpose of this toxicity study is to determine the potential toxicity of the test articles, 50 mpk AT1014, 200 mpk AT1014, 50 mpk AT1011, 200 mpk AT1011, 50 mpk FDC (AT1019) and 200 mpk FDC (AT1019), 1 day and 7 days post single dose via subcutaneous (sc) injection to Sprague Dawley (SD) rats. This study was conducted by HDB at its facility. The study was conducted at AAALAC accredited facility, and all animal study procedures have been approved by the Institutional Animal Care and Use Committee (IACUC) of HDB. [288] The test compounds were provided by sponsors as shown in Table 28. Table 28 Sample label Test article Concentration (mg/mL) Volume (mL) AR20230330-G1 Vehicle NA 31.2

[289] SD rats (M/F, 6~8 weeks old) were purchased from Zhejiang Vital River Laboratory Animal Technology Co. Ltd (License NO.: 91330482MA28BDKP5U). Animals were acclimatized prior to the experiment. Animals were kept under a standard condition with room temperature at 21-23 ºC, 40-70% relative humidity, and a 12/12h light: dark cycle. Chow and water are available ad libitum. All the in vivo experimental procedures were approved by the institutional animal care and use committee (IACUC) at HDB. All euthanasia was performed using carbon dioxide inhalation and all efforts were made to minimize animal suffering. The AUF number for this study at HDB is 118. [290] Group information is listed in Table 29. A total of 112 animals were used for this study. Table Error! No text of specified style in document.9 D^ose Dose Dose Termination

^{G1 SD} 1.25 8^{M/8F AR20230330-G1 NA s.c.} 2, 8 mL/kg × 4 ¹ i_l h lf h lf

Blood Sample Collection and Hematology Analysis [291] Blood samples were collected for comprehensive hematology analysis using tubes that had been pre-treated with EDTA-K2 (KANG JIAN, Cat#KJ202). The analysis aimed to evaluate a broad spectrum of hematological parameters, including: White Blood Cell Count (WBC) Red Blood Cell Count (RBC) Hemoglobin (HGB) Hematocrit (HCT) Mean Corpuscular Volume (MCV) Mean Corpuscular Hemoglobin Concentration (MCHC) Platelet Count (PLT) Percentage of Neutrophils (NEUT%) Percentage of Lymphocytes (LYMPH%) Percentage of Monocytes (MONO%) Percentage of Eosinophils (EO%) Percentage of Basophils (BASO%) Absolute Neutrophil Count (NEUT#) Absolute Lymphocyte Count (LYMPH#) Absolute Monocyte Count (MONO#) Absolute Eosinophil Count (EO#) Absolute Basophil Count (BASO#) Red Cell Distribution Width - Standard Deviation (RDW-SD) Red Cell Distribution Width - Coefficient of Variation (RDW-CV) Platelet Distribution Width (PDW) Mean Platelet Volume (MPV) Platelet-Large Cell Ratio (P-LCR) Plateletcrit (PCT) Reticulocyte Percentage (RET%) Immature Reticulocyte Fraction (IRF) Low Fluorescence Ratio (LFR) Medium Fluorescence Ratio (MFR) High Fluorescence Ratio (HFR) Major Organ Collection and Organ Index Analysis [292] Animals at their respective end points were euthanized by CO₂. Gross pathology should be assessed in all animals at termination and in all animals dying during the study period. Necropsies included inspection for gross lesions as well as collection and weighing of critical organs (brain, heart, kidney, liver, lungs, spleen, adrenal glands, thymus, testis, epididymites, ovaries and uterus) plus any visible lesion tissues/organs. The collected tissues/ organs were fixed in 10% neutral buffered formalin (NBF) or Davidson’ fixative for potential pathological examination. Testis were fixed in Davidson’s buffer. Organ index was calculated by the weight of specific organ divided by body weight. Data Analysis [293] In vivo animal data were expressed as mean±SEM. Statistical analysis was performed using a One Way ANOVA followed by Dunnett’s multiple comparison or a Two Way ANOVA followed by Bonferroni’s multiple comparison. Nonparametric tests like Mann-Whitney were used when the N was too small, or data did not follow Gaussian distribution. The difference was considered significant when p< 0.05. Summary [294] In this report, the potential toxicity of the test articles, 50 mpk AT1014, 200 mpk AT1014, 50 mpk AT1011, 200 mpk AT1011, 50 mpk FDC and 200 mpk FDC, was determined 1 day and 7 days post single dose via subcutaneous (sc) injection to Sprague Dawley (SD) rats. [295] For clinical observation: all the animals survived and maintained in good condition during the whole study period. [296] For body weight change: there was no significant difference in G2-G7 compared with G1 (vehicle group). [297] For representative major organ weight index: a significant female-tp7d liver weight index increase was found in G3 (AT1014, 200 mpk) and G5 (AT1011, 200 mpk) compared with G1 (vehicle group). Example 46: Integrated safety study of ACNPs in a bilateral CT26 tumor model [298] The integrated safety of ACNPs was investigated in a bilateral CT26 tumor model. The mice were treated with anti-PD-1 antibody through IP injection on day 0 and 3. The primary tumor received irradiation on day 0, 1 and 2 and treated with ACNPs through intratumor injection on day 2, 3 and 4. The tumor sizes of both treated tumor and untreated tumor were monitored to investigate the tumor growth inhibition effects. When the mice were dead during the study or the tumor size was larger than 2000 mm³, gross necropsy was performed to investigate the potential safety issues. The body weights were also monitored during the study. Table 30. Sample information in the integrated toxicity study.

[299] Group 1 was the control group treated with PBS and without anti-PD1 and irradiation; Group 2 was the baseline treatment group treated with anti-PD1 and irradiation; Group 3 was treated with anti-PD1 + irradiation + AT1011 (PLGA-PEG-Mal) at 2 mg/mouse; Group 4 was treated with anti-PD1 + irradiation + AT1014 (PLGA) at 2 mg/mouse; Group 5 was treated with anti-PD1 + irradiation + AT1011 (PLGA-PEG-Mal) at 1 mg/mouse + AT1014 (PLGA) at 1 mg/mouse; Group 6 was treated with anti-PD1 + irradiation + AT1011 (PLGA-PEG-Mal) at 0.1 mg/mouse + AT1014 (PLGA) at 0.1 mg/mouse. [300] Body weights in Group 3-6 were comparable to Group 2. Fig. 21. shows the changes of body weight in the integrated safety study. Necropsy results [301] The necropsy results were summarized in the attached Excel file. The main abnormal events found in all the groups were shown in Table 31. Tabel 31. The main abnormal events in all the groups Events N=43 (%) 8 5

Uterine horn: discolored 21 48.8 Liver: pale 15 34.9 3 6 [302] The total numb

erage number of abnormal events per mouse in each group are summarized in Table 32. Compared to Group 1 (control group), Group 2 (baseline treatment) increased the average number of abnormal events per mouse from 1.9 to 4.5. While Group 3-6 showed lower average numbers of abnormal events to Group 2. Table 32. The total number and the average number of abnormal events in each group # Treatment Total Number of Average Number of Adverse Event Adverse Event

Example 47: Efficacy study in a bilateral CT26 tumor model 1. Materials and Methods 1.1 Cell Culture [303] Murine colorectal carcinoma CT26 WT cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum at 37 ºC in an atmosphere of 5% CO₂. The cells in exponential growth phase were harvested and quantitated by cell counter before tumor inoculation. 1.2 Tumor Inoculation and Animal Randomization [304] Six- to eight-week-old female Balb/c mice (Shanghai Lingchang Bio-Technology.co., LTD) were used in this study. CT26 WT cancer cells (5 x 10⁵) in 0.1 mL of PBS were subcutaneously injected to the left lower flank of the mice (i.e. primary tumors). After 3 days, the same number of CT26 WT cells were injected to the right lower flank (i.e. secondary tumors). [305] The randomization was performed when the mean tumor size of the right tumors reached approximately 30mm³ (30-40mm³).48 mice were selected and enrolled in the study. The criteria for the mouse selection are shown below: [306] Step 1: Select mice with primary tumors of 80-120mm³, then check to see if there are 48 mice among them with secondary tumors of 30-40mm³. If not, go to the next step. [307] Step 2: Increase primary tumor size range to 75-130mm³ and see if there are enough mice with secondary tumor within 30-40mm³. If not, go to the next step. [308] Step 3: Select mice with primary tumors of 80-120mm³, then check to see if there are 48 mice among them with secondary tumors of 25-45mm³. If not, go to the next step. [309] Step 4: Increase primary tumor size range to 75-130mm³ and see if there are enough mice with secondary tumor within 25-45mm³. [310] All animals that met the selection criteria were randomly allocated to six study groups (n = 8 per group). Randomization was performed using “Matched distribution” method (StudyDirectorTM software, version 3.1.399.19) and was based on both the right tumors and left tumors. The date of randomization was denoted as day 0. 1.3. Animal Treatment [311] Fig. 22 shows the treatment schedule. Group 1 received PBS through intraperitoneally (i.p.) injection on days 0 and 3 and vehicle through intratumorally (i.t.) injection at left tumors on days 2, 3 and 4. For group 2, the left tumors were irradiated with 8 Gy on days 0, 1 and 2 using SmART-Precision X-Ray IGRT (Precision X-Ray Inc.). In addition, the mice were treated with anti- PD-1 antibody (5 mg/kg) through Intraperitoneally (i.p.) injection on days 0 and 3 and vehicle through intratumorally (i.t.) injection at left tumors on days 2, 3 and 4. These two groups served as control groups. For group 3-6, all the mice received irritation, anti-PD-1 antibody and ACNPs (AT1019). The left tumors (primary tumors) were irradiated with 8 Gy on days 0, 1 and 2. The anti-PD-1 antibody (5 mg/kg) was intraperitoneally injected into the animals on days 0 and 3. ACNPs (AT1019) were intratumorally injected to the left tumors on days 2 ,3 and 4 at different doses (1 mg, 0.3 mg, 0.1 mg and 0.01 mg per mouse for group 3, 4, 5 and 6, respectively). [312] Fig.22 schematically depicts the treatment timelines for in vivo cancer immunotherapy experiments. 1.4. Observation and Data Collection [313] After tumor inoculation, the animals were checked daily for morbidity and mortality. During routine monitoring, the animals were checked for any effects of tumor growth and treatments on behavior such as mobility, food and water consumption, body weight gain/loss (body weights were measured three times per week after randomization), eye/hair matting and any other abnormalities. Mortality and observed clinical signs will be recorded for individual animals in detail. [314] Tumor volumes were measured three times per week after randomization in two dimensions using a caliper, and the volume will be expressed in mm³ using the formula: V = (L x W x W)/2, where V is tumor volume, L is tumor length (the longest tumor dimension), and W is tumor width (the longest tumor dimension perpendicular to L). 2. Results [315] The abscopal effect of ACNPs (AT1019) at different dose levels were evaluated by measuring the tumor volume of right tumors (secondary tumors) over time. It was shown that AT1019 at all four dose levels combined with radiation and anti-PD-1 antibody significantly inhibited the secondary tumor growth compared to vehicle plus radiation and anti-PD-1 antibody (Fig.20A and 20B). On day 21, groups 3-6 showed strong tumor growth inhibition (TGI) effects to the control group (group 2) (Table 33). Impressively, there were 3 out of 8 (37.5 %), 2 out of 8 (25 %), 4 out of 8 (50 %) and 3 out of 8 (37.5 %) mice had right tumor completely cured (Fig.20A). Additionally, groups that received the treatment of AT1019 at various dose levels showed significant survival benefits compared to the control groups (Fig.23). Groups 3-6 had significantly longer median survival time to group 2 (Table 33). [316] Fig.23: ACNPs improved immunotherapy and the abscopal effect in CT26 WT xenografts. (A) Growth curves of irradiated (primary) and unirradiated (secondary) tumors in individual mice treated with immunotherapy and AT1019 at various dose levels; (B) Average tumor-growth curves of unirradiated (secondary) tumors in the mice treated in (A); (C) Survival curves of the mice in various groups. Data represent mean ± s.e.m. Differences in survival were determined for each group by the Kaplan–Meier method and the overall p value was calculated by the log- rank test. *p < 0.05, vs group 2 (RT+anti-PD-1). Table 33. Summaries of TGI on day 21 and number of complete response mice, number of survival mice and median survival time by day 61. Abscopal% TGI #Complete Median Group Treatment compared to G2 Response # Survival Survival (n=8 D61)

[317] The body weights of mice were monitored to evaluate the potential toxicity of ACNPs. Compared to the control groups, all the other groups that received ACNPs did not show significant changes in body weight (Fig.24). In addition, no clinical signs were observed for all the mice in this study. These results indicated that ACNPs were well tolerated in mice. Fig.24: Body weight changes over time for various groups. Example 48: CT26-WT and EMT6 rechallenge Study in ACNP treated tumor-free mice [318] Cured mice (tumor-free) from example above were rechallenged with CT26-WT or EMT6 cancer cells to evaluate the tumor growth. 1. Animal housing Ca e T e Pol sulfone IVC ca e (325mm × 210mm × 180mm;)

Water 0.2 ^m filtered, reverse osmosis (RO) water, autoclaved er

. Ulceration Cancer Cachectic Observed in

3. Experiment design [319] Treatment plan of this study is shown in Table 34 below. Table 34. Treatment Plan for Re-challenge Study Group Mouse ID Rechallenge Duration

1 5169 CT26-WT TBD 2 5221 EMT6 TBD

4. Experiment methods 4.1 Cell Culture [320] The CT26 WT tumor cells were maintained in vitro with RPMI1640 medium supplemented with 10% fetal bovine serum at 37ºC in an atmosphere of 5% CO₂ in air. The cells in exponential growth phase were harvested and quantitated by cell counter before tumor inoculation. The EMT6 tumor cells were maintained in vitro with DMEM medium supplemented with 10% fetal bovine serum at 37ºC in an atmosphere of 5% CO₂ in air. The cells in exponential growth phase were harvested and quantitated by cell counter before tumor inoculation. 4.2 Tumor Inoculation [321] Each mouse was inoculated subcutaneously in the right front flank region with CT26 WT tumor cells (5x 10⁵) and EMT6 tumor cells (5x 10⁵) in 0.1 ml of PBS for tumor development. 4.3 Randomization [322] The randomization started at the beginning of the study. No randomization was needed for the tumor-free mice from example 47. Those mice were inoculated according to Table 34. The 8 control mice will be randomly allocated to 2 study groups as shown in Table 34. The day of randomization was denoted as day 0. 4.4 Observation and Data Collection [323] After tumor inoculation, the animals were checked daily for morbidity and mortality. During routine monitoring, the animals were checked for any effects of tumor growth and treatments on behavior such as mobility, food and water consumption, body weight gain/loss (Body weights will be measured two times per week after randomization), eye/hair matting and any other abnormalities. Mortality and observed clinical signs were recorded for individual animals in detail. [324] Tumor volumes were measured two times per week after randomization in two dimensions using a caliper, and the volume was expressed in mm³ using the formula: “V = (L x W x W)/2, where V is tumor volume, L is tumor length (the longest tumor dimension) and W is tumor width (the longest tumor dimension perpendicular to L). Dosing as well as tumor and body weight measurements were conducted in a Laminar Flow Cabinet. [325] The body weights and tumor volumes were measured by using StudyDirector^TM software (version 3.1.399.19). [326] Fig.25 shows the tumor growth curves. And results were summarized below: Group 1: Eight cured mice received CT26 cells; no tumors for all mice. Group 2: Four cured mice received EMT26 cells; two mice have tumors while the other two do not have tumors. Group 3: Four healthy mice received CT26 cells; all have tumor grow up. Group 4: Four healthy mice received EMT26 cells; all have tumor grow up. [327] Mice cured by ACNP treatment in example 47 showed no tumor growth in all eight mice challenged by CT26 cells indicating strong immune protection against the same type of tumor.

Claims

What is claimed is: 1. A nanoparticle comprising a core, wherein the core comprises Polymer-X-J, in which X is PEG', linker, PEG’-Linker, or absent; J is a reactive group or absent; and when J is a reactive group, a portion or all of J is optionally covalently bound to PEP-PEG" in which PEP is a protease sensitive protein sequence, and the Polymer comprises poly(lactic-co-glycolic acid) (“PLGA”) and the ratio of lactic acid-to-glycolic acid (“LA:GA”) in the Polymer is about 25:75, about 50:50, or about 75: 25 (weight/weight).

2. The nanoparticle of claim 1, wherein the ratio of lactic acid-to-glycolic acid (“LA:GA”) is about 75:25.

3. The nanoparticle of claim 1, wherein molecular weight of the Polymer is in a range of 10- 100 KDa, 30-70 KDa, or 40-65 KDa.

4. The nanoparticle of claim 3, wherein molecular weight of the Polymer is in a range of 55- 65KDa, 42-62KDa or 45-55KDa.

5. The nanoparticle of claim 1, wherein the nanoparticle is an antigen-capturing nanoparticle (ACNP).

6. The nanoparticle of claim 1, wherein the nanoparticle is free from any antigen.

7. The nanoparticle of claim 1, wherein J is maleimide (“Mal”).

8. The nanoparticle of claim 1, wherein the core of the nanoparticle comprises PLGA-X- Maleimide

9. The nanoparticle of claim 1, wherein J is absent.

10. The nanoparticle of claim 1, wherein the core of the nanoparticle comprises PLGA or PLGA-X.

11. The nanoparticle of claim 1, wherein the nanoparticle comprises a mixture of PLGA-X-J and PLGA-X at a mass ratio of 0.05: 1 to 1:0.05, wherein X is PEG', linker, PEG’-Linker, or absent, and a portion or all of J is optionally covalently bound to PEP-PEG" in which PEP is a protease sensitive protein sequence.

12. The nanoparticle of claim 11, wherein the nanoparticle comprises a mixture of PLGA-X- Maleimide and PLGA at a mass ratio of 0.05: 1 to 1:0.05, wherein maleimide is optionally covalently bound to PEP-PEG" in which PEP is a protease sensitive protein sequence.

13. The nanoparticle of claim 12, wherein the mass ratio of PLGA-X-Maleimide to PLGA is 1:1.

14. The nanoparticle of claim 1, wherein PEP is capable of being cleaved by Caspase, Cathepsin, or MMP2.

15. The nanoparticle of claim 1, further comprising an adjuvant.

16. The nanoparticle of claim 15, wherein the adjuvant is a small molecule, a double-stranded RNA molecule, or a single-stranded DNA molecule.

17. The nanoparticle of claim 16, wherein the small molecule is imiquimod, resiquimod, or gardiquimod.

18. The nanoparticle of claim 16, wherein the double-stranded RNA molecule is poly(inosinic- cytidylic acid) (“Poly IC”), poly IC, Riboxxol RGI®50, poly IC mixed with the stabilizers, poly ICLC, complexes between poly IC and poly(ethylene imine) (“PEI”), or PEI.

19. The nanoparticle of claim 16, wherein the single-stranded DNA molecule is CpG oligodeoxynucleotides (CpG ODN).

20. The nanoparticle of claim 16, wherein the adjuvant is poly IC, PEI or poly IC/PEI.

21. The nanoparticle of claim 1, wherein the nanoparticle is optionally lyophilized by a process comprising freeze-drying or spray-drying in the presence of a lyoprotectant.

22. The nanoparticle of claim 21, wherein the lyoprotectant is a buffer, sugar molecule, polymer, or a mixture thereof.

23. The nanoparticle of claim 22, wherein the lyoprotectant is HEPES buffered saline (“HBS”), mannose, sucrose, trehalose, mannitol, poly(ethylene glycol) (“PEG”), poly(ethyleneimines) (“PEI”), Poly(vinyl alcohol) (“PVA”), or a mixture thereof.

24. The nanoparticle of claim 23, wherein the lyoprotectant is HEPES buffered saline (“HBS”), sucrose, PVA or a mixture thereof.

25. A pharmaceutical formulation comprising the nanoparticle of any one of claims 1-24 and a pharmaceutically acceptable carrier and/or excipient thereof.

26. The pharmaceutical formulation of claim 25, further comprising Poly(vinyl alcohol) (“PVA”) associated with the nanoparticles.

27. The pharmaceutical formulation of claim 26, wherein the mass ratio of the associated PVA to the core of the nanoparticle is 20-70% or 30-50%.

28. The pharmaceutical formulation of claim 25, wherein the formulation further comprises a buffer with pH range of 7-8.

29. The pharmaceutical formulation of claim 28, wherein the buffer is HEPES buffer at pH7.4 or HEPES buffered saline (HBS) at pH 7.4.

30. A method for enhancing effectiveness of cancer treatment in a subject in need thereof, comprising administering to the subject the nanoparticle of any one of claims 1-24 or the pharmaceutical composition of any one of claims 25-29.

31. The method of claim 30, wherein the composition is administrated after a previous treatment of the subject with an anti-cancer therapy.

32. The method of claim 31, wherein the previous treatment is radiation.

33. The method of any one of claims 30-32, wherein the composition is used in combination with a second therapeutic agent.

34. The method of claim 33, wherein the second therapeutic agent is an immune checkpoint inhibitor.

35. The method of claim 34, wherein the immune checkpoint inhibitor is PD-1 antibody.

36. The method of claim 30, wherein the cancer is brain cancer, non-small cell lung cancer, small cell lung cancer, esophageal cancer, gastric cancer, pancreatic cancer, colorectal cancer, renal cell carcinoma, bladder cancer, prostate cancer, breast cancer, non- hodgkins lymphoma, hodgkin's lymphoma, anal cancer, head and neck cancer, or melanoma.