Engineering antigen-specific t cells for car t cell therapy by antigen-presenting lipid nanoparticles

Abstract

Antigen-presenting nanoparticles and methods of engineering CAR T cells and treating and / or preventing cancer using the same.

Description

ENGINEERING ANTIGEN- SPECIFIC T CELLS FOR CAR T CELL THERAPY BY ANTIGEN-PRESENTING LIPID NANOPARTICLESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 589,821 filed October 12, 2023, which is incorporated by reference herein in its entirety.GOVERNMENT SUPPORT CLAUSE

[0002] This invention was made with government support under Grant No. CA260247 and CA276890 awarded by the National Institutes of Health. The Government has certain rights in the invention.BACKGROUND

[0003] In the wake of the first two FDA-approved aCD19 CAR T-cell therapies, KYMRIAH™ (Novartis) and YESCARTA™ (Kite), the first-in-class BCMA-directed CAR T cell therapy (Abecma, Bristol Meyers Squibb) recently received FDA approval for multiple myeloma (MM). However, the high price ($350K-450K per treatment), long lead time (3-4 weeks), and complex ex vivo manufacturing procedures remain major obstacles for implementing CAR T cell therapy as standard-of-care for cancer treatment. The multi-step manufacturing process includes cell isolation, stimulation, genetic modification, expansion, characterization, and infusion. Emerging strategies to solve these challenges include in vivo engineering of circulating T cells, using viral vectors or polymeric / lipid nanoparticles functionalized with pan-T cell antibodies to deliver plasmid DNA or mRNA encoding CAR transgenes. While promising, the limited specificity of these approaches can lead to transfection of autoimmune and regulatory T cells, which can increase off-target toxicity, break self-tolerance and reduce treatment efficacy.SUMMARY

[0004] In one aspect, disclosed herein are antigen-presenting nanoparticles (APN) (including, but not limited to a lipid nanoparticle, a liposome, or a polymeric nanoparticle), comprising: a major histocompatibility complex (MHC) (such as, for example an MHC class I or MHC class II molecule) presented on a surface of the APN and comprising a peptide epitope (such as, for example an epitope of a virus, bacteria, fungus, or parasite); and an encapsulatednucleic acid encoding a chimeric antigen receptor (CAR), antibody, or antibody fragment (such as for example, a ScFv or VHH also known as a nanobody). In some aspects, the CAR, antibody, or antibody fragment encoded by the encapsulated nucleic acid recognizes a cancer or tumor antigen (such as, for example, a B cell maturation antigen (BCMA), G protein-coupled receptor class C group 5 member D (GPRC5D), CD 19, guanylate cyclase-C (GUCY2C), human epidermal growth factor receptor 2 (HER2), CD20, CD22, mucin- 1 (MUC1), mesothelin, or VHH (e.g., a sequence comprising 80% similarity or more to SEQ ID NO: 8)). In some aspects, the CAR encoded by the encapsulated nucleic acid comprises 80% similarity or more to SEQ ID NO: 2 or SEQ ID NO: 5.

[0005] Also disclosed herein are APN of any preceding aspect, wherein the peptide epitope is a viral epitope from a virus selected from the group consisting of Herpes Simplex virus-1, Herpes Simplex virus-2, Varicella-Zoster virus, Epstein-Barr virus, Cytomegalovirus, Human Herpes virus-6, Variola virus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus (such as, for example, a SARS-CoV-2 infection including, but not limited to the SARS-CoV-2 Bl.351 variant, SARS-CoV-2B.1.1.7 (alpha), SARS-CoV-2B.1.1.7 variant mutant N501Y (alpha), SARS-CoV-2 delta variant, SARS-CoV-2 P.l variant, SARS-CoV-2 with T487K, P681R, and L452R mutations in B.1.617.2 (Delta), SARS-CoV-2 with K417N mutation in AY.1 / AY.2 (Delta plus), SARS-CoV-2 with D614G, P681H, and D950N mutations in B.1.621 (Mu), SARS-CoV-2 with G75V, T76I, A246-252, L452Q, F490S, D614G, and T859N mutations in C.37 (Lambda), SARS-CoV-2 with T478K, Q498R, and H655Y mutations in B.1.1.529 (Omicron)), Influenza virus A, Influenza virus B, Measles virus, Polyomavirus, Human Papilomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Reovirus, Yellow fever virus, Zika virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, Rotavirus A, Rotavirus B, Rotavirus C, Sindbis virus, Simian Immunodeficiency virus, Human T-cell Leukemia virus type-1, Hantavirus, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type-1, and Human Immunodeficiency virus type-2.

[0006] In one aspect, disclosed herein are APN of any preceding aspect, wherein the peptide epitope is a bacterial epitope from a bacteria selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis strain BCG, BCG sub strains, Mycobacterium avium, Mycobacterium intracellular, Mycobacterium africanum,Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium ulcerans, Mycobacterium avium subspecies paratuberculosis, Nocardia asteroides, other Nocardia species, Legionella pneumophila, other Legionella species, Acetinobacter baumanii, Salmonella typhi, Salmonella enterica, other Salmonella species, Shigella boydii, Shigella dysenteriae, Shigella sonnei, Shigella flexneri, other Shigella species, Yersinia pestis, Pasteurella haemolytica, Pasteurella multocida, other Pasteurella species, Actinobacillus pleuropneumoniae, Listeria monocytogenes, Listeria ivanovii, Brucella abortus, other Brucella species, Cowdria ruminantium, Borrelia burgdorferi, Bordetella avium, Bordetella pertussis, Bordetella bronchiseptica, Bordetella trematum, Bordetella hinzii, Bordetella pteri, Bordetella parapertussis, Bordetella ansorpii other Bordetella species, Burkholderia mallei, Burkholderia psuedomallei, Burkholderia cepacian, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Coxiella burnetii, Rickettsial species, Ehrlichia species, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Escherichia coli, Vibrio cholerae, Campylobacter species, Neiserria meningitidis, Neiserria gonorrhea, Pseudomonas aeruginosa, other Pseudomonas species, Haemophilus influenzae, Haemophilus ducreyi, other Hemophilus species, Clostridium tetani, other Clostridium species, Yersinia enterolitica, and other Yersinia species.

[0007] Also disclosed herein are APN of any preceding aspect, wherein the peptide epitope is a fungal epitope from a fungi selected from the group consisting of Candida albicans, Cryptococcus neoformans, Histoplama capsulatum, Aspergillus fumigatus, Coccidiodes immitis, Paracoccidiodes brasiliensis, Blastomyces dermitidis, Pneumocystis camii, Penicillium marneffi, and Alternaria alternata.

[0008] In one aspect disclosed herein are APN of any preceding aspect, wherein the peptide epitope is a parasitic epitope from a parasite selected from the group consisting of Toxoplasma gondii, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, other Plasmodium species, Entamoeba histolytica, Naegleria fowleri, Rhinosporidium seeberi, Giardia lamblia, Enterobius vermicularis, Enterobius gregorii, Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus, Cryptosporidium spp., Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, other Leishmania species, Diphyllobothrium latum, Hymenolepis nana, Hymenolepis diminuta, Echinococcus granulosus, Echinococcus multilocularis, Echinococcus vogeli, Echinococcus oligarthrus, Diphyllobothrium latum, Clonorchis sinensis; Clonorchis viverrini, Fasciola hepatica, Fasciola gigantica, Dicrocoelium dendriticum, Fasciolopsis buski, Metagonimus yokogawai, Opisthorchis viverrini, Opisthorchis felineus, Clonorchis sinensis, Trichomonas vaginalis, Acanthamoeba species, Schistosoma intercalatum, Schistosomahaematobium, Schistosoma japonicum, Schistosoma mansoni, other Schistosoma species, Trichobilharzia regenti, Trichinella spiralis, Trichinella britovi, Trichinella nelsoni, Trichinella nativa, and Entamoeba histolytica.

[0009] Also disclosed herein are APN of any preceding aspect, wherein the APN is a lipid nanoparticle, and wherein the APN comprises at least one ionizable lipid (such as, for example, cKK-E12, SM102, MC3, Lipid-5, Alc-0315, LP01, Lipid A9, or any combination thereof), cholesterol, phospholipid (such as, for example, DSPC, DOPE, or any combination thereof), PEGylated lipid (such as, for example, ALC-0159, DMG-PEG, DSPE-PEG, PEG14-2000, or any combination thereof). In some aspects, the nanoparticle comprises from about 30 mol% to about 60 mol% of the ionizable lipid, from about 35 mol% to about 50 mol% of the cholesterol, from about 5 mol% to about 20 mol% of the phospholipid, and / or from about 0.5 mol% to about 5 mol% of the PEGylated lipid. In some aspects the ratio of PEGylated lipid to total lipids is from about 0.01 : 1 to about 0.05: 1.

[0010] In one aspect, disclosed herein are methods of engineering a CAR T cell, the method comprising exposing an antigen-specific T cell (including, but not limited to a human T cell) to the APN of any preceding aspect. For example, disclosed herein are methods of engineering a CAR T cell, the method comprising exposing an antigen-specific T cell to an antigen presenting nanoparticle (APN) (including, but not limited to a lipid nanoparticle, a liposome, or a polymeric nanoparticle), the APN comprising: a major histocompatibility complex (MHC) (such as, for example an MHC class I or MHC class II molecule) presented on a surface of the APN and comprising a peptide epitope (such as, for example an epitope of a virus, bacteria, fungus, or parasite); and an encapsulated nucleic acid encoding a chimeric antigen receptor (CAR), antibody, or antibody fragment (such as for example, a ScFv or VHH also known as a nanobody). In some aspects, the CAR, antibody, or antibody fragment encoded by the encapsulated nucleic acid recognizes a cancer or tumor antigen (such as, for example, a B cell maturation antigen (BCMA), G protein-coupled receptor class C group 5 member D (GPRC5D), CD 19, guanylate cyclase-C (GUCY2C), human epidermal growth factor receptor 2 (HER2), CD20, CD22, mucin-1 (MUC1), mesothelin, or VHH (e.g., a sequence comprising 80% similarity or more to SEQ ID NO: 8)).

[0011] Also disclosed herein are methods of engineering a CAR T cell of any preceding aspect, wherein the T cell is a CD8+ T cell, and wherein the MHC is MHC class I or a CD4+ T cell, and wherein the MHC is MHC class II.

[0012] In one aspect disclosed herein are methods of engineering a CAR T cell of any preceding aspect, wherein the method is carried out in vitro, in vivo, or ex vivo.

[0013] Also disclosed herein are CAR T cells produced by the method of any preceding aspect.

[0014] In one aspect, disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating and / or preventing a cancer, tumor, and / or metastasis (such as, for example, lymphoma, multiple myeloma, myeloid leukemia, colorectal cancer, breast cancer, giloblastoma, ovarian cancer, sarcoma, pancreatic cancer, mesothelioma, lung cancer, gastric cancer, or cervical cancer) in a subject, the method comprising administering to the subject the APN of any preceding aspect. For example, disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating and / or preventing a cancer, tumor, and / or metastasis (such as, for example, lymphoma, multiple myeloma, myeloid leukemia, colorectal cancer, breast cancer, giloblastoma, ovarian cancer, sarcoma, pancreatic cancer, mesothelioma, lung cancer, gastric cancer, or cervical cancer), the method comprising administering to the subject an antigen-presenting nanoparticle (APN) (including, but not limited to a lipid nanoparticle, a liposome, or a polymeric nanoparticle), comprising: a major histocompatibility complex (MHC) (such as, for example an MHC class I or MHC class II molecule) presented on a surface of the APN and comprising a peptide epitope (such as, for example an epitope of a virus, bacteria, fungus, or parasite); and an encapsulated nucleic acid encoding a chimeric antigen receptor (CAR), antibody, or antibody fragment (such as for example, a ScFv or VHH also known as a nanobody) that recognizes a cancer or tumor antigen (such as, for example, a B cell maturation antigen (BCMA), G protein-coupled receptor class C group 5 member D (GPRC5D), CD 19, guanylate cyclase-C (GUCY2C), human epidermal growth factor receptor 2 (HER2), CD20, CD22, mucin-1 (MUC1), mesothelin, or VHH (e.g., a sequence comprising 80% similarity or more to SEQ ID NO: 8)). In some aspects, the CAR encoded by the encapsulated nucleic acid comprises 80% similarity or more to SEQ ID NO: 2 or SEQ ID NO: 5. In some aspects, after administration of the APN, at least about 40% of the antigen-specific T cells express the CAR

[0015] Also disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating and / or preventing a cancer, tumor, and / or metastasis of any preceding aspect, further comprising administering to the subject a peptide pulse or vaccine to the subject prior to administering the APN; wherein the peptide comprises an epitope for a microbe to which the subject has been exposed; wherein the vaccine expresses the same epitope as the APN.

[0016] Also disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating and / or preventing a cancer, tumor, and / or metastasis of any preceding aspect, wherein the peptide epitope is a viral epitope from a virus selected from the group consistingof Herpes Simplex virus-1, Herpes Simplex virus-2, Varicella-Zoster virus, Epstein-Barr virus, Cytomegalovirus, Human Herpes virus-6, Variola virus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus (such as, for example, a SARS-CoV-2 infection including, but not limited to the SARS-CoV-2 Bl.351 variant, SARS-CoV-2B.1.1.7 (alpha), SARS-CoV-2B.1.1.7 variant mutant N501Y (alpha), SARS-CoV-2 delta variant, SARS-CoV-2 P. l variant, SARS-CoV-2 with T487K, P681R, and L452R mutations in B.1.617.2 (Delta), SARS-CoV-2 with K417N mutation in AY.1 / AY.2 (Delta plus), SARS-CoV-2 with D614G, P681H, and D950N mutations in B.1.621 (Mu), SARS-CoV-2 with G75V, T76I, A246-252, L452Q, F490S, D614G, and T859N mutations in C.37 (Lambda), SARS-CoV-2 with T478K, Q498R, and H655Y mutations in B.1.1.529 (Omicron)), Influenza virus A, Influenza virus B, Measles virus, Polyomavirus, Human Papilomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Reovirus, Yellow fever virus, Zika virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, Rotavirus A, Rotavirus B, Rotavirus C, Sindbis virus, Simian Immunodeficiency virus, Human T-cell Leukemia virus type-1, Hantavirus, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type-1, and Human Immunodeficiency virus type-2.

[0017] In one aspect, disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating and / or preventing a cancer, tumor, and / or metastasis of any preceding aspect, wherein the peptide epitope is a bacterial epitope from a bacteria selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis strain BCG, BCG substrains, Mycobacterium avium, Mycobacterium intracellular, Mycobacterium africanum, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium ulcerans, Mycobacterium avium subspecies paratuberculosis, Nocardia asteroides, other Nocardia species, Legionella pneumophila, other Legionella species, Acetinobacter baumanii, Salmonella typhi, Salmonella enterica, other Salmonella species, Shigella boydii, Shigella dysenteriae, Shigella sonnei, Shigella flexneri, other Shigella species, Yersinia pestis, Pasteurella haemolytica, Pasteurella multocida, other Pasteurella species, Actinobacillus pleuropneumoniae, Listeria monocytogenes, Listeria ivanovii, Brucella abortus, other Brucella species, Cowdria ruminantium, Borrelia burgdorferi, Bordetella avium, Bordetella pertussis, Bordetella bronchiseptica, Bordetella trematum, Bordetella hinzii, Bordetella pteri, Bordetella parapertussis, Bordetella ansorpii other Bordetella species,Burkholderia mallei, Burkholderia psuedomallei, Burkholderia cepacian, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Coxiella burnetii, Rickettsial species, Ehrlichia species, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Escherichia coli, Vibrio cholerae, Campylobacter species, Neiserria meningitidis, Neiserria gonorrhea, Pseudomonas aeruginosa, other Pseudomonas species, Haemophilus influenzae, Haemophilus ducreyi, other Hemophilus species, Clostridium tetani, other Clostridium species, Yersinia enterolitica, and other Yersinia species.

[0018] Also disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating and / or preventing a cancer, tumor, and / or metastasis of any preceding aspect, wherein the peptide epitope is a fungal epitope from a fungi selected from the group consisting of Candida albicans, Cryptococcus neoformans, Histoplama capsulatum, Aspergillus fumigatus, Coccidiodes immitis, Paracoccidiodes brasiliensis, Blastomyces dermitidis, Pneumocystis carnii, Penicillium marneffi, and Alternaria altemata.

[0019] In one aspect disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating and / or preventing a cancer, tumor, and / or metastasis of any preceding aspect, wherein the peptide epitope is a parasitic epitope from a parasite selected from the group consisting of Toxoplasma gondii, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, other Plasmodium species, Entamoeba histolytica, Naegleria fowleri, Rhinosporidium seeberi, Giardia lamblia, Enterobius vermicularis, Enterobius gregorii, Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus, Cryptosporidium spp., Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, other Leishmania species, Diphyllobothrium latum, Hymenolepis nana, Hymenolepis diminuta, Echinococcus granulosus, Echinococcus multilocularis, Echinococcus vogeli, Echinococcus oligarthrus, Diphyllobothrium latum, Clonorchis sinensis; Clonorchis viverrini, Fasciola hepatica, Fasciola gigantica, Dicrocoelium dendriticum, Fasciolopsis buski, Metagonimus yokogawai, Opisthorchis viverrini, Opisthorchis felineus, Clonorchis sinensis, Trichomonas vaginalis, Acanthamoeba species, Schistosoma intercalatum, Schistosoma haematobium, Schistosoma japonicum, Schistosoma mansoni, other Schistosoma species, Trichobilharzia regenti, Trichinella spiralis, Trichinella britovi, Trichinella nelsoni, Trichinella nativa, and Entamoeba histolytica.

[0020] Also disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating and / or preventing a cancer, tumor, and / or metastasis of any preceding aspect, wherein the APN is a lipid nanoparticle, and wherein the APN comprises at least one ionizablelipid (such as, for example, cKK-E12, SM102, MC3, Lipid-5, Alc-0315, LP01, Lipid A9, or any combination thereof), cholesterol, phospholipid (such as, for example, DSPC, DOPE, or any combination thereof), PEGylated lipid (such as, for example, ALC-0159, DMG-PEG, DSPE-PEG, PEG14-2000, or any combination thereof). In some aspects, the nanoparticle comprises from about 30 mol% to about 60 mol% of the ionizable lipid, from about 35 mol% to about 50 mol% of the cholesterol, from about 5 mol% to about 20 mol% of the phospholipid, and / or from about 0.5 mol% to about 5 mol% of the PEGylated lipid. In some aspects the ratio of PEGylated lipid to total lipids is from about 0.01 : 1 to about 0.05: 1.

[0021] In one aspect, disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating and / or preventing a cancer, tumor, and / or metastasis of any preceding aspect, wherein from about 0.1 mg / kg to about 5 mg / kg APN is administered. In some aspects, the APN is repeatedly administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times at an interval of about every 5 days.

[0022] Other systems, methods, features and / or advantages will be or may become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features and / or advantages be included within this description and be protected by the accompanying claims.BRIEF DESCRIPTION OF DRAWINGS

[0023] FIGURE 1 depicts bar graphs showing APNs specifically targeted to flu-specific T cells (flu tet+, flu tetramer stained cells), but not other CD8+ T cells present in the human PBMC.

[0024] FIGURE 2 depicts the transfection of mouse CD8+ T cells and human CD3+ T cells with firefly luciferase mRNA delivered by LNPs formulated by various ionizable lipids.

[0025] FIGURE 3 depicts in vitro transfection of human lAV-specific T cells by luciferase mRNA-loaded APNs.0.0001, two-way ANOVA and Si dak post-test.

[0026] FIGURES 4A-4D depict that APNs transfect human flu-specific T cells in NSG mice. FIG. 4A is an illustration of human flu-specific T cell expansion and adoptive transfer to NSG mice, which subsequently received APN injection to transfect flu-specific T cells with a model VHH mRNA. FIG. 4B shows the frequency of flu -specific T cells in splenocytes at 48 hours after adoptive transfer of flu peptide-pulsed PBMC. FIGS. 4C-4D show representative flow plots (FIG. 4C) and a bar graph (FIG. 4D) showing APNs transfected human flu-specific T cells with VHH model protein in spleen of NSG mice. N=4-5. ****p < 0.0001. NS: Not significant. Mean ± SD. One-way ANOVA.

[0027] FIGURES 5A-5C depict the validation of clinical grade BCMA CAR mRNA by electroporating primary human T cells. FIG. 5A shows a human BCMA CAR construct. FIG. 5B shows BCMA CAR expression on primary human T cells. Cells were stained by fluorophore-labelled BCMA proteins. FIG. 5C shows cytotoxicity induced by BCMA CAR T cells (effector) after incubation with BCMA-expressing MM.1R cancer cells (target). Mock: Mock electroporated T cells. N=4. ****p < 0.0001. Mean ± SD. Two-way AN0VA & Tukey’s test.

[0028] FIGURES 6A-6E depict that APNs transfect human lAV-specific T cells with aBCMA CAR mRNA in NSG mice. FIG. 6A shows the workflow for validating anti-cancer activity of in vivo programed BCMA CAR using ex vivo cytotoxicity assay by quantifying cancer cell death using bioluminescence. FIG. 6B shows flow plots showing dose-dependent transfection of aBCMA CAR mRNA delivered by APNs. FIGS. 6C-6D show representative flow plots (FIG. 6C) and a bar graph (FIG. 6D) showing APNs transfected human IAV- specific T cells with aBCMA CAR mRNA in spleen of NSG mice. N=4-5. FIG. 6E shows ex vivo cytotoxicity assay showing CD8 T cells isolated from NSG mice treated with aBCMA CAR APNs demonstrated significantly higher cytotoxicity against cMM.1R multiple myeloma cell line. **P < 0.01. ****p < 0.0001. NS: Not significant. Mean ± SD. One-way ANOVA.

[0029] FIGURES 7A-7B depict that a single dose of aBCMA CAR APNs resulted in tumor regression in NSG mice bearing BCMA+ U266 tumor. FIG. 7A sows NSG mice were systemically inoculated with U266 MM cells. At 11 days after tumor inoculation, the mice received lAV-specific T cells, followed by intravenous injection of APNs at 24 hours after the T cell transfer. FIG. 7B shows in vivo luminescence imaging time course by an IVIS Spectrum CT system representative of U266 tumor burden. ****P<0.0001. Mean ± SD. N=3. Two-way ANOVA and Sidak test.

[0030] FIGURES 8A-8E depict that APNs transfect human lAV-specific T cells with aBCMA CAR mRNA in vitro. FIG. 8A is an illustration of human lAV-specific T cell transfection by APNs, followed by co-incubation with human multiple myeloma (MM) cancer cells, MM1R, to evaluate the effector functions of CAR T cells. FIG. 8B shows APNs transfected lAV-specific T cells with nano-luciferase (nLuc) that generated bioluminescence in the presence of nLuc substrate. N=3. FIGS. 8C-8D show representative flow plots (FIG. 8C) and bar graph (FIG. 8D) showing APNs transfected human lAV-specific T cells with aBCMA CAR mRNA in vitro. N=2 FIG. 8E shows an in vitro assay comparing cytotoxicity of lAV-specific T cells transfected by nLuc or CAR APNs against MM1R MM cells. T cellswere co-cultured with MM cells at a 2: 1 ratio. N=3. ****p < 0.0001. NS: Not significant. Mean ± SD. One-way ANOVA.

[0031] FIGURES 9A-9D depict validation of human GPRC5D CAR mRNA by electroporating primary human T cells. FIG. 9A shows a human GPRC5D CAR construct. FIG. 9B shows GPRC5D CAR expression on primary human T cells. Cells were stained by fluorophore-labelled anti-strep tag. FIG. 9C shows cytotoxicity induced by electroporated T cells after incubation with GPRC5D-expressing MM.1R cancer cells. Mock: Mock electroporated T cells. GFP: GFP mRNA-electroporated T cells. GPRC5D: GPRC5D mRNA- electroporated T cells. FIG. 9D shows IFNy concentration in the supernatant of T cell-MMIR cancer cell culture. N=3. ****p < 0.0001. Mean ± SD. Two-way ANOVA & Tukey’s test.

[0032] FIGURES 10A-10B depict that APNs deliver BCMA CAR and GPRC5D CAR to virus-specific T cells enriched from patients with multiple myeloma. FIG. 10A is a schematic showing two different APNs, HLA / Flu BCMA CAR APN and HLA / CMV GPRC5D CAR APN, designed to transfect Flu-specific T cells and CMV-specific T cells with BCMA CAR and GPRC5D CAR, respectively. FIG. 10B shows that a mixture of flu-specific T cells and CMV-specific T cells was transfected with either HLA / Flu BCMA CAR APN, HLA / CMV GPRC5D CAR APN, or a combination of these two APNs. The CAR transfection was evaluated by fluorescent staining of strep-tag expression as a surrogate transfection marker. N=3. ****p < 0.0001. Mean ± SD. Two-way ANOVA & Tukey’s test.

[0033] FIGURES 11A-11C depict that intravenous injections of aBCMA CAR APNs resulted in tumor regression in NSG mice bearing systemic BCMA+ U266 tumor. FIG. HA shows that NSG mice were systemically inoculated with 2 million U266 MM cells. At 6 days after tumor inoculation, the mice received lAV-specific T cells, followed by intravenous injection of APNs at 7, 12, 17, 22, and 27 days after tumor inoculation. A total of 5 doses of APNs were given to mice. FIG. 11B shows representative luminescence images of U266 tumor-bearing mice on day 10, 20, and 30. Signal indicate luciferase activity as an indicator of tumor burden. FIG. 11C shows in vivo luminescence imaging time course by an IVIS Spectrum CT system representative of U266 tumor burden. ****P<0.0001. Mean ± SD. N=5-6. Two- way ANOVA and Sidak test.

[0034] FIGURES 12A-12B depict that human monocyte-derived dendritic cell (mDC) vaccination expanded lAV-specific T cells in vitro and enhanced the anticancer efficacy of in vivo CAR. FIG. 12A shows representative flow plots showing that after 7 and 14 days coincubation, peptide pulsed mDCs expanded lAV-specific T cells compared to the untreated PBMC control. FIG. 12B shows a treatment timeline and in vivo luminescence imaging timecourse by an IVIS Spectrum CT system representative of U266 tumor burden. The black arrow indicates the APN injection day (D14). *P<0.05. Mean ± SD. N=6. Two-way ANOVA and Sidak test.DETAILED DESCRIPTION

[0035] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate aspects, can also be provided in combination with a single aspect. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single aspect, can also be provided separately or in any suitable subcombination. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure.DEFINITIONS

[0036] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:

[0037] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of’ and “consisting of.” Similarly, the term “consisting essentially of’ is intended to include examples encompassed by the term “consisting of.

[0038] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound”, “a composition”, or “a cancer”, includes, but is not limited to, two or more such compounds, compositions, or cancers, and the like.

[0039] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It can be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself.For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it can be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0040] When a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of Tess than x’, less than y’, and Tess than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0041] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the subranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0042] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at orabout” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0043] As used herein, the term “effective amount” refers to an amount that is sufficient to achieve the desired modification of a physical property of the composition or material. For example, an “effective amount” of a monomer refers to an amount that is sufficient to achieve the desired improvement in the property modulated by the formulation component, e.g. desired antioxidant release rate or viscoelasticity. The specific level in terms of wt% in a composition required as an effective amount will depend upon a variety of factors including the amount and type of monomer, amount and type of polymer, e.g., acrylamide, amount of antioxidant, and desired release kinetics.

[0044] As used herein, the term “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors within the knowledge and expertise of the health practitioner and which may be well known in the medical arts. In the case of treating a particular disease or condition, in some instances, the desired response can be inhibiting the progression of the disease or condition. This may involve only slowing the progression of the disease temporarily. However, in other instances, it may be desirable to halt the progression of the disease permanently. This can be monitored by routine diagnostic methods known to one of ordinary skill in the art for any particular disease. The desired response to treatment of the disease or condition also can be delaying the onset or even preventing the onset of the disease or condition.

[0045] For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dosecompositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. It is generally preferred that a maximum dose of the pharmacological agents of the invention (alone or in combination with other therapeutic agents) be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.

[0046] A response to a therapeutically effective dose of a disclosed drug delivery composition can be measured by determining the physiological effects of the treatment or medication, such as the decrease or lack of disease symptoms following administration of the treatment or pharmacological agent. Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response. The amount of a treatment may be varied for example by increasing or decreasing the amount of a disclosed compound and / or pharmaceutical composition, by changing the disclosed compound and / or pharmaceutical composition administered, by changing the route of administration, by changing the dosage timing and so on. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.

[0047] As used herein, the term “prophylactically effective amount” refers to an amount effective for preventing onset or initiation of a disease or condition.

[0048] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0049] An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase so long as the increase is statistically significant.

[0050] A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previouslyobserved. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.

[0051] Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

[0052] By “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control.

[0053] By “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed.

[0054] The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

[0055] As used herein, the terms "treating" and "treatment" can refer generally to obtaining a desired pharmacological and / or physiological effect. The effect can be, but does not necessarily have to be, prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof. The effect can be therapeutic in terms of a partial or completecure of a disease, condition, symptom or adverse effect attributed to the disease, disorder, or condition. The term "treatment" as used herein can include any treatment of a disease disorder in a subject, particularly a human and can include any one or more of the following: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., mitigating or ameliorating the disease and / or its symptoms or conditions. The term "treatment" as used herein can refer to both therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment. Those in need of treatment (subjects in need thereof) can include those already with the disorder and / or those in which the disorder is to be prevented. As used herein, the term "treating", can include inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and / or condition. Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, e.g., such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.

[0056] As used herein, “dose,” “unit dose,” or “dosage” can refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of a disclosed compound and / or a pharmaceutical composition thereof calculated to produce the desired response or responses in association with its administration.

[0057] As used herein, “therapeutic” can refer to treating, healing, and / or ameliorating a disease, disorder, condition, or side effect, or to decreasing in the rate of advancement of a disease, disorder, condition, or side effect.

[0058] The terms “nucleic acid” and “nucleic acid sequence” refer to a nucleotide, oligonucleotide, polynucleotide (which terms may be used interchangeably), or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin (which may be single-stranded or double-stranded and may represent the sense or the antisense strand).

[0059] Reference also is made herein to peptides, polypeptides, proteins and compositions comprising peptides, polypeptides, and proteins. As used herein, a polypeptide and / or protein is defined as a polymer of amino acids, typically of length>100 amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks / Cole, 110). A peptide is defined as a short polymer of amino acids, of a length typically of 20 or less amino acids, and more typically of a length of 12 or less amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks / Cole, 110).

[0060] A “functional fragment” as referred to herein comprises a portion of a polypeptide which retains its functional ability. In this case, the functional fragment would retain the ability to perform as a telomerase.

[0061] As disclosed herein, exemplary peptides, polypeptides, proteins may comprise, consist essentially of, or consist of any reference amino acid sequence disclosed herein, or variants of the peptides, polypeptides, and proteins may comprise, consist essentially of, or consist of an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any amino acid sequence disclosed herein. Variant peptides, polypeptides, and proteins may include peptides, polypeptides, and proteins having one or more amino acid substitutions, deletions, additions and / or amino acid insertions relative to a reference peptide, polypeptide, or protein. Also disclosed are nucleic acid molecules that encode the disclosed peptides, polypeptides, and proteins (e.g., polynucleotides that encode any of the peptides, polypeptides, and proteins disclosed herein and variants thereof).

[0062] The term “amino acid,” includes but is not limited to amino acids contained in the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (He or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Vai or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues. The term “amino acid residue” also may include amino acid residues contained in the group consisting of homocysteine, 2- Aminoadipic acid, N-Ethylasparagine, 3 -Aminoadipic acid, Hydroxylysine, P-alanine, P-Amino-propionic acid, allo-Hydroxylysine acid, 2-Aminobutyric acid, 3-Hydroxyproline, 4-Aminobutyric acid, 4-Hydroxyproline, piperidinic acid, 6- Aminocaproic acid, Isodesmosine, 2-Aminoheptanoic acid, allo-Isoleucine, 2- Aminoisobutyric acid, N-Methylglycine, sarcosine, 3 -Aminoisobutyric acid, N- Methylisoleucine, 2-Aminopimelic acid, 6-N-Methyllysine, 2,4-Diaminobutyric acid, N- Methylvaline, Desmosine, Norvaline, 2,2'-Diaminopimelic acid, Norleucine, 2,3- Diaminopropionic acid, Ornithine, and N-Ethylglycine. Typically, the amide linkages of the peptides are formed from an amino group of the backbone of one amino acid and a carboxyl group of the backbone of another amino acid.

[0063] The peptides, polypeptides, and proteins disclosed herein may be modified to include non-amino acid moieties. Modifications may include but are not limited to carboxylation (e.g., N-terminal carboxylation via addition of a di-carboxylic acid having 4-7 straight-chain or branched carbon atoms, such as glutaric acid, succinic acid, adipic acid, and4,4-dimethylglutaric acid), amidation (e.g., C-terminal amidation via addition of an amide or substituted amide such as alkylamide or dialkylamide), PEGylation (e.g., N-terminal or C- terminal PEGylation via additional of polyethylene glycol), acylation (e.g., O-acylation (esters), N-acylation (amides), S-acylation (thioesters)), acetylation (e.g., the addition of an acetyl group, either at the N-terminus of the protein or at lysine residues), formylation lipoylation (e.g., attachment of a lipoate, a C8 functional group), myristoylation (e.g., attachment of myristate, a C14 saturated acid), palmitoylation (e.g., attachment of palmitate, a C16 saturated acid), alkylation (e.g., the addition of an alkyl group, such as an methyl at a lysine or arginine residue), isoprenylation or prenylation (e.g., the addition of an isoprenoid group such as farnesol or geranylgeraniol), amidation at C-terminus, glycosylation (e.g., the addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine, resulting in a glycoprotein). Distinct from glycation, which is regarded as a nonenzymatic attachment of sugars, polysialylation (e.g., the addition of polysialic acid), glypiation (e.g., glycosylphosphatidylinositol (GPI) anchor formation, hydroxylation, iodination (e.g., of thyroid hormones), and phosphorylation (e.g., the addition of a phosphate group, usually to serine, tyrosine, threonine or histidine).

[0064] Variants comprising deletions relative to a reference amino acid sequence or nucleotide sequence are contemplated herein. A “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides relative to a reference sequence. A deletion removes at least 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 amino acids residues or nucleotides. A deletion may include an internal deletion or a terminal deletion (e.g., an N-terminal truncation or a C-terminal truncation or both of a reference polypeptide or a 5 '-terminal or 3 '-terminal truncation or both of a reference polynucleotide).

[0065] Variants comprising a fragment of a reference amino acid sequence or nucleotide sequence are contemplated herein. A “fragment” is a portion of an amino acid sequence or a nucleotide sequence which is identical in sequence to but shorter in length than the reference sequence. A fragment may comprise up to the entire length of the reference sequence, minus at least one nucleotide / amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or contiguous amino acid residues of a reference polynucleotide or reference polypeptide, respectively. In some embodiments, a fragment may comprise at least 5, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 contiguous nucleotides or contiguous amino acid residues of a reference polynucleotide or reference polypeptide, respectively. Fragments may be preferentially selected from certainregions of a molecule, for example the N-terminal region and / or the C-terminal region of a polypeptide or the 5 '-terminal region and / or the 3' terminal region of a polynucleotide. The term “at least a fragment” encompasses the full length polynucleotide or full length polypeptide.

[0066] Variants comprising insertions or additions relative to a reference sequence are contemplated herein. The words “insertion” and “addition” refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides. An insertion or addition may refer to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 amino acid residues or nucleotides.

[0067] Fusion proteins and fusion polynucleotides also are contemplated herein. A “fusion protein” refers to a protein formed by the fusion of at least one peptide, polypeptide, protein or variant thereof as disclosed herein to at least one molecule of a heterologous peptide, polypeptide, protein or variant thereof. The heterologous protein(s) may be fused at the N- terminus, the C-terminus, or both termini. A fusion protein comprises at least a fragment or variant of the heterologous protein(s) that are fused with one another, preferably by genetic fusion (i.e., the fusion protein is generated by translation of a nucleic acid in which a polynucleotide encoding all or a portion of a first heterologous protein is joined in-frame with a polynucleotide encoding all or a portion of a second heterologous protein). The heterologous protein(s), once part of the fusion protein, may each be referred to herein as a “portion”, “region” or “moiety” of the fusion protein.

[0068] A fusion polynucleotide refers to the fusion of the nucleotide sequence of a first polynucleotide to the nucleotide sequence of a second heterologous polynucleotide (e.g., the 3' end of a first polynucleotide to a 5' end of the second polynucleotide). Where the first and second polynucleotides encode proteins, the fusion may be such that the encoded proteins are in-frame and results in a fusion protein. The first and second polynucleotide may be fused such that the first and second polynucleotide are operably linked (e.g., as a promoter and a gene expressed by the promoter as discussed below).

[0069] A “full length” polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon. A “full length” polynucleotide sequence encodes a “full length” polypeptide sequence.

[0070] A “variant,” “mutant,” or “derivative” of a particular nucleic acid sequence may be defined as a nucleic acid sequence having at least 50% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastnwith the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences — a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250). In some embodiments a variant polynucleotide may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to a reference polynucleotide.

[0071] Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.

[0072] “Operably linked” refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0073] A “recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1 3, Cold Spring Harbor Press, Plainview N.Y. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.

[0074] Transformation” describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment. The term “transformed cells” includes stably transformed cells inwhich the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.

[0075] “Substantially isolated or purified” nucleic acid or amino acid sequences are contemplated herein. The term “substantially isolated or purified” refers to nucleic acid or amino acid sequences that are removed from their natural environment, and are at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which they are naturally associated.ANTIGEN-PRESENTING NANOPARTICLES

[0076] In one aspect, disclosed herein are antigen-presenting nanoparticles (APN) (including, but not limited to a lipid nanoparticle, a liposome, or a polymeric nanoparticle), including: a major histocompatibility complex (MHC) (such as, for example an MHC class I or MHC class II molecule) presented on a surface of the APN and including a peptide epitope (such as, for example an epitope of a virus, bacteria, fungus, or parasite); and an encapsulated nucleic acid encoding a chimeric antigen receptor (CAR), antibody, or antibody fragment (such as for example, a ScFv or VHH also known as a nanobody). In some aspects, the CAR, antibody, or antibody fragment encoded by the encapsulated nucleic acid can recognize a cancer or tumor antigen (such as, for example, a B cell maturation antigen (BCMA), G protein- coupled receptor class C group 5 member D (GPRC5D), CD 19, guanylate cyclase-C (GUCY2C), human epidermal growth factor receptor 2 (HER2), CD20, CD22, mucin-1 (MUC1), mesothelin, or VHH (e.g., a sequence comprising 80% similarity or more to SEQ ID NO: 8)).

[0077] In some aspects, the CAR, antibody, or antibody fragment encoded by the encapsulated nucleic acid can recognize a cancer or tumor antigen comprising 80% similarity or more (e.g., 81% similarity or more, 82% similarity or more, 83% similarity or more, 84% similarity or more, 85% similarity or more, 86% similarity or more, 87% similarity or more, 88% similarity or more, 89% similarity or more, 90% similarity or more, 91% similarity or more, 92% similarity or more, 93% similarity or more, 94% similarity or more, 95% similarity or more, 96% similarity or more, 97% similarity or more, 98% similarity or more, 99% similarity or more) to SEQ ID NO: 8. In some aspects, the CAR, antibody, or antibody fragment encoded by the encapsulated nucleic acid can recognize a cancer or tumor antigen comprising SEQ ID NO: 8. In some aspects, the CAR, antibody, or antibody fragment encoded by the encapsulated nucleic acid can recognize a cancer or tumor antigen consisting of SEQ ID NO: 8.Chimeric antigen receptors

[0078] In some aspects, the CAR contains an extracellular antigen-recognition domain that specifically binds to an antigen. In some aspects, the antigen is a protein expressed on the surface of cells. In some aspects, the CAR is a TCR-like CAR and the antigen is a processed peptide antigen, such as a peptide antigen of an intracellular protein, which, like a TCR, is recognized on the cell surface in the context of a major histocompatibility complex (MHC) molecule.

[0079] In some aspects, the chimeric antigen receptors (CARs), include activating or stimulatory CARs, costimulatory CARs (see WO2014 / 055668), and / or inhibitory CARs (iCARs, see Fedorov el al.. Sci. TransL Medicine, 5(215) (2013). The CARs generally include an extracellular antigen (or ligand) binding domain linked to one or more intracellular signaling components, in some aspects via linkers and / or transmembrane domain(s). Such molecules typically mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and / or a signal through a costimulatory receptor alone.

[0080] In some aspects, CAR is constructed with a specificity for a particular antigen (or marker or ligand), such as an antigen expressed in a particular cell type to be targeted by adoptive therapy, e.g., a cancer marker, and / or an antigen intended to induce a dampening response, such as an antigen expressed on a normal or non-diseased cell type. Thus, the CAR typically includes in its extracellular portion one or more antigen binding molecules, such as one or more antigen-binding fragment, domain, or portion, or one or more antibody variable domains, and / or antibody molecules. In some aspects, the CAR includes an antigen-binding portion or portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb).

[0081] In some aspects, the antigen-specific binding, or recognition component is linked to one or more transmembrane and intracellular signaling domains. In some aspects, the CAR includes a transmembrane domain fused to the extracellular domain of the CAR. In one aspect, the transmembrane domain that naturally is associated with one of the domains in the CAR is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0082] The transmembrane domain in some aspects is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e., comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD 154. Alternatively, the transmembrane domain in some aspects is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.

[0083] The CAR generally includes at least one intracellular signaling component or components. In some aspects, the CAR includes an intracellular component of the TCR complex, such as a TCR CD3+chain that mediates T-cell activation and cytotoxicity, e.g., CD3 zeta chain. Thus, in some aspects, the antigen binding molecule is linked to one or more cell signaling modules. In some aspects, cell signaling modules include CD3 transmembrane domain, CD3 intracellular signaling domains, and / or other CD transmembrane domains. In some aspects, the CAR further includes a portion of one or more additional molecules such as Fc receptor y, CD8, CD4, CD25, or CD16. For example, in some aspects, the CAR includes a chimeric molecule between CD3-zeta (CD3-Q orFc receptor y and CD8, CD4, CD25 or CD16.

[0084] In some aspects, the CAR can further include an intracellular co-stimulatory signaling motif. As used herein, “intracellular co-stimulatory signaling motifs” are defined as components or domains within the engineered receptors (CARs) that provide additional signals to T cells upon engagement with their target antigens. Co-stimulatory motifs are often derived from natural signaling proteins involved in T cell activation, such as CD28, 4 IBB, 0X40, Myd88, ICOS, CD2, CD226, BAFF-R, TACI, or IL2RB. Co-stimulatory motifs used herein, enhance T cell proliferation, cytokine secretion, cytotoxicity, and memory formation, ultimately improving the efficacy of CAR therapy.AntibodiesAntibodies Generally

[0085] The term “antibodies” is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term “antibodies” are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules or fragments thereof. Theantibodies can be tested for their desired activity using the in vitro assays described herein, or by analogous methods, after which their in vivo therapeutic and / or prophylactic activities are tested according to known clinical testing methods. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. One skilled in the art would recognize the comparable classes for mouse. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.

[0086] The term “monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules. The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity.

[0087] The disclosed monoclonal antibodies can be made using any procedure which produces mono clonal antibodies. For example, disclosed monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.

[0088] The monoclonal antibodies may also be made by recombinant DNA methods. DNA encoding the disclosed monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques, e.g., as described in U.S. Patent No. 5,804,440 to Burton et al. and U.S. Patent No. 6,096,441 to Barbas et al.

[0089] In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplishedusing routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94 / 29348 published Dec. 22, 1994 and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment that has two antigen combining sites and is still capable of cross-linking antigen.

[0090] As used herein, the term “antibody or fragments thereof’ encompasses chimeric antibodies and hybrid antibodies, with dual or multiple antigen or epitope specificities, and fragments, such as F(ab’)2, Fab’, Fab, Fv, sFv, scFv, VHH (i.e., nanobodies) diabodies,, and the like, including hybrid fragments. Thus, fragments of the antibodies that retain the ability to bind their specific antigens are provided. For example, fragments of antibodies which maintain binding activity are included within the meaning of the term “antibody or fragment thereof.” Such antibodies and fragments can be made by techniques known in the art and can be screened for specificity and activity according to the methods set forth in the Examples and in general methods for producing antibodies and screening antibodies for specificity and activity (See Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988)).

[0091] Also included within the meaning of “antibody or fragments thereof’ are conjugates of antibody fragments and antigen binding proteins (single chain antibodies).

[0092] The fragments, whether attached to other sequences or not, can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove / add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the antibody or antibody fragment must possess a bioactive property, such as specific binding to its cognate antigen. Functional or active regions of the antibody or antibody fragment may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to a skilled practitioner in the art and can include site-specific mutagenesis of the nucleic acid encoding the antibody or antibody fragment. (Zoller, M.J. Curr. Opin. Biotechnol. 3:348-354, 1992).

[0093] As used herein, the term “antibody” or “antibodies” can also refer to a human antibody and / or a humanized antibody. Many non-human antibodies (e.g., those derived frommice, rats, or rabbits) are naturally antigenic in humans, and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods serves to lessen the chance that an antibody administered to a human will evoke an undesirable immune response.Human antibodies

[0094] The disclosed human antibodies can be prepared using any technique. The disclosed human antibodies can also be obtained from transgenic animals. For example, transgenic, mutant mice that are capable of producing a full repertoire of human antibodies, in response to immunization, have been described (see, e.g., Jakobovits et al., Proc. Natl. Acad. Set. USA, 90:2551-255 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993)). Specifically, the homozygous deletion of the antibody heavy chain joining region (J(77)) gene in these chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production, and the successful transfer of the human germ-line antibody gene array into such germ-line mutant mice results in the production of human antibodies upon antigen challenge. Antibodies having the desired activity are selected using Env-CD4-co-receptor complexes as described herein.Humanized antibodies

[0095] Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule. Accordingly, a humanized form of a non-human antibody (or a fragment thereof) is a chimeric antibody or antibody chain (or a fragment thereof, such as an sFv, Fv, Fab, Fab’, F(ab’)2, or other antigen-binding portion of an antibody) which contains a portion of an antigen binding site from a non-human (donor) antibody integrated into the framework of a human (recipient) antibody.

[0096] To generate a humanized antibody, residues from one or more complementarity determining regions (CDRs) of a recipient (human) antibody molecule are replaced by residues from one or more CDRs of a donor (non-human) antibody molecule that is known to have desired antigen binding characteristics (e.g., a certain level of specificity and affinity for the target antigen). In some instances, Fv framework (FR) residues of the human antibody are replaced by corresponding non-human residues. Humanized antibodies may also contain residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. In practice, humanized antibodies aretypically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. Humanized antibodies generally contain at least a portion of an antibody constant region (Fc), typically that of a human antibody (Jones et al., Nature, 321 :522-525 (1986), Reichmann et al., Nature, 332:323-327 (1988), and Presta, Curr. Opin. Struct. Biol., 2:593-596 (1992)).

[0097] Methods for humanizing non-human antibodies are well known in the art. For example, humanized antibodies can be generated according to the methods of Winter and co-workers (Jones et al., Nature, 321 :522-525 (1986), Riechmann et al., Nature, 332:323-327 (1988), Verhoeyen et al., Science, 239: 1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Methods that can be used to produce humanized antibodies are also described in U.S. Patent No. 4,816,567 (Cabilly et al.), U.S. PatentNo. 5,565,332 (Hoogenboom et al.), U.S. Patent No. 5,721,367 (Kay et al.), U.S. Patent No. 5,837,243 (Deo et al.), U.S. Patent No. 5, 939,598 (Kucherlapati et al.), U.S. Patent No. 6,130,364 (Jakobovits et al.), and U.S. Patent No. 6,180,377 (Morgan et al.).

[0098] In some aspects, the CAR encoded by the encapsulated nucleic acid can include 80% similarity or more (e.g., 81% similarity or more, 82% similarity or more, 83% similarity or more, 84% similarity or more, 85% similarity or more, 86% similarity or more, 87% similarity or more, 88% similarity or more, 89% similarity or more, 90% similarity or more, 91% similarity or more, 92% similarity or more, 93% similarity or more, 94% similarity or more, 95% similarity or more, 96% similarity or more, 97% similarity or more, 98% similarity or more, 99% similarity or more) to SEQ ID NO: 2 or SEQ ID NO: 5. In some aspects, the CAR encoded by the encapsulated nucleic acid can include SEQ ID NO: 2 or SEQ ID NO: 5. In some aspects, the CAR encoded by the encapsulated nucleic acid can consist of SEQ ID NO: 2 or SEQ ID NO: 5.

[0099] In some aspects, the peptide epitope can be a viral epitope from a virus selected from the group consisting of Herpes Simplex virus-1, Herpes Simplex virus-2, Varicella-Zoster virus, Epstein-Barr virus, Cytomegalovirus, Human Herpes virus-6, Variola virus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus (such as, for example, a SARS-CoV-2 infection including, but not limited to the SARS-CoV-2 Bl.351 variant, SARS-CoV-2B.1.1.7 (alpha), SARS-CoV-2B.1.1.7 variant mutant N501Y (alpha), SARS-CoV-2 delta variant, SARS-CoV- 2 P. l variant, SARS-CoV-2 with T487K, P681R, and L452R mutations in B.1.617.2 (Delta), SARS-CoV-2 with K417N mutation in AY.1 / AY.2 (Delta plus), SARS-CoV-2 with D614G, P681H, and D950N mutations in B.1.621 (Mu), SARS-CoV-2 with G75V, T76I, A246-252,L452Q, F490S, D614G, and T859N mutations in C.37 (Lambda), SARS-CoV-2 with T478K, Q498R, and H655Y mutations in B.1.1.529 (Omicron)), Influenza virus A, Influenza virus B, Measles virus, Polyomavirus, Human Papilomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Reovirus, Yellow fever virus, Zika virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, Rotavirus A, Rotavirus B, Rotavirus C, Sindbis virus, Simian Immunodeficiency virus, Human T-cell Leukemia virus type-1, Hantavirus, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type-1, and Human Immunodeficiency virus type-2.

[0100] In some aspects, the peptide epitope can be a bacterial epitope from a bacteria selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis strain BCG, BCG substrains, Mycobacterium avium, Mycobacterium intracellular, Mycobacterium africanum, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium ulcerans, Mycobacterium avium subspecies paratuberculosis, Nocardia asteroides, other Nocardia species, Legionella pneumophila, other Legionella species, Acetinobacter baumanii, Salmonella typhi, Salmonella enterica, other Salmonella species, Shigella boydii, Shigella dysenteriae, Shigella sonnei, Shigella flexneri, other Shigella species, Yersinia pestis, Pasteurella haemolytica, Pasteurella multocida, other Pasteurella species, Actinobacillus pleuropneumoniae, Listeria monocytogenes, Listeria ivanovii, Brucella abortus, other Brucella species, Cowdria ruminantium, Borrelia burgdorferi, Bordetella avium, Bordetella pertussis, Bordetella bronchiseptica, Bordetella trematum, Bordetella hinzii, Bordetella pteri, Bordetella parapertussis, Bordetella ansorpii other Bordetella species, Burkholderia mallei, Burkholderia psuedomallei, Burkholderia cepacian, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Coxiella burnetii, Rickettsial species, Ehrlichia species, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Escherichia coli, Vibrio cholerae, Campylobacter species, Neiserria meningitidis, Neiserria gonorrhea, Pseudomonas aeruginosa, other Pseudomonas species, Haemophilus influenzae, Haemophilus ducreyi, other Hemophilus species, Clostridium tetani, other Clostridium species, Yersinia enterolitica, and other Yersinia species.

[0101] In some aspects, the peptide epitope can be a fungal epitope from a fungi selected from the group consisting of Candida albicans, Cryptococcus neoformans, Histoplamacapsulatum, Aspergillus fumigatus, Coccidiodes immitis, Paracoccidiodes brasiliensis, Blastomyces dermitidis, Pneumocystis carnii, Penicillium marneffi, and Alternaria alternata.

[0102] In some aspects, the peptide epitope can be a parasitic epitope from a parasite selected from the group consisting of Toxoplasma gondii, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, other Plasmodium species, Entamoeba histolytica, Naegleria fowleri, Rhinosporidium seeberi, Giardia lamblia, Enterobius vermicularis, Enterobius gregorii, Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus, Cryptosporidium spp., Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, other Leishmania species, Diphyllobothrium latum, Hymenolepis nana, Hymenolepis diminuta, Echinococcus granulosus, Echinococcus multilocularis, Echinococcus vogeli, Echinococcus oligarthrus, Diphyllobothrium latum, Clonorchis sinensis; Clonorchis viverrini, Fasciola hepatica, Fasciola gigantica, Dicrocoelium dendriticum, Fasciolopsis buski, Metagonimus yokogawai, Opisthorchis viverrini, Opisthorchis felineus, Clonorchis sinensis, Trichomonas vaginalis, Acanthamoeba species, Schistosoma intercalatum, Schistosoma haematobium, Schistosoma japonicum, Schistosoma mansoni, other Schistosoma species, Trichobilharzia regenti, Trichinella spiralis, Trichinella britovi, Trichinella nelsoni, Trichinella nativa, and Entamoeba histolytica.

[0103] As used herein, the term “ionizable lipid” refers to a lipid, e.g., cationic lipid or anionic lipid, having at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g., pH 7.4), and neutral at a second pH, preferably at or above physiological pH. It will be understood by one of ordinary skill in the art that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of the lipid be present in the charged or neutral form. Generally, ionizable lipids have a pKa of the protonatable group in the range of about 4 to about 7. In some aspects, the ionizable lipid can include cKK-E12, SM102, MC3, Lipid-5, Alc- 0315, LP01, Lipid A9, or any combination thereof.

[0104] In some aspects, the nanoparticle can include at least about 30 mol% (e.g., at least about 32 mol%, at least about 34 mol%, at least about 36 mol%, at least about 38 mol%, at least about 40 mol%, at least about 42 mol%, at least about 44 mol%, at least about 46 mol%, at least about 48 mol%, at least about 50 mol%, at least about 52 mol%, at least about 54 mol%, at least about 56 mol%, at least about 58 mol%, at least about 60 mol%) of the ionizable lipid. In some aspects, the nanoparticle can include up to about 60 mol% (e.g., up to about 58 mol%, up to about 56 mol%, up to about 54 mol%, up to about 52 mol%, up to about 50 mol%, up toabout 48 mol%, up to about 46 mol%, up to about 44 mol%, up to about 42 mol%, up to about 40 mol%, up to about 38 mol%, up to about 36 mol%, up to about 34 mol%, up to about 32 mol%, up to about 30 mol%) of the ionizable lipid.

[0105] It is considered that the nanoparticle can include an amount of the ionizable lipid ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the nanoparticle can include from about 30 mol% to about 60 mol% (e.g., from about 32 mol% to about 58 mol%, from about 34 mol% to about 56 mol%, from about 36 mol% to about 54 mol%, from about 38 mol% to about 52 mol%, from about 40 mol% to about 50 mol%, from about 42 mol% to about 48 mol%, from about 44 mol% to about 46 mol%, from about 30 mol% to about 46 mol%, from about 32 mol% to about 44 mol%, from about 34 mol% to about 42 mol%, from about 36 mol% to about 40 mol%, from about 44 mol% to about 60 mol%, from about 46 mol% to about 58 mol%, from about 48 mol% to about 56 mol%, from about 50 mol% to about 54 mol%) of the ionizable lipid.

[0106] In some aspects, the nanoparticle can include at least about 35 mol% (e.g., at least about 36 mol%, at least about 37 mol%, at least about 38 mol%, at least about 39 mol%, at least about 40 mol%, at least about 41 mol%, at least about 42 mol%, at least about 43 mol%, at least about 44 mol%, at least about 45 mol%, at least about 46 mol%, at least about 47 mol%, at least about 48 mol%, at least about 49 mol%, at least about 50 mol%) of the cholesterol. In some aspects, the nanoparticle can include up to about 50 mol% (e.g., up to about 49 mol%, up to about 48 mol%, up to about 47 mol%, up to about 46 mol%, up to about 45 mol%, up to about 44 mol%, up to about 43 mol%, up to about 42 mol%, up to about 41 mol%, up to about 40 mol%, up to about 39 mol%, up to about 38 mol%, up to about 37 mol%, up to about 36 mol%, up to about 35 mol%) of the cholesterol.

[0107] It is considered that the nanoparticle can include an amount of the cholesterol ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the nanoparticle can include from about 35 mol% to about 50 mol% (e.g., from about 36 mol% to about 49 mol%, from about 37 mol% to about 48 mol%, from about 38 mol% to about 47 mol%, from about 39 mol% to about 46 mol%, from about 40 mol% to about 45 mol%, from about 41 mol% to about 44 mol%, from about 42 mol% to about 43 mol%, from about 35 mol% to about 43 mol%, from about 36 mol% to about 42 mol%, from about 37 mol% to about 41 mol%, from about 38 mol% to about 40 mol%, from about 42 mol% to about 50 mol%, from about 43 mol% to about 49 mol%, from about 44 mol% to about 48 mol%, from about 45 mol% to about 47 mol%) of the cholesterol.

[0108] In some aspects, the phospholipid can include DSPC, DOPE, or any combination thereof.

[0109] In some aspects, the nanoparticle can include at least about 5 mol% (e.g., at least about 6 mol%, at least about 7 mol%, at least about 8 mol%, at least about 9 mol%, at least about 10 mol%, at least about 11 mol%, at least about 12 mol%, at least about 13 mol%, at least about 14 mol%, at least about 15 mol%, at least about 16 mol%, at least about 17 mol%, at least about 18 mol%, at least about 19 mol%, at least about 20 mol%) of the phospholipid. In some aspects, the nanoparticle can include up to about 20 mol% (e.g., up to about 19 mol%, up to about 18 mol%, up to about 17 mol%, up to about 16 mol%, up to about 15 mol%, up to about 14 mol%, up to about 13 mol%, up to about 12 mol%, up to about 11 mol%, up to about 10 mol%, up to about 9 mol%, up to about 8 mol%, up to about 7 mol%, up to about 6 mol%, up to about 5 mol%) of the phospholipid.

[0110] It is considered that the nanoparticle can include an amount of the phospholipid ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the nanoparticle can include from about 5 mol% to about 20 mol% (e.g., from about 6 mol% to about 19 mol%, from about 7 mol% to about 18 mol%, from about 8 mol% to about 17 mol%, from about 9 mol% to about 16 mol%, from about 10 mol% to about 15 mol%, from about 11 mol% to about 14 mol%, from about 12 mol% to about 13 mol%, from about 5 mol% to about 13 mol%, from about 6 mol% to about 12 mol%, from about 7 mol% to about 11 mol%, from about 8 mol% to about 10 mol%, from about 12 mol% to about 20 mol%, from about 13 mol% to about 19 mol%, from about 14 mol% to about 18 mol%, from about 15 mol% to about 17 mol%) of the phospholipid.

[0111] In some aspects, the PEGylated lipid can include ALC-0159, DMG-PEG, DSPE- PEG, PEG14-2000, or any combination thereof.

[0112] In some aspects, the nanoparticle can include at least about 0.5 mol% (e.g., at least about 1 mol%, at least about 1.5 mol%, at least about 2 mol%, at least about 2.5 mol%, at least about 3 mol%, at least about 3.5 mol%, at least about 4 mol%, at least about 4.5 mol%, at least about 5 mol%) of the PEGylated lipid. In some aspects, the nanoparticle can include up to about 5 mol% (e.g., up to about 4.5 mol%, up to about 4 mol%, up to about 3.5 mol%, up to about 3 mol%, up to about 2.5 mol%, up to about 2 mol%, up to about 1.5 mol%, up to about 1 mol%, up to about 0.5 mol%) of the PEGylated lipid.

[0113] It is considered that the nanoparticle can include an amount of the PEGylated lipid ranging from any of the minimum values described above. For example, in some aspects, the nanoparticle can include from about 0.5 mol% to about 5 mol% (e.g. from about 1 mol% toabout 4.5 mol%, from about 1.5 mol% to about 4 mol%, from about 2 mol% to about 3.5 mol%, from about 2.5 mol% to about 3 mol%, from about 0.5 mol% to about 3 mol%, from about 1 mol% to about 2.5 mol%, from about 1.5 mol% to about 2 mol%, from about 2.5 mol% to about 5 mol%, from about 3 mol% to about 4.5 mol%, from about 3.5 mol% to about 4 mol%) of the PEGylated lipid.

[0114] In some aspects, the ratio of PEGylated lipid to total lipids can be at least about 0.01 :1 (e.g., at least about 0.015: 1, at least about 0.02: 1, at least about 0.025: 1, at least about 0.03:1, at least about 0.035:1, at least about 0.04: 1, at least about 0.045: 1, at least about 0.05: 1). In some aspects, the ratio of PEGylated lipid to total lipids can be up to about 0.05: 1 (e.g., up to about 0.045: 1, up to about 0.04: 1, up to about 0.035: 1, up to about 0.03: 1, up to about 0.025:1, up to about 0.02: 1, up to about 0.015: 1, up to about 0.01 : 1)

[0115] It is considered that the ratio of PEGylated lipid to total lipids can range from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the ratio of PEGylated lipid to total lipids can be from about 0.01 : 1 to about 0.05: 1 (e.g., from about 0.015: 1 to about 0.045: 1, from about 0.02: 1 to about 0.04: 1, from about 0.025: 1 to about 0.035: 1, from about 0.01 : 1 to about 0.03: 1, from about 0.015:1 to about 0.025: 1, from about 0.03: 1 to about 0.05: 1, from about 0.035: 1 to about 0.045: 1).

[0116] In some aspects, the APN can be a lipid nanoparticle, a liposome, or a polymeric nanoparticle. As used herein, “lipid nanoparticles” refer to particles having at least one dimension on the order of nanometers (e.g., 1-1000 nm) and including one or more lipids. In the context of the present invention, a lipid nanoparticle typically serves to transport a desired nucleic acid to a target cell or tissue. The process of incorporation of a desired nucleic acid into a lipid nanoparticle is often referred to as “loading”. The lipids and the nucleic acid can create a self-assembled structure via counterion interactions. The purpose of incorporating a nucleic acid into a transfer vehicle, such as a lipid nanoparticle, is often to protect the nucleic acid from an environment which may contain enzymes or chemicals that degrade nucleic acids and / or systems or receptors that cause the rapid excretion of the nucleic acids. Accordingly, in some aspects, a suitable delivery vehicle is capable of enhancing the stability of the nucleic acid contained therein and / or facilitate the delivery of nucleic acid to the target cell or tissue.METHODS

[0117] In one aspect, disclosed herein are methods of engineering a CAR T cell, the method including exposing an antigen-specific T cell (including, but not limited to a human T cell) to the APN of any preceding aspect. For example, disclosed herein are methods of engineering a CAR T cell, the method including exposing an antigen-specific T cell to an antigen presentingnanoparticle (APN) (including, but not limited to a lipid nanoparticle, a liposome, or a polymeric nanoparticle), the APN comprising: a major histocompatibility complex (MHC) (such as, for example an MHC class I or MHC class II molecule) presented on a surface of the APN and comprising a peptide epitope (such as, for example an epitope of a virus, bacteria, fungus, or parasite); and an encapsulated nucleic acid encoding a chimeric antigen receptor (CAR), antibody, or antibody fragment (such as for example, a ScFv or VHH also known as a nanobody). In some aspects, the CAR, antibody, or antibody fragment encoded by the encapsulated nucleic acid can recognize a cancer or tumor antigen (such as, for example, a B cell maturation antigen (BCMA), G protein-coupled receptor class C group 5 member D (GPRC5D), CD 19, guanylate cyclase-C (GUCY2C), human epidermal growth factor receptor 2 (HER2), CD20, CD22, mucin-1 (MUC1), mesothelin, or VHH (e.g., a sequence comprising 80% similarity or more to SEQ ID NO: 8)).

[0118] Also disclosed herein are methods of engineering a CAR T cell of any preceding aspect, wherein the T cell is a CD8+ T cell, and wherein the MHC is MHC class I or a CD4+ T cell, and wherein the MHC is MHC class II.

[0119] In one aspect, disclosed herein are methods of engineering a CAR T cell of any preceding aspect, wherein the method is carried out in vitro, in vivo, or ex vivo.

[0120] Also disclosed herein are CAR T cells produced by the method of any preceding aspect.

[0121] In one aspect, disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating and / or preventing a cancer, tumor, and / or metastasis (such as, for example, lymphoma, multiple myeloma, myeloid leukemia, colorectal cancer, breast cancer, giloblastoma, ovarian cancer, sarcoma, pancreatic cancer, mesothelioma, lung cancer, gastric cancer, or cervical cancer) in a subject, the method including administering to the subject the APN of any preceding aspect. For example, disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating and / or preventing a cancer, tumor, and / or metastasis (such as, for example, lymphoma, multiple myeloma, myeloid leukemia, colorectal cancer, breast cancer, giloblastoma, ovarian cancer, sarcoma, pancreatic cancer, mesothelioma, lung cancer, gastric cancer, or cervical cancer), the method comprising administering to the subject an antigen-presenting nanoparticle (APN) (including, but not limited to a lipid nanoparticle, a liposome, or a polymeric nanoparticle), including: a major histocompatibility complex (MHC) (such as, for example an MHC class I or MHC class II molecule) presented on a surface of the APN and comprising a peptide epitope (such as, for example an epitope of a virus, bacteria, fungus, or parasite); and an encapsulated nucleic acid encoding a chimeric antigen receptor(CAR), antibody, or antibody fragment (such as for example, a ScFv or VHH also known as a nanobody) that recognizes a cancer or tumor antigen (such as, for example, a B cell maturation antigen (BCMA), G protein-coupled receptor class C group 5 member D (GPRC5D), CD 19, guanylate cyclase-C (GUCY2C), human epidermal growth factor receptor 2 (HER2), CD20, CD22, mucin-1 (MUC1), mesothelin, or VHH (e.g., a sequence comprising 80% similarity or more to SEQ ID NO: 8)).

[0122] In some aspects, the CAR encoded by the encapsulated nucleic acid can include 80% similarity or more (e.g., 81% similarity or more, 82% similarity or more, 83% similarity or more, 84% similarity or more, 85% similarity or more, 86% similarity or more, 87% similarity or more, 88% similarity or more, 89% similarity or more, 90% similarity or more, 91% similarity or more, 92% similarity or more, 93% similarity or more, 94% similarity or more, 95% similarity or more, 96% similarity or more, 97% similarity or more, 98% similarity or more, 99% similarity or more) to SEQ ID NO: 2 or SEQ ID NO: 5. In some aspects, the CAR encoded by the encapsulated nucleic acid can include SEQ ID NO: 2 or SEQ ID NO: 5. In some aspects, the CAR encoded by the encapsulated nucleic acid can consist of SEQ ID NO: 2 or SEQ ID NO: 5.

[0123] In some aspects, after administration of the APN, at least about 40% (e.g., at least about 42%, at least about 44%, at least about 46%, at least about 48%, at least about 50%, at least about 52%, at least about 54%, at least about 56%, at least about 58%, at least about 60%, at least about 62%, at least about 64%, at least about 66%, at least about 68%, at least about 70%, at least about 72%, at least about 74%, at least about 76%, at least about 78%, at least about 80%, at least about 82%, at least about 84%, at least about 86%, at least about 88%, at least about 90%, at least about 92%, at least about 94%, at least about 96%, at least about 98%, about 100%) of the antigen-specific T cells may express the CAR.

[0124] Also disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating and / or preventing a cancer, tumor, and / or metastasis of any preceding aspect, further including administering to the subject a peptide pulse or vaccine to the subject prior to administering the APN; wherein the peptide includes an epitope for a microbe to which the subject has been exposed; wherein the vaccine expresses the same epitope as the APN.

[0125] In some aspects, the peptide epitope can be a viral epitope from a virus selected from the group consisting of Herpes Simplex virus-1, Herpes Simplex virus-2, Varicella-Zoster virus, Epstein-Barr virus, Cytomegalovirus, Human Herpes virus-6, Variola virus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus (such as, for example, a SARS-CoV-2 infectionincluding, but not limited to the SARS-CoV-2 Bl.351 variant, SARS-CoV-2B.1.1.7 (alpha), SARS-CoV-2B.1.1.7 variant mutant N501Y (alpha), SARS-CoV-2 delta variant, SARS-CoV- 2 P. l variant, SARS-CoV-2 with T487K, P681R, and L452R mutations in B.1.617.2 (Delta), SARS-CoV-2 with K417N mutation in AY.1 / AY.2 (Delta plus), SARS-CoV-2 with D614G, P681H, and D950N mutations in B.1.621 (Mu), SARS-CoV-2 with G75V, T76I, A246-252, L452Q, F490S, D614G, and T859N mutations in C.37 (Lambda), SARS-CoV-2 with T478K, Q498R, and H655Y mutations in B.1.1.529 (Omicron)), Influenza virus A, Influenza virus B, Measles virus, Polyomavirus, Human Papilomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Reovirus, Yellow fever virus, Zika virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, Rotavirus A, Rotavirus B, Rotavirus C, Sindbis virus, Simian Immunodeficiency virus, Human T-cell Leukemia virus type-1, Hantavirus, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type-1, and Human Immunodeficiency virus type-2.

[0126] In some aspects, the peptide epitope can be a bacterial epitope from a bacteria selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis strain BCG, BCG substrains, Mycobacterium avium, Mycobacterium intracellular, Mycobacterium africanum, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium ulcerans, Mycobacterium avium subspecies paratuberculosis, Nocardia asteroides, other Nocardia species, Legionella pneumophila, other Legionella species, Acetinobacter baumanii, Salmonella typhi, Salmonella enterica, other Salmonella species, Shigella boydii, Shigella dysenteriae, Shigella sonnei, Shigella flexneri, other Shigella species, Yersinia pestis, Pasteurella haemolytica, Pasteurella multocida, other Pasteurella species, Actinobacillus pleuropneumoniae, Listeria monocytogenes, Listeria ivanovii, Brucella abortus, other Brucella species, Cowdria ruminantium, Borrelia burgdorferi, Bordetella avium, Bordetella pertussis, Bordetella bronchiseptica, Bordetella trematum, Bordetella hinzii, Bordetella pteri, Bordetella parapertussis, Bordetella ansorpii other Bordetella species, Burkholderia mallei, Burkholderia psuedomallei, Burkholderia cepacian, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Coxiella burnetii, Rickettsial species, Ehrlichia species, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Escherichia coli, Vibrio cholerae, Campylobacter species, Neiserria meningitidis, Neiserria gonorrhea, Pseudomonas aeruginosa, other Pseudomonas species, Haemophilus influenzae, Haemophilus ducreyi, otherHemophilus species, Clostridium tetani, other Clostridium species, Yersinia enterolitica, and other Yersinia species.

[0127] In some aspects, the peptide epitope can be a fungal epitope from a fungi selected from the group consisting of Candida albicans, Cryptococcus neoformans, Histoplama capsulatum, Aspergillus fumigatus, Coccidiodes immitis, Paracoccidiodes brasiliensis, Blastomyces dermitidis, Pneumocystis carnii, Penicillium marneffi, and Alternaria alternata.

[0128] In some aspects, the peptide epitope can be a parasitic epitope from a parasite selected from the group consisting of Toxoplasma gondii, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, other Plasmodium species, Entamoeba histolytica, Naegleria fowleri, Rhinosporidium seeberi, Giardia lamblia, Enterobius vermicularis, Enterobius gregorii, Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus, Cryptosporidium spp., Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, other Leishmania species, Diphyllobothrium latum, Hymenolepis nana, Hymenolepis diminuta, Echinococcus granulosus, Echinococcus multilocularis, Echinococcus vogeli, Echinococcus oligarthrus, Diphyllobothrium latum, Clonorchis sinensis; Clonorchis viverrini, Fasciola hepatica, Fasciola gigantica, Dicrocoelium dendriticum, Fasciolopsis buski, Metagonimus yokogawai, Opisthorchis viverrini, Opisthorchis felineus, Clonorchis sinensis, Trichomonas vaginalis, Acanthamoeba species, Schistosoma intercalatum, Schistosoma haematobium, Schistosoma japonicum, Schistosoma mansoni, other Schistosoma species, Trichobilharzia regenti, Trichinella spiralis, Trichinella britovi, Trichinella nelsoni, Trichinella nativa, and Entamoeba histolytica.

[0129] In some aspects, the ionizable lipid can include cKK-E12, SM102, MC3, Lipid-5, Alc-0315, LP01, Lipid A9, or any combination thereof.

[0130] In some aspects, the nanoparticle can include at least about 30 mol% (e.g., at least about 32 mol%, at least about 34 mol%, at least about 36 mol%, at least about 38 mol%, at least about 40 mol%, at least about 42 mol%, at least about 44 mol%, at least about 46 mol%, at least about 48 mol%, at least about 50 mol%, at least about 52 mol%, at least about 54 mol%, at least about 56 mol%, at least about 58 mol%, at least about 60 mol%) of the ionizable lipid. In some aspects, the nanoparticle can include up to about 60 mol% (e.g., up to about 58 mol%, up to about 56 mol%, up to about 54 mol%, up to about 52 mol%, up to about 50 mol%, up to about 48 mol%, up to about 46 mol%, up to about 44 mol%, up to about 42 mol%, up to about 40 mol%, up to about 38 mol%, up to about 36 mol%, up to about 34 mol%, up to about 32 mol%, up to about 30 mol%) of the ionizable lipid.

[0131] It is considered that the nanoparticle can include an amount of the ionizable lipid ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the nanoparticle can include from about 30 mol% to about 60 mol% (e.g., from about 32 mol% to about 58 mol%, from about 34 mol% to about 56 mol%, from about 36 mol% to about 54 mol%, from about 38 mol% to about 52 mol%, from about 40 mol% to about 50 mol%, from about 42 mol% to about 48 mol%, from about 44 mol% to about 46 mol%, from about 30 mol% to about 46 mol%, from about 32 mol% to about 44 mol%, from about 34 mol% to about 42 mol%, from about 36 mol% to about 40 mol%, from about 44 mol% to about 60 mol%, from about 46 mol% to about 58 mol%, from about 48 mol% to about 56 mol%, from about 50 mol% to about 54 mol%) of the ionizable lipid.

[0132] In some aspects, the nanoparticle can include at least about 35 mol% (e.g., at least about 36 mol%, at least about 37 mol%, at least about 38 mol%, at least about 39 mol%, at least about 40 mol%, at least about 41 mol%, at least about 42 mol%, at least about 43 mol%, at least about 44 mol%, at least about 45 mol%, at least about 46 mol%, at least about 47 mol%, at least about 48 mol%, at least about 49 mol%, at least about 50 mol%) of the cholesterol. In some aspects, the nanoparticle can include up to about 50 mol% (e.g., up to about 49 mol%, up to about 48 mol%, up to about 47 mol%, up to about 46 mol%, up to about 45 mol%, up to about 44 mol%, up to about 43 mol%, up to about 42 mol%, up to about 41 mol%, up to about 40 mol%, up to about 39 mol%, up to about 38 mol%, up to about 37 mol%, up to about 36 mol%, up to about 35 mol%) of the cholesterol.

[0133] It is considered that the nanoparticle can include an amount of the cholesterol ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the nanoparticle can include from about 35 mol% to about 50 mol% (e.g., from about 36 mol% to about 49 mol%, from about 37 mol% to about 48 mol%, from about 38 mol% to about 47 mol%, from about 39 mol% to about 46 mol%, from about 40 mol% to about 45 mol%, from about 41 mol% to about 44 mol%, from about 42 mol% to about 43 mol%, from about 35 mol% to about 43 mol%, from about 36 mol% to about 42 mol%, from about 37 mol% to about 41 mol%, from about 38 mol% to about 40 mol%, from about 42 mol% to about 50 mol%, from about 43 mol% to about 49 mol%, from about 44 mol% to about 48 mol%, from about 45 mol% to about 47 mol%) of the cholesterol.

[0134] In some aspects, the phospholipid can include DSPC, DOPE, or any combination thereof.

[0135] In some aspects, the nanoparticle can include at least about 5 mol% (e.g., at least about 6 mol%, at least about 7 mol%, at least about 8 mol%, at least about 9 mol%, at least about 10 mol%, at least about 11 mol%, at least about 12 mol%, at least about 13 mol%, at least about 14 mol%, at least about 15 mol%, at least about 16 mol%, at least about 17 mol%, at least about 18 mol%, at least about 19 mol%, at least about 20 mol%) of the phospholipid. In some aspects, the nanoparticle can include up to about 20 mol% (e.g., up to about 19 mol%, up to about 18 mol%, up to about 17 mol%, up to about 16 mol%, up to about 15 mol%, up to about 14 mol%, up to about 13 mol%, up to about 12 mol%, up to about 11 mol%, up to about 10 mol%, up to about 9 mol%, up to about 8 mol%, up to about 7 mol%, up to about 6 mol%, up to about 5 mol%) of the phospholipid.

[0136] It is considered that the nanoparticle can include an amount of the phospholipid ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the nanoparticle can include from about 5 mol% to about 20 mol% (e.g., from about 6 mol% to about 19 mol%, from about 7 mol% to about 18 mol%, from about 8 mol% to about 17 mol%, from about 9 mol% to about 16 mol%, from about 10 mol% to about 15 mol%, from about 11 mol% to about 14 mol%, from about 12 mol% to about 13 mol%, from about 5 mol% to about 13 mol%, from about 6 mol% to about 12 mol%, from about 7 mol% to about 11 mol%, from about 8 mol% to about 10 mol%, from about 12 mol% to about 20 mol%, from about 13 mol% to about 19 mol%, from about 14 mol% to about 18 mol%, from about 15 mol% to about 17 mol%) of the phospholipid.

[0137] In some aspects, the PEGylated lipid can include ALC-0159, DMG-PEG, DSPE- PEG, PEG14-2000, or any combination thereof.

[0138] In some aspects, the nanoparticle can include at least about 0.5 mol% (e.g., at least about 1 mol%, at least about 1.5 mol%, at least about 2 mol%, at least about 2.5 mol%, at least about 3 mol%, at least about 3.5 mol%, at least about 4 mol%, at least about 4.5 mol%, at least about 5 mol%) of the PEGylated lipid. In some aspects, the nanoparticle can include up to about 5 mol% (e.g., up to about 4.5 mol%, up to about 4 mol%, up to about 3.5 mol%, up to about 3 mol%, up to about 2.5 mol%, up to about 2 mol%, up to about 1.5 mol%, up to about 1 mol%, up to about 0.5 mol%) of the PEGylated lipid.

[0139] It is considered that the nanoparticle can include an amount of the PEGylated lipid ranging from any of the minimum values described above. For example, in some aspects, the nanoparticle can include from about 0.5 mol% to about 5 mol% (e.g. from about 1 mol% to about 4.5 mol%, from about 1.5 mol% to about 4 mol%, from about 2 mol% to about 3.5 mol%, from about 2.5 mol% to about 3 mol%, from about 0.5 mol% to about 3 mol%, from about 1mol% to about 2.5 mol%, from about 1.5 mol% to about 2 mol%, from about 2.5 mol% to about 5 mol%, from about 3 mol% to about 4.5 mol%, from about 3.5 mol% to about 4 mol%) of the PEGylated lipid.

[0140] In some aspects, the ratio of PEGylated lipid to total lipids can be at least about 0.01 :1 (e.g., at least about 0.015: 1, at least about 0.02: 1, at least about 0.025: 1, at least about 0.03:1, at least about 0.035:1, at least about 0.04: 1, at least about 0.045: 1, at least about 0.05: 1). In some aspects, the ratio of PEGylated lipid to total lipids can be up to about 0.05: 1 (e.g., up to about 0.045: 1, up to about 0.04: 1, up to about 0.035: 1, up to about 0.03: 1, up to about 0.025:1, up to about 0.02: 1, up to about 0.015: 1, up to about 0.01 : 1)

[0141] It is considered that the ratio of PEGylated lipid to total lipids can range from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the ratio of PEGylated lipid to total lipids can be from about 0.01 : 1 to about 0.05: 1 (e.g., from about 0.015: 1 to about 0.045: 1, from about 0.02: 1 to about 0.04: 1, from about 0.025: 1 to about 0.035: 1, from about 0.01 : 1 to about 0.03: 1, from about 0.015:1 to about 0.025: 1, from about 0.03: 1 to about 0.05: 1, from about 0.035: 1 to about 0.045: 1).

[0142] In some aspects, the APN can be a lipid nanoparticle, a liposome, or a polymeric nanoparticle.

[0143] In some aspects, at least about 0.1 mg / kg (e.g., at least about 0.2 mg / kg, at least about 0.3 mg / kg, at least about 0.4 mg / kg, at least about 0.5 mg / kg, at least about 1 mg / kg, at least about 1.5 mg / kg, at least about 2 mg / kg, at least about 2.5 mg / kg, at least about 3 mg / kg, at least about 3.5 mg / kg, at least about 4 mg / kg, at least about 4.5 mg / kg, at least about 5 mg / kg) APN can be administered. In some aspects, up to about 5 mg / kg (e.g., up to about 4.5 mg / kg, up to about 4 mg / kg, up to about 3.5 mg / kg, up to about 3 mg / kg, up to about 2.5 mg / kg, up to about 2 mg / kg, up to about 1.5 mg / kg, up to about 1 mg / kg, up to about 0.5 mg / kg, up to about 0.4 mg / kg, up to about 0.3 mg / kg, up to about 0.2 mg / kg, up to about 0.1 mg / kg) APN can be administered.

[0144] It is considered that an amount of APN ranging from any of the minimum values described above to any of the maximum values described above can be administerd. For example, in some aspects, from about 0.1 mg / kg to about 5 mg / kg (e.g., from about 0.2 mg / kg to about 4.5 mg / kg, from about 0.3 mg / kg to about 4 mg / kg, from about 0.4 mg / kg to about 3.5 mg / kg, from about 0.5 mg / kg to about 3 mg / kg, from about 1 mg / kg to about 2.5 mg / kg, from about 1.5 mg / kg to about 2 mg / kg, from about 0.1 mg / kg to about 2 mg / kg, from about 0.2 mg / kg to about 1.5 mg / kg, from about 0.3 mg / kg to about 1 mg / kg, from about 0.4 mg / kg to about 0.5 mg / kg, from about 1.5 mg / kg to about 5 mg / kg, from about 2 mg / kg to about 4.5mg / kg, from about 2.5 mg / kg to about 4 mg / kg, from about 3 mg / kg to about 3.5 mg / kg) APN can be administered.

[0145] In some aspects, the APN can be repeatedly administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times at an interval of about every 5 days, or every 4 days, or every 3 days, or every 2 days, or every day, or twice per day, or every 6 days, or every 7 days, or every 8 days, or every 9 days, or every 10 days.

[0146] Also disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating and / or preventing a cancer, tumor, and / or metastasis of any preceding aspect, further including subsequently administering to the subject an anti-cancer agent or an immunotherapy.

[0147] In some aspects, the immunotherapy can include an immune checkpoint inhibitor. In some such aspects, the immune checkpoint can include an antibody that blocks PD-1 (such as, for example, Nivolumab (BMS-936558 or MDX1106), pembrolizumab, cemiplimab , CT- 011, MK-3475), PD-L1 (such as, for example, atezolizumab, avelumab, durvalwmab, MDX- 1105 (BMS-936559), MPDL3280A, or MSB0010718C), PD-L2 (such as, for example, rHIgM12B7), CTLA-4 (such as, for example, Ipilimumab (MDX-010), Tremelimumab (CP- 675,206)), IDO, B7-H3 (such as, for example, MGA271, MGD009, omburtamab), B7-H4, B7- H3, T cell immunoreceptor with Ig and ITIM domains (TIGIT)(such as, for example BMS- 986207, OMP-313M32, MK-7684, AB-154, ASP-8374, MTIG7192A, or PVSRIPO), CD96, B- and T-lymphocyte attenuator (BTLA), V-domain Ig suppressor of T cell activation (VISTA)(such as, for example, JNJ-61610588, CA-170), TIM3 (such as, for example, TSR- 022, MBG453, Sym023, INCAGN2390, LY3321367, BMS-986258, SHR-1702, RO7121661), LAG-3 (such as, for example, BMS-986016, LAG525, MK-4280, REGN3767, TSR-033, BI754111, Sym022, FS118, MGD013, and Immutep).

[0148] In some aspects, the immunotherapy can include an adoptive cell therapy. In some such aspects, the adoptive cell therapy can include the administration of tumor infiltrating lymphocytes (TILs), marrow infiltrating lymphocytes (NILs), chimeric antigen receptor (CAR) T cells, CAR natural killer (NK) (CAR NK) cells, CAR macrophage (CARMACs), or CAR NK T cells expressing a CAR, T cell receptor, or NK receptor which recognizes the target antigen.

[0149] It is understood and herein contemplated that the disclosed treatment regimens can used alone or in combination with any anti-cancer therapy known in the art including, but not limited to Abemaciclib, Abiraterone Acetate, ABITREXATE® (Methotrexate), ABRAXANE® (Paclitaxel Albumin-stabilized Nanoparticle Formulation), ABVD, ABVE,ABVE-PC, AC, AC-T, ADCETRIS® (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, ADRIAMYCIN® (Doxorubicin Hydrochloride), Afatinib Dimaleate, AFINITOR® (Everolimus), AKYNZEO® (Netupitant and Palonosetron Hydrochloride), ALDARA® (Imiquimod), Aldesleukin, ALECENSA® (Alectinib), Alectinib, Alemtuzumab, ALIMTA® (Pemetrexed Disodium), ALIQOPA® (Copanlisib Hydrochloride), ALKERAN™ for Injection (Melphalan Hydrochloride), ALKERAN™ Tablets (Melphalan), ALOXI® (Palonosetron Hydrochloride), ALUNBRIG® (Brigatinib), AMBOCHLORIN® (Chlorambucil), AMBOCLORIN® (Chlorambucil), Amifostine, Aminolevulinic Acid, Anastrozole, Aprepitant, AREDIA® (Pamidronate Disodium), ARIMIDEX® (Anastrozole), AROMASIN® (Exemestane),ARRANON® (Nelarabine), Arsenic Trioxide, ARZERRA® (Ofatumumab), Asparaginase Erwinia chrysanthemi, Atezolizumab, AVASTIN® (Bevacizumab), Avelumab, Axitinib, Azacitidine, BAVENCIO® (Avelumab), BEACOPP, BECENUM® (Carmustine), BELEODAQ® (Belinostat), Belinostat, Bendamustine Hydrochloride, BEP, BESPONSA® (Inotuzumab Ozogamicin) , Bevacizumab, Bexarotene, BEXXAR® (Tositumomab and Iodine I 131 Tositumomab), Bicalutamide, BICNU® (Carmustine), Bleomycin, Blinatumomab, BLINCYTO® (Blinatumomab), Bortezomib, BOSULIF® (Bosutinib), Bosutinib, Brentuximab Vedotin, Brigatinib, BuMel, Busulfan, BUSULFEX® (Busulfan), Cabazitaxel, CABOMETYX® (Cabozantinib-S-Malate), Cabozantinib-S-Malate, CAF, CAMPATH® (Alemtuzumab), CAMPTOSAR® (Irinotecan Hydrochloride), Capecitabine, CAPOX, CARAC® (Fluorouracil— Topical), Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, CARMUBRIS® (Carmustine), Carmustine, Carmustine Implant, CASODEX® (Bicalutamide), CEM, Ceritinib, CERUBIDINE® (Daunorubicin Hydrochloride), CERVARIX® (Recombinant HPV Bivalent Vaccine), Cetuximab, CEV, Chlorambucil, CHLORAMBUCIL-PREDNISONE, CHOP, Cisplatin, Cladribine, CLAFEN® (Cyclophosphamide), Clofarabine, CLOFAREX® (Clofarabine), CLOLAR® (Clofarabine), CMF, Cobimetinib, COMETRIQ® (Cabozantinib-S-Malate), Copanlisib Hydrochloride, COPDAC, COPP, COPP-ABV, COSMEGEN® (Dactinomycin), COTELLIC® (Cobimetinib), Crizotinib, CVP, Cyclophosphamide, CYFOS® (Ifosfamide), CYRAMZA® (Ramucirumab), Cytarabine, Cytarabine Liposome, CYTOSAR-U® (Cytarabine), CYTOXAN® (Cyclophosphamide), Dabrafenib, Dacarbazine, DACOGEN® (Decitabine), Dactinomycin, Daratumumab, DARZALEX® (Daratumumab), Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposome, Decitabine, Defibrotide Sodium, DEFITELIO® (Defibrotide Sodium), Degarelix, Denileukin Diftitox, Denosumab, DEPOCYT® (Cytarabine Liposome), Dexamethasone, DexrazoxaneHydrochloride, Dinutuximab, Docetaxel, DOXIL® (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome, DOX-SL® (Doxorubicin Hydrochloride Liposome), DTIC-DOME® (Dacarbazine), Durvalumab, EFUDEX® (Fluorouracil— Topical), ELITEK® (Rasburicase), ELLENCE® (Epirubicin Hydrochloride), Elotuzumab, ELOXATIN® (Oxaliplatin), Eltrombopag Olamine, EMEND® (Aprepitant), EMPLICITI® (Elotuzumab), Enasidenib Mesylate, Enzalutamide, Epirubicin Hydrochloride , EPOCH, ERBITUX® (Cetuximab), Eribulin Mesylate, ERIVEDGE® (Vismodegib), Erlotinib Hydrochloride, ERWINAZE® (Asparaginase Erwinia chrysanthemi), ETHYOL® (Amifostine), Etopophos ETOPOPHOS® (Etoposide Phosphate), Etoposide, Etoposide Phosphate, EV ACET® (Doxorubicin Hydrochloride Liposome), Everolimus, EVISTA® (Raloxifene Hydrochloride), EVOMELA® (Melphalan Hydrochloride), Exemestane, 5-FU® (Fluorouracil Injection), 5-FU® (Fluorouracil— Topical), FARESTON® (Toremifene), FARYDAK® (Panobinostat), FASLODEX® (Fulvestrant), FEC, FEMARA® (Letrozole), Filgrastim, FLUDARA® (Fludarabine Phosphate), Fludarabine Phosphate, FLUOROPLEX® (Fluorouracil— Topical), Fluorouracil Injection, Fluorouracil— Topical, Flutamide, FOLEX® (Methotrexate), FOLEX PFS® (Methotrexate), FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRI-CETUXIMAB, FOLFIRINOX, FOLFOX, FOLOTYN® (Pralatrexate), FU-LV, Fulvestrant, GARDASIL® (Recombinant HPV Quadrivalent Vaccine), GARDASIL 9® (Recombinant HPV Nonavalent Vaccine), GAZYVA® (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINEOXALIPLATIN, Gemtuzumab Ozogamicin, GEMZAR® (Gemcitabine Hydrochloride), GILOTRIF® (Afatinib Dimaleate), GLEEVEC® (Imatinib Mesylate), GLIADEL® (Carmustine Implant), GLIADEL WAFER® (Carmustine Implant), Glucarpidase, Goserelin Acetate, HALAVEN® (Eribulin Mesylate), HEMANGEOL® (Propranolol Hydrochloride), HERCEPTIN® (Trastuzumab), HPV Bivalent Vaccine, Recombinant, HPV Nonavalent Vaccine, Recombinant, HPV Quadrivalent Vaccine, Recombinant, HYCAMTIN® (Topotecan Hydrochloride), HYDREA® (Hydroxyurea), Hydroxyurea, Hyper-CVAD, IBRANCE® (Palbociclib), Ibritumomab Tiuxetan, Ibrutinib, ICE, ICLUSIG® (Ponatinib Hydrochloride), IDAMYCIN® (Idarubicin Hydrochloride), Idarubicin Hydrochloride, Idelalisib, IDHIFA® (Enasidenib Mesylate), IFEX® (Ifosfamide), Ifosfamide, IFOSFAMfDUM® (Ifosfamide), IL- 2 (Aldesleukin), Imatinib Mesylate, IMBRUVICA® (Ibrutinib), IMFINZI® (Durvalumab), Imiquimod, IMLYGIC® (Talimogene Laherparepvec), INLYTA® (Axitinib), Inotuzumab Ozogamicin, Interferon Alfa-2b, Recombinant, Interleukin-2 (Aldesleukin), INTRON A® (Recombinant Interferon Alfa-2b), Iodine 1 131 Tositumomab and Tositumomab, Ipilimumab,IRESSA® (Gefitinib), Irinotecan Hydrochloride, Irinotecan Hydrochloride Liposome, ISTODAX® (Romidepsin), Ixabepilone, Ixazomib Citrate, IXEMPRA® (Ixabepilone), JAKAFI® (Ruxolitinib Phosphate), JEB, JEVTANA® (Cabazitaxel), KADCYLA® (Ado- Trastuzumab Emtansine), KEOXIFENE® (Raloxifene Hydrochloride), KEPIVANCE® (Palifermin), KEYTRUDA® (Pembrolizumab), KISQALI® (Ribociclib), KYMRIAH® (Tisagenlecleucel), KYPROLIS® (Carfilzomib), Lanreotide Acetate, Lapatinib Ditosylate, LARTRUVO® (Olaratumab), Lenalidomide, Lenvatinib Mesylate, LENVIMA® (Lenvatinib Mesylate), Letrozole, Leucovorin Calcium, LEUKERAN® (Chlorambucil), Leuprolide Acetate, LEUSTATIN® (Cladribine), LEVULAN® (Aminolevulinic Acid), LINFOLIZIN® (Chlorambucil), LIPODOX® (Doxorubicin Hydrochloride Liposome), Lomustine, LONSURF® (Trifluridine and Tipiracil Hydrochloride), LUPRON® (Leuprolide Acetate), LUPRON DEPOT® (Leuprolide Acetate), LUPRON DEPOT-PED® (Leuprolide Acetate), LYNPARZA® (Olaparib), MARQIBO® (Vincristine Sulfate Liposome), MATULANE® (Procarbazine Hydrochloride), Mechlorethamine Hydrochloride, Megestrol Acetate, MEKINIST® (Trametinib), Melphalan, Melphalan Hydrochloride, Mercaptopurine, Mesna, MESNEX® (Mesna), METHAZOLASTONE® (Temozolomide), Methotrexate, METHOTREXATE LPF® (Methotrexate), Methylnaltrexone Bromide, MEXATE® (Methotrexate), MEXATE-AQ® (Methotrexate), Midostaurin, Mitomycin C, Mitoxantrone Hydrochloride, MITOZYTREX® (Mitomycin C), MOPP, MOZOBIL® (Plerixafor), MUSTARGEN® (Mechlorethamine Hydrochloride) , MUTAMYCIN® (Mitomycin C), MYLERAN® (Busulfan), MYLOSAR® (Azacitidine), MYLOTARG® (Gemtuzumab Ozogamicin), NANOPARTICLE PACLITAXEL® (Paclitaxel Albumin-stabilized Nanoparticle Formulation), NAVELBINE® (Vinorelbine Tartrate), Necitumumab, Nelarabine, NEOSAR® (Cyclophosphamide), Neratinib Maleate, NERLYNX® (Neratinib Maleate), Netupitant and Palonosetron Hydrochloride, NEULASTA® (Pegfilgrastim), NEUPOGEN® (Filgrastim), NEXAVAR® (Sorafenib Tosylate), NILANDRON® (Nilutamide), Nilotinib, Nilutamide, NINLARO® (Ixazomib Citrate), Niraparib Tosylate Monohydrate, Nivolumab, NOLVADEX® (Tamoxifen Citrate), NPLATE® (Romiplostim), Obinutuzumab, ODOMZO® (Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Olaratumab, Omacetaxine Mepesuccinate, ONCASPAR® (Pegaspargase), Ondansetron Hydrochloride, ONIVYDE® (Irinotecan Hydrochloride Liposome), ONTAK® (Denileukin Diftitox), OPDIVO® (Nivolumab), OPP A, Osimertinib, Oxaliplatin, Paclitaxel, Paclitaxel Albumin- stabilized Nanoparticle Formulation, PAD, Palbociclib, Palifermin, Palonosetron Hydrochloride, Palonosetron Hydrochloride and Netupitant, Pamidronate Disodium,Panitumumab, Panobinostat, PARAPLAT® (Carboplatin), PARAPLATIN® (Carboplatin), Pazopanib Hydrochloride, PCV, PEB, Pegaspargase, Pegfilgrastim, Peginterferon Alfa-2b, PEG-INTRON® (Peginterferon Alfa-2b), Pembrolizumab, Pemetrexed Disodium, PERJETA® (Pertuzumab), Pertuzumab, PLATINOL® (Cisplatin), PLATINOL-AQ® (Cisplatin), Plerixafor, Pomalidomide, POMALYST® (Pomalidomide), Ponatinib Hydrochloride, PORTRAZZA® (Necitumumab), Pralatrexate, Prednisone, Procarbazine Hydrochloride, PROLEUKIN® (Aldesleukin), PROLIA® (Denosumab), PROMACTA® (Eltrombopag Olamine), Propranolol Hydrochloride, PROVENGE® (Sipuleucel-T), PURTNETHOL® (Mercaptopurine), PURIXAN® (Mercaptopurine), Radium 223 Dichloride, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, R-CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nonaval ent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Interferon Alfa-2b, Regorafenib, RELISTOR® (Methylnaltrexone Bromide), R- EPOCH, REVLIMID® (Lenalidomide), RHEUMATREX® (Methotrexate), Ribociclib, R- ICE, RITUXAN® (Rituximab), RITUXAN HYCELA® (Rituximab and Hyaluronidase Human), Rituximab, Rituximab and , Hyaluronidase Human, ,Rolapitant Hydrochloride, Romidepsin, Romiplostim, RUBIDOMYCIN® (Daunorubicin Hydrochloride), RUBRACA® (Rucaparib Camsylate), Rucaparib Camsylate, Ruxolitinib Phosphate, RYDAPT® (Midostaurin), Sclerosol Intrapleural Aerosol (Talc), Siltuximab, Sipuleucel-T, SOMATULINE DEPOT® (Lanreotide Acetate), Sonidegib, Sorafenib Tosylate, SPRYCEL® (Dasatinib), STANFORD V, Sterile Talc Powder (Talc), STERITALC® (Talc), STIVARGA® (Regorafenib), Sunitinib Malate, SUTENT® (Sunitinib Malate), SYLATRON® (Peginterferon Alfa-2b), SYLVANT® (Siltuximab), Synribo SYNRIBO® (Omacetaxine Mepesuccinate), TABLOID® (Thioguanine), TAC, TAFINLAR® (Dabrafenib), TAGRISSO® (Osimertinib), Talc, Talimogene Laherparepvec, Tamoxifen Citrate, T ARABINE PFS® (Cytarabine), TARCEVA® (Erlotinib Hydrochloride), TARGRETIN® (Bexarotene), TASIGNA® (Nilotinib), TAXOL® (Paclitaxel), TAXOTERE® (Docetaxel), TECENTRIQ® (Atezolizumab), TEMODAR® (Temozolomide), Temozolomide, Temsirolimus, Thalidomide, THALOMID® (Thalidomide), Thioguanine, Thiotepa, Tisagenlecleucel, TOLAK® (Fluorouracil— Topical), Topotecan Hydrochloride, Toremifene, TORISEL® (Temsirolimus), Tositumomab and Iodine I 131 Tositumomab, TOTECT® (Dexrazoxane Hydrochloride), TPF, Trabectedin, Trametinib, Trastuzumab, TREANDA® (Bendamustine Hydrochloride), Trifluridine and Tipiracil Hydrochloride, TRISENOX® (Arsenic Trioxide), TYKERB® (Lapatinib Ditosylate) , UNITUXIN® (Dinutuximab), UridineTriacetate, VAC, Vandetanib, VAMP, VARUBI® (Rolapitant Hydrochloride), VECTIBIX® (Panitumumab), VelP, VELBAN® (Vinblastine Sulfate), VELCADE® (Bortezomib), VELSAR® (Vinblastine Sulfate), Vemurafenib, VENCLEXTA® (Venetoclax), Venetoclax, VERZENIO® (Abemaciclib), VIADUR® (Leuprolide Acetate), VIDAZA® (Azacitidine), Vinblastine Sulfate, VINCASAR PFS® (Vincristine Sulfate), Vincristine Sulfate, Vincristine Sulfate Liposome, Vinorelbine Tartrate, VIP, Vismodegib, VISTOGARD® (Uridine Triacetate), VORAXAZE® (Glucarpidase), Vorinostat, VOTRIENT® (Pazopanib Hydrochloride), VYXEOS® (Daunorubicin Hydrochloride and Cytarabine Liposome), WELLCOVORIN® (Leucovorin Calcium), XALKORI® (Crizotinib), XELODA® (Capecitabine), XELIRI, XELOX, XGEVA® (Denosumab), XOFIGO® (Radium 223 Dichloride), XTANDI® (Enzalutamide), YERVOY® (Ipilimumab), YONDELIS® (Trabectedin), ZALTRAP® (Ziv-Aflibercept), ZARXIO® (Filgrastim), ZEJULA® (Niraparib Tosylate Monohydrate), ZELBORAF® (Vemurafenib), ZEVALIN® (Ibritumomab Tiuxetan), ZINECARD® (Dexrazoxane Hydrochloride), Ziv-Aflibercept, ZOFRAN® (Ondansetron Hydrochloride), ZOLADEX® (Goserelin Acetate), Zoledronic Acid, ZOLINZA® (Vorinostat), ZOMETA® (Zoledronic Acid), ZYDELIG® (Idelalisib), ZYKADIA® (Ceritinib), and / or ZYTIGA® (Abiraterone Acetate).

[0150] In some aspects, the cancer or tumor can be hematologic cancer, lymphoma, colorectal cancer, colon cancer, lung cancer, head and neck cancer, ovarian cancer, prostate cancer, testicular cancer, renal cancer, skin cancer, melanoma, cervical cancer, brain cancer, glioblastoma, pancreatic cancer, gastric cancer, or breast cancer. In some aspects, the subject can have a solid tumor. In other aspects, cancer can be acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia, acute lymphoblastic leukemia, myelofibrosis, or multiple myeloma. In yet other aspects, cancer or solid tumor can be leukemia, lymphoma, sarcoma, or carcinoma, any of which may originate in the marrow, brain, lung, breast, pancreas, liver, head and neck, skin, reproductive tract, prostate, colon, liver, kidney, intraperitoneum, bone, joint, or eye.EXAMPLESExample 1: Engineering antigen-specific T cells for CAR T cell therapy by antigen- presenting lipid nanoparticles

[0151] Antigen (Ag)-specific T cells express T cell receptors (TCRs) that recognize processed peptide Ags bound to major histocompatibility complex (MHC) molecules, which forms the basis for their exquisite specificity. A study was conducted which developed Ag- presenting lipid nanoparticles (APNs) to deliver CAR mRNA to defined T cell subsets for CART cell therapy. APNs include lipid nanoparticles (LNP) encapsulated with CAR mRNA and surface-decorated with major histocompatibility complex (MHC) molecules. The data shown below focus on B-cell maturation antigen (BCMA) CAR in the context of multiple myeloma (MM). It is anticipated that in situ production of aBCMA CAR T cells will be amenable for translation to other CAR constructs and indications including CD 19+ cancers. As demonstrated by the data below, the disclosed APNs can directly target and program human antigen-specific T cells in vivo with functional CAR mRNA to circumvent the costly and lengthy ex vivo manufacturing process.

[0152] Antigen-specific targeting to human T cells ex vivo'. The nanoparticle matrix of this approach includes but not limited to liposomes, lipid nanoparticles, and polymeric nanoparticles. Surfaces of the nanoparticles are functionalized with MHC carrying peptide antigens of interest, and the resulting product is abbreviated as antigen-presenting nanoparticles (APNs) henceforth. The method described here is demonstrated in MHC class 1 for CD8+ T cell modulation, but it can broadly use for both class 1 (target CD8+ T cells) and class 2 (target CD4+ T cells) MHC. To achieve this surface pMHC conjugation in a selective manner, the study engineered the C-terminal of the heavy chain, a major component of pMHC, with a cysteine to allow for selective conjugations based on thiol-maleimide chemistry. This bioconjugation approach is applicable to both murine and human MHC by engineering human pMHC with C-terminal cysteine. LNPs functionalized with human pMHC (a.k.a. human leukocyte antigen, HLA) carrying influenza (flu) peptide antigens specifically targeted fluspecific CD8+ T cells isolated from human blood in the presence of other untargeted CD8+ T cells (FIG. 1).

[0153] LNP formulation screening for primary T cell transfection '. The study screened 8 different ionizable lipids for their ability to transfect primary mouse CD8+ mouse T cells and human CD3+ T cells. The study used mRNA encoding firefly luciferase (flue) as the model reporter to evaluate the transfection by bioluminescence. It was found that most LNP formulations resulted in over 2-log-fold transfection of CD3+ human T cells and CD8+ mouse T cells (FIG. 2). The ALC0315 LNP formulation demonstrated highest transfection potency in human T cells. The study therefore selected the ALC0315 LNP formulation to formulate APNs for primary human T cell transfection.

[0154] Optimization of pMHC-PEG lipid amount in APN for I AV-specific T cell transfection'. Molar percentage of PEG-conjugated lipids in the final LNP formulations has shown to impact the mRNA transfection efficiency in Jurkat cells. Therefore, to achieve IAV- specific T cell transfection, the study optimized the weight ratio of pMHC-PEG to total lipidin the APN formulation (FIG. 3). To target human lAV-specific T cells, the study functionalized APNs with HLA-A2.1 - an allele expressed by >30% of the US population to present an immunodominant IAV peptide (GILGFVFTL (SEQ ID NO: 1), denoted as HLA / IAV). The study formulated APNs with HLA / CMV as a non-cognate control. It was found that primary human T cells treated with HLA / IAV APNs show significantly higher expression of model firefly luciferase mRNA than T cells treated with bare LNPs and HLA / CMV APNs. Moreover, the transfection efficiency peaked at pMHC-PEG to total lipid ratio at 0.75: 1 and reduced when the pMHC-PEG was reduced.

[0155] APNs transfected human flu-specific T cells with model VHH mRNA in vivo'. To target human flu-specific T cells, the study functionalized APNs with HLA-A2.1 - an allele expressed by >30% of the US population- to present an immunodominant flu peptide (GILGFVFTL (SEQ ID NO: 1)). Since the frequency of flu-specific T cells in PBMCs isolated from healthy donors are usually low (<0.1%), the study first expanded flu-specific T cells by peptide pulse to mimic the influenza vaccine that will be given to patients before APN injection in the intended use of the proposed approach. To mimic the flu-specific T cell frequency after vaccination in human (-0.5-1%), NSG mice were infused with 12 million flu-peptide pulsed PBMC (containing -1.75 million lAV-specific CD8 T cells), resulting in lAV-specific CD8 T cells to be -1% of total splenocytes at 48 hr after the cell transfer (FIGS. 4A-4B). Even at such low frequency, intravenous injection of HLA / flu APNs to these NSG mice resulted in -90% transfection of lAV-specific CD8 T cells with a surface-anchored VHH model protein. By contrast, PBS and APNs carrying nano-Luciferase (nLuc) mRNA did not result in detectable VHH transfection in flu-specific CD8 T cells (FIGS. 4C-4D).

[0156] Design and characterization of aBCMA CAR-encoded mRNA for functional CAR expression by human T cells'. The study designed and synthesized a mRNA construct based on a clinical grade aBCMA CAR (courtesy of Riddell Lab, Fred Hutch) (SEQ ID NO: 2) (FIG. 5A). The study validated the mRNA sequence by electroporating primary human T cells followed by staining with fluorophore-labeled recombinant BCMA proteins to evaluate aBCMA CAR expression. It was observed that -99% of the human T cells electroporated with aBCMA CAR mRNA were CAR+ at 24 hr after electroporation (FIG. 5B). To test the effector functions of the aBCMA CAR T cells, the study performed an in vitro killing assay where BCMA+ CAG multiple myeloma tumor cells (target cells) were co-incubated with either CAR- mRNA electroporated human T cells (denoted as CAR) or mock electroporated human T cells (denoted as mock). Significantly higher cytotoxicity was observed resulting from CAR-mRNA transfected human T cells than from mock electroporated T cells, indicating that the CARs caninduce cytotoxicity against cancer cells (FIG. 5C). These results show the established protocols for synthesis of CAR mRNA and for characterizing CAR T cells using ex vivo cytotoxicity assays.

[0157] APNs transfect human flu-specific T cells with aBCMA CAR mRNA in immunodeficient NSG mice'. Intravenous injection of HLA / flu APNs to these NSG mice preinfused with peptide pulsed PBMC resulted in a dose-dependent aBCMA CAR transfection with -40% transfection of flu-specific T cells at 1 mg / kg dose (FIGS. 6A-6B). By contrast, no aBCMA CAR expression was observed in NSG mice treated with non-cognate HLA / CMV APNs displaying a CMV peptide epitope. Similarly, PBS and APNs carrying aCD19 CAR mRNA did not result in detectable aBCMA CAR transfection in flu-specific CD8 T cells (FIGS. 6B-6C) To study whether those in vivo transfected aBCMA CAR T cells were functional against multiple myeloma cancer cells expressing BCMA, the study isolated CD8 T cells from the spleens of mice treated with PBS (carrier control), APNs loaded with VHH mRNA (mRNA control), and APNs loaded with aBCMA CAR mRNA (FIG. 6A). The isolated CD8 T cells were subsequently co-incubated with U266 cells, a human multiple myeloma cell line. The U266 cells were luciferized to enable cytotoxicity evaluation based on bioluminescence. It was found that CD8 T cells isolated from mice treated with aBCMA CAR APNs showed significantly higher cytotoxicity against U266 cells (FIG. 6D), indicating that the in vivo transfected aBCMA CAR were functional against BCMA-expressing U266 cells.

[0158] APN-engineered human IA V-specific T cells reduced tumor burden in NSG mice bearing human multiple myeloma'. Having achieved the aBCMA CAR transfection in IAV- specific T cells in vivo, the study then tested whether those CAR T cells were functional against MM. To test this, NSG mice were first systematically inoculated with BCMA+ U266 cancer cells which were luciferized to allow tracking tumor growth kinetics by live animal imaging using an IVIS Spectrum CT system. At 11 days after tumor inoculation, lAV-specific T cells were adoptively transferred to the tumor bearing mice, followed by intravenous APN injection at 24 hours after the cell transfer (FIG. 7A). It was found that a single dose (0.5 mg / kg) of APNs carrying BCMA CAR mRNA resulted in tumor regression as compared to the PBS control group (FIG. 7B). These data suggest that APNs can transfect lAV-specific T cells with functional aBCMA CAR in vivo.

[0159] APN transfected human influenza-specific T cells with functional aBCMA CAR in vitro'. To strengthen the translational potential of antigen-presenting nanoparticles (APNs) for in vivo chimeric antigen receptor (CAR) T cell production, the study tested the activity of APNs in transfecting human influenza (flu)-specific T cells with mRNA encoding a clinical gradeaBCMA CAR. As a first proof-of-concept experiment, the study first tested whether APNs could transfect human lAV-specific T cells with functional mRNA in vitro. To do this, human peptide major histocompatibility complex (pMHC) composed of human HLA-A*02:01 class I heavy chain engineered with C-terminal cysteine (SEQ ID NO: 3), human beta-2 microglobulin (SEQ ID NO: 4), and a peptide derived from either influenza (Flu, GILGFVFTL (SEQ ID NO: 1)) was expressed. This pMHC was functionalized with lipid nanoparticles (LNPs) loaded with mRNA encoding either nano-Luciferase (nLuc) or aBCMA CAR to form HLA / Flu APNs that deliver mRNA to human Flu-specific T cells. The study incubated HLA / Flu APNs with enriched human lAV-specific T cells in vitro for 24 hours (FIG. 8A). It was found that HLA / GIL APNs not only resulted in the bioluminescence expression from nLuc transfection in lAV-specific T cells (FIG. 8B), but also induced a dose-dependent CAR expression in lAV-specific T cells. Notably, non-cognate T cells incubated with HLA / GIL APNs only show aBCMA CAR close to background levels (FIGS. 8C-8D). The study next tested the anti-cancer efficacy of the APN-transfected aBCMA CAR T cells by co-incubating the CAR T cells with BCMA+ MM1R MM cancer cells that constitutively express renilla luciferase for evaluating cytotoxicity (FIG. 8A). After 24-hour co-incubation, MM1R coincubated with T cells transfected nLuc mRNA-loaded APNs showed comparable viability as PBS-treated MM1R (FIG. 8E). In contrast, T cells transfected aBCMA CAR mRNA-loaded APNs resulted in significant lower viability of MM1R. These data indicate that APN can transfect human influenza-specific T cells with functional aBCMA CAR in vitro.

[0160] Design and characterization of anti-human GPRC5D CAR-encoded mRNA for functional CAR expression by human T cells'. CAR T cell therapy targeting a single tumor antigen can induce remissions of hematologic cancers but relapses often occur due to the outgrowth of tumor cells with absent or low expression of the antigen. To mitigate this issue, strategies engineering CAR T cells ex vivo with two CARs against two tumor antigens (BCMA and GPRC5D) have shown promising results for patients with relapsed or refractory multiple myeloma (MM). As shown in FIG. 8, APNs transfect human influenza (flu)-specific T cells with functional BCMA CAR mRNA both in vitro and in vivo. To test the feasibility of using APNs to deliver both BCMA CAR mRNA and GPRC5D-CAR mRNA to mitigate the antigen escape-mediated treatment failure for MM patients, the study first designed and synthesized a mRNA construct encoding anti-human GPRC5D (aGPRC5D) CAR (SEQ ID NO: 5) (FIG. 9A). To validate the mRNA sequence, the study electroporated primary human T cells with the mRNA. As the CAR construct include three strep-tag peptide sequences in the hinge domain, the study used anti-strep tag antibody to stain for the strep-tag as a surrogate marker for CARexpression. It was observed that -99% of the human T cells electroporated with aGPRC5D CAR mRNA expressed CAR at 24 hr after electroporation (FIG. 9B). To test the effector functions of the aGPRC5D CAR T cells, the study included an in vitro killing assay where GPRC5D+ MM1R multiple myeloma tumor cells were co-incubated with either CAR-mRNA electroporated human T cells (denoted as CAR), GFP-mRNA electroporated human T cells (denoted as GFP), or mock electroporated human T cells (denoted as mock). Significantly higher cytotoxicity (FIG. 9C) and human IFNy (h IFNy) secretion (FIG. 9D) was observed resulting from CAR-mRNA transfected human T cells than from GFP mRNA or mock electroporated T cells. Those data indicate that aGPRC5D CAR mRNA can induce cytotoxicity of T cells against cancer cells.

[0161] APNs deliver aBCMA CAR mRNA and aGPRC5D CAR mRNA to virus-specific T cells enriched from a multiple myeloma patient. Leveraging the ability of APNs to target different antigen-specific T cell subsets in the body, the study tested APNs for co-delivery of aBCMA CAR mRNA and aGPRC5D CAR mRNA to flu-specific T cells and cytomegalovirus (CMV)-specific T cells (FIG. 10A), respectively, to mitigate the antigen escape-mediated treatment failure in MM patients. The study first pulsed the frozen peripheral blood mononuclear cells (PBMCs) isolated MM patients with peptides to expand flu-specific T cells and CMV-specific T cells. In the in vitro cell culture setting, equal numbers of Flu-specific T cells and CMV-specific T cells were mixed, and treated with one of the following four conditions: (1) untransfected, (2) APNs with pMHC to target Flu-specific T cells and aBCMA CAR mRNA (denoted as HLA / Flu BCMA CAR APN), (3) APNs with pMHC to target CMV- specific T cells and aGPRC5D CAR mRNA (denoted as HLA / CMV GPRC5D CAR APN), or (4) a combination of HLA / Flu BCMA CAR APN and HLA / CMV GPRC5D CAR APN (FIG. 10B) The CMV-derived peptide sequence that was used to refold and form HLA / CMV APN is NLVPMVATV (SEQ ID NO: 6). It was found that HLA / Flu BCMA CAR APN preferentially transfected Flu-specific T cells, while HLA / CMV GPRC5D CAR APN selectively transfected CMV-specific T cells. Moreover, the combination of HLA / Flu BCMA CAR APN and HLA / CMV GPRC5D CAR APN resulted in CAR expression in both Fluspecific T cells and CMV-specific T cells. Those results indicate that APNs can transfect two virus-specific T cells isolated from MM patients with CAR mRNA.

[0162] In vivo transfected aBCMA CAR T cells transfected by APNs induced anti-cancer efficacy in NSG mice bearing human MM cancer'. To test whether the aBCMA CAR T cells transfected by APNs are functional against MM in NSG mice, the study first systemically inoculated NSG mice with BCMA+ U266 cancer cells which were luciferized to allow trackingtumor growth kinetics by live animal imaging using an IVIS Spectrum CT system. At 6 days after tumor inoculation, the study adoptively transferred enriched lAV-specific T cells to the tumor bearing mice, followed by intravenous APN injection (0.5 mg / kg mRNA dose) at 24 hours after the cell transfer (FIG. 11 A). A total of 5 doses of APNs were given to the mice every 5 days. Similar to the gold standard aBCMA CAR transduced by lentivirus ex vivo (ex vivo CAR), it was found that APNs carrying aBCMA CAR mRNA resulted in tumor regression as compared to the PBS control group (FIGS. 11B-11C). These data suggest that APNs can transfect lAV-specific T cells with functional aBCMA CAR in vivo.

[0163] Dendritic cell vaccination improved in vivo CAR T cell therapy. Increasing the in vivo proliferation and persistence of CAR T cells has been suggested as one of the approaches to improve the treatment outcome of aBCMA CAR T cell therapy. Vaccinations have been developed as approaches to expand CAR T cells and enhance their anti-cancer responses by stimulating T cells through CAR or their endogenous TCRs. Here, leveraging the endogenous T cell receptors on lAV-specific T cells, it was hypothesized that seasonal influenza vaccines could be used to expand and improve the persistence of aBCMA CAR-expressing, IAV- specific T cells, thereby further enhancing the treatment outcome of CAR T cell therapy. To test this hypothesis, the study used dendritic cells pulsed with IAV peptides to model the seasonal influenza vaccination in immunodeficient NSG mice bearing xenograft human MM cancers. Prior to in vivo testing, the study first incubated IAV peptide-pulsed, monocyte- derived DCs (mDCs) with human PBMC to see if DC vaccination could enhance lAV-specific T cell expansion in the in vitro setting. lAV-specific CD8 T cells (CD8+ IAV tetramer+, top right quadrant) expanded overtime as compared to PBMC alone without co-incubation with DCs (FIG. 12A). In the preliminary study testing whether DC vaccination can enhance in vivo APN-transfected CAR T cell therapy, a single dose of APN loaded with aBCMA CAR mRNA at 7 days after DC vaccination significantly reduced tumor burden as compared to mice treated with PBS alone or APNs and DCs without peptide pulse. These data suggest the potential of using vaccination to boost the CAR T cell therapy produced by APNs in vivo (FIG. 12B).

[0164] Any patents, applications and publications as listed throughout this document are hereby incorporated by reference in their entirety herein.SEQUENCESSEQ ID NO: 1GILGFVFTLSEQ ID NO: 2 (mRNA sequence of anti-human BCMA CAR)MLLLVTSLLLCELPHPAFLLIPQIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWV KRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTAT YFCALDYSYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVLTQSPPSLAMSLG KRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDF TLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIKNWSHPQFEKGGGSGGGSGGNW SHPQFEKGGGGSGGGGSNWSHPQFEKGGGSGGGSGGESKYGPPCPPCPMFWVLVV VGGVLACYSLLVTVAFIIFWVRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK PRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA LHMQALPPRSEQ ID NO: 3 (DNA sequence of human HLA-A*02:01 class I heavy chain engineered with C-terminal cysteine)ATGGGTTCTCATTCTATGAGATATTTCTTCACATCCGTGTCCCGGCCCGGCCGCG GGGAGCCCCGCTTCATCGCAGTGGGCTACGTGGACGACACGCAGTTCGTGCGGT TCGACAGCGACGCCGCGAGCCAGAGGATGGAGCCGCGGGCGCCGTGGATAGAG CAGGAGGGTCCGGAGTATTGGGACGGGGAGACACGGAAAGTGAAGGCCCACTC ACAGACTCACCGAGTGGACCTGGGGACCCTGCGCGGCTACTACAACCAGAGCGA GGCCGGTTCTCACACCGTCCAGAGGATGTATGGCTGCGACGTGGGGTCGGACTG GCGCTTCCTCCGCGGGTACCACCAGTACGCCTACGACGGCAAGGATTACATCGCCCTGAAAGAGGACCTGCGCTCTTGGACCGCGGCGGACATGGCAGCTCAGACCACC AAGCACAAGTGGGAGGCGGCCCATGTGGCGGAGCAGTTGAGAGCCTACCTGGAG GGCACGTGCGTGGAGTGGCTCCGCAGATACCTGGAGAACGGGAAGGAGACGCTG CAGCGCACGGACGCCCCCAAAACGCATATGACTCACCACGCTGTCTCTGACCAT GAAGCCACCCTGAGGTGCTGGGCCCTGAGCTTCTACCCTGCGGAGATCACACTG ACCTGGCAGCGGGATGGGGAGGACCAGACCCAGGACACGGAGCTCGTGGAGAC CAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGGCGGCTGTGGTGGTGCCTTCTGGACAGGAGCAGAGATACACCTGCCATGTGCAGCATGAGGGTTTGCCCAAGCCCCTCACCCTGAGATGGGAGCCGGCTAGCTGCSEQ ID NO: 4 (DNA sequence of human beta-2 microglobulin)ATCCAGCGTACCCCGAAGATTCAGGTGTACTCACGCCATCCAGCTGAGAACGGCAAAAGTAACTTTCTGAATTGCTATGTCTCGGGCTTCCACCCGTCCGATATCGAAGTGGACCTTCTGAAAAACGGTGAACGGATCGAAAAAGTCGAGCATTCTGACTTATCATTCAGCAAAGATTGGTCTTTTTACTTGTTATATTATACCGAATTTACCCCGACAGAAAAAGATGAGTATGCATGCCGTGTCAACCACGTTACGCTGTCCCAGCCGAAAATCGTGAAATGGGATCGTGATATGTAASEQ ID NO: 5 (mRNA sequence of anti-human GPRC5D CAR)AUGCUGCUGCUCGUGACAAGCCUGCUGCUGUGCGAGCUGCCCCACCCUGCCUUUCUGCUGAUCCCCAUGAAGAAAACGGCGAUUGCCAUAGCCGUGGCGCUCGCCGGGUUUGCGACAGUUGCACAAGCUGCGGAACUUCAAAGCGUAGUCACGCAACCACCAAGUGUUUCAGCCGCUCCCGGCCAACGGGUUACAAUAUCAUGUUCCGGAGGGUCUAGCAACAUCGGAAAUAAUUACGUAUCCUGGUUCCAACAGCUCCCAAGGACAGCUCCGAAAUUGCUGAUUUACGACAAUAAUAAGAGGCCCUCAGGGAUACCAGAUAGGUUCUCAGGCAGCAAAUCCGGUACAAGCGCGGCACUGGACAUUACAGUAUUGCAAACGGGCGAUGAAGCUGAUUACUACUGUGGAACGUGGGAUUCUUCUCUCCGAAAUUGGGUUUUUGGAGGCGGAACCAAGCUCACUGUACUUGGGUCAAGAGGGGGUGGGGGGUCUGGAGGCGGUGGGUCUGGAGGAGGCGGCAGCCUCGAAAUGGCACAAAUGCAGCUCGUACAGAGUGGCGCUGAAGUCAAAAAACCCGGGGCCUCAGUAAAAGUCUCCUGCAAAGCCUCAGGGUAUACGUUCACAAGCUACUACAUGCACUGGGUUAGGCAAGCUCCAGGCCAAGGACUCGAAUGGAUGGGGAUAAUCAAUCCGUCCGGAGGAAGUACGUCAUACGCUCAGAAGUUCCAAGGCCGCGUUACGAUGACUCGGGACACAUCAACCUCUACAGUAUACAUGGAACUCUCCAGCCUCAGGAGUGAAGAUACAGCAGUUUAUUAUUGCGCCCGGGGUCAGAAGUACCACAGCCAGUAUUCUCGGGGGGGCACCGGGGGCGGAAUGACGCAAGAUAUGUGGGGUCAAGGUACACUGGUAACUGUCUCAAGCAAUUGGAGUCAUCCUCAAUUCGAAAAAGGAGGCGGUUCUGGAGGUGGAAGCGGUGGCAACUGGAGCCACCCACAGUUCGAAAAAGGAGGUGGAGGUUCAGGUGGUGGAGGCUCUAACUGGUCCCAUCCGCAGUUCGAGAAGGGUGGAGGCUCUGGCGGAGGUAGCGGUGGCGAAUCUAAGUACGGACCGCCCUGCCCCCCUUGCCCUAUGUUCUGGGUGCUGGUGGUGGUCGGAGGCGUGCUGGCCUGCUACAGCCUGCUGGUCACCGUGGCCUUCAUCAUCUU UUGGGUGCGCAGCAAACGGGGCAGAAAGAAACUCCUGUAUAUAUUCAAACAA CCAUUUAUGAGACCAGUACAAACUACUCAAGAGGAAGAUGGCUGUAGCUGCC GAUUUCCAGAAGAAGAAGAAGGAGGAUGUGAACUGCGGGUGAAGUUCAGCAG AAGCGCCGACGCCCCUGCCUACCAGCAGGGCCAGAAUCAGCUGUACAACGAGCUGAACCUGGGCAGAAGGGAAGAGUACGACGUCCUGGAUAAGCGGAGAGGCCG GGACCCUGAGAUGGGCGGCAAGCCUCGGCGGAAGAACCCCCAGGAAGGCCUGU AUAACGAACUGCAGAAAGACAAGAUGGCCGAGGCCUACAGCGAGAUCGGCAU GAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGCCUGUAUCAGGGCCUGU CCACCGCCACCAAGGAUACCUACGACGCCCUGCACAUGCAGGCCCUGCCCCCAAGGSEQ ID NO: 6NLVPMVATVSEQ ID NO: 7 atgaaatgggtcacatttatatctctgctcttccttttctcttcagcctacagccaggtacagttgcaggagtccggaggtggtctggtaca accaggtgggtccctcagattgtcttgtgcagctagtggctttacgctcgactactattatatcgggtggtttcggcaagcaccgggtaaa gagagggaggctgttagctgtatcagcggctcttcagggtccacgtattaccctgacagtgttaaagggagatttaccatatcccgcgat aacgcaaagaacactgtgtacttgcagatgaatagcctgaagcccgaggacacagccgtttactactgtgccacgattcgctcctcttc atggggaggatgcgttcattacgggatggattactggggcaaaggcactcaggtgacggttagctctggaggcgggggcagccacg agaccacccccaacaaggggagcgggaccacgtccggcacaactagactgctttccggccatacatgctttacacttactgggctgct ggggactcttgtaactatggggctcctcacatgaSEQ ID NO: 8MKWVTFISLLFLFSSAYSQVQLQESGGGLVQPGGSLRLSCAASGFTLDYYYIGWFRQ APGKEREAVSCISGSSGSTYYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC ATIRSSSWGGCVHYGMDYWGKGTQVTVSSGGGGSHETTPNKGSGTTSGTTRLLSGH TCFTLTGLLGTLVTMGLLT

Claims

What is claimed is:

1. An antigen-presenting nanoparticle (APN), comprising: a major histocompatibility complex (MHC) presented on a surface of the APN and comprising a peptide epitope; and an encapsulated nucleic acid encoding a chimeric antigen receptor (CAR), antibody, or antibody fragment.

2. The APN of claim 1, wherein the MHC is MHC class I or MHC class II.

3. The APN of claim 1 or 2, wherein the peptide epitope is an epitope of a virus, bacteria, fungus, or parasite.

4. The APN of claim 3, wherein the peptide epitope is a viral epitope from a virus selected from the group consisting of Herpes Simplex virus- 1, Herpes Simplex virus-2, Varicella-Zoster virus, Epstein-Barr virus, Cytomegalovirus, Human Herpes virus-6, Variola virus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus (such as, for example, a SARS- CoV-2 infection including, but not limited to the SARS-CoV-2 Bl.351 variant, SARS-CoV- 2B.1.1.7 (alpha), SARS-CoV-2B.1.1.7 variant mutant N501 Y (alpha), SARS-CoV-2 delta variant, SARS-CoV-2 P.l variant, SARS-CoV-2 with T487K, P681R, and L452R mutations in B.1.617.2 (Delta), SARS-CoV-2 with K417N mutation in AY.1 / AY.2 (Delta plus), SARS- CoV-2 with D614G, P681H, and D950N mutations in B.1.621 (Mu), SARS-CoV-2 with G75V, T76I, A246-252, L452Q, F490S, D614G, and T859N mutations in C.37 (Lambda), SARS-CoV-2 with T478K, Q498R, and H655Y mutations in B.1.1.529 (Omicron)), Influenza virus A, Influenza virus B, Measles virus, Polyomavirus, Human Papilomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Reovirus, Yellow fever virus, Zika virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valley fever virus, West Nile virus,Rift Valley fever virus, Rotavirus A, Rotavirus B, Rotavirus C, Sindbis virus, Simian Immunodeficiency virus, Human T-cell Leukemia virus type-1, Hantavirus, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type-1, and Human Immunodeficiency virus type-2.

5. The APN of claim 3, wherein the peptide epitope is a bacterial epitope from a bacteria selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis strain BCG, BCG substrains, Mycobacterium avium, Mycobacterium intracellular, Mycobacterium africanum, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium ulcerans, Mycobacterium avium subspecies paratuberculosis, Nocardia asteroides, other Nocardia species, Legionella pneumophila, other Legionella species, Acetinobacter baumanii, Salmonella typhi, Salmonella enterica, other Salmonella species, Shigella boydii, Shigella dysenteriae, Shigella sonnei, Shigella flexneri, other Shigella species, Yersinia pestis, Pasteurella haemolytica, Pasteurella multocida, other Pasteurella species, Actinobacillus pleuropneumoniae, Listeria monocytogenes, Listeria ivanovii, Brucella abortus, other Brucella species, Cowdria ruminantium, Borrelia burgdorferi, Bordetella avium, Bordetella pertussis, Bordetella bronchiseptica, Bordetella trematum, Bordetella hinzii, Bordetella pteri, Bordetella parapertussis, Bordetella ansorpii other Bordetella species, Burkholderia mallei, Burkholderia psuedomallei, Burkholderia cepacian, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Coxiella burnetii, Rickettsial species, Ehrlichia species, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Escherichia coli, Vibrio cholerae, Campylobacter species, Neiserria meningitidis, Neiserria gonorrhea, Pseudomonas aeruginosa, other Pseudomonas species, Haemophilus influenzae, Haemophilus ducreyi, other Hemophilus species, Clostridium tetani, other Clostridium species, Yersinia enterolitica, and other Yersinia species.

6. The APN of claim 3, wherein the peptide epitope is a fungal epitope from a fungi selected from the group consisting of Candida albicans, Cryptococcus neoformans, Histoplama capsulatum, Aspergillus fumigatus, Coccidiodes immitis, Paracoccidiodes brasiliensis, Blastomyces dermitidis, Pneumocystis camii, Penicillium marneffi, and Alternaria altemata.

7. The APN of claim 3, wherein the peptide epitope is a parasitic epitope from a parasite selected from the group consisting of Toxoplasma gondii, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, other Plasmodium species, Entamoeba histolytica, Naegleria fowleri, Rhinosporidium seeberi, Giardia lamblia, Enterobius vermicularis, Enterobius gregorii, Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus, Cryptosporidium spp., Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, other Leishmania species, Diphyllobothrium latum, Hymenolepis nana, Hymenolepis diminuta, Echinococcus granulosus, Echinococcus multilocularis, Echinococcus vogeli, Echinococcus oligarthrus, Diphyllobothrium latum, Clonorchis sinensis; Clonorchis viverrini, Fasciola hepatica, Fasciola gigantica, Dicrocoelium dendriticum, Fasciolopsis buski, Metagonimus yokogawai, Opisthorchis viverrini, Opisthorchis felineus, Clonorchis sinensis, Trichomonas vaginalis, Acanthamoeba species, Schistosoma intercalatum, Schistosoma haematobium, Schistosoma japoni cum, Schistosoma mansoni, other Schistosoma species, Trichobilharzia regenti, Trichinella spiralis, Trichinella britovi, Trichinella nelsoni, Trichinella nativa, and Entamoeba histolytica.

8. The APN of any one of claims 1-7, wherein the CAR, antibody, or antibody fragment encoded by the encapsulated nucleic acid recognizes a cancer or tumor antigen.

9. The APN of claim 8, wherein the cancer antigen is B cell maturation antigen (BCMA), G protein-coupled receptor class C group 5 member D (GPRC5D), CD 19, guanylate cyclase-C (GUCY2C), human epidermal growth factor receptor 2 (HER2), CD20, CD22, mucin-1 (MUC1), mesothelin, or VHH (e.g., a sequence comprising 80% similarity or more to SEQ ID NO: 8).

10. The APN of claim 9, wherein the CAR encoded by the encapsulated nucleic acid comprises 80% similarity or more to SEQ ID NO: 2 or SEQ ID NO: 5.

11. The APN of any one of claims 1-10, wherein the APN is a lipid nanoparticle, a liposome, or a polymeric nanoparticle.

12. The APN of claim 11, wherein the APN comprises at least one ionizable lipid, at least one PEGylated lipid, or a combination thereof.

13. The APN of claim 12, wherein the at least one ionizable lipid comprises cKK-E12, SM102, MC3, Lipid-5, Alc-0315, LP01, Lipid A9, or any combination thereof.

14. The APN of any one of claims 12-13, wherein the APN comprises from about 30 mol% to about 60 mol% of the ionizable lipid.

15. The APN of any one of claims 12-14, wherein the at least one PEGylated lipid comprises ALC-0159, DMG-PEG, DSPE-PEG, PEG14-2000, or any combination thereof.

16. The APN of any one of claims 12-15, wherein the APN comprises from about 0.5 mol% to about 5 mol% of the PEGylated lipid.

17. The APN of any one of claims 12-16, wherein a ratio of PEGylated lipid to total lipids is from about 0.01:1 to about 0.05:1.

18. A method of engineering a CAR T cell, the method comprising exposing an antigenspecific T cell to the APN of any one of claims 1-17.

19. A method of engineering a CAR T cell, the method comprising exposing an antigenspecific T cell to an antigen presenting nanoparticle (APN), the APN comprising:a major histocompatibility complex (MHC) presented on a surface of the APN and comprising a peptide epitope which is recognized by the antigen-specific T cell; and an encapsulated nucleic acid encoding a chimeric antigen receptor (CAR), antibody, or antibody fragment.

19. The method of claim 18, wherein the T cell is a CD8+ T cell, and wherein the MHC is MHC class I.

20. The method of claim 18, wherein the T cell is a CD4+ T cell, and wherein the MHC is MHC class II.

22. The method of any one of claims 18-21, wherein the T cell is a human T cell.

23. The method of any one of claims 18-22, wherein the method is carried out in vitro, in vivo, or ex vivo.

24. An engineered CAR T cell produced by the method of any one of claims 18-23.

25. A method of treating and / or preventing a cancer or tumor in a subject, the method comprising administering to the subject the APN of any one of claims 1-17.

26. A method of treating and / or preventing a cancer or tumor in a subject, the method comprising administering to the subject an antigen-presenting nanoparticle (APN) comprising: a major histocompatibility complex (MHC) presented on a surface of the APN and comprising a peptide epitope which is recognized by the antigen-specific T cells; andan encapsulated nucleic acid encoding a chimeric antigen receptor (CAR), antibody, or antibody fragment that recognizes a cancer or tumor antigen.

27. The method treating and / or preventing a cancer or tumor of claim 25 or 26, further comprising administering to the subject a peptide pulse or vaccine to the subject prior to administering the APN; wherein the peptide comprises an epitope for a microbe to which the subject has been exposed; wherein the vaccine expresses the same epitope as the APN.

28. The method treating and / or preventing a cancer or tumor of claim 27, wherein the peptide epitope or epitope expressed in the vaccine comprise an epitope of a virus, bacteria, fungus, or parasite.

29. The method of treating and / or preventing a cancer or tumor of claim 28, wherein the peptide epitope is a viral epitope from a virus selected from the group consisting of Herpes Simplex virus- 1, Herpes Simplex virus-2, Varicella-Zoster virus, Epstein-Barr virus, Cytomegalovirus, Human Herpes virus-6, Variola virus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus (such as, for example, a SARS-CoV-2 infection including, but not limited to the SARS-CoV-2 B 1.351 variant, SARS-CoV-2B.1.1.7 (alpha), SARS-CoV-2B.1.1.7 variant mutant N501Y (alpha), SARS-CoV-2 delta variant, SARS-CoV-2 P.l variant, SARS-CoV-2 with T487K, P681R, and L452R mutations in B.1.617.2 (Delta), SARS-CoV-2 with K417N mutation in AY.1 / AY.2 (Delta plus), SARS-CoV-2 with D614G, P681H, and D950N mutations in B.1.621 (Mu), SARS-CoV-2 with G75V, T76I, A246-252, L452Q, F490S, D614G, and T859N mutations in C.37 (Lambda), SARS-CoV-2 with T478K, Q498R, and H655Y mutations in B.1.1.529 (Omicron)), Influenza virus A, Influenza virus B, Measles virus, Polyomavirus, Human Papilomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Reovirus, Yellow fever virus, Zika virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, Rotavirus A, Rotavirus B, Rotavirus C, Sindbis virus, Simian Immunodeficiency virus, Human T-cell Leukemiavirus type-1, Hantavirus, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type-1, and Human Immunodeficiency virus type-2.

30. The method of treating and / or preventing a cancer or tumor of claim 28, wherein the peptide epitope is a bacterial epitope from a bacteria selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis strain BCG, BCG substrains, Mycobacterium avium, Mycobacterium intracellular, Mycobacterium africanum, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium ulcerans, Mycobacterium avium subspecies paratuberculosis, Nocardia asteroides, other Nocardia species, Legionella pneumophila, other Legionella species, Acetinobacter baumanii, Salmonella typhi, Salmonella enterica, other Salmonella species, Shigella boydii, Shigella dysenteriae, Shigella sonnei, Shigella flexneri, other Shigella species, Yersinia pestis, Pasteurella haemolytica, Pasteurella multocida, other Pasteurella species, Actinobacillus pleuropneumoniae, Listeria monocytogenes, Listeria ivanovii, Brucella abortus, other Brucella species, Cowdria ruminantium, Borrelia burgdorferi, Bordetella avium, Bordetella pertussis, Bordetella bronchiseptica, Bordetella trematum, Bordetella hinzii, Bordetella pteri, Bordetella parapertussis, Bordetella ansorpii other Bordetella species, Burkholderia mallei, Burkholderia psuedomallei, Burkholderia cepacian, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Coxiella burnetii, Rickettsial species, Ehrlichia species, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Escherichia coli, Vibrio cholerae, Campylobacter species, Neiserria meningitidis, Neiserria gonorrhea, Pseudomonas aeruginosa, other Pseudomonas species, Haemophilus influenzae, Haemophilus ducreyi, other Hemophilus species, Clostridium tetani, other Clostridium species, Yersinia enterolitica, and other Yersinia species.

31. The method of treating and / or preventing a cancer or tumor of claim 28, wherein the peptide epitope is a fungal epitope from a fungi selected from the group consisting of Candida albicans, Cryptococcus neoformans, Histoplama capsulatum, Aspergillus fumigatus, Coccidiodes immitis, Paracoccidiodes brasiliensis, Blastomyces dermitidis, Pneumocystis carnii, Penicillium mameffi, and Altemaria altemata.

32. The method of treating and / or preventing a cancer or tumor of claim 28, wherein the peptide epitope is a parasitic epitope from a parasite selected from the group consisting of Toxoplasma gondii, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, other Plasmodium species, Entamoeba histolytica, Naegleria fowleri, Rhinosporidium seeberi, Giardia lamblia, Enterobius vermicularis, Enterobius gregorii, Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus, Cryptosporidium spp., Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, other Leishmania species, Diphyllobothrium latum, Hymenolepis nana, Hymenolepis diminuta, Echinococcus granulosus, Echinococcus multilocularis, Echinococcus vogeli, Echinococcus oligarthrus, Diphyllobothrium latum, Clonorchis sinensis; Clonorchis viverrini, Fasciola hepatica, Fasciola gigantica, Dicrocoelium dendriticum, Fasciolopsis buski, Metagonimus yokogawai, Opisthorchis viverrini, Opisthorchis felineus, Clonorchis sinensis, Trichomonas vaginalis, Acanthamoeba species, Schistosoma intercalatum, Schistosoma haematobium, Schistosoma japoni cum, Schistosoma mansoni, other Schistosoma species, Trichobilharzia regenti, Trichinella spiralis, Trichinella britovi, Trichinella nelsoni, Trichinella nativa, and Entamoeba histolytica.

32. The method of treating and / or preventing a cancer or tumor of any one of claims 25-31, wherein from about 0.1 mg / kg to about 5 mg / kg APN is administered.

33. The method of treating and / or preventing a cancer or tumor of any one of claims 25-32, wherein the APN is repeatedly administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times at an interval of about every 5 days.

34. The method of treating and / or preventing a cancer or tumor of any one of claims 25-33, wherein, after administration of the APN, at least about 40% of the antigen-specific T cells express the CAR.

35. The method of treating and / or preventing a cancer or tumor of any one of claims 25-34, wherein the CAR, antibody, or antibody fragment encoded by the encapsulated nucleic acid recognizes a cancer or tumor antigen.

36. The method of treating and / or preventing a cancer or tumor of claim 35, wherein the cancer antigen is B cell maturation antigen (BCMA), G protein-coupled receptor class C group 5 member D (GPRC5D), CD19, guanylate cyclase-C (GUCY2C), human epidermal growth factor receptor 2 (HER2), CD20, CD22, mucin-1 (MUC1), mesothelin, or VHH (e.g., a sequence comprising 80% similarity or more to SEQ ID NO: 8).

40. The method of treating and / or preventing a cancer or tumor of any one of claims 25- 36, wherein the APN is a lipid nanoparticle, a liposome, or a polymeric nanoparticle.

41. The method of treating and / or preventing a cancer or tumor of claim 40, wherein the APN comprises at least one ionizable lipid, at least one PEGylated lipid, or a combination thereof.

42. The method of treating and / or preventing a cancer or tumor of claim 41, wherein the at least one ionizable lipid comprises cKK-E12, SM102, MC3, Lipid-5, Alc-0315, LP01, Lipid A9, or any combination thereof.

43. The method of treating and / or preventing a cancer or tumor of claim 41 or 42, wherein the APN comprises from about 30 mol% to about 60 mol% of the ionizable lipid.

44. The method of treating and / or preventing a cancer or tumor of any one of claims 41-43, wherein the at least one PEGylated lipid comprises ALC-0159, DMG-PEG, DSPE-PEG, or any combination thereof.

45. The method of treating and / or preventing a cancer or tumor of any one of claims 41-44, wherein the APN comprises from about 0.5 mol% to about 5 mol% of the PEGylated lipid.

46. The method of treating and / or preventing a cancer or tumor of any one of claims 41-45, wherein the phospholipid comprises DSPC, DOPE, or any combination thereof.

47. The method of treating and / or preventing a cancer or tumor of any one of claims 41-46, wherein the nanoparticle comprises from about 5 mol% to about 20 mol% of the phospholipid.

48. The method of treating and / or preventing a cancer or tumor of any one of claims 41-47, wherein a ratio of PEGylated lipid to total lipids is from about 0.01 : 1 to about 0.05: 1.

49. The method of treating and / or preventing a cancer or tumor of any one of claims 25-48, wherein the cancer or tumor is lymphoma, multiple myeloma, myeloid leukemia, colorectal cancer, breast cancer, giloblastoma, ovarian cancer, sarcoma, pancreatic cancer, mesothelioma, lung cancer, gastric cancer, or cervical cancer.