Antigen-presenting tumor cells (APTCS) and tumor-derived antigen-presenting vesicles from aptcs compositions and methods of use

Abstract

Disclosed herein are compositions of antigen-presenting tumor cells (APTCs) and tumor-derived antigen-presenting vesicles (Tumor-APVs) derived from APTCs and methods of their use.

Description

WSGR Docket No. 48295-719.601ANTIGEN-PRESENTING TUMOR CELLS (APTCS) AND TUMOR-DERIVED ANTIGEN- PRESENTING VESICLES FROM APTCS COMPOSITIONS AND METHODS OF USECROSS REFERENCE

[0001] This application claims the benefit of the U.S. Provisional Application No. 63 / 688,758 filed August 29, 2024, which is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 48295-719_601_SL. xml, created on August 21, 2025, which is 123,337 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.INCORPORATION BY REFERENCE

[0003] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BACKGROUND

[0004] Despite the tremendous progress made in therapies and prevention, cancer remains one of the largest health problems in the world. Recent breakthroughs in cancer immunotherapy, such as chimeric antigen-receptor (CAR) T cells and immune checkpoint blockade (ICB) therapies, led to cures for a small fraction of the treated patients. These results demonstrate the curative potential of engaging patients’ anti-tumor T cells to treat cancer and beg for further development to engage patients’ immune systems to eliminate all cancers. Indeed, among various therapeutic and prevention approaches, cancer vaccines are designed to create immune memory against cancer cells and have tremendous potential for cancer prevention and treatment. However, there remain significant barriers to establishing vaccines as effective approaches for cancer prevention and treatment due to the intrinsic limitations of cancer vaccine platforms.BRIEF SUMMARY

[0005] Described herein, in some embodiments, is an antigen-presenting tumor cell (APTC) comprising, a first fusion protein that comprises the displayed costimulatory ligand molecule CD80 or CD86, a second fusion protein that comprises the displayed costimulatory ligand moleculeWSGR Docket No. 48295-719.601GITRL, and an endogenous protein that comprises an endogenous peptide major histocompatibility complex class I (pMHC-I), wherein the first fusion protein, the second fusion protein, or the endogenous protein, is expressed at a valency of at least about 1000 copies on a surface of the APTC. In some embodiments, the first fusion protein, the second fusion protein, and the endogenous protein are all expressed on the cell surface of the APTC. In some embodiments, the APTC comprises the first fusion protein that comprises the displayed costimulatory ligand molecule CD80 or CD86. In some embodiments, the APTC comprises the second fusion protein that comprises the displayed costimulatory ligand molecule GITRL. In some embodiments, the APTC comprises displayed multivalent, oligomerized costimulatory ligand molecules CD80 and GITRL alongside endogenous pMHC-I. In some embodiments, the first fusion protein of the displayed costimulatory ligand molecule CD80 or CD86, or the second fusion protein of the displayed costimulatory ligand molecule GITRL on the surface of the APTC binds specifically to the cognate costimulatory receptors on a T-cell surface. In some embodiments, the APTC further comprises a third fusion protein comprising at least one, two, three, four, five, six, seven, eight, nine, ten, or more than ten separate fusion proteins, wherein the at least one, two, three, four, five, six, seven, eight, nine, ten, or more than ten separate fusion proteins each comprises 0X40 Ligand, CD48, CD30 Ligand, ICOS Ligand, CD70, CD40, CD40 Ligand, 4-BB Ligand, TLIA, TIM4, SLAM, CD48, CD58, CD155, CD112, LIGHT, or ICAM-1 . In some embodiments, the endogenous pMHC-I on the surface of the APTC binds specifically to an antigen -specific T cell receptor (TCR) that recognizes an antigenic peptide derived from an endogenous antigen, a viral antigen, a mutated tumor antigen, a tumor-specific antigen, a differentiated tumor antigen, an over-expressed tumor antigen, or a neoantigen within tumor cells. Alternatively, the endogenous pMHC-I on the surface of the APTC binds specifically to a CD8+ T cell bearing an antigen -specific T cell receptor (TCR) recognizing an antigenic peptide derived from an endogenous antigen, a viral antigen, a mutated tumor antigen, a tumor-specific antigen, a differentiated tumor antigen, an over-expressed tumor antigen, or a neoantigen within tumor cells. Described herein, in some embodiments, is an APTC comprising an endogenous pMHC-I that comprises an endogenous antigenic peptide, an endogenous P-2 -microglobulin (P2m), and an endogenous MHC-I alpha chain. In some embodiments, the endogenous antigenic peptide binds to the endogenous MHC-I alpha chain. In some embodiments, the endogenous antigenic peptide in the endogenous pMHC-I is derived from an endogenous antigen, a viral antigen, a mutated tumor antigen, a tumor-specific antigen, a differentiated tumor antigen, an over-expressed tumor antigen, or a neoantigen within tumor cells. In some embodiments, the APTC comprises an endogenous pMHC-I that comprises an endogenous antigenic peptide, an endogenous P -2 -microglobulin (P2m), and an endogenous MHC-I alpha chainWSGR Docket No. 48295-719.601 wherein the endogenous antigenic peptide comprises an endogenous antigenic peptide from a tumor cell. The present disclosure provides in some embodiments the APTC that is derived from pancreatic cancer, rectal cancer, colorectal cancer, lung cancer, endometrial cancer, liver cancer, testicular cancer, prostate cancer, cervical cancer, breast cancer, bladder cancer, head and neck cancer, skin cancer, brain cancer, ovarian cancer, salivary gland cancer, appendiceal cancer, esophagogastric cancer, thyroid cancer, soft tissue sarcoma, kidney cancer, leukemia, myeloma, lymphoma, melanoma, non-small cell lung cancer, Hodgkin’s lymphoma, or non-Hodgkin’s lymphoma. Described herein, in some embodiments, is an APTC comprising an endogenous pMHC-I that comprises an endogenous antigenic peptide. In some embodiments, the antigenic peptide is derived from pancreatic cancer, rectal cancer, colorectal cancer, lung cancer, endometrial cancer, liver cancer, testicular cancer, prostate cancer, cervical cancer, breast cancer, b ladder cancer, head and neck cancer, skin cancer, brain cancer, ovarian cancer, salivary gland cancer, appendiceal cancer, esophagogastric cancer, thyroid cancer, soft tissue sarcoma, kidney cancer, leukemia, myeloma, lymphoma, melanoma, non-small cell lung cancer, Hodgkin’s lymphoma, or non-Hodgkin’s lymphoma. In some embodiments, each of the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules comprises an amino acid sequence with at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 23 - 58. Described herein, in some embodiments, is an APTC comprising a first, a second, or a third fusion protein of displayed costimulatory ligand molecules wherein each of the first, second, or third fusion protein of the displayed costimulatory ligand molecules further comprises a transmembrane domain. In some embodiments, the transmembrane domain of the fusion protein of the displayed costimulatory ligand molecule comprises a transmembrane domain derived from VSV-G, Dengue E protein, influenza hemagglutinin, influenza neuraminidase, spike protein SI, spike protein S2, Sindbis virus envelope (SINDBIS) protein, hemagglutinin envelope protein from measles virus, envelope glycoprotein of measles virus fusion (F) protein, RD114, BaEV, GP41, GP120, or the wild-type transmembrane domain of costimulatory ligand molecules. In some embodiments, the transmembrane domain of the fusion protein of the displayed costimulatory ligand molecule comprises a transmembrane domain derived from VSV-G wherein the VSV-G comprises a transmembrane domain and cytoplasmic tail. In some embodiments, transmembrane domain of the fusion protein comprisesan amino acid sequence with at least 90% sequence identity to an amino acid sequence of SEQ ID NOs: 59-78. Described herein, in some embodiments, is an APTC comprising a first, a second, or a third fusion protein of displayed costimulatory ligand molecules wherein each of the first, second, or third fusion protein further comprises an oligomerization domain. In some embodiments, the oligomerization domain of the displayedWSGR Docket No. 48295-719.601 costimulatory ligand molecule is a dimerization domain, a trimerization domain, a tetramerization domain, a hexamerization domain, a heterohexamerization domain, a heptamerization domain, or an octamerization domain. In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecule comprises a leucine zipper dimerization domain, a post-fusion oligomerization domain of viral surface protein, a D4 post-fusion trimerization domain of VSV-G protein, a Dengue E protein post-fusion trimerization domain, a foldon trimerization domain, a coiled coil domain, a LINEl retrotransposon coiled coil domain (Llcc), a collagen XV coiled coil domain (CXVcc), a tetranectin coiled coil domain (TNTNcc), a cc-hex domain, a L24D domain, a L24DH domain, a cc-Hept domain, a wazOct domain, or an influenza neuraminidase stem domain. In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecule comprises an amino acid sequence set forth in any one of SEQ ID NO: 79 to SEQ ID NO: 93 with at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 79 - 93. Described herein, in some embodiments, is an APTC capable of activating or stimulating an antigen-specific T cell wherein the APTC comprises a costimulatory ligand molecule of a fusion protein is expressed at least about 1000 copies on a surface of the APTC. In some embodiments, the APTC comprises the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules wherein when the first fusion protein, the second fusion protein, or the third fusion protein of the costimulatory ligand molecules is expressed on the surface of the antigen- presenting tumor cell (APTC), the oligomerization domain is outside of the antigen-presenting tumor cell (APTC). In some embodiments, the APTC comprises the first, second, or third fusion protein of the displayed costimulatory ligand molecules wherein when the first fusion protein, the second fusion protein, or third fusion protein of the displayed costimulatory ligand molecules is expressed on the surface of the antigen-presenting tumor cell (APTC), the oligomerization domain is inside of the antigen-presenting tumor cell (APTC). Described herein, in some embodiments, is an APTC capable of activating or stimulating an antigen -specific T cell wherein the APTC comprises a first, a second, or a third fusion protein of displayed costimulatory ligand molecules and an endogenous peptide major histocompatibility complex class I (pMHC-I). In some embodiments, the APTC comprises the first, second, or third fusion protein of the displayed costimulatory ligand molecules and the endogenous peptide major histocompatibility complex class I (pMHC-I). The first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 1000 copies or more on a surface of the antigen-presenting tumor cell (APTC). In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency ofWSGR Docket No. 48295-719.601 about 2500 copies or more on a surface of the antigen-presenting tumor cell (APTC). In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 5000 copies or more on a surface of the antigen-presenting tumor cell (APTC). In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 10,000 copies or more on a surface of the antigen-presenting tumor cell (APTC). In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 20,000 copies or more on a surface of the antigen-presenting tumor cell (APTC). In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 40,000 copies or more on a surface of the antigen-presenting tumor cell (APTC). In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 60,000 copies or more on a surface of the antigen-presenting tumor cell (APTC). In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 80,000 copies or more on a surface of the antigen-presenting tumor cell (APTC). In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 120,000 copies or more on a surface of the antigen-presenting tumor cell (APTC). In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 200,000 copies or more on a surface of the antigen-presenting tumor cell (APTC). In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 250,000 copies or more on a surface of the antigen-presenting tumor cell (APTC). In some embodiments, the APTC comprises the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous peptide major histocompatibility complex class I (pMHC-I). The first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of at least 1000 copies on the cell surface of the APTC. In some embodiments, the APTC comprises the first fusion protein, the second fusion protein, or the third fusion protein of the displayedWSGR Docket No. 48295-719.601 costimulatory ligand molecules, or the endogenous pMHC-I wherein the endogenous pMHC-I comprises a natural pMHC-I. In some embodiments, the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules comprises an amino acid sequence with at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 94-111. In some embodiments, the APTC comprises a first fusion protein that comprises the costimulatory ligand molecule CD80 or CD86, and a second fusion protein that comprises the costimulatory ligand molecule GITRL, and a recombinant peptide major histocompatibility complex class I (pMHC-I), wherein the APTC lacks an endogenous pMHC-I or expresses a dysfunctional endogenous pMHC-I, wherein the first fusion protein, the second fusion protein, or the recombinant pMHC-I is expressed at a valency of at least about 1000 copies on a surface of the APTC. Described herein, in some embodiments, is a fusion protein comprising a costimulatory ligand molecule that binds specifically to a T cell surface costimulatory receptor, an oligomerization domain, and a transmembrane domain, wherein the fusion protein is expressed at a valency of at least about 1000 copies on a surface of an antigen-presenting tumor cell (APTC). In some embodiments, the fusion protein comprises at least two separate fusion proteins, wherein the at least two separate fusion proteins each comprises a different costimulatory ligand molecule. In some embodiments, the costimulatory molecule of the fusion protein comprises one or more selected from the group comprising GITR Ligand, CD80, CD86, 0X40 Ligand, CD48, CD30 Ligand, ICOS Ligand, CD70, CD40, CD40 Ligand, 4-BB Ligand, TLIA, TIM4, SLAM, CD48, CD58, CD155, CD112, LIGHT, and ICAM1.

[0006] In some embodiments, an antigen-presenting tumor cell (APTC) comprising the fusion protein, wherein the APTC is capable of activating or stimulating an antigen -specific T cell and wherein the fusion protein is expressed at least about 1000 copies on a surface of the APTC. Described herein, in some embodiments, are methods of preventing or treating cancer in a subject in need thereof comprising administering to the subject an autologous cell comprising the APTC. Described herein, in some embodiments, are methods of preventing or treating established cancer in a subject in need thereof comprising administering to the subject an irradiated autologous cell comprising the APTC. Described herein, in some embodiments, are methods of inducing protective immunity against cancer in a subject in need thereof comprising administering to the subject an autologous cell comprising the APTC. Described herein, in some embodiments, are methods of inducing protective immunity against cancer in a subject in need thereof comprising administering to the subject an irradiated autologous cell comprising the APTC. In some embodiments, the methods of preventing or treating cancer in a subject or inducing protective immunity against cancer in a subject further comprising administering the subject a second therapy wherein theWSGR Docket No. 48295-719.601 second therapy comprises an adjuvant. In some embodiments, the second therapy comprises Poly (I:C) or lipopolysaccharide (LPS). In some embodiments, the second therapy comprises an immune checkpoint blocker therapy. In some embodiments, the second therapy comprises an anti-PDl antibody, anti-PDLl antibody, or an anti-CTLA-4 antibody. In some embodiments, the second therapy comprises a Toll-like receptor agonist for TLR3, TLR4, TLR7, TLR8, TLR9, CpG, or a STING agonist. In some embodiments, the second therapy comprises a cytokine. In some embodiments, the second therapy comprises the cytokine which comprises IL-2, IL-7, IL-12, IL-15, IL-21, or interferon -gamma (IFN-gamma). In some embodiments, the second therapy comprises a targeted therapy, and optionally a tyrosine kinase inhibitor or PARP inhibitor or RAS inhibitor. In some embodiments, the second therapy comprises T cell therapy, and optionally a chimeric antigen-receptor T cell therapy or tumor-infiltrated lymphocyte therapy. In some embodiments, the second therapy comprises standard care therapy, and optionally surgery or chemotherapy or radiation therapy. Described herein, in some embodiments, are methods of preventing or treating established cancer in a subject comprising administering to the subject a nucleic acid encoding the fusion protein of co-stimulatory molecules, wherein the administering results in the expression of the fusion protein on a surface of a cancer cell in the subject. Described herein, in some embodiments, are methods of inducing protective immunity against cancer in a subject comprising administering to the subject a nucleic acid encoding the fusion protein of co-stimulatory molecules, wherein the administering results in expression of the fusion protein on a surface of a cancer cell in the subject. In some embodiments, the cancer cell is reprogrammed into an APTC that activates or stimulates a T cell in the subject. Described herein, in some embodiments, are methods of preventing cancer relapse in a subject in need thereof comprising administering to the subject an autologous cell comprising the APTC. In some embodiments, the subject undergoes cancer treatment before the administering and does not have cancer after the treatment. In some embodiments, the autologous cell comprising the APTC is produced in culture. In some embodiments, the autologous cell comprising the APTC is a live cell, an irradiated cell, or an inactivated cell. Described herein, in some embodiments, are methods of preventing cancer relapse in a subject comprising administering to the subject a Toll-like receptor agonist and the autologous cell comprising the APTC. In some embodiments, the administering further comprises administering an anti-PDl, anti-PDLl, an anti-CTLA4 immune checkpoint blockade therapy, a targeted therapy, an antibody-drug therapy, a tyrosine kinase inhibitor, a chemotherapy, surgery, or a therapy of standard care. Described herein, in some embodiments, are methods of preventing cancer relapse in a subject comprising introducing, before the administering, into the autologous cell a nucleic acid molecule encoding the first fusion protein, the second fusion protein, or the thirdWSGR Docket No. 48295-719.601 fusion protein of the displayed costimulatory ligand molecule, wherein the autologous cell is cultured ex vivo. In some embodiments, the nucleic acid comprises mRNA, DNA, or a viral vector. In some embodiments, the nucleic acid comprises mRNA, DNA, or the viral vector wherein the viral vector comprises a lentiviral vector, a retroviral vector, an adeno -associated viral (AAV) vector, an adenoviral vector, a vector derived from herpes simplex virus (HSV), a vector derived from parvoviruses, or a vector derived from poxviruses. In some embodiments, the vector derived from parvoviruses wherein the poxviruses comprise vaccinia virus (VACV) or myxoma virus. Described herein, in some embodiments, are methods of preventing or treating established cancer in a subject or inducing protective immunity against cancer in a subject wherein the introducing comprises intratumoral delivery, intravenous delivery, intramuscular delivery, intraperitoneal delivery, or transdermal delivery. Described herein, in some embodiments, are methods of preventing or treating established cancer in a subject or inducing protective immunity against cancer in a subject wherein the administering comprises administering a Toll-like receptor agonist. In some embodiments, the methods of preventing or treating established cancer in a subject or inducing protective immunity against cancer in a subject further comprise administering an anti- PD1, anti-PDLl, an anti-CTLA4 immune checkpoint blockade therapy, a targeted therapy, an antibody -drug therapy, a tyrosine kinase inhibitor, a chemotherapy, surgery, or a therapy of standard care. Described herein, in some embodiments, are methods of inducing protective immunity against cancer in a subject comprising administering to the subject an irradiated autologous cell comprising the APTC. Described herein, in some embodiments, are methods of preventing or treating cancer in a subject in need thereof comprising obtaining a T cell, a tumor- infiltrated lymphocyte (TIL), or a cancer cell from the subject; introducing ex vivo into the cancer cell a nucleic acid encoding the fusion protein of co -stimulatory molecules to express the fusion protein on a surface of the cancer cell; contacting ex vivo the T cell or TIL with the cancer cell to activate or stimulate the T cell or TIL; and administering the activated T cell or TIL to the subject.

[0007] In some embodiments, the method of producing APTCs, comprises introducing into a tumor cell a nucleic acid molecule encoding the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecule. In some embodiments, the introduction of the nucleic acid into the tumor cell is carried out ex vivo after the tumor cell is obtained from a subject. Alternatively, in some embodiments, the introduction of the nucleic acid into the tumor cell is carried out in vivo or intratumorally in a subject. In some embodiments, the introduction of the nucleic acid into the tumor cell comprises intratumoral delivery, intravenous delivery, intramuscular delivery, intraperitoneal delivery, or transdermal delivery, or intranasal delivery, or inhalation. In some embodiments, the nuclei acid molecule comprises an ectodomain ofWSGR Docket No. 48295-719.601 the costimulatory ligand molecule, an oligomerization domain, a transmembrane domain and a cytosolic domain. In some embodiments, the nucleic acid molecule encoding the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecule comprises mRNA or DNA. In some embodiments, the nucleic acid molecule encoding the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules comprises a DNA vector, an mRNA vector, or a viral vector. In some embodiments, the nucleic acid comprises mRNA, DNA, or a viral vector. In some embodiments, the nucleic acid comprises mRNA, DNA, or the viral vector wherein the viral vector comprises a lentiviral vector, a retroviral vector, an adeno -associated viral (AAV) vector, an adenoviral vector, a vector derived from herpes simplex virus (HSV), a vector derived from parvoviruses, or a vector derived from poxviruses. In some embodiments, the vector derived from parvoviruses wherein the poxviruses comprise vaccinia virus (VACV) or myxoma virus. In some embodiments, the pharmaceutical composition comprises the APTC, and a pharmaceutically acceptable excipient.

[0008] Described herein, in some embodiments, is a tumor-derived antigen-presenting vesicle (tumor-APV) comprising, a first fusion protein that comprises the displayed costimulatory ligand molecule CD80 or CD86, a second fusion protein that comprises the displayed costimulatory ligand molecule GITRL, and an endogenous protein that comprises an endogenous peptide major histocompatibility complex class I (pMHC-I), wherein the tumor-APV is derived from an antigen- presenting tumor cell (APTC) comprising the first fusion protein, the second fusion protein, and the endogenous protein, wherein the first fusion protein, the second fusion protein, or the endogenous protein, is expressed at a valency of at least about 100 copies on a surface of the APV or at least 1000 copies on a surface of the APTC. In some embodiments, the tumor-APV is derived from the APTC comprising the first fusion protein that comprises the displayed costimulatory ligand molecule CD80 or CD86. In some embodiments, the tumor-APV is derived from the APTC comprising the second fusion protein that comprises the displayed costimulatory ligand molecule GITRL. In some embodiments, the tumor-APV is derived from the APTC comprising the endogenous protein that comprises the endogenous peptide major histocompatibility complex class I (pMHC-I). In some embodiments, the tumor-APV is derived from the APTC comprising the first fusion protein of costimulatory ligand molecule CD80 or CD86, the second fusion protein of costimulatory ligand molecule GITRL, and an endogenous peptide major histocompatibility complex class I (pMHC-I). In some embodiments, the tumor-APV is a viral-like particle. In some embodiments, the multivalent particle is an extracellular vesicle derived from APTC. In some embodiments, the multivalent particle is an exosome derived from APTC. In some embodiments,WSGR Docket No. 48295-719.601 the multivalent particle is an ectosome derived from APTC. In some embodiments, the tumor-APV is a vesicle generated from physically disrupted APTC by using nitrogen cavitation, sonication, or size extrusion. In some embodiments, the first or the second fusion protein of the displayed costimulatory ligand molecule on the surface of the tumor-APV binds specifically to the cognate costimulatory receptors on a T-cell surface. In some embodiments, the tumor-APV or the APTC further comprises a third fusion protein comprising at least one, two, three, four, five, six, seven, eight, nine, ten, or more than ten separate fusion proteins, wherein the at least one, two, three, four, five, six, seven, eight, nine, ten, or more than ten separate fusion proteins each comprises 0X40 Ligand, CD48, CD30 Ligand, ICOS Ligand, CD70, CD40, CD40 Ligand, 4-BB Ligand, TLIA, TIM4, SLAM, CD48, CD58, CD155, CD112, LIGHT, or IC AM- 1. A tumor-APV derived from an APTC expressing an endogenous pMHC-I binds specifically to an antigen-specific T cell receptor (TCR) that recognizes an antigenic peptide derived from a viral antigen, a mutated tumor antigen, a tumor-specific antigen, a differentiated tumor antigen, an over-expressed tumor antigen, or a neoantigen within tumor cells. Alternatively, in some embodiments, the expression of the endogenous pMHC-I on the surface of the tumor-APV binds specifically to a CD8+ T cell bearing an antigen-specific T cell receptor (TCR) recognizing an antigenic peptide derived from an endogenous peptide, a viral antigen, a mutated tumor antigen, a tumor-specific antigen, a differentiated tumor antigen, an over-expressed tumor antigen, or a neoantigen within tumor cells. In some embodiments, the endogenous pMHC-I on the surface of the tumor-APV comprises an endogenous antigenic peptide, an endogenous P-2-microglobulin (P2m), and an endogenous MHC-I alpha chain. In some embodiments, the endogenous antigenic peptide binds to the endogenous MHC-I alpha chain. In some embodiments, the endogenous antigenic peptide in the endogenous pMHC-I is derived from an endogenous antigen, a viral antigen, a mutated tumor antigen, a tumorspecific antigen, a differentiated tumor antigen, an over-expressed tumor antigen, or a neoantigen within tumor cells. In some embodiments, the tumor-APV is derived from the APTC comprising an endogenous pMHC-I that comprises an endogenous antigenic peptide, an endogenous P-2- microglobulin (P2m), and an endogenous MHC-I alpha chain wherein the endogenous antigenic peptide comprises an endogenous antigenic protein from a tumor cell. The present disclosure provides in some embodiments a tumor-APV or its parental APTC that is derived from pancreatic cancer, rectal cancer, colorectal cancer, lung cancer, endometrial cancer, liver cancer, testicular cancer, prostate cancer, cervical cancer, breast cancer, bladder cancer, head and neck cancer, skin cancer, brain cancer, ovarian cancer, salivary gland cancer, appendiceal cancer, esophagogastric cancer, thyroid cancer, soft tissue sarcoma, kidney cancer, leukemia, myeloma, lymphoma, melanoma, non-small cell lung cancer, Hodgkin’s lymphoma, or non-Hodgkin’s lymphoma.WSGR Docket No. 48295-719.601Described herein, in some embodiments, is a tumor- APV or its parental APTC comprising an endogenous pMHC-I that comprises an endogenous antigenic peptide. In some embodiments, the antigenic peptide is derived from pancreatic cancer, rectal cancer, colorectal cancer, lung cancer, endometrial cancer, liver cancer, testicular cancer, prostate cancer, cervical cancer, breast cancer, bladder cancer, head and neck cancer, skin cancer, brain cancer, ovarian cancer, salivary gland cancer, appendiceal cancer, esophagogastric cancer, thyroid cancer, soft tissue sarcoma, kidney cancer, leukemia, myeloma, lymphoma, melanoma, non-small cell lung cancer, Hodgkin’s lymphoma, or non-Hodgkin’s lymphoma. In some embodiments, the endogenous antigenic peptide comprises an endogenous antigenic peptide from a tumor cell. In some embodiments, the costimulatory ligand molecule comprises an amino acid sequence with at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 23 -58. Described herein, in some embodiments, is a tumor-APV derived from an APTC comprising a first, a second, or a third fusion protein of displayed costimulatory ligand molecules wherein each of the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules further comprises a transmembrane domain. In some embodiments, the transmembrane domain of the fusion protein of the displayed costimulatory ligand molecule comprises a transmembrane domain derived from VSV-G, Dengue E protein, influenza hemagglutinin, influenza neuraminidase, spike protein SI, spike protein S2, Sindbis virus envelope (SINDBIS) protein, hemagglutinin envelope protein from measles virus, envelope glycoprotein of measles virus fusion (F) protein, RD114, BaEV, GP41, GP120, or the wild-type transmembrane domain of costimulatory ligand molecules. In some embodiments, the transmembrane domain of the fusion protein comprises a transmembrane domain derived from VSV-G wherein the VSV-G comprises a transmembrane domain and cytoplasmic tail. In some embodiments, the transmembrane domain of the displayed costimulatory ligand molecules comprises an amino acid sequence with at least 90% sequence identity to an amino acid sequence of SEQ ID NOs: 59-78. Described herein, in some embodiments, is a tumor-APV derived from a APTC comprising a first, a second, or a third fusion protein of displayed costimulatory ligand molecules wherein each of the first, the second, or the third fusion protein further comprises an oligomerization domain. In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecules is a dimerization domain, a trimerization domain, a tetramerization domain, a hexamerization domain, a heterohexamerization domain, a heptamerization domain, or an octamerization domain . In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecules comprises a leucine zipper dimerization domain, a post-fusion oligomerization domain of viral surface protein, a D4 post-fusion trimerization domain of VSV-G protein, a Dengue E protein post-WSGR Docket No. 48295-719.601 fusion trimerization domain, a foldon trimerization domain, a coiled coil domain, a LINE! retrotransposon coiled coil domain (Llcc), a collagen XV coiled coil domain (CXVcc), a tetranectin coiled coil domain (TNTNcc), a cc-hex domain, a L24D domain, a L24DH domain, a cc-Hept domain, a wazOct domain, or an influenza neuraminidase stem domain. In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecules comprises an amino acid sequence that has at least 90% sequence identity to an amino acid sequence according to SEQ ID NOs: 79-93. Described herein, in some embodiments, is a tumor- APV capable of activating or stimulating an antigen -specific T cell wherein a parental APTC comprises a first fusion protein, a second fusion protein, a third fusion protein of the displayed costimulatory ligand molecules, or an endogenous pMHC-I. In some embodiments, the parental APTC comprises the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules wherein when the first fusion protein, the second fusion protein, or the third fusion protein of the costimulatory ligand molecules is expressed on the surface of the tumor-APV, the oligomerization domain is outside of the tumor-APV. In some embodiments, the APTC comprises the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules wherein when the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules is expressed on the surface of the tumor- APV, the oligomerization domain is inside of the tumor-APV. In some embodiments, the APTC comprises the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules and the endogenous peptide major histocompatibility complex class I (pMHC-I). The first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 100 copies or more on a surface of the tumor-APV. In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 250 copies or more on a surface of the tumor-APV. In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 500 copies or more on a surface of the tumor-APV. In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 1,000 copies or more on a surface of the tumor-APV. In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 2,000 copies or more on a surface of the tumor-APV. In some embodiments, the first fusion protein, the second fusion protein,WSGR Docket No. 48295-719.601 or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 4,000 copies or more on a surface of the tumor-APV. In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 6,000 copies or more on a surface of the tumor-APV. In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 8,000 copies or more on a surface of the tumor-APV. In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 10,000 copies or more on a surface of the tumor-APV. In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 20,000 copies or more on a surface of the tumor-APV. In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 50,000 copies or more on a surface of the tumor-APV. In some embodiments, the tumor- APV is derived from its parental APTC comprising the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I wherein the endogenous pMHC-I comprises a natural pMHC-I. In some embodiments, the parental APTC comprises the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC- I wherein the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules comprises an amino acid sequence with at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 94-111. In some embodiments, the tumor-derived antigen-presenting vesicle (tumor-APV) comprising a first fusion protein that comprises the costimulatory ligand molecule CD80 or CD86, and a second fusion protein that comprises the costimulatory ligand molecule GITRL, and a recombinant peptide major histocompatibility complex class I (pMHC-I), wherein the tumor-APV is derived from its parental antigen-presenting tumor cell (APTC) comprising the first fusion protein, the second fusion protein, and the recombinant pMHC-I, wherein the tumor-APV or its parental APTC lacks an endogenous pMHC-I or expresses a dysfunctional endogenous pMHC-I, wherein first fusion protein, the second fusion protein, or the recombinant pMHC-I is expressed at a valency of at least about 100 copies on a surface of the tumor-APV. In some embodiments, a fusion protein comprising a costimulatory ligand molecule that binds specifically to a T cell surface costimulatory receptor, anWSGR Docket No. 48295-719.601 oligomerization domain, and a transmembrane domain, wherein the fusion protein is expressed at a valency of at least about 100 copies on a surface of a tumor-APV, wherein the tumor- APV is derived from its parental APTC. In some embodiments, the fusion protein comprises at least two separate fusion proteins, wherein the at least two separate fusion proteins each comprises a different costimulatory ligand molecule. In some embodiments, the costimulatory molecule comprises one or more selected from the group comprising GITR Ligand, CD80, CD86, 0X40 Ligand, CD48, CD30 Ligand, ICOS Ligand, CD70, CD40, CD40 Ligand, 4-BB Ligand, TLIA, TIM4, SLAM, CD48, CD58, CD155, CD112, LIGHT, and ICAM1.

[0009] Described herein, in some embodiments, is a tumor-APV capable of activating or stimulating an antigen-specific T cell wherein the tumor-APV is derived from its parental APTC comprising a first fusion protein, a second fusion protein, a third fusion protein of displayed costimulatory ligand molecules wherein any one of the fusion proteins is expressed at least about 100 copies on a surface of the tumor-APV. Described herein, in some embodiments, are methods of preventing or treating cancer in a subject in need thereof comprising administering to the subject an antigen-presenting vesicle (APV) wherein the APV is derived from the APTC that is obtained from a subject. Described herein, in some embodiments, are methods of inducing protective immunity against cancer in a subject in need thereof comprising administering to the subject the tumor- derived antigen-presenting vesicle (tumor-APV), wherein the tumor-APV is derived from its parental APTC that is derived from the subject. In some embodiments, the method of preventing or treating cancer in a subject in need thereof comprises administering to the subject a T cell or a TIL, wherein the T cell or the TIL is activated or stimulated by the tumor-APV before the administering to the subject. In some embodiments, the method of preventing cancer relapse in a subject in need thereof comprising administering to the subject tumor-APVs. In some embodiments, the method of preventing or treating established cancer in a subject in need thereof comprising administering to the subject autologous tumor-APVs. In some embodiments, the method of inducing protective immunity against cancer in a subject in need thereof comprising administering to the subject autologous tumor-APVs. In some embodiments, the autologous tumor-APVs is derived from a cancer cell produced in culture. In some embodiments, the subject undergoes cancer treatment before the administering and does not have cancer after the treatment. In some embodiments, the methods of preventing or treating cancer in a subject or inducing protective immunity against cancer in a subject comprise administering of tumor-APVs wherein the administering of tumor- APVs comprises administering a Toll-like receptor agonist in the subject. In some embodiments, the methods of preventing or treating cancer in a subject or inducing protective immunity against cancer in a subject comprise administering of tumor-APVs wherein the administering of tumor-WSGR Docket No. 48295-719.601APVs comprises administering an anti-PDl, anti-PDLl, an anti-CTLA4 immune checkpoint blockade therapy, a targeted therapy, an antibody -drug therapy, a tyrosine kinase inhibitor, a chemotherapy, surgery, or a therapy of standard care. Described herein, in some embodiments, are methods of enhancing anti-tumor T cell immunity in a subject, comprising administering to a subject in need thereof the tumor- APV or its parental APTC. In some embodiments, the methods of preventing or treating cancer in a subject, inducing protective immunity against cancer in a subject, or enhancing anti -tumor T cell immunity in a subject comprise administering a therapeutic agent wherein the therapeutic agent comprises an inflammatory cytokine, a dendritic cell targeting signal, or an innate modulating signal. In some embodiments, the tumor APVs induce T cell mediated cytotoxicity against tumor cells. In some instances, the subject is a mammal.

[0010] Described herein, in some embodiments, is a method of producing the tumor-APV, comprises administering to a subject a nucleic acid encoding a fusion protein comprising a costimulatory ligand molecule that binds specifically to a T cell surface costimulatory receptor, an oligomerization domain, and a transmembrane domain, wherein the administering results in the expression of the fusion protein on a surface of a cancer cell in the subject. Described herein, in some embodiments, is a method of producing the tumor-APV comprising: (A) generating an antigen-presenting tumor cell (APTC) by introducing ex vivo into the cancer cell a nucleic acid encoding a fusion protein comprising a costimulatory ligand molecule that binds specifically to a T cell surface costimulatory receptor, an oligomerization domain, and a transmembrane domain to express the fusion protein on a surface of the cancer cell; and (B) culturing and expanding APTCs in culture dishes as a suspension, adherent, or organoid cell culture; and (C) generating tumor- derived antigen-presenting vesicles (tumor- APVs) by disrupting the antigen-presenting tumor cells (APTCs) into tumor-APVs using physical processes, including nitrogen cavitation, freeze and thaw, sonication, or size-exclusion filtration. In some embodiments, the method of producing the tumor- APV comprises disrupting the antigen -presenting tumor cell (APTC) expressing the fusion protein into the tumor-APV wherein the disrupting of APTCs is carried out using a physical method. In some embodiments, the physical method comprises nitrogen cavitation, sonication, freeze and thaw, or membrane exclusion. In some embodiments, the method of producing the tumor-APV further comprise purifying the tumor-APV to remove cellular debris, nucleic acid impurities, or protein impurities. In some embodiments, the tumor-APV described herein comprises a size from about lOOnm to about 1 pm. In some embodiments, a pharmaceutical composition comprises the tumor-APV, and a pharmaceutically acceptable excipient.WSGR Docket No. 48295-719.601BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0012] Figures 1A-1C illustrate stimulation antigen-specific T cells by antigen-presenting cells (APCs) and tumor cells. (A) effective activation of antigen -specific T cells by APCs requires both pMHC and TCR engagement as the signal 1 and costimulatory signals as signal 2. (B) pMHC and TCR engagement between a T cell and a tumor cell or a somatic cell that lack of the expression of costimulatory molecules results in inappropriate T cell stimulation and leads to T cell anergy and tolerance. (C) Symbol legends for the key molecules involved in T cell activation.

[0013] Figures 2A-2C illustrate lack of co -stimulatory molecule expression in mouse and human cancer cells. (A) Relative expression level of class I MHC molecules and T cell co- stimulatory molecules in a panel of murine tumor cell lines, as determined via FACS. Data is representative of three experiments. (B) Relative expression level of class I HLA molecules and T cell co-stimulatory molecules as determined by mRNA microarray in human tumor cell samples from a panel of cancer types. Data was provided by the cancer cell line encyclopedia (Novartis / Broad, Nature 2012). (C) Quantification of MHC I molecule expression on a panel of murine cancer cell lines, as determined by FACS analysis using PE quantibrite beads. Composite data of 2 experiments is shown.

[0014] Figures 3A-3C illustrate in vivo rejection of MC38 tumor cells expressing wild -type CD80 and GITRL. (A) Rejection of MC38 expressing wt-CD80 or wt-GITRL cells in mice. (B) Average tumor growth of mice bearing MC38, orMC38-wtCD80, or MC38-wtGITRL tumors and (C) individual mouse tumor growth curves in C57BL / 6 mice (n=5 / group).

[0015] Figures 4 A-4C illustrate in vivo rejection of Lewis lung cancer cells (LLC) expressing wild-type CD80 and GITRL. (A) Rejection of LLC expressing wt-CD80 or wt-GITRL cells in mice. (B) Average tumor growth of mice baring LLC, or LLC-wt-CD80 or LLC-wt-GITRL tumors and (C) individual mouse tumor growth curves in C57BL / 6 mice (n=5 / group)

[0016] Figures 5 illustrates that T cells employ multiple co-stimulatory molecules to modulate T cell activation, proliferation, differentiation, and function.

[0017] Figures 6A-6B illustrate strategy to define the costimulatory signals required to reprogram tumor cells into antigen-presenting tumor cells (APTCs). (A) Schematic illustrating the antigen- presenting vesicles (APVs) that can be used to selectively and potently activate antigen -specific T cells. The composition of APVs consists of high density, multivalent, oligomerized, surfaceWSGR Docket No. 48295-719.601 displayed single-chain trimer peptide MHC class I (SCT-pMHC-I) and costimulatory ligand molecules, CD80 / CD86 and GITRL on exosomes or virus-like particles (VLPs). (B) Antigen- presenting tumor cells (APTCs) expressing high density, multivalent, surface displayed co- stimulatory molecules CD80 / CD86 and GITRL alongside endogenous peptide -MHC complexes presenting tumor antigens.

[0018] Figures 7A-7B illustrate vesicle display vector with oligomerization domains. (A) Schematics of vesicle display vector. (B) List of oligomerization domains utilized in the vesicle display vector.

[0019] Figures 8A-8C illustrate production of vesicles displaying dimeric peptide on (A) viral- like particles (VLPs) with viral genomes, (B) VLPs without viral genome, and (C) extracellular vesicles (EVs).

[0020] Figures 9A-9C illustrate characterization of trimeric ACE2-MVPs. (A) Quantitative Western-blot analyses of trimeric ACE2-MVPs. (B) Western-blot analyses of trimeric ACE2- MVPs in the reducing and non -reducing conditions. (C) Viral neutralization activity of the trimeric ACE2-MVPs determined in a pseudovirus neutralization assay.

[0021] Figures 10A-10D illustrate characterization of ACE2-MVPs with various oligomerization domains. (A) Quantitative Western-blot analyses of ACE2-MVPs. (B) Westernblot analyses of ACE2-MVPs under reducing and non-reducing conditions. (C) Viral neutralization activity of ACE2-MVPs determined in a pseudovirus neutralization assay. (D) Effects of oligomerization on ACE2 display on VLPs and its correlation with ACE2-MVPs neutralization function.

[0022] Figure 11 illustrates schematics of neuraminidase stem-based tetrameric vector for type II transmembrane protein display on vesicles. Neuraminidase stem-based tetrameric domain linked to an optional additional oligomerization domain and an extracellular domain of a type II transmembrane protein.

[0023] Figures 12A-12C illustrate production of vesicles displaying tetrameric type II peptide on (A) VLPs with viral genomes, (B) VLPs without viral genome, and (C) EVs.

[0024] Figures 13A-13C illustrate characterization of DPP4-MVPs with variations of neuraminidase-stem tetrameric domains. (A) Quantitative Western-blot analyses of DPP4-MVPs. (B) Western-blot analyses ofDPP4-MVPs under reducing and non-reducing conditions. (C) Viral neutralization activity of DPP4-MVPs determined in a pseudovirus neutralization assay.

[0025] Figures 14A-14C illustrate production of vesicles displaying mixed oligomer, combination peptide on (A) VLPs with viral genomes, (B) VLPs without viral genome, and (C) EVs.WSGR Docket No. 48295-719.601

[0026] Figures 15A-15C illustrate characterization of mixed oligomer, bi-specific Ab-MVPs displaying monomeric and trimeric scFv targeting SARS CoV-2 spike proteins. (A) Quantitative Western -blot analyses of Ab-MVPs. (B) Western-blot analyses of Ab-MVPs under reducing and non-reducing conditions. (C) Viral neutralization activity of Ab-MVPs determined in a pseudovirus neutralization assay.

[0027] Figures 16A-16D illustrate production and characterization of EVs displaying trimeric ACE2 targeting SARS CoV-2 spike proteins. (A) Production of ACE2-D4-EVs with D4-trimeric display vector.(B) Quantitative We stern -blot analyses of ACE2-D4-EVs. (C) Particle size analyses of ACE2-D4-EVs using qNano. (D) Viral neutralization activity of H2A / ACE2-D4-EVs determined in a pseudovirus neutralization assay.

[0028] Figures 17A-17C illustrates schematics of Linel coiled-coil (Llcc)-based trimeric vector for vesicle display. (A). Schematics of LINE1 open reading frame protein 1. (B) The monomeric and trimeric structure of LINE1 orf 1 coiled-coil peptide. (C) Design of LI cc proteinbased trimeric display vector with Llcc linked to the LI transmembrane and cytosolic domains in the vector.

[0029] Figures 18A-18C illustrate production of vesicles displaying Llcc-trimeric peptide on (A) VLPs with viral genomes, (B) VLPs without viral genome, and (C) EVs.

[0030] Figures 19A-19C illustrate production and characterization of VLPs displaying trimeric Llcc-ACE2 targeting SARS CoV-2 spike proteins. (A) Quantitative Western-blot analyses of ACE2-Llcc-MVPs. (B) Particle size analyses of ACE2-Llcc-MVPs using qNano. (C) Viral neutralization activity of ACE2-Llcc-MVPs determined in a pseudovirus neutralization assay.

[0031] Figures 20A-20C illustrate schematics depicting the molecular construction of SINGLE-CHAIN TRIMER PMHC-I antigen-presenting vesicles (SCT-APVs). (A) T cells employ multiple co -stimulatory molecules to modulate T cell activation, proliferation, differentiation, and function. (B) Molecular components of the displayed molecules for TCR engagement. (C) Schematics of the displayed single-chain trimer (SCT), consisting of linked peptide, MHC-I, and P2m, and displayed type I and type II costimulatory (costim) molecules with corresponding oligomerization domains and membrane anchoring domain. “C” stands for the C-terminus of type I protein and “N” stands for the N-terminus of type II protein.

[0032] Figure 21 illustrates production of SINGLE-CHAIN TRIMER PMHC-I antigen- presenting vesicles (SCT-APV) by displaying high -density oligomerized SCT-pMHC-I and combination of costimulatory molecules on VLPs or EVs.

[0033] Figures 22A-22C illustrate effects of costimulatory subtraction on naive OT-1 T cell activation by EV -based ova-APVs displaying ova-SCT and combination of costimulatoryWSGR Docket No. 48295-719.601 molecules. (A) A matrix of costim molecules displayed on ova-APVs. (B) Effects of ova-APVs displaying varied costim molecules on T cell activation. Naive OT1 T cells were stimulated with ova-APVs and analyzed by FACS to determine the up-regulation of CD69 and CD25 expression at day 1 post stimulation. (C) Representative FACS plots are shown.

[0034] Figures 23A-23C illustrate effects of costimulatory addition on T cell activation by EV- based ova-APVs displaying ova-SCT and combination of costimulatory molecules. (A) A matrix of costimulatory molecules displayed on ova-APVs. All ova-APVs displayed ova-SCT, GITRL, and ICAM-1 but with varied CD80, CD86, XO40L, and CD48. (B) Effects of ova-APVs displaying varied costimulatory molecules on T cell activation. Pan OT1 and Pmel T cells, including both naive and antigen-experienced T cells were stimulated with ova-APVs and analyzed by FACS to determined the up-regulation of CD69 and CD25 expression at day 1 post stimulation. (C) Representative FACS plots are shown.

[0035] Figures 24A-24B illustrate production of ova-APVs displaying SCT-pMHC-I with oligomerization domains with varied degree of oligomerization. (A) Schematics of SCT-pMHC-I display vector with varied oligomerization domain. (B) The list of display vectors used to produce ova-APVs displaying ova-SCT, CD86, GITRL, and ICAM-1 and their corresponding oligomerization domains. Coiled-coil helix domains with varied degree of oligomerization were used to optimize SCT-pMHC-I display on EVs.

[0036] Figures 25A-25D illustrate effects of oligomerization domain for SCT-pMHC-I display on the function of ova-APVs on T cell activation and proliferation. (A) FACS gating to distinguish the effects of ova-APVs on anti gen -specific OT-1 T cells (Ly5.2) and non-specific T cells (Ly5.1). (B) Effects of ova-APVs displaying SCT-MHC-I with varied oligomerization domain on T cell activation. Naive OT1 and non-specific Ly5.1 cells were stimulated with various ova-APVs and analyzed by FACS to determine the up-regulation of CD69 and CD25 expression at day 2 post stimulation. Representative FACS plots are shown. (C) Effects of ova-APVs with varied oligomerization domain on T cell activation as indicated by the percent of CD69 positive cells or CD69 and CD25 double-positive cells among the stimulated T cell mixture. (D) Effects of ova- APVs with varied oligomerization domain on T cell proliferation as determined by the fold of T cell expansion or total cell increases.

[0037] Figures 26A-26C illustrate optimized EV- and VLP-based APVs with displayed high- density oligomerized SCT, CD80, ICAM1, and GITRL. (A, B) Schematics of the displayed SCT / D4, consisting of linked peptide, MHC-I, P2m and D4, and displayed CD80 / D4, ICAM1 / D4, and GITRL / NA costimulatory molecules. “C” stands for the C-terminus of type I protein and “N”WSGR Docket No. 48295-719.601 stands for the N-terminus of type II protein. (C) Production of APVs by displaying high-density oligomerized SCT, CD80, ICAM1, and GITRL on VLPs or EVs.

[0038] Figures 27A-27E illustrate production and characterization of APV(86IG) displaying high-density oligomerized SCT-pMHC-I and combination of chimeric costimulatory molecules, including CD86 / D4, GITRL / NA, and ICAM1 / D4. (A, B) Particle size analyses of (A) exosome- based ova-APV(86IG) and (B) VLP-based ova-APV(86IG) using qNano. (C) Quantitative westernblot analyses to determine the copies of ova-H2Kb SCT class I MHC molecules on EV- or VLP- based ova-APV(86IG). (D) Quantitative western-blot analyses to determine the copies of CD86 / D4, GITRL / NA, and ICAM1 / D4 molecules on EV- or VLP-based ova-APV(86IG). (E) The copies of ova-H2Kb SCT, CD86 / D4, GITRL / NA, and ICAM1 / D4 on EV- or VLP-based ova- APV(86IG).

[0039] Figures 28A-28E illustrate potent and specific activation of ova-specific OT-1 T cells with EV- or VLP-based ova-APVs displaying high-density oligomerized ova-H2Kb SCT-pMHC-I and combination of chimeric costimulatory molecules, including CD86 / D4, GITRL / NA, and ICAM1 / D4. (A, B) EV- or VLP-based ova-APVs are as potent as anti-CD3 / CD28 Dynavbeads at activating OT1 T cells. (A) Representative FACS plots of CD69 and CD25 expression or (B) total percent of CD69 and CD25 positive cells at day 1 after ova-specific OT-1 T cells were activated with anti-CD3 / CD28 Dyna beads, or EV-ova-APVs or VLP-ova-APVs. (C, D). EV-ova-APVs selectively activate ova-specific OT-1 T cells but not non-specific Ly5.1 T cells. (E) The ratio of fully-activated (CD69 and CD25 double-positive) and partially-activated T cells (CD69 positive only) among the EV-ova-APVs activated OT-1 and Ly5.1 T cells at different time point post stimulation.

[0040] Figures 29A-29C illustrates reprogramming cancer cells into antigen-presenting tumor cells (APTCs) by displaying oligomerized multivalent co -stimulatory ligand molecules on tumor cells. (A) Schematic illustrating design of antigen-presenting tumor cells (APTCs) expressing multivalent, displayed co-stimulatory molecules CD80 and GITRL with D4 oligomerization domain alongside endogenous peptide-MHC complexes presenting tumor antigens. (B) FACS characterization of a panel of murine APTC cell lines expressing displayed CD80 and GITRL. Composite data of 3 experiments is shown. (C) FACS quantification of CD80 and GITRL expression on a panel of murine APTC cell lines determined using PE quantibrite beads. Composite data of 2 experiments is shown.

[0041] Figure 30 illustrates MC38-APTCs selectively activate antigen -specific T cells in vitro. Expression of early T cell activation markers CD69 and CD25 in naive OT-1 vs Ly5.1 T cells andWSGR Docket No. 48295-719.601 proportion of CD69+ / CD25+ OT-1 vs Ly5.1 T cells after 24 hours of co-culture with ova MC38 APTCs. Data representative of 2 experiments is shown.

[0042] Figure 31 illustrates PancO2-APTCs selectively activate antigen-specific T cells in vitro. Expression of early T cell activation markers CD69 and CD25 in naive OT-1 vs Ly5.1 T cells and proportion of CD69+ / CD25+ OT-1 vs Ly5.1 T cells after 24 hours of co-culture with ova Panc02 APTCs. Data representative of 2 experiments is shown.

[0043] Figures 32A-32D illustrate in vivo rejection of MC38 tumor cells expressing displayedCD80 and GITRL (MC38-APTCs). (A) Schematic illustrating design of MC38-APTC in vivo rejection study in mice. (B) Average tumor growth of WT MC38 tumors (n=5) vs MC38 APTC tumors (n=5) and (C) individual mouse tumor growth curves in C57BL / 6 mice. Representative data from 5 experiments are shown. (D) Probability of survival of mice implanted with WT MC38 vs MC38 APTCs. Representative data from 5 experiments are shown.

[0044] Figures 33A-33C illustrate MC38-APTCs are equally tumorigenic as wild-type MC38 tumors in athymic nude mice. (A) Schematic illustrating design of MC38-APTC in vivo tumor growth study in athymic nude mice. (B) Average tumor growth of WT MC38 tumors (n=3) vs MC38 APTC tumors (n=4) and (C) individual mouse tumor growth curves in athymic nude mice.

[0045] Figures 34A-34C illustrate MC38-APTCs mediate the rejection of co-injected wildtype MC38 tumor cells. (A) Schematic illustrating design of MC38-APTC and WT tumor cell mixing titration in vivo rejection study. MC38-APTCs were mixed with increasing numbers of WT MC38 cells at the indicated ratios and tumor growth was observed. (B) Average tumor growth of MC38-APTC and WT MC38 tumor mixtures at various ratios (n=5) and (C) individual mouse tumor growth curves in C57BL / 6 mice.

[0046] Figures 35A-35D illustrate rejection of MC38-APTCs induces robust anti -tumor immunity against wild -type MC38 re-challenge. (A) Schematic illustrating design of MC38-APTC vaccinated mouse re-challenge in vivo study. (B) Average MC38 tumor growth and (C) individual mouse tumor growth curves in naive vs MC38 -APTC vaccinated mice re-challenged with wild-type tumor cells. Data is representative of three experiments. (D) Probability of survival of naive mice vs APTC-vaccinated mice re-challenged with WT MC38 cells. Representative data from three experiments are shown.

[0047] Figures 36A-36C illustrate anti-tumor immunity from MC38-APTC vaccination mediates rejection of re-challenge with a wide range of wild type tumor cells. (A) Schematic illustrating design of MC38-APTC vaccinated mouse wild-type MC38 re-challenge titration study. (B, C) Individual mouse MC38 tumor growth curves (n = 5) in naive vs MC38-APTC vaccinated mice re-challenged with increasing numbers of WT MC38 tumor cells.WSGR Docket No. 48295-719.601

[0048] Figures 37A-37C illustrate vaccination with irradiated MC38-APTCs induces robust anti-tumor immunity against re-challenge. (A) Schematic illustrating design of irradiated MC38- APTC vaccination study. (B) Average growth curves of WT MC38 tumors in naive mice vs irradiated MC38-APTC vaccinated mice. (C) Individual mouse tumor growth curves from naive vs irradiated MC38-APTC vaccinated mice.

[0049] Figures 38A-38D illustrate Renca-APTCs are effectively rejected in syngeneic mice. (A) Schematic illustrating design of Renca-APTC in vivo rejection study in mice. (B) Average tumor growth of WT Renca tumors (n=5) vs Renca-APTC tumors (n=5) and (C) individual mouse tumor growth curves in BALB / C mice. Representative data from two experiments are shown. (D) Probability of survival of mice implanted with WT Renca vs Renca-APTCs. Representative data from two experiments are shown.

[0050] Figures 39A-39D illustrate rejection of Renca-APTCs induces robust anti-tumor immunity against wild-type Renca re-challenge. (A) Schematic illustrating design of Renca-APTC vaccinated mouse re-challenge in vivo study. (B) Average Renca tumor growth and (C) individual mouse tumor growth curves in naive vs Renca-APTC vaccinated mice re-challenged with wild-type tumor cells. (D) Probability of survival of naive mice vs APTC-vaccinated mice re-challenged with WT Renca cells. Representative data from two experiments are shown.

[0051] Figures 40A-40D illustrate Panc02 cells expressing displayed CD80 and GITRL molecules (PancO2-APTCs) are rejected in mice. (A) Schematic illustrating design of Panc02- APTC in vivo rejection study in mice. (B) Average tumor growth of WT Panc02 tumors (n=5) vs PancO2-APTC tumors (n=5) and (C) individual mouse tumor growth curves in BL / 6 mice. (D) Probability of survival of mice implanted with WT Panc02 vs PancO2-APTCs (n=5).

[0052] Figures 41A-41C illustrate rejection of PancO2-APTCs induces robust anti-tumor immunity against wild-type Panc02 re-challenge. (A) Schematic illustrating design of Panc02- APTC vaccinated mouse re-challenge in vivo study. (B) Average Panc02 tumor growth and (C) individual mouse tumor growth curves in naive vs PancO2-APTC vaccinated mice re-challenged subcutaneously with wild-type tumor cells; and Probability of survival of naive mice vs APTC- vaccinated mice re-challenged with WT Panc02 cells. Representative data from two experiments are shown.

[0053] Figures 42A-42C illustrate GL261-APTCs are rejected in mice. (A) Schematic illustrating design of GL261-APTC in vivo rejection study in mice. (B) Average tumor growth of WT GL261 tumors (n=5) vs GL261 -APTC tumors (n=5) and (C) individual mouse tumor growth curves in BL / 6 mice and probability of survival of mice implanted subcutaneously with WT GL261 vs GL261-APTCs (n=5).WSGR Docket No. 48295-719.601

[0054] Figures 43A-43C illustrate rejection of GL261-APTCs induces complete anti -tumor immunity against wild-type GL261 re-challenge. (A) Schematic illustrating design of GL261- APTC vaccinated mouse re-challenge in vivo study. (B) Average GL261 tumor growth and (C) individual mouse tumor growth curves in naive vs GL261 -APTC vaccinated mice re-challenged subcutaneously with wild-type tumor cells.

[0055] Figures 44A-44C illustrate synergistic control and elimination of established MC38 tumors with combined autologous APTCs and LPS adjuvant treatments. (A) Schematic illustrating design of established MC38 treatment study by APTCs and LPS adjuvant. (B) Average tumor growth curves and (C) individual mouse tumor growth curves of WT MC38 tumors after treatment with APTCs, LPS adjuvant, or a combination of APTC and adjuvant (n=5).

[0056] Figures 45A-45C illustrate synergistic control and elimination of established MC38 tumors with combined autologous APTCs and poly(LC) (Nexavant) adjuvant treatments. (A) Schematic illustrating treatment of established MC38 tumors by APTCs and poly(LC) Nexavant adjuvant. (B) Average tumor growth curves and (C) individual mouse tumor growth curves of WT MC38 tumors after treatment with APTCs, poly(I:C) Nexavant adjuvant, or a combination of APTC and adjuvant (n=5).

[0057] Figures 46A-46C illustrate synergistic control and elimination of established MC38 tumors with combined autologous APTCs and HMW poly(LC) adjuvant treatments. (A) Schematic illustrating treatment of established MC38 tumors by APTCs and High-molecular weight (HMW) poly(LC) adjuvant. (B) Average tumor growth curves and (C) individual mouse tumor growth curves of WT MC38 tumors after treatment with APTCs, HMW poly(LC) adjuvant, or a combination of APTC and adjuvant (n=5).

[0058] Figures 47A-47C illustrate synergistic control of established MC38 tumors with combined autologous APTCs and checkpoint blockade treatments. (A) Schematic illustrating treatment of established MC38 tumors by APTCs and immune checkpoint blockade (ICB). (B) Average tumor growth curves and (C) individual mouse tumor growth curves of WT MC38 tumors after treatment with APTCs, anti-PD-1 ICB, or a combination of APTC and ICB (n=5).Representative data from two experiments are shown.

[0059] Figures 48A-48C illustrate synergistic control and elimination of established MC38 tumors with APTC, checkpoint blockade and adjuvant treatments. (A) Schematic illustrating treatment of established MC38 tumors using APTCs, immune checkpoint blockade (ICB) and poly(LC) adjuvant treatment. (B) Average tumor growth curves and (C) individual tumor growth curves for mice with established WT MC38 tumors after treatment with anti-PD-1 ICB, anti-PD-1WSGR Docket No. 48295-719.601 in combination with APTCs, or anti-PD-1 in combination with APTCs and HMW poly(I:C) adjuvant treatments.

[0060] Figures 49A-49E illustrate generation of tumor-activated T cells (tac-T) from A375- APTCs. (A) Expression of early T cell activation markers CD69 and CD25 on human tac-T cells 9 days post-stimulation by A375-APTCs. (B) Proportion of CD4+ vs CD8+ T cells after no stimulation vs stimulation with Dyna beads or A375-APTCs. (C) Proportion of CD25 expression in T cells after no stimulation vs stimulation with Dyna beads or A375-APTCs. (D) Fold proliferation of T cells after no stimulation vs stimulation with Dyna beads, wild-type A375 tumor cells, or A375 -APTCs. (E) In vitro CTL activity of tac-T cells against wild-type A375 tumor cells. Specific killing activity of T cells after no stimulation vs stimulation with Dyna beads or A375 -APTCs is shown. Data is representative of 3 experiments.

[0061] Figures 50A-50C illustrate control and elimination of established A375 tumors with tac-T adoptive cell transfer. (A) Schematic illustrating treatment of established A375 tumors in NCG immunodeficient mice using tac-T cell adoptive transfer. tac-T cells were generated via A375-APTC stimulation. Mice were given regular infusions of IL-2 post-adoptive transfer to support human T cell survival. (B) Average tumor growth curves and (C) individual mouse tumor growth curves of NCG mice with WT A375 tumors after treatment with tac-T cells vs non-specific T cells stimulated with Dyna beads.

[0062] Figures 51A-51C illustrate MC38-APTC organoids are rejected in vivo. (A) Workflow of tumor growth in vivo of MC38 tumors derived from 3 -D cultured wild type or MC38-APTC organoids. (B) Average tumor growth curves and (C) individual mouse tumor growth curves for mice implanted with tumor cells from wild-type MC38 organoids vs MC38-APTC organoids (n=5).

[0063] Figures 52A-52C illustrate rejection of MC38-APTC organoid tumors mediates antitumor immunity against re-challenge. (A) Schematic illustrating design of WT MC38 re-challenge of mice vaccinated with MC38-APTC organoids. (B) Average tumor growth curves and (C) individual mouse tumor growth curves for naive vs MC38-APTC organoid (n=5) vaccinated mice after re-challenge with WT MC38 tumor cells.

[0064] Figures 53A-53C illustrate tumor-APVs: Antigen-presenting vesicles derived from APTCs. (A) Schematic illustrating generation of tumor-APVs as EVs secreted from APTCs or vesicles from physically -disrupted APTCs. Tumor-APVS display necessary membrane proteins for antigen-specific T cell activation, including endogenous tumor pMHC-I, CD80 / D4 and GITRL / NA. (B, C) Particle diameter distribution of tumor-APVs from APTC-EVs (B), or vesicles from disrupted APTCs (C) determined via TRPS analysis.WSGR Docket No. 48295-719.601

[0065] Figures 54A-54C illustrate EV-based Antigen-presenting vesicles secreated from ova MC38-APTCs selectively activate antigen -specific OT-1 T cells. (A) Expression of early T cell activation markers CD69 and CD25 in co-culturedOT-1 and Ly5.1 T cells. Antigen-specific OT-1 T cells were mixed with equal numbers of non-specific Ly5.1 T cells and stimulated with (ova MC38-APTC) EVs at a 5000:1 particle to cell ratio, or Dynabeads. (B) Proportion of fully activated, CD69+ / CD25+ OT-1 vs Ly5.1 T cells after stimulation by (ova MC38-APTC) EVs vs Dynabeads. (C) Total fold T cell proliferation of co-cultured OT-1 vs Ly5.1 T cells after stimulation by (ova MC38-APTC) EVs vs Dynabeads.

[0066] Figures 55A-55C illustrate antigen-presenting EVs produced by ova PancO2-APTCs selectively activate antigen -specific OT-1 T cells. (A) Expression of early T cell activation markers CD69 and CD25 in co-cultured OT-1 and Ly5.1 T cells. Antigen-specific OT-1 T cells were mixed with equal numbers of non -specif icLy 5.1 T cells and stimulated with (ova-Panc02-APTC) EVs at a 5000:1 particle to cell ratio, or Dynabeads. (B) Proportion of fully activated, CD69+ / CD25+ OT-1 vs Ly5.1 T cells after stimulation by (ova-Panc02-APTC) EVs vs Dynabeads. (C) Total fold T cell proliferation of co-cultured OT-1 vs Ly5.1 T cells after stimulation by (ova-Panc02-APTC) EVs vs Dynabeads.

[0067] Figures 56A-56C illustrate selective activation of antigen-specific OT-1 T cells by tumor-derived antigen-presenting vesicles (tumor-APVs) produced by nitrogen cavitation of ova- MC38-APTCs. (A) Expression of early T cell activation markers CD69 and CD25 in co-cultured OT-1 and Ly5.1 T cells. Antigen-specific OT-1 T cells were mixed with equal numbers of nonspecific Ly 5.1 T cells and stimulated with (ova-MC38-APTC) tumor-APVs produced via nitrogen cavitation at a 2000:1 particle to cell ratio. (B) Proportion of fully activated, CD69+ / CD25+ OT-1 vs Ly 5.1 T cells after stimulation by (ova-MC38-APTC) tumor-APVs vs Dynabeads. (C) Total fold T cell proliferation of co-cultured OT-1 vs Ly5.1 T cells after stimulation by (ova-MC38-APTC) tumor-APVs vs Dynabeads.

[0068] Figures 57A-57C illustrate selective activation of antigen-specific OT-1 T cells by tumor-derived antigen-presenting vesicles (tumor-APVs) produced by nitrogen cavitation of ova PancO2-APTCs. (A) Expression of early T cell activation markers CD69 and CD25 in co-cultured OT-1 and Ly5.1 T cells. Antigen-specific OT-1 T cells were mixed with equal numbers of nonspecific Ly5.1 T cells and stimulated with (ova-Panc02-APTC) tumor-APVs produced from via nitrogen cavitation at a 10000:1 particle to cell ratio. (B) Proportion of fully activated, CD69+ / CD25+ OT-1 vs Ly5.1 T cells after stimulation by (ova-Panc02-APTC) tumor-APVs vs Dynabeads. (C) Total fold T cell proliferation of co-cultured OT-1 vs Ly5.1 T cells after stimulation by (ova-Panc02-APTC) tumor-APVs vs Dynabeads.WSGR Docket No. 48295-719.601

[0069] Figures 58A-58C illustrate tumor-derived antigen-presenting vesicles (tumor-APVs) produced by nitrogen cavitation of ova PancO2-APTCs selectively activate antigen-specific OT-1 T cells. (A) Expression of early T cell activation markers CD69 and CD25 in co -cultured OT-1 and Ly5.1 T cells. Antigen-specific OT-1 T cells were mixed with equal numbers of non-specific Ly 5.1 T cells and stimulated with ova PancO2-APTC derived tumor-APVs produced from via nitrogen cavitation ata 10000:1 particle to cell ratio. (B) Proportion of fully activated, CD69+ / CD25+ OT-1 vs Ly5.1 T cells after stimulation by antigen presenting tumor-APVs vs Dynabeads. (C) Total fold T cell proliferation of co-cultured OT-1 vs Ly5.1 T cells after stimulation by tumor-APVs vs Dynabeads.

[0070] Figures 59A-59C illustrate that tumor-APVs improve treatment of established MC38 tumors compared to MC38 APTCs. (A) Schematic illustrating treatment of established MC38 tumors in mice using MC38 APTCs vs tumor-APVs derived from MC38-APTCs via nitrogen cavitation. C57BL / 6 mice with established subcutaneous MC38 tumors were given 2 intratumoral injections of MC38 APTCs or tumor-APVs. (B) Average mouse tumor growth curves and (C) individual mouse tumor growth curves after treatment with tumor-APVs, as compared to untreated mice or mice receiving MC38 APTCs.

[0071] Figures 60A-60D illustrate that tumor-APVs synergize with immune checkpoint blockade to treat established MC38 tumors. (A) Schematic illustrating treatment of established MC3 8 tumors in mice using combination aPD-1 checkpoint therapy and tumor-APVs derived from MC38-APTCs via nitrogen cavitation. C57BL / 6 mice with established subcutaneous MC38 tumors were given 2 intratumoral injections of tumor-APVs. 150pg aPD-1 mAb was delivered weekly via intraperitoneal injection. (B) Average mouse tumor growth curves and (C) individual mouse tumor growth curves after treatment with tumor-APVs in combination with aPD-1, as compared to untreated mice or mice receiving aPD-1 or tumor-APVs alone.

[0072] Figures 61A-61C illustrate induction of systemic anti-tumor immunity in mice immunized with tumor APVs (t-APVs) derived from B16F0 APTCs are resistant to rechallenge. (A) Schematic illustrating t-APV immunization and re-challenge study design. t-APVs derived from corresponding APTCs were injected into mice intramuscularly with poly IC as adjuvant following the immunization schedule depicted. Immunized mice were challenged with wild-type tumor cells at day 35 post-primary immunization. (B, C) Mice immunized with (B) no t-APV, or (C) t-APV derived from B16F0-APTCs treated with interferon gramma.

[0073] Figures 62A-62C illustrate induction of systemic anti-tumor immunity in mice immunized with tumor APVs (t-APVs) derived from MC38-APTCs are resistant to rechallenge. (A) Schematic illustrating t-APV immunization and re-challenge study design. t-APVs derivedWSGR Docket No. 48295-719.601 from corresponding APTCs were injected into mice intramuscularly with poly IC as adjuvant following the immunization schedule depicted. Immunized mice were challenged with wild -type tumor cells at day 35 post-primary immunization. (B, C) Mice immunized with (B) no t-APV, or (C) t-APV derived from MC38-APTCs with interferon gramma treatment.

[0074] Figure 63 illustrates schematic depicting the in situ APTC as therapeutic cancer vaccine.

[0075] Figures 64A-64C illustrate eliminate established colon cancer (MC38) with in situ APTCs. (A) Schematic illustrating treatment of established MC38 tumors in mice using in situ APTCs through intratumoral infection of tumor cells with lentivirus expressing oligomerized costimulatory molecules CD80 and GITRL. C57BL / 6 mice with established subcutaneou s MC38 tumors were given 3 consecutive intratumoral injections of costim lentivirus. lOpg of poly-IC was delivered weekly via intraperitoneal injection. (B) Average mouse tumor growth curves and (C) individual mouse tumor growth curves after treatment with in situ APTC in combination with poly- IC, as compared to untreated mice or mice receiving poly-IC alone.

[0076] Figures 65A-65C illustrate eliminate established pancreatic tumors (Panc02) with in situ APTCs. (A) Schematic illustrating treatment of established Panc02 tumors in mice using in situ APTCs through intratumoral infection of tumor cells with lentivirus expressing oligomerized costimulatory molecules CD80 and GITRL. C57BL / 6 mice with established subcutaneous Panc02 tumors were given 3 consecutive intratumoral injections of costim lentivirus. lOpg of polylC was delivered weekly via intraperitoneal injection. (B) Average mouse tumor growth curves and (C) individual mouse tumor growth curves after treatment with in situ APTC in combination with polylC, as compared to untreated mice or mice receiving polylC alone.DETAILED DESCRIPTION

[0077] The present disclosure employs, unless otherwise indicated, conventional molecular biology techniques, which are within the skill of the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art.Definitions

[0078] Throughout this disclosure, various embodiments are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of any embodiments. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range to the tenth of the unit of theWSGR Docket No. 48295-719.601 lower limit unless the context clearly dictates otherwise. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual values within that range, for example, 1.1, 2, 2.3, 5, and 5.9. This applies regardless of the breadth of the range. The upper and lower limits of these intervening ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. 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, unless the context clearly dictates otherwise.

[0079] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of any embodiment. As used herein, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and / or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0080] Unless specifically stated or obvious from context, as used herein, the term “about” in reference to a number or range of numbers is understood to mean the stated number and numbers + / - 10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range.

[0081] Antigen-Presenting Tumor Cells (APTCs) and Tumor-Derived Antigen-Presenting Vesicles (Tumor-APVs)

[0082] Among various therapeutic and prevention approaches, cancer vaccines are designed to create immune memory against cancer cells and have tremendous potential for cancer prevention and treatment. It is now well-established that successful anti -tumor immunity may be induced against tumor-specific antigens. These antigens include viral antigens with tumorigenic potential, tumor-associated antigens expressed in cancer cells, cancer-driving mutant proteins, and neoantigens from non-synonymous somatic mutations. Cancer vaccines are typically designed to exogenously administer selected tumor antigens either directly combined with adjuvants or loaded into dendritic cells (DCs) to induce T-cell and humoral immunity against the specific tumor antigens. Alternatively, whole tumor lysates may be formulated or loaded into DCs and injected as vaccines. Finally, DC recruiting and activation signals may be delivered into tumors to generate cancer vaccines in situ. So far, tremendous progress has been made in recent decades, and it hasWSGR Docket No. 48295-719.601 been encouraging to observe vaccine-induced anti-tumor immune response and tumor regression and / or survival benefits in some model systems and small clinical trials. However, there remain significant barriers to establishing vaccines as effective approaches for cancer prevention and treatment due to the intrinsic limitations of cancer vaccine platforms.

[0083] The conventional vaccine platforms are suboptimal for the induction of T-cell immunity against cancer. Since tumor antigens are generally intracellular and are hidden from antibody -based humoral immunity, cancer vaccines can generate robust CD8 and CD4 T cell immunity against tumor antigens. However, the existing cancer vaccine platforms are derived from successful vaccines for pathogens, which are highly effective at generating long-lasting humoral immunity but less so for the induction of T-cell immunity. To facilitate T cell priming, various ex -vivo or in situ DC-based cancer vaccines have been developed to prime anti -tumor T cells selectively. However, ex-vivo DC-based cancer vaccines with pre-loaded cancer antigens, which can prime T cell responses against cancer antigens, also seem to have limited clinical efficacy despite generating tumor-specific immune responses. It is possible that the complexity of DC vaccine manufacturing, including the type of DCs used in the vaccines, tedious large-scale manufacturing, antigen selection, and antigen loading into DCs, may further limit the potential of this cancer vaccine platform.

[0084] Besides the T cell immunity, the choice of cancer antigens may also have a major impact on the potency and durability of cancer vaccines. So far, most vaccines are formulated with selected tumor antigens, such as TAA, TSA, or neoantigens; they may have limited potency to only those antigens and may be sensitive to immune escape as tumors evolve. Interestingly, some have attempted to use whole tumor lysates to formulate vaccines or loaded into DCs. These approaches are still constrained by the general limitations of the DC vaccine platforms in inducing T-cell immunity and complex DC biology and manufacturing.

[0085] Beyond the vaccine-intrinsic issues, cancer vaccines need to be formulated to choreograph sequential responses in the host immune systems and, to reprogram the suppressive immune environment within established tumors. Also established tumors have tolerized host immunity systems so that tumor cells are treated as self and not foreign, thus are protected and not attacked by host immune cells. Therefore, an effective cancer vaccine must break the tolerance barriers and the suppressive tumor microenvironment by re-educating the immune systems to recognize cancer as foreign.

[0086] Described herein are compositions and methods of use for tumor-derived antigen- presenting cells, dubbed antigen-presenting tumor cells (APTCs). Compositions and methods ofWSGR Docket No. 48295-719.601 use as described herein are antigen-presenting tumor cells (APTCs) and tumor-derived antigen- presenting vesicles (Tumor-APVs) from APTCs.

[0087] Antigen-Presenting Tumor Cells (APTCs)

[0088] Described herein, in some embodiments, is an antigen-presenting tumor cell (APTC) comprising, a first fusion protein that comprises the displayed costimulatory ligand molecule CD80 or CD86, a second fusion protein that comprises the displayed costimulatory ligand molecule GITRL, and an endogenous protein that comprises an endogenous peptide major histocompatibility complex class I (pMHC-I), wherein the first fusion protein, the second fusion protein, or the endogenous protein, is expressed at a valency of at least about 1000 copies on a surface of the APTC. In some embodiments, the first fusion protein, the second fusion protein, and the endogenous protein are all expressed on the cell surface of the APTC. In some embodiments, the APTC comprises the first fusion protein that comprises the displayed costimulatory ligand molecule CD80 or CD86. In some embodiments, the APTC comprises the second fusion protein that comprises the displayed costimulatory ligand molecule GITRL. In some embodiments, the APTC comprises displayed multivalent, oligomerized costimulatory ligand molecules CD80 and GITRL alongside endogenous MHC I.

[0089] In some embodiments, the first fusion protein of the displayed costimulatory ligand molecule CD80 or CD86, or the second fusion protein of the displayed costimulatory ligand molecule GITRL on the surface of the APTC binds specifically to the cognate costimulatory receptors on a T-cell surface.

[0090] In some embodiments, the APTC further comprises a third fusion protein comprising at least one, two, three, four, five, six, seven, eight, nine, ten, or more than ten separate fusion proteins, wherein the at least one, two, three, four, five, six, seven, eight, nine, ten, or more than ten separate fusion proteins each comprises 0X40 Ligand, CD48, CD30 Ligand, ICOS Ligand, CD70, CD40, CD40 Ligand, 4-BB Ligand, TLIA, TIM4, SLAM, CD48, CD58, CD155, CD112, LIGHT, or ICAM-1.

[0091] In some embodiments, the endogenous pMHC-I on the surface of the APTC binds specifically to an antigen -specific T cell receptor (TCR) that recognizes an antigenic peptide derived from an endogenous antigen, a viral antigen, a mutated tumor antigen, a tumor-specific antigen, a differentiated tumor antigen, an over-expressed tumor antigen, or a neoantigen within tumor cells. Alternatively, the endogenous pMHC-I on the surface of the APTC binds specifically to a CD8+ T cell bearing an antigen-specific T cell receptor (TCR) recognizing an antigenic peptide derived from an endogenous antigen, a viral antigen, a mutated tumor antigen, a tumor-WSGR Docket No. 48295-719.601 specific antigen, a differentiated tumor antigen, an over-expressed tumor antigen, or a neoantigen within tumor cells.

[0092] Described herein, in some embodiments, is an APTC comprising an endogenous pMHC-I that comprises an endogenous antigenic peptide, an endogenous P -2 -microglobulin (P2m), and an endogenous MHC-I alpha chain. In some embodiments, the endogenous antigenic peptide binds to the endogenous MHC-I alpha chain. In some embodiments, the endogenous antigenic peptide in the endogenous pMHC-I is derived from an endogenous antigen, a viral antigen, a mutated tumor antigen, a tumor-specific antigen, a differentiated tumor antigen, an over-expressed tumor antigen, or a neoantigen within tumor cells.

[0093] In some embodiments, the APTC comprises an endogenous pMHC-I that comprises an endogenous antigenic peptide, an endogenous P-2-microglobulin (P2m), and an endogenous MHC-I alpha chain wherein the endogenous antigenic peptide comprises an endogenous antigenic peptide from a tumor cell.

[0094] The present disclosure provides in some embodiments the APTC that is derived from pancreatic cancer, rectal cancer, colorectal cancer, lung cancer, endometrial cancer, liver cancer, testicular cancer, prostate cancer, cervical cancer, breast cancer, bladder cancer, head and neck cancer, skin cancer, brain cancer, ovarian cancer, salivary gland cancer, appendiceal cancer, esophagogastric cancer, thyroid cancer, soft tissue sarcoma, kidney cancer, leukemia, myeloma, lymphoma, melanoma, non-small cell lung cancer, Hodgkin’s lymphoma, or non -Hodgkin’s lymphoma.

[0095] Described herein, in some embodiments, is an APTC comprising an endogenous pMHC-I that comprises an endogenous antigenic peptide. In some embodiments, the antigenic peptide is derived from pancreatic cancer, rectal cancer, colorectal cancer, lung cancer, endometrial cancer, liver cancer, testicular cancer, prostate cancer, cervical cancer, breast cancer, bladder cancer, head and neck cancer, skin cancer, brain cancer, ovarian cancer, salivary gland cancer, appendiceal cancer, esophagogastric cancer, thyroid cancer, soft tissue sarcoma, kidney cancer, leukemia, myeloma, lymphoma, melanoma, non-small cell lung cancer, Hodgkin’s lymphoma, or non-Hodgkin’s lymphoma.

[0096] In some embodiments, each of the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules comprises an amino acid sequence with at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 23 -58. In some embodiments, the costimulatory ligand molecule comprises an amino acid sequence set forth in any one of SEQ ID NO: 23 to SEQ ID NO: 58 in Table 2, or an amino acid sequence that isWSGR Docket No. 48295-719.601 substantially identical to an amino acid sequence set forth in any one of SEQ ID NO: 23 to SEQ ID NO: 58 (e.g. 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity).

[0097] Described herein, in some embodiments, is an APTC comprising a first, a second, or a third fusion protein of displayed costimulatory ligand molecules wherein each of the first, second, or third fusion protein of the displayed costimulatory ligand molecules further comprises a transmembrane domain. In some embodiments, the transmembrane domain of the fusion protein of the displayed costimulatory ligand molecule comprises a transmembrane domain derived from VSV-G, Dengue E protein, influenza hemagglutinin, influenza neuraminidase, spike protein SI, spike protein S2, Sindbis virus envelope (SINDBIS) protein, hemagglutinin envelope protein from measles virus, envelope glycoprotein of measles virus fusion (F) protein, RD114, BaEV, GP41, GP120, or the wild-type transmembrane domain of costimulatory ligand molecules. In some embodiments, the transmembrane domain of the fusion protein of the displayed costimulatory ligand molecule comprises a transmembrane domain derived from VSV-G wherein the VSV-G comprises a transmembrane domain and cytoplasmic tail.

[0098] In some embodiments, transmembrane domain of the fusion protein comprises an amino acid sequence with at least 90% sequence identity to an amino acid sequence of SEQ ID NOs: 59- 78. In some embodiments, transmembrane domain of the fusion protein comprises an amino acid sequence set forth in any one of SEQ ID NO: 59 to SEQ ID NO: 78 in Table 3, or an amino acid sequence that is substantially identical to an amino acid sequence set forth in any one of SEQ ID NO: 59 to SEQ ID NO: 78 (e.g. 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity).

[0099] Described herein, in some embodiments, is an APTC comprising a first, a second, or a third fusion protein of displayed costimulatory ligand molecules wherein each of the first, second, or third fusion protein further comprises an oligomerization domain. In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecule is a dimerization domain, a trimerization domain, a tetramerization domain, a hexamerization domain, a heterohexamerization domain, a heptamerization domain, or an octamerization domain. In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecule comprises a leucine zipper dimerization domain, a post-fusion oligomerization domain of viral surface protein, a D4 postfusion trimerization domain of VSV-G protein, a Dengue E protein post -fusion trimerization domain, a foldon trimerization domain, a coiled coil domain, a LINE1 retrotransposon coiled coil domain (Lice), a collagen XV coiled coil domain (CXVcc), a tetranectin coiled coil domain (TNTNcc), a cc-hex domain, a L24D domain, a L24DH domain, a cc-Hept domain, a wazOct domain, or an influenza neuraminidase stem domain.WSGR Docket No. 48295-719.601

[0100] In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecule comprises an amino acid sequence set forth in any one of SEQ ID NO: 79 to SEQ ID NO: 93 in Table 4, or an amino acid sequence that is substantially identical to an amino acid sequence set forth in any one of SEQ ID NO: 79 to SEQ ID NO: 93 (e.g. 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity). In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecule comprises an amino acid sequence set forth in any one of SEQ ID NO: 79 to SEQ ID NO: 93 with at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 79-93.

[0101] Described herein, in some embodiments, is an APTC capable of activating or stimulating an antigen-specific T cell wherein the APTC comprises a costimulatory ligand molecule of a fusion protein is expressed at least about 1000 copies on a surface of the APTC.

[0102] In some embodiments, the APTC comprises the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules wherein when the first fusion protein, the second fusion protein, or the third fusion protein of the costimulatory ligand molecules is expressed on the surface of the antigen-presenting tumor cell (APTC), the oligomerization domain is outside of the antigen-presenting tumor cell (APTC). In some embodiments, the APTC comprises the first, second, or third fusion protein of the displayed costimulatory ligand molecules wherein when the first fusion protein, the second fusion protein, or third fusion protein of the displayed costimulatory ligand molecules is expressed on the surface of the antigen-presenting tumor cell (APTC), the oligomerization domain is inside of the antigen-presenting tumor cell (APTC).

[0103] Described herein, in some embodiments, is an APTC capable of activating or stimulating an antigen-specific T cell wherein the APTC comprises a first, a second, or a third fusion protein of displayed costimulatory ligand molecules and an endogenous peptide major histocompatibility complex class I (pMHC-I).

[0104] In some embodiments, the APTC comprises the first, second, or third fusion protein of the displayed costimulatory ligand molecules and the endogenous peptide major histocompatibility complex class I (pMHC-I). The first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 1000 copies or more on a surface of the antigen-presenting tumor cell (APTC). In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 2500 copies or more on a surface of the antigen-presenting tumor cell (APTC). In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valencyWSGR Docket No. 48295-719.601 of about 5000 copies or more on a surface of the antigen-presenting tumor cell (APTC). In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 10,000 copies or more on a surface of the antigen-presenting tumor cell (APTC). In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 20,000 copies or more on a surface of the antigen-presenting tumor cell (APTC). In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 40,000 copies or more on a surface of the antigen-presenting tumor cell (APTC). In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 60,000 copies or more on a surface of the antigen-presenting tumor cell (APTC). In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 80,000 copies or more on a surface of the antigen-presenting tumor cell (APTC). In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 120,000 copies or more on a surface of the antigen-presenting tumor cell (APTC). In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 200,000 copies or more on a surface of the antigen-presenting tumor cell (APTC). In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 250,000 copies or more on a surface of the antigen-presenting tumor cell (APTC).

[0105] In some embodiments, the APTC comprises the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous peptide major histocompatibility complex class I (pMHC-I). The first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC- I is expressed at a valency of at least 1000 copies on the cell surface of the APTC.

[0106] In some embodiments, the APTC comprises the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I wherein the endogenous pMHC-I comprises a natural pMHC-I.WSGR Docket No. 48295-719.601

[0107] In some embodiments, the APTC comprises the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I wherein the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules comprises an amino acid sequence set forth in any one of SEQ ID NO: 94 to SEQ ID NO: 111 in Table 5, or an amino acid sequence that is substantially identical to an amino acid sequence set forth in any one of SEQ ID NO: 94 to SEQ ID NO: 111 (e.g. 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity). In some embodiments, the first, second, or third fusion protein of the displayed costimulatory ligand molecules comprises an amino acid sequence with at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 94-111.

[0108] In some embodiments, the APTC comprises a first fusion protein that comprises the costimulatory ligand molecule CD80 or CD86, and a second fusion protein that comprises the costimulatory ligand molecule GITRL, and a recombinant peptide major histocompatibility complex class I (pMHC-I), wherein the APTC lacks an endogenous pMHC-I or expresses a dysfunctional endogenous pMHC-I, wherein the first fusion protein, the second fusion protein, or the recombinant pMHC-I is expressed at a valency of at least about 1000 copies on a surface of the APTC.

[0109] Described herein, in some embodiments, is a fusion protein comprising a costimulatory ligand molecule that binds specifically to a T cell surface costimulatory receptor, an oligomerization domain, and a transmembrane domain, wherein the fusion protein is expressed at a valency of at least about 1000 copies on a surface of an antigen-presenting tumor cell (APTC). In some embodiments, the fusion protein comprises at least two separate fusion proteins, wherein the at least two separate fusion proteins each comprises a different costimulatory ligand molecule.

[0110] In some embodiments, the costimulatory molecule of the fusion protein comprises one or more selected from the group comprising GITR Ligand, CD80, CD86, 0X40 Ligand, CD48, CD30 Ligand, ICOS Ligand, CD70, CD40, CD40 Ligand, 4-BB Ligand, TLIA, TIM4, SLAM, CD48, CD58, CD155, CD112, LIGHT, and ICAM1.

[0111] Costimulatory Ligand Molecules

[0112] Effective activation of antigen-specific T cells by antigen-presenting cells (APCs), including B cells and dendritic cells (DCs), requires signal 1 from pMHC and TCR engagement and signal 2 from the interaction of costimulatory molecules. In contrast, cancer cells, as well as normal somatic cells, generally lack the expression of costimulatory molecules. Thus, despite producing and presenting many mutant antigens and neoantigens, tumor cells cannot activate anti-tumor T cells properly. In fact, pMHC and TCR engagement without costimulatory signalsWSGR Docket No. 48295-719.601 induces tolerance and T cell anergy. It renders them unresponsive to further stimulation, resulting in tolerance of tumors by our immune systems.

[0113] In some embodiments, the first fusion protein of costimulatory ligand molecule CD80 or CD86, or the second fusion protein of costimulatory ligand molecule GITRL on the surface of the APTC binds specifically to the cognate costimulatory receptors on a T-cell surface.

[0114] In some embodiments, each of the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules comprises an amino acid sequence with at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 23 -58. In some embodiments, the costimulatory ligand molecule comprisesan amino acid sequence set forth in any one of SEQ ID NO: 23 to SEQ ID NO: 58 in Table 2, or an amino acid sequence that is substantially identical to an amino acid sequence set forth in any one of SEQ ID NO: 23 to SEQ ID NO: 58 (e.g. 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity).

[0115] In some embodiments, the first, the second, or the third costimulatory ligand molecule comprises an amino acid sequence of at least 80% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 23-58. In some embodiments, the costimulatory ligand molecule of the fusion protein comprises an amino acid sequence of at least 85% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 23-58. In some embodiments, the costimulatory ligand molecule of the fusion protein comprises an amino acid sequence of at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 23-58. In some embodiments, the costimulatory ligand molecule of the fusion protein comprises an amino acid sequence of at least 95% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 23-58. In some embodiments, the costimulatory ligand molecule of the fusion protein comprises an amino acid sequence of at least 96% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 23-58. In some embodiments, the costimulatory ligand molecule of the fusion protein comprises an amino acid sequence of at least 97% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 23-58. In some embodiments, the costimulatory ligand molecule of the fusion protein comprises an amino acid sequence of at least 98% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 23-58. In some embodiments, the costimulatory ligand molecule of the fusion protein comprises an amino acid sequence of at least 99% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 23-58.

[0116] The term “sequence identity” means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison. The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window ofWSGR Docket No. 48295-719.601 comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Typically, techniques for determining sequence identity include comparing two nucleotide or amino acid sequences and the determining their percent identity. Sequence comparisons, such as for the purpose of assessing identities, may be performed by any suitable alignment algorithm, including but not limited to the Needleman-Wunsch algorithm (see, e.g., the EMBOSS Needle aligner available atwww.ebi.ac.uk / Tools / psa / emboss_needle / , optionally with default settings), the BLAST algorithm (see, e.g., the BLAST alignment tool available at blast.ncbi.nlm.nih.gov / Blast.cgi, optionally with default settings), and the Smith -Waterman algorithm (see, e.g., the EMBOSS Water aligner available at www.ebi.ac.uk / Tools / psa / emboss_water / , optionally with default settings). Optimal alignment may be assessed using any suitable parameters of a chosen algorithm, including default parameters. The “percent identity”, also referred to as “percent homology”, between two sequences may be calculated as the number of exact matches between two optimally aligned sequences divided by the length of the reference sequence and multiplied by 100. Percent identity may also be determined, for example, by comparing sequence information using the advanced BLAST computer program, including version 2.2.9, available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and as discussed in Altschul, et al., J. Mol. Biol. 215 :403 -410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873 -5877 (1993); and Altschul et al., Nucleic Acids Res. 25 :3389- 3402 (1997). Briefly, the BLAST program defines identity as the number of identical aligned symbols (i.e., nucleotides or amino acids), dividedby the total number of symbols in the shorter of the two sequences. The program maybe used to determine percent identity over the entire length of the sequences being compared. Default parameters are provided to optimize searches with short query sequences, for example, with the blastp program. The program also allows use of an SEG filter to mask-off segments of the query sequences as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17: 149-163 (1993). High sequence identity generally includes ranges of sequence identity of approximately 80% to 100% and integer values there between. pMHC-I Peptides

[0117] The assembling of Class I major histocompatibility complex (MHC) molecules to the cell surface requires association of the class I heavy chain with endogenous peptide and the class IWSGR Docket No. 48295-719.601 light chain, p2-microglobulin (P2M). Antigen-presenting cells (APCs) express pMHCs and many activating costimulatory molecules that participate in TCR engagement. During antigen recognition by T cells, pMHC and TCR engagement leads to the formation of immunological synapses, which include highly condensed aggregates of the TCR subunits and signaling proteins, adhesion molecules, costimulatory receptors, and inhibitor receptors on the T cell membrane.

[0118] In some embodiments, the expression of the endogenous pMHC-I on the APTC binds specifically to an antigen -specific T cell receptor (TCR) that recognizes an antigenic peptide derived from a viral antigen, a mutated tumor antigen, a tumor-specific antigen, a differentiated tumor antigen, an over-expressed tumor antigen, or a neoantigen within tumor cells. Alternatively, the expression of the endogenous pMHC-I on the APTC binds specifically to a CD8+ T cell bearing an antigen-specific T cell receptor (TCR) recognizing an antigenic peptide derived from a viral antigen, a mutated tumor antigen, a tumor-specific antigen, a differentiated tumor antigen, an over-expressed tumor antigen, or a neoantigen within tumor cells.

[0119] Described herein, in some embodiments, is an APTC comprising an endogenous pMHC-I that comprises an endogenous antigenic peptide, an endogenous P -2 -microglobulin (P2m), and an endogenous MHC-I alpha chain. In some embodiments, the endogenous antigenic peptide binds to the endogenous MHC-I alpha chain. In some embodiments, the endogenous antigenic peptide in the endogenous pMHC-I is derived from a viral antigen, a mutated tumor antigen, a tumor-specific antigen, a differentiated tumor antigen, an over-expressed tumor antigen, or a neoantigen within tumor cells.

[0120] In some embodiments, the APTC comprises an endogenous pMHC-I that comprises an endogenous antigenic peptide, an endogenous P-2-microglobulin (P2m), and an endogenous MHC-I alpha chain wherein the endogenous antigenic peptide comprises an endogenous antigenic peptide from a tumor cell.

[0121] In some embodiments, the APTC comprises an endogenous pMHC-I that comprises an endogenous antigenic peptide, an endogenous P-2-microglobulin (P2m), and an endogenous MHC-I alpha chain wherein the endogenous MHC-I alpha chain comprises an amino acid sequence set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 20 in Table 1, or an amino acid sequence that is substantially identical to an amino acid sequence set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 20 (e.g. 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity).

[0122] In some embodiments, the APTC comprises an endogenous pMHC-I that comprises an endogenous antigenic peptide, an endogenous P-2-microglobulin (P2m), and an endogenous MHC-I alpha chain wherein the endogenous P -2 -microglobulin (P2m) comprises an amino acid sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 22 in Table 1, or an amino acid sequence that isWSGR Docket No. 48295-719.601 substantially identical to an amino acid sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 22 (e.g. 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity).

[0123] The present disclosure provides in some embodiments the APTC that is derived from pancreatic cancer, rectal cancer, colorectal cancer, lung cancer, endometrial cancer, liver cancer, testicular cancer, prostate cancer, cervical cancer, breast cancer, bladder cancer, head and neck cancer, skin cancer, brain cancer, ovarian cancer, salivary gland cancer, appendiceal cancer, esophagogastric cancer, thyroid cancer, soft tissue sarcoma, kidney cancer, leukemia, myeloma, lymphoma, melanoma, non-small cell lung cancer, Hodgkin’s lymphoma, or non -Hodgkin’s lymphoma.

[0124] Described herein, in some embodiments, is an APTC comprising an endogenous pMHC-I that comprises an endogenous antigenic peptide. In some embodiments, the antigenic peptide is derived from pancreatic cancer, rectal cancer, colorectal cancer, lung cancer, endometrial cancer, liver cancer, testicular cancer, prostate cancer, cervical cancer, breast cancer, bladder cancer, head and neck cancer, skin cancer, brain cancer, ovarian cancer, salivary gland cancer, appendiceal cancer, esophagogastric cancer, thyroid cancer, soft tissue sarcoma, kidney cancer, leukemia, myeloma, lymphoma, melanoma, non-small cell lung cancer, Hodgkin’s lymphoma, or non-Hodgkin’s lymphoma.Transmembrane Domain

[0125] Described herein, in some embodiments, is an APTC comprising a first, a second, or a third fusion protein of displayed costimulatory ligand molecules wherein each of the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules further comprises a transmembrane domain. In some embodiments, the transmembrane domain of the fusion protein of the displayed costimulatory ligand molecules comprises a transmembrane domain derived from VSV-G, Dengue E protein, influenza hemagglutinin, influenza neuraminidase, spike protein SI, spike protein S2, Sindbis virus envelope (SINDBIS) protein, hemagglutinin envelope protein from measles virus, envelope glycoprotein of measles virus fusion (F) protein, RD114, BaEV, GP41, GP120, or the wild-type transmembrane domain of costimulatory ligand molecules. In some embodiments, the transmembrane domain of the fusion protein of the displayed costimulatory ligand molecule comprises a transmembrane domain derived from VSV-G wherein the VSV-G comprises a transmembrane domain and cytoplasmic tail.

[0126] In some embodiments, transmembrane domain of the fusion protein comprises an amino acid sequence with at least 90% sequence identity to an amino acid sequence of SEQ ID NOs: 59- 78. In some embodiments, transmembrane domain of the fusion protein comprises an amino acid sequence set forth in any one of SEQ ID NO: 59 to SEQ ID NO: 78 in Table 3, or an amino acidWSGR Docket No. 48295-719.601 sequence that is substantially identical to an amino acid sequence set forth in any one of SEQ ID NO: 59 to SEQ ID NO: 78 (e.g. 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity). In some embodiments, the transmembrane domain comprises an amino acid sequence set forth in any one of SEQ ID NO: 59 to SEQ ID NO: 78 with at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 59-78.

[0127] In some embodiments, the transmembrane domain of the fusion protein comprises an amino acid sequence of at least 80% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 59-78. In some embodiments, the transmembrane domain of the fusion protein comprises an amino acid sequence of at least 85% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 59-78. In some embodiments, the transmembrane domain of the fusion protein comprises an amino acid sequence of at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NO: 59-78s. In some embodiments, the transmembrane domain of the fusion protein comprises an amino acid sequence of at least 95% sequence identity to an amino acid sequence according to any one of SEQ ID NO: 59-78s. In some embodiments, the transmembrane domain of the fusion protein comprises an amino acid sequence of at least 96% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 59-78. In some embodiments, the transmembrane domain of the fusion protein comprises an amino acid sequence of at least 97% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 59-78. In some embodiments, the transmembrane domain of the fusion protein comprises an amino acid sequence of at least 98% sequence identity to an amino acid sequence accordingto any one of SEQ ID NOs: 59-78. In some embodiments, the transmembrane domain of the fusion protein comprises an amino acid sequence of at least 99% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 59-78.

[0128] In some embodiments, the transmembrane domain of the fusion protein comprises an amino acid sequence comprising at least a portion having at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or more than 150 amino acids of any one of SEQ ID NOs: 59-78.Oligomerization Domain

[0129] Described herein, in some embodiments, is an APTC comprising a first, a second, or a third fusion protein of displayed costimulatory ligand molecules wherein each of the first, second, or third fusion protein further comprises an oligomerization domain. In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecule is a dimerization domain, a trimerization domain, a tetramerization domain, a hexamerization domain, a heterohexamerization domain, a heptamerization domain, or an octamerization domain. In some embodiments, theWSGR Docket No. 48295-719.601 oligomerization domain of the displayed costimulatory ligand molecule comprises a leucine zipper dimerization domain, a post-fusion oligomerization domain of viral surface protein, a D4 postfusion trimerization domain of VSV-G protein, a Dengue E protein post -fusion trimerization domain, a foldon trimerization domain, a coiled coil domain, a LINE! retrotransposon coiled coil domain (Ll cc), a collagen XV coiled coil domain (CXVcc), a tetranectin coiled coil domain (TNTNcc), a cc-hex domain, a L24D domain, a L24DH domain, a cc-Hept domain, a wazOct domain, or an influenza neuraminidase stem domain.

[0130] In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecule comprises an amino acid sequence set forth in any one of SEQ ID NO: 79 to SEQ ID NO: 93 in Table 4, or an amino acid sequence that is substantially identical to an amino acid sequence set forth in any one of SEQ ID NO: 79 to SEQ ID NO: 93 (e.g. 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity). In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecule comprises an amino acid sequence set forth in any one of SEQ ID NO: 79 to SEQ ID NO: 93 with at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 79-93.

[0131] In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecule comprises an amino acid sequence of at least 80% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 79-93. In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecule comprises an amino acid sequence of at least 85% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 79-93. In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecule comprises an amino acid sequence of at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 79-93. In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecule comprises an amino acid sequence of at least 95% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 79-93. In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecule comprises an amino acid sequence of at least 96% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 79-93. In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecule comprises an amino acid sequence of at least 97% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 79-93. In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecule comprises an amino acid sequence of at least 98% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 79-93. In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecule comprises an amino acidWSGR Docket No. 48295-719.601 sequence of at least 99% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 79-93.

[0132] In some embodiments, the APTC comprises the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules wherein when the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules is expressed on the surface of the antigen-presenting tumor cell (APTC), the oligomerization domain is outside of the antigen-presenting tumor cell (APTC). In some embodiments, the APTC comprises the first, second, or third fusion protein of the displayed costimulatory ligand molecules wherein when the first fusion protein, the second fusion protein, or third fusion protein of the displayed costimulatory ligand molecules is expressed on the surface of the antigen-presenting tumor cell (APTC), the oligomerization domain is inside of the antigen-presenting tumor cell (APTC).

[0133] Described herein, in some embodiments, is an APTC capable of activating or stimulating an antigen-specific T cell wherein the APTC comprises a first, a second, or a third fusion protein of displayed costimulatory ligand molecules and an endogenous peptide major histocompatibility complex class I (pMHC-I). For example, expression of multivalent CD80 and GITRL on tumor cells enables potent, neoantigen -specific CD8+T cell activation.Expression of Displayed Costimulatory Ligand Molecules and Endogenous pMHC-I on The Cell Surface

[0134] In some embodiments, the APTC comprises the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules and the endogenous peptide major histocompatibility complex class I (pMHC-I). The first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC- I is expressed at a valency of about 1000 copies or more on a surface of the antigen-presenting tumor cell (APTC). In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 2500 copies or more on a surface of the antigen-presenting tumor cell (APTC). In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 5000 copies or more on a surface of the antigen-presenting tumor cell (APTC). In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 10,000 copies or more on a surface of the antigen-presenting tumor cell (APTC). In some embodiments, the first fusion protein, the second fusion protein, or the thirdWSGR Docket No. 48295-719.601 fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 20,000 copies or more on a surface of the antigen-presenting tumor cell (APTC). In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 40,000 copies or more on a surface of the antigen-presenting tumor cell (APTC). In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 60,000 copies or more on a surface of the antigen-presenting tumor cell (APTC). In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 80,000 copies or more on a surface of the antigen-presenting tumor cell (APTC). In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 120,000 copies or more on a surface of the antigen-presenting tumor cell (APTC). In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed co stimulatory ligand molecules, or the endogenou s pMHC-I is expressed at a valency of about 200,000 copies or more on a surface of the antigen-presenting tumor cell (APTC). In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 250,000 copies or more on a surface of the antigen-presenting tumor cell (APTC).

[0135] In some embodiments, the APTC comprises the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous peptide major histocompatibility complex class I (pMHC-I). The first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC- I is expressed at a valency of at least 1000 copies on the cell surface of the APTC.

[0136] In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 1000 copies to about 250,000 copies or more on the surface of the antigen-presenting tumor cell (APTC) herein, e.g., about 1000 copies to about 120,000 copies, about 1000 copies to about 80,000 copies, about 1000 copies to about 60,000 copies, about 1000 copies to about 40,000 copies, about 1000 copies to about 20,000 copies, about 1000 copies to about 10,000 copies, about 1000 copies to about 5,000 copies, about 1000 copies to about 2,500 copies, about 2500 copies to about 250,000 copies, about 2500 copies to about 120,000 copies,WSGR Docket No. 48295-719.601 about 2500 copies to about 80,000 copies, about 2500 copies to about 60,000 copies, about 2500 copies to about 40,000 copies, about 2500 copies to about 20,000 copies, about 2500 copies to about 10,000 copies, about2500 copies to about 5,000 copies, about 5,000 copiesto about250,000 copies, about 5,000 copies to about 120,000 copies, about 5,000 copies to about 80,000 copies, about 5,000 copies to about 60,000 copies, about 5,000 copies to about 40,000 copies, about 5,000 copies to about 20,000 copies, about 5,000 copies to about 10,000 copies, about 10,000 copies to about 250,000 copies, about 10,000 copies to about 120,000 copies, about 10,000 copies to about 80,000 copies, about 10,000 copies to about 60,000 copies, about 10,000 copies to about 40,000 copies, about 10,000 copies to about 20,000 copies, about 20,000 copies to about 250,000 copies, about 20,000 copiesto about 120,000 copies, about 20,000 copies to about 80,000 copies, about 20,000 copies to about 60,000 copies, about 20,000 copies to about 40,000 copies, about 40,000 copies to about 250,000 copies, about 40,000 copies to about 120,000 copies, about 40,000 copies to about 80,000 copies, about 40,000 copies to about 60,000 copies, about 60,000 copies to about 250,000 copies, about 60,000 copies to about 120,000 copies, about 60,000 copies to about 80,000 copies, about 80,000 copies to about 250,000 copies, about 80,000 copies to about 120,000 copies, about 120,000 copies to about 250,000 copies.

[0137] In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of at least about 1000 copies on a surface of the APTC.

[0138] In some embodiments, the APTC comprises the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I wherein the endogenous pMHC-I comprises a natural pMHC-I.

[0139] In some embodiments, the APTC comprises the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I wherein the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules comprises an amino acid sequence set forth in any one of SEQ ID NO: 94 to SEQ ID NO: 111 in Table 5, or an amino acid sequence that is substantially identical to an amino acid sequence set forth in any one of SEQ ID NO: 94 to SEQ ID NO: 111 (e.g. 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity). In some embodiments, the first, second, or third fusion protein of the displayed costimulatory ligand molecules comprises an amino acid sequence with at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 94-111.

[0140] In some embodiments, the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules comprises an amino acid sequence of at least 80% sequenceWSGR Docket No. 48295-719.601 identity to an amino acid sequence according to any one of SEQ ID NOs: 94-111. In some embodiments, the costimulatory molecule of the fusion protein comprises an amino acid sequence of at least 85% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 94-111. In some embodiments, the costimulatory molecule of the fusion protein comprises an amino acid sequence of at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 94-111. In some embodiments, the costimulatory molecule of the fusion protein comprises an amino acid sequence of at least 95% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 94-111 . In some embodiments, the costimulatory molecule of the fusion protein comprises an amino acid sequence of at least 96% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 94-111. In some embodiments, the costimulatory molecule of the fusion protein comprises an amino acid sequence of at least 97% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 94-111. In some embodiments, the costimulatory molecule of the fusion protein comprises an amino acid sequence of at least 98% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 94-111 . In some embodiments, the costimulatory molecule of the fusion protein comprises an amino acid sequence of at least 99% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 94-111 .

[0141] In some embodiments, the APTC comprises a first fusion protein that comprises the costimulatory ligand molecule CD80 or CD86, and a second fusion protein that comprises the costimulatory ligand molecule GITRL, and a recombinant peptide major histocompatibility complex class I (pMHC-I), wherein the APTC lacks an endogenous pMHC-I or expresses a dysfunctional endogenous pMHC-I, wherein the first fusion protein, the second fusion protein, or the recombinant pMHC-I is expressed at a valency of at least about 1000 copies on a surface of the APTC.

[0142] Described herein, in some embodiments, is a fusion protein comprising a costimulatory ligand molecule that binds specifically to a T cell surface costimulatory receptor, an oligomerization domain, and a transmembrane domain, wherein the fusion protein is expressed at a valency of at least about 1000 copies on a surface of an antigen-presenting tumor cell (APTC). In some embodiments, the fusion protein comprises at least two separate fusion proteins, wherein the at least two separate fusion proteins each comprises a different costimulatory ligand molecule.

[0143] In some embodiments, the costimulatory molecule of the fusion protein comprises one or more selected from the group comprising GITR Ligand, CD80, CD86, 0X40 Ligand, CD48, CD30 Ligand, ICOS Ligand, CD70, CD40, CD40 Ligand, 4-BB Ligand, TLIA, TIM4, SLAM, CD48, CD58, CD155, CD112, LIGHT, and ICAM1. In some embodiments, the costimulatoryWSGR Docket No. 48295-719.601 molecule of the fusion protein comprises an amino acid sequence set forth in any one of SEQ ID NO: 23 to SEQ ID NO: 58 in Table 2, or an amino acid sequence that is substantially identical to an amino acid sequence set forth in any one of SEQ ID NO: 23 to SEQ ID NO: 58 (e.g. 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity).

[0144] Described herein, in some embodiments, is an APTC capable of activating or stimulating an antigen-specific T cell wherein the APTC comprises a costimulatory ligand molecule of a fusion protein is expressed at least about 1000 copies on a surface of the APTC. Methods of Use

[0145] APTCs can effectively activate tumor-specific T cells in culture and in vivo. Activation of anti-tumor T cells often results in robust anti-tumor T cell immune response to prevent cancer development and the induction of robust memory response against rechallenges. Autologous APTCs can be used as therapeutic T-cell vaccines for the prevention of cancer relapse and treatment of established tumors.

[0146] Described herein, in some embodiments, are methods of preventing or treating cancer in a subject in need thereof comprising administering to the subject an autologous cell comprising the APTC. Described herein, in some embodiments, are methods of preventing or treating established cancer in a subject in need thereof comprising administering to the subject an irradiated autologous cell comprising the APTC. Described herein, in some embodiments, are methods of inducing protective immunity against cancer in a subject in need thereof comprising administering to the subject an autologous cell comprising the APTC. Described herein, in some embodiments, are methods of inducing protective immunity against cancer in a subject in need thereof comprising administering to the subject an irradiated autologous cell comprising the APTC.

[0147] In some embodiments, the methods of preventing or treating cancer in a subject or inducing protective immunity against cancer in a subject further comprising administering the subject a second therapy wherein the second therapy comprises an adjuvant. In some embodiments, the second therapy comprises Poly (EC) or lipopolysaccharide (LPS). In some embodiments, the second therapy comprises an immune checkpoint blocker therapy. In some embodiments, the second therapy comprises an anti-PDl antibody, anti-PDLl antibody, or an anti-CTLA-4 antibody. In some embodiments, the second therapy comprises a Toll-like receptor agonist for TLR3, TLR4, TLR7, TLR8, TLR9, CpG, or a STING agonist. In some embodiments, the second therapy comprises a cytokine. In some embodiments, the second therapy comprises the cytokine which comprises IL-2, IL-7, IL-12, IL-15, IL-21, or interferon-gamma (IFN-gamma). In some embodiments, the second therapy comprises a targeted therapy, and optionally a tyrosine kinase inhibitor or PARP inhibitor orRAS inhibitor. In some embodiments, the second therapy comprisesWSGR Docket No. 48295-719.601T cell therapy, and optionally a chimeric antigen -receptor T cell therapy or tumor-infiltrated lymphocyte therapy. In some embodiments, the second therapy comprises standard care therapy, and optionally surgery or chemotherapy or radiation therapy.

[0148] Described herein, in some embodiments, are methods of preventing or treating established cancer in a subject comprising administering to the subject a nucleic acid encoding the fusion protein of co-stimulatory molecules, wherein the administering results in the expression of the fusion protein on a surface of a cancer cell in the subject. Described herein, in some embodiments, are methods of inducing protective immunity against cancer in a subject comprising administering to the subject a nucleic acid encoding the fusion protein of co-stimulatory molecules, wherein the administering results in expression of the fusion protein on a surface of a cancer cell in the subject. In some embodiments, the cancer cell is reprogrammed into an APTC that activates or stimulates a T cell in the subject. Described herein, in some embodiments, are methods of preventing cancer relapse in a subject in need thereof comprising administering to the subject an autologous cell comprising the APTC.

[0149] In some embodiments, the subject undergoes cancer treatment before the administering and does not have cancer after the treatment. In some embodiments, the autologous cell comprising the APTC is produced in culture. In some embodiments, the autologous cell comprising the APTC is a live cell, an irradiated cell, or an inactivated cell.

[0150] Combined adjuvant treatment with APTC therapeutic vaccination results in strong, synergistic tumor control effects, likely mediated by the combination of tumor-specific, APTC- activated T cells functioning in concert with adjuvant-activated, pro-inflammatory innate immune cells that make the tumor microenvironment less tolerogenic. Described herein, in some embodiments, are methods of preventing cancer relapse in a subject comprising administering to the subject a Toll-like receptor agonist and the autologous cell comprising the APTC. In some embodiments, the administering further comprises administering an anti-PDl, anti-PDLl, an anti- CTLA4 immune checkpoint blockade therapy, a targeted therapy, an antibody -drug therapy, a tyrosine kinase inhibitor, a chemotherapy, surgery, or a therapy of standard care.

[0151] Described herein, in some embodiments, are methods of preventing cancer relapse in a subject comprising introducing, before the administering, into the autologous cell a nucleic acid molecule encoding the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecule, wherein the autologous cell is cultured ex vivo. In some embodiments, the nucleic acid comprises mRNA, DNA, or a viral vector. In some embodiments, the nucleic acid comprises mRNA, DNA, or the viral vector wherein the viral vector comprises a lentiviral vector, a retroviral vector, an adeno -associated viral (AAV) vector, anWSGR Docket No. 48295-719.601 adenoviral vector, a vector derived from herpes simplex virus (HSV), a vector derived from parvoviruses, or a vector derived from poxviruses. In some embodiments, the vector derived from parvoviruses wherein the poxviruses comprise vaccinia virus (VACV) or myxoma virus.

[0152] Described herein, in some embodiments, are methods of preventing or treating established cancer in a subject or inducing protective immunity against cancer in a subject wherein the introducing comprises intratumoral delivery, intravenous delivery, intramuscular delivery, intraperitoneal delivery, or transdermal delivery. Described herein, in some embodiments, are methods of preventing or treating established cancer in a subject or inducing protective immunity against cancer in a subject wherein the administering comprises administering a Toll -like receptor agonist. In some embodiments, the methods of preventing or treating established cancer in a subject or inducing protective immunity against cancer in a subject further comprise administering an anti- PD1, anti-PDLl, an anti-CTLA4 immune checkpoint blockade therapy, a targeted therapy, an antibody -drug therapy, a tyrosine kinase inhibitor, a chemotherapy, surgery, or a therapy of standard care.

[0153] Described herein, in some embodiments, are methods of inducing protective immunity against cancer in a subject comprising administering to the subject an irradiated autologous cell comprising the APTC.

[0154] Described herein, in some embodiments, are methods of preventing or treating cancer in a subject in need thereof comprising obtaining a T cell, a tumor-infiltrated lymphocyte (TIL), or a cancer cell from the subject; introducing ex vivo into the cancer cell a nucleic acid encoding the fusion protein of co-stimulatory molecules to express the fusion protein on a surface of the cancer cell; contacting ex vivo the T cell or TIL with the cancer cell to activate or stimulate the T cell or TIL; and administering the activated T cell or TIL to the subject.

[0155] In some embodiments, administration of the APTCs reduces or eliminates the cancer. In some embodiments, administration of the APTCs increase cancer cell death, decrease tumor size, decrease cancer metastasis, or combinations thereof. In some embodiments, cell death is increased by about 1-fold to about 2.5-fold, about 1-fold to about 5-fold, about 2-fold to about 10-fold. In some embodiments, cell death is increased by at least 5 -fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70 -fold, at least 80-fold, at least 90-fold, at least 95 -fold, 100-fold, or greater than 100-fold. In some embodiments, tumor size is decreased by about 1 -fold to about 2.5 -fold, about 1-fold to about 5 -fold, about 2-fold to about 10-fold. In some embodiments, tumor size is decreased by at least 5 -fold, at least 10-fold, at least 20-fold, at least 3 O-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 95-fold, 100-fold, or greater than 100-fold. In some embodiments,WSGR Docket No. 48295-719.601 cancer metastasis is decreased by about 1 -fold to about 2.5-fold, about 1 -fold to about 5-fold, about 2-fold to about 10-fold. In some embodiments, cancer metastasis is decreased by at least 5 -fold, at least 10-fold, at least 20 -fold, at least 30 -fold, at least 40-fold, at least 50 -fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 95 -fold, 100-fold, or greater than 100-fold.

[0156] In some embodiments, administration of the APTCs reduces or eliminates the cancer as compared to a level prior to administration of the multivalent particles in the subject. In some embodiments, administration of the APTCs reduces or eliminates the cancer as compared to a level if the subject had not received the multivalent particles. In some embodiments, administration of the APTCs reduces or eliminates the cancer as compared to a level if the subject had received a different cancer treatment including but not limited to, radiation, surgery, and chemotherapy.

[0157] In some embodiments, the APTCs induce T cell mediated cytotoxicity against tumor cells.

[0158] In some instances, the subject is a mammal. In some instances, the subject is a mouse, rabbit, dog, pig, cattle, or human. Subjects treated by methods described herein may be infants, adults, or children.Compositions for Generation of Antigen-presenting Tumor Cells (APTCs)

[0159] Described herein, in some embodiments, are compositions comprising a APTC expressing a fusion protein of a co-stimulatory molecule and an endogenous peptide major histocompatibility complex class I (pMHC-I) on a surface of the APTC. In some embodiments, the composition of the APTC comprises a first fusion protein that comprises the costimulatory ligand molecule CD80 or CD86, a second fusion protein that comprises the costimulatory ligand molecule GITRL, and a recombinant peptide major histocompatibility complex class I (pMHC-I).

[0160] In some embodiments, the method of producing APTCs, comprises introducing into a tumor cell a nucleic acid molecule encoding the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecule. Compositions for producing APTCs, in some embodiments, comprise introducing into a tumor cell a nucleic acid molecule encoding the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecule. In some embodiments, the introduction of the nucleic acid into the tumor cell is carried out ex vivo after the tumor cell is obtained from a subject. Alternatively, in some embodiments, the introduction of the nucleic acid into the tumor cell is carried out in vivo or intratumorally in a subject.

[0161] In some embodiments, the nuclei acid molecule comprises an ectodomain of the costimulatory ligand molecule, an oligomerization domain, a transmembrane domain and a cytosolic domain. In some embodiments, the nucleic acid molecule encoding the first fusionWSGR Docket No. 48295-719.601 protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecule comprises mRNA or DNA. In some embodiments, the nucleic acid molecule encoding the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules comprises a DNA vector, an mRNA vector, or a viral vector. In some embodiments, the nucleic acid comprises mRNA, DNA, or a viral vector. In some embodiments, the nucleic acid comprises mRNA, DNA, or the viral vector wherein the viral vector comprises a lentiviral vector, a retroviral vector, an adeno -associated viral (AAV) vector, an adenoviral vector, a vector derived from herpes simplex virus (HSV), a vector derived from parvoviruses, or a vector derived from poxviruses. In some embodiments, the vector derived from parvoviruses wherein the poxviruses comprise vaccinia virus (VACV) or myxoma virus.

[0162] Various vectors, in some embodiments, are used herein. In some embodiments, the vector is a eukaryotic or prokaryotic vector. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus vector. Exemplary vectors include, without limitation, mammalian expression vectors: pSF- CMV-NEO-NH2-PPT-3XFLAG, pSF-CMV-NEO-COOH-3XFLAG, pSF-CMV-PURO-NH2- GST-TEV, pSF-OXB20-COOH-TEV-FLAG(R)-6His, pCEP4 pDEST27, pSF-CMV-Ub-KrYFP, pSF-CMV-FMDV-daGFP, pEFla-mCherry-Nl Vector, pEFla-tdTomato Vector, pSF-CMV- FMDV-Hygro, pSF-CMV-PGK-Puro, pMCP-tag(m), and pSF-CMV-PURO-NH2-CMYC; bacterial expression vectors: pSF-OXB20-BetaGal,pSF-OXB20-Fluc, pSF-OXB20, and pSF-Tac; plant expression vectors: pRI 101 -AN DNA and pCambia2301; and yeast expression vectors: pTYB21 and pKLAC2, and insect vectors: pAc5.1 / V5 -His A and pDEST8

[0163] The tumor cell for generating the APTC comprises an ex vivo cultured cell. In some embodiments, the cultured tumor cell comprises an adherent cell, a suspension cell, or an organoid.

[0164] To generate the APTC, the introduction of the nucleic acid into the tumor cell comprises intratumoral delivery, intravenous delivery, intramuscular delivery, intraperitoneal delivery, or transdermal delivery.Compositions and Pharmaceutical Compositions

[0165] Described herein, in some embodiments, are compositions comprising a APTC expressing a fusion protein of a displayed costimulatory ligand molecule and an endogenous peptide major histocompatibility complex class I (pMHC-I) on a surface of the APTC. In some embodiments, the composition of the APTC comprises a first fusion protein that comprises the costimulatory ligand molecule CD80 or CD86, a second fusion protein that comprises the costimulatory ligand molecule GITRL, and a recombinant peptide major histocompatibility complex class I (pMHC-I).WSGR Docket No. 48295-719.601

[0166] Described herein, in some embodiments, are compositions comprising a nucleic acid molecule encoding a first fusion protein, a second fusion protein, or a third fusion protein of displayed costimulatory ligand molecules. In some embodiments, the composition comprises the nucleic acid molecule encoding a first fusion protein of a displayed costimulatory ligand molecule CD80 or CD86, a second fusion protein of a displayed costimulatory ligand molecule GITRL.

[0167] Described herein, in some embodiments, are pharmaceutical compositions comprising a APTC expressing one or more fusion proteins of co-stimulatory molecules and an endogenous peptide major histocompatibility complex class I (pMHC-I) on a surface of the APTC. The generation of the composition comprises introducing into a tumor cell a nucleic acid molecule encoding the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecule wherein the introducing is carried out ex vivo. In some embodiments, the pharmaceutical composition comprises an APTC and a pharmaceutically acceptable excipient.

[0168] Described herein, in some embodiments, are pharmaceutical compositions comprising a nucleic acid molecule encoding a first fusion protein, a second fusion protein, or a third fusion protein of the displayed costimulatory ligand molecules. The generation of the composition comprising the APTC is carried out in vivo or intratumorally in a subject.

[0169] For administration to a subject, the APTCs as disclosed herein, may be provided in a pharmaceutical composition together with one or more pharmaceutically acceptable carriers or excipients. In some embodiments, the APTCs as disclosed herein, may be provided in a composition together with one or more carriers or excipients. The term "pharmaceutically acceptable carrier" includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the ingredients and that is not toxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil / water emulsions, various types of wetting agents, sterile solutions etc. Such carriers can be formulated by conventional methods and can be administered to the subject at a suitable dose. Preferably, the compositions are sterile. These compositions may also contain adjuvants such as preservative, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents.

[0170] The pharmaceutical composition may be in any suitable form, (depending upon the desired method of administration). It may be provided in unit dosage form, may be provided in a sealed container and may be provided as part of a kit. Such a kit may include in structions for use. It may include a plurality of said unit dosage forms.WSGR Docket No. 48295-719.601

[0171] The pharmaceutical composition maybe adapted for administration by any appropriate route, including a parenteral (e.g., subcutaneous, intramuscular, intravenous, or inhalation) route. Such compositions may be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.

[0172] Dosages of the substances of the present disclosure can vary between wide limits, depending upon the disease or disorder to be treated, the age and condition of the individual to be treated, etc. and a physician will ultimately determine appropriate dosages to be used.Tumor-Derived Antigen-presenting Vesicles (Tumor-APVs)

[0173] Vesicles, such as exosomes (EVs) and virus-like particles (VLPs), can be reprogrammed into antigen-presenting vesicles (APVs) which can effectively activate antigenspecific T cells without sensitivity to negative environmental cues. The engineered vesicles displaying high copies of oligomerized molecular machinery required for TCR engagement.

[0174] Described herein, in some embodiments, is a tumor-derived antigen-presenting vesicle (tumor-APV) comprising, a first fusion protein that comprises the displayed costimulatory ligand molecule CD80 or CD86, a second fusion protein that comprises the displayed costimulatory ligand molecule GITRL, and an endogenous protein that comprises an endogenous peptide major histocompatibility complex class I (pMHC-I), wherein the tumor-APV is derived from an antigen- presenting tumor cell (APTC) comprising the first fusion protein, the second fusion protein, and the endogenous protein, wherein the first fusion protein, the second fusion protein, or the endogenous protein, is expressed at a valency of at least about 1000 copies on a surface of the APV or APTC. In some embodiments, the tumor-APV is derived from the APTC comprising the first fusion protein that comprises the displayed costimulatory ligand molecule CD80 or CD86. In some embodiments, the tumor-APV is derived from the APTC comprising the second fusion protein that comprises the displayed costimulatory ligand molecule GITRL. In some embodiments, the tumor-APV is derived from the APTC comprising the endogenous protein that comprises the endogenous peptide major histocompatibility complex class I (pMHC-I). In some embodiments, the tumor-APV is derived from the APTC comprising the first fusion protein of costimulatory ligand molecule CD80 or CD86, the second fusion protein of costimulatory ligand molecule GITRL, and an endogenous peptide major histocompatibility complex class I (pMHC-I).

[0175] In some embodiments, the tumor-APV is a viral-like particle. In some embodiments, the multivalent particle is an extracellular vesicle. In some embodiments, the multivalent particle is an exosome. In some embodiments, the multivalent particle is an ectosome.WSGR Docket No. 48295-719.601

[0176] In some embodiments, the first or the second fusion protein of the displayed costimulatory ligand molecule on the surface of the tumor-APV binds specifically to the cognate costimulatory receptors on a T-cell surface.

[0177] In some embodiments, the tumor-APV or the APTC further comprises a third fusion protein comprising at least one, two, three, four, five, six, seven, eight, nine, ten, or more than ten separate fusion proteins, wherein the at least one, two, three, four, five, six, seven, eight, nine, ten, or more than ten separate fusion proteins each comprises 0X40 Ligand, CD48, CD30 Ligand, ICOS Ligand, CD70, CD40, CD40 Ligand, 4-BB Ligand, TLIA, TIM4, SLAM, CD48, CD58, CD155, CD112, LIGHT, or ICAM-1.

[0178] A tumor-APV derived from an APTC expressing an endogenous pMHC-I binds specifically to an antigen -specific T cell receptor (TCR) that recognizes an antigenic peptide derived from a viral antigen, a mutated tumor antigen, a tumor-specific antigen, a differentiated tumor antigen, an over-expressed tumor antigen, or a neoantigen within tumor cells. Alternatively, in some embodiments, the expression of the endogenous pMHC-I on the surface of the tumor-APV binds specifically to a CD8+ T cell bearing an antigen -specific T cell receptor (TCR) recognizing an antigenic peptide derived from a viral antigen, a mutated tumor antigen, a tumor-specific antigen, a differentiated tumor antigen, an over-expressed tumor antigen, or a neoantigen within tumor cells.

[0179] In some embodiments, the endogenous pMHC-I on the surface of the tumor-APV comprises an endogenous antigenic peptide, an endogenous P -2 -microglobulin (P2m), and an endogenous MHC-I alpha chain. In some embodiments, the endogenous antigenic peptide binds to the endogenous MHC-I alpha chain. In some embodiments, the endogenous antigenic peptide in the endogenous pMHC-I is derived from a viral antigen, a mutated tumor antigen, a tumor-specific antigen, a differentiated tumor antigen, an over-expressed tumor antigen, or a neoantigen within tumor cells.

[0180] In some embodiments, the tumor-APV is derived from the APTC comprising an endogenous pMHC-I that comprises an endogenous antigenic peptide, an endogenous P-2- microglobulin (P2m), and an endogenous MHC-I alpha chain wherein the endogenous antigenic peptide comprises an endogenous antigenic peptide from a tumor cell.

[0181] The present disclosure provides in some embodiments a tumor-APV or its parental APTC that is derived from pancreatic cancer, rectal cancer, colorectal cancer, lung cancer, endometrial cancer, liver cancer, testicular cancer, prostate cancer, cervical cancer, breast cancer, bladder cancer, head and neck cancer, skin cancer, brain cancer, ovarian cancer, salivary gland cancer, appendiceal cancer, esophagogastric cancer, thyroid cancer, soft tissue sarcoma, kidneyWSGR Docket No. 48295-719.601 cancer, leukemia, myeloma, lymphoma, melanoma, non-small cell lung cancer, Hodgkin’s lymphoma, or non-Hodgkin’s lymphoma.

[0182] Described herein, in some embodiments, is a tumor- APV or its parental APTC comprising an endogenous pMHC-I that comprises an endogenous antigenic peptide. In some embodiments, the antigenic peptide is derived from pancreatic cancer, rectal cancer, colorectal cancer, lung cancer, endometrial cancer, liver cancer, testicular cancer, prostate cancer, cervical cancer, breast cancer, bladder cancer, head and neck cancer, skin cancer, brain cancer, ovarian cancer, salivary gland cancer, appendiceal cancer, esophagogastric cancer, thyroid cancer, soft tissue sarcoma, kidney cancer, leukemia, myeloma, lymphoma, melanoma, non-small cell lung cancer, Hodgkin’s lymphoma, or non-Hodgkin’s lymphoma. In some embodiments, the endogenous antigenic peptide comprises an endogenous antigenic peptide from a tumor cell.

[0183] In some embodiments, the costimulatory ligand molecule comprises an amino acid sequence with at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 23-58. In some embodiments, the first, the second, or the third fusion protein of the costimulatory ligand molecules comprises an amino acid sequence set forth in any one of SEQ ID NO: 23 to SEQ ID NO: 58 in Table 2, or an amino acid sequence that is substantially identical to an amino acid sequence setforthin any oneof SEQ IDNO: 23 to SEQ IDNO: 58 (e.g. 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity).

[0184] Described herein, in some embodiments, is a tumor-APV derived from an APTC comprising a first, a second, or a third fusion protein of displayed costimulatory ligand molecules wherein each of the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules further comprises a transmembrane domain. In some embodiments, the transmembrane domain of the fusion protein of the displayed costimulatory ligand molecule comprises a transmembrane domain derived from VSV-G, Dengue E protein, influenza hemagglutinin, influenza neuraminidase, spike protein SI, spike protein S2, Sindbis virus envelope (SINDBIS) protein, hemagglutinin envelope protein from measles virus, envelope glycoprotein of measles virus fusion (F) protein, RD114, BaEV, GP41, GP120, or the wild-type transmembrane domain of costimulatory ligand molecules. In some embodiments, the transmembrane domain of the fusion protein comprises a transmembrane domain derived from VSV-G wherein the VSV-G comprises a transmembrane domain and cytoplasmic tail.

[0185] In some embodiments, the transmembrane domain of the displayed costimulatory ligand molecules comprises an amino acid sequence with at least 90% sequence identity to an amino acid sequence of SEQ ID NOs: 59-78. In some embodiments, the transmembrane domain of the displayed costimulatory ligand molecules comprises an amino acid sequence set forth in any one ofWSGR Docket No. 48295-719.601SEQ ID NO: 59 to SEQ ID NO: 78 in Table 3, or an amino acid sequence that is substantially identical to an amino acid sequence set forth in any one of SEQ ID NO: 59 to SEQ ID NO: 78 (e.g. 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity). In some embodiments, the transmembrane domain of the displayed costimulatory ligand molecules comprises an amino acid sequence set forth in any one of SEQ ID NO: 59 to SEQ ID NO: 78 with at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 59-78.

[0186] Described herein, in some embodiments, is a tumor-APV derived from a APTC comprising a first, a second, or a third fusion protein of displayed costimulatory ligand molecules wherein each of the first, the second, or the third fusion protein further comprises an oligomerization domain. In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecules is a dimerization domain, a trimerization domain, a tetramerization domain, a hexamerization domain, a heterohexamerization domain, a heptamerization domain, or an octamerization domain. In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecules comprises a leucine zipper dimerization domain, a post-fusion oligomerization domain of viral surface protein, a D4 post-fusion trimerization domain of VSV-G protein, a Dengue E protein post-fusion trimerization domain, a foldon trimerization domain, a coiled coil domain, a LINEl retrotransposon coiled coil domain (Llcc), a collagen XV coiled coil domain (CXVcc), a tetranectin coiled coil domain (TNTNcc), a cc-hex domain, a L24D domain, a L24DH domain, a cc-Hept domain, a wazOct domain, or an influenza neuraminidase stem domain.

[0187] In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecules comprises an amino acid sequence that has at least 90% sequence identity to an amino acid sequence according to SEQ ID NOs: 79-93. In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecules comprises an amino acid sequence set forth in any one of SEQ ID NO: 79 to SEQ ID NO: 93 in Table 4, or an amino acid sequence that is substantially identical to an amino acid sequence set forth in any one of SEQ ID NO: 79 to SEQ ID NO: 93 (e.g. 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity). In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecules comprises an amino acid sequence set forth in any one of SEQ ID NO: 79 to SEQ ID NO: 93 with at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 79 - 93.

[0188] Described herein, in some embodiments, is a tumor-APV capable of activating or stimulating an antigen -specific T cell wherein a parental APTC comprises a first fusion protein, a second fusion protein, a third fusion protein of the displayed costimulatory ligand molecules, or an endogenous pMHC-I.WSGR Docket No. 48295-719.601

[0189] In some embodiments, the parental APTC comprises the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules wherein when the first fusion protein, the second fusion protein, or the third fusion protein of the costimulatory ligand molecules is expressed on the surface of the tumor- APV, the oligomerization domain is outside of the tumor- APV. In some embodiments, the APTC comprises the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules wherein when the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules is expressed on the surface of the tumor-APV, the oligomerization domain is inside of the tumor- APV.

[0190] In some embodiments, the APTC comprises the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules and the endogenous peptide major histocompatibility complex class I (pMHC-I). The first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC- I is expressed at a valency of about 100 copies or more on a surface of the tumor-APV. In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 250 copies or more on a surface of the the tumor-APV. In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 500 copies or more on a surface of the the tumor-APV. In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 1,000 copies or more on a surface of the tumor-APV. In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 2,000 copies or more on a surface of the tumor-APV. In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 4,000 copies or more on a surface of the tumor-APV. In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 6,000 copies or more on a surface of the tumor-APV. In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 8,000 copies or more on a surface of the tumor-APV. In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein ofWSGR Docket No. 48295-719.601 the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 10,000 copies or more on a surface of the tumor-APV. In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-Iis expressed at a valency of about 20,000 copies or more on a surface of the tumor-APV. In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 50,000 copies or more on a surface of the tumor-APV.

[0191] In some embodiments, the tumor-APV is derived from its parental APTC comprising the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I wherein the endogenous pMHC-I comprises a natural pMHC-I.

[0192] In some embodiments, the parental APTC comprises the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I wherein the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules comprises an amino acid sequence with at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 94-111. In some embodiments, the parental APTC comprises the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC- I wherein the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules comprises an amino acid sequence set forth in any one of SEQ ID NO: 94 to SEQ ID NO: 111 in Table 5, or an amino acid sequence that is substantially identical to an amino acid sequence set forth in any one of SEQ ID NO: 94 to SEQ ID NO: 111 (e.g. 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity). In some embodiments, the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules comprises an amino acid sequence with at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 94-111.

[0193] In some embodiments, the tumor-derived antigen-presenting vesicle (tumor-APV) comprising a first fusion protein that comprises the costimulatory ligand molecule CD80 or CD86, and a second fusion protein that comprises the costimulatory ligand molecule GITRL, and a recombinant peptide major histocompatibility complex class I (pMHC-I), wherein the tumor-APV is derived from its parental antigen-presenting tumor cell (APTC) comprising the first fusion protein, the second fusion protein, and the recombinant pMHC-I, wherein the tumor-APV or its parental APTC lacks an endogenous pMHC-I or expresses a dysfunctional endogenous pMHC-I,WSGR Docket No. 48295-719.601 wherein first fusion protein, the second fusion protein, or the recombinant pMHC-I is expressed at a valency of at least about 100 copies on a surface of the tumor -APV. In some embodiments, a fusion protein comprising a costimulatory ligand molecule that binds specifically to a T cell surface costimulatory receptor, an oligomerization domain, and a transmembrane domain, wherein the fusion protein is expressed at a valency of at least about 100 copies on a surface of a tumor-APV, wherein the tumor-APV is derived from its parental APTC. In some embodiments, the fusion protein comprises at least two separate fusion proteins, wherein the at least two separate fusion proteins each comprises a different costimulatory ligand molecule. In some embodiments, the costimulatory molecule comprises one or more selected from the group comprising GITR Ligand, CD80, CD86, 0X40 Ligand, CD48, CD30L, ICOS Ligand, CD70, CD40, CD40 Ligand, 4-BB Ligand, TLIA, TIM4, SLAM, CD48, CD58, CD155, CD112, LIGHT, and ICAM1.Costimulatory Ligand Molecules

[0194] In some embodiments, the first or the second fusion protein of the displayed costimulatory ligand molecules on the surface of the tumor-APV binds specifically to the cognate costimulatory receptors on a T-cell surface.

[0195] In some embodiments, the tumor-APV or the APTC further comprises a third fusion protein comprising at least one, two, three, four, five, six, seven, eight, nine, ten, or more than ten separate fusion proteins, wherein the at least one, two, three, four, five, six, seven, eight, nine, ten, or more than ten separate fusion proteins each comprises 0X40 Ligand, CD48, CD30 Ligand, ICOS Ligand, CD70, CD40, CD40 Ligand, 4-BB Ligand, TLIA, TIM4, SLAM, CD48, CD58, CD155, CD112, LIGHT, or ICAM-1.

[0196] In some embodiments, the costimulatory ligand molecule comprises an amino acid sequence with at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 23-58. In some embodiments, the first, the second, or the third fusion protein of the costimulatory ligand molecules comprises an amino acid sequence set forth in any one of SEQ ID NO: 23 to SEQ ID NO: 58 in Table 2, or an amino acid sequence that is substantially identical to an amino acid sequence setforthin any one of SEQ ID NO: 23 to SEQ ID NO: 58 (e.g. 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity).

[0197] In some embodiments, the costimulatory ligand molecule of the fusion protein comprises an amino acid sequence of at least 70% sequence identity to an amino acid sequence accordingto any one of SEQ ID NO: 23-58, e.g., at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, or any percentage therebetween. In some embodiments, the costimulatory ligand molecule of the fusion protein comprises an amino acid sequence of at leastWSGR Docket No. 48295-719.60180% sequence identity to an amino acid sequence according to any one of SEQ ID NO: 23-58. In some embodiments, the costimulatory ligand molecule of the fusion protein comprises an amino acid sequence of at least 85% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 23-58. In some embodiments, the costimulatory ligand molecule of the fusion protein comprises an amino acid sequence of at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 23-58. In some embodiments, the costimulatory ligand molecule of the fusion protein comprises an amino acid sequence of at least 95% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 23-58. In some embodiments, the costimulatory ligand molecule of the fusion protein comprises an amino acid sequence of at least 96% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 23-58. In some embodiments, the costimulatory ligand molecule of the fusion protein comprises an amino acid sequence of at least 97% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 23-58. In some embodiments, the costimulatory ligand molecule of the fusion protein comprises an amino acid sequence of at least 98% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 23-58. In some embodiments, the costimulatory ligand molecule of the fusion protein comprises an amino acid sequence of at least 99% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 23-58. pMHC-I Peptides

[0198] A tumor-APV derived from an APTC expressing an endogenous pMHC-I binds specifically to an antigen -specific T cell receptor (TCR) that recognizes an antigenic peptide derived from a viral antigen, a mutated tumor antigen, a tumor-specific antigen, a differentiated tumor antigen, an over-expressed tumor antigen, or a neoantigen within tumor cells. Alternatively, in some embodiments, the expression of the endogenous pMHC-I on the surface of the tumor-APV binds specifically to a CD8+ T cell bearing an antigen -specific T cell receptor (TCR) recognizing an antigenic peptide derived from a viral antigen, a mutated tumor antigen, a tumor-specific antigen, a differentiated tumor antigen, an over-expressed tumor antigen, or a neoantigen within tumor cells.

[0199] In some embodiments, the endogenous pMHC-I on the surface of the tumor-APV comprises an endogenous antigenic peptide, an endogenous P -2 -microglobulin (P2m), and an endogenous MHC-I alpha chain. In some embodiments, the endogenous antigenic peptide binds to the endogenous MHC-I alpha chain. In some embodiments, the endogenous antigenic peptide in the endogenous pMHC-I is derived from a viral antigen, a mutated tumor antigen, a tumor-specific antigen, a differentiated tumor antigen, an over-expressed tumor antigen, or a neoantigen within tumor cells.WSGR Docket No. 48295-719.601

[0200] In some embodiments, the tumor-APV is derived from the APTC comprising an endogenous pMHC-I that comprises an endogenous antigenic peptide, an endogenous P-2- microglobulin (P2m), and an endogenous MHC-I alpha chain wherein the endogenous antigenic peptide comprises an endogenous antigenic peptide from a tumor cell.

[0201] In some embodiments, the tumor-APV is derived from the APTC comprising an endogenous pMHC-I that comprises an endogenous antigenic peptide, an endogenous P-2- microglobulin (P2m), and an endogenous MHC-I alpha chain wherein the endogenous MHC-I alpha chain comprises an amino acid sequence set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 20 in Table 1, or an amino acid sequence that is substantially identical to an amino acid sequence set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 20 (e.g. 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity).

[0202] In some embodiments, the tumor-APV is derived from the APTC comprising an endogenous pMHC-I that comprises an endogenous antigenic peptide, an endogenous P-2- microglobulin (P2m), and an endogenous MHC-I alpha chain wherein the endogenous MHC-I alpha chain comprises an amino acid sequence with at least 70% sequence identity to any one of SEQ ID NOs: 1-20, e.g., at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, or any percentage therebetween.

[0203] In some embodiments, the tumor-APV is derived from the APTC comprising an endogenous pMHC-I that comprises an endogenous antigenic peptide, an endogenous P-2- microglobulin (P2m), and an endogenous MHC-I alpha chain wherein the endogenous P-2- microglobulin (P2m) comprises an amino acid sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 22 in Table 1, or an amino acid sequence that is substantially identical to an amino acid sequence setforth in SEQ ID NO: 21 or SEQ ID NO: 22 (e.g. 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity).

[0204] The present disclosure provides in some embodiments a tumor-APV or its parental APTC that is derived from pancreatic cancer, rectal cancer, colorectal cancer, lung cancer, endometrial cancer, liver cancer, testicular cancer, prostate cancer, cervical cancer, breast cancer, bladder cancer, head and neck cancer, skin cancer, brain cancer, ovarian cancer, salivary gland cancer, appendiceal cancer, esophagogastric cancer, thyroid cancer, soft tissue sarcoma, kidney cancer, leukemia, myeloma, lymphoma, melanoma, non-small cell lung cancer, Hodgkin’s lymphoma, or non-Hodgkin’s lymphoma.

[0205] Described herein, in some embodiments, is a tumor- APV or its parental APTC comprising an endogenous pMHC-I that comprises an endogenous antigenic peptide. In someWSGR Docket No. 48295-719.601 embodiments, the antigenic peptide is derived from pancreatic cancer, rectal cancer, colorectal cancer, lung cancer, endometrial cancer, liver cancer, testicular cancer, prostate cancer, cervical cancer, breast cancer, bladder cancer, head and neck cancer, skin cancer, brain cancer, ovarian cancer, salivary gland cancer, appendiceal cancer, esophagogastric cancer, thyroid cancer, soft tissue sarcoma, kidney cancer, leukemia, myeloma, lymphoma, melanoma, non -small cell lung cancer, Hodgkin’s lymphoma, or non-Hodgkin’s lymphoma.Transmembrane Domain

[0206] Described herein, in some embodiments, is a tumor-APV derived from an APTC comprising a first, a second, or a third fusion protein of displayed costimulatory ligand molecules wherein each of the first, second, or third fusion protein of the displayed costimulatory ligand molecules further comprises a transmembrane domain. In some embodiments, the transmembrane domain of the fusion protein of the displayed costimulatory ligand molecule comprises a transmembrane domain derived from VSV-G, Dengue E protein, influenza hemagglutinin, influenza neuraminidase, spike protein SI, spike protein S2, Sindbis virus envelope (SINDBIS) protein, hemagglutinin envelope protein from measles virus, envelope glycoprotein of measles virus fusion (F) protein, RD114, BaEV, GP41, GP120, or the wild-type transmembrane domain of costimulatory ligand molecules. In some embodiments, the transmembrane domain of the fusion protein comprises a transmembrane domain derived from VSV-G wherein the VSV-G comprises a transmembrane domain and cytoplasmic tail.

[0207] In some embodiments, the transmembrane domain of the displayed costimulatory ligand molecules comprises an amino acid sequence with at least 90% sequence identity to an amino acid sequence of SEQ ID NOs: 59-78. In some embodiments, the transmembrane domain of the displayed costimulatory ligand molecules comprises an amino acid sequence set forth in any one of SEQ ID NO: 59 to SEQ ID NO: 78 in Table 3, or an amino acid sequence that is substantially identical to an amino acid sequence set forth in any one of SEQ ID NO: 59 to SEQ ID NO: 78 (e.g. 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity). In some embodiments, the transmembrane domain of the displayed costimulatory ligand molecules comprises an amino acid sequence set forth in any one of SEQ ID NO: 59 to SEQ ID NO: 78 with at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 59-78.

[0208] In some embodiments, the transmembrane domain of the displayed costimulatory ligand molecules comprisesan amino acid sequence of at least 70% sequence identity to an amino acid sequence accordingto any one of SEQ ID NOs: 59-78, e.g., at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, or any percentage therebetween. In someWSGR Docket No. 48295-719.601 embodiments, the transmembrane domain of the displayed costimulatory ligand molecules comprises an amino acid sequence of at least 80% sequence identity to an amino acid sequence accordingto any one of SEQ ID NOs: 59-78. In some embodiments, the transmembrane domain of the displayed costimulatory ligand molecules comprises an amino acid sequence of at least 85% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 59-78. In some embodiments, the transmembrane domain of the displayed costimulatory ligand molecules comprises an amino acid sequence of at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 59-78. In some embodiments, the transmembrane domain of the displayed costimulatory ligand molecules comprises an amino acid sequence of at least 95% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 59-78. In some embodiments, the transmembrane domain of the displayed costimulatory ligand molecules comprises an amino acid sequence of at least 96% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 59-78. In some embodiments, the transmembrane domain of the displayed costimulatory ligand molecules comprises an amino acid sequence of at least 97% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 59-78. In some embodiments, the transmembrane domain of the displayed costimulatory ligand molecules comprises an amino acid sequence of at least 98% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 59-78. In some embodiments, the transmembrane domain of the displayed costimulatory ligand molecules comprises an amino acid sequence of at least 99% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 59-78.Oligomerization Domain

[0209] Described herein, in some embodiments, is a tumor-APV derived from a APTC comprising a first, a second, or a third fusion protein of displayed costimulatory ligand molecules wherein each of the first, second, or third fusion protein further comprises an oligomerization domain. In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecules is a dimerization domain, a trimerization domain, a tetramerization domain, a hexamerization domain, a heterohexamerization domain, a heptamerization domain, or an octamerization domain. In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecules comprises a leucine zipper dimerization domain, a post-fusion oligomerization domain of viral surface protein, a D4 post-fusion trimerization domain of VSV-G protein, a Dengue E protein post-fusion trimerization domain, a foldon trimerization domain, a coiled coil domain (Table 6), a LINE1 retrotransposon coiled coil domain (Llcc), a collagen XV coiled coil domain (CXVcc), a tetranectin coiled coil domain (TNTNcc), a cc-hex domain, a L24DWSGR Docket No. 48295-719.601 domain, a L24DH domain, a cc-Hept domain, a wazOct domain, or an influenza neuraminidase stem domain.

[0210] In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecules comprises an amino acid sequence that has at least 90% sequence identity to an amino acid sequence according to SEQ ID NOs: 79-93. In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecules comprises an amino acid sequence set forth in any one of SEQ ID NO: 79 to SEQ ID NO: 93 in Table 4, or an amino acid sequence that is substantially identical to an amino acid sequence set forth in any one of SEQ ID NO: 79 to SEQ ID NO: 93 (e.g. 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity). In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecules comprises an amino acid sequence set forth in any one of SEQ ID NO: 79 to SEQ ID NO: 93 with at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 79 - 93.

[0211] In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecules comprises an amino acid sequence of at least 70% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 79-93, e.g., at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, or any percentage therebetween. In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecules comprises an amino acid sequence of at least 80% sequence identity to an amino acid sequence accordingto any one of SEQ ID NOs: 79-93. In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecules comprises an amino acid sequence of at least 85% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 79-93. In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecules comprises an amino acid sequence of at least 90% sequence identity to an amino acid sequence accordingto any one of SEQ ID NOs: 79-93. In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecules comprises an amino acid sequence of at least 95% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 79-93. In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecules comprises an amino acid sequence of at least 96% sequence identity to an amino acid sequence accordingto any one of SEQ ID NOs: 79-93. In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecules comprises an amino acid sequence of at least 97% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 79-93. In some embodiments, the oligomerization domain of the displayed costimulatory ligand moleculesWSGR Docket No. 48295-719.601 comprises an amino acid sequence of at least 98% sequence identity to an amino acid sequence accordingto any one of SEQ ID NOs: 79-93. In some embodiments, the oligomerization domain of the displayed costimulatory ligand molecules comprises an amino acid sequence of at least 99% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 79-93.

[0212] Described herein, in some embodiments, is a tumor-APV capable of activating or stimulating an antigen -specific T cell wherein a parental APTC comprises a first fusion protein, a second fusion protein, a third fusion protein of the displayed costimulatory ligand molecules, or an endogenous pMHC-I.

[0213] In some embodiments, the parental APTC comprises the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules wherein when the first fusion protein, the second fusion protein, or the third fusion protein of the costimulatory ligand molecules is expressed on the surface of the tumor-APV, the oligomerization domain is outside of the tumor- APV. In some embodiments, the APTC comprises the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules wherein when the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules is expressed on the surface of the tumor-APV, the oligomerization domain is inside of the tumor- APV.

[0214] Expression of Displayed Costimulatory LigandMolecules And Endogenous pMHC-I onThe Surface

[0215] Costimulatory ligand molecules which are required to build highly potent and specific APVs can be introduced into tumor cells to reprogram them into antigen-presenting tumor cells (APTCs). Described herein, in some embodiments, are tumor-APVs displaying high copies of oligomerized proteins on the surface of exosomes or viral -like particles.

[0216] In some embodiments, the APTC comprises the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules and the endogenous peptide major histocompatibility complex class I (pMHC-I). The first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC- I is expressed at a valency of about 100 copies or more on a surface of the tumor-APV. In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 250 copies or more on a surface of the the tumor-APV. In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 500 copies or more on a surface of the the tumor-APV. In some embodiments, the first fusion protein, the second fusionWSGR Docket No. 48295-719.601 protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 1,000 copies or more on a surface of the tumor- APV. In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 2,000 copies or more on a surface of the tumor-APV. In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 4,000 copies or more on a surface of the tumor-APV. In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 6,000 copies or more on a surface of the tumor-APV. In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 8,000 copies or more on a surface of the tumor-APV. In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 10,000 copies or more on a surface of the tumor-APV. In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 20,000 copies or more on a surface of the tumor-APV. In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 50,000 copies or more on a surface of the tumor-APV.

[0217] In some embodiments, the APTC comprises the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous peptide major histocompatibility complex class I (pMHC-I). The first fusion protein, the second fusion protein, the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC- I is expressed at a valency of at least 1000 copies on the cell surface of the tumor-APV.

[0218] Tumor-APV as described herein, in some embodiments, comprises the first fusion protein, the second fusion protein, the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I expressed on a surface of the tumor-APV. In some embodiments, the first fusion protein, the second fusion protein, the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of at least or about 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000,WSGR Docket No. 48295-719.60111000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000, 25000, 30000, 35000, 40000, 45000, 50000, or more than 50000 copies on a surface of the multivalent particle.

[0219] In some embodiments, the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 100 copies to about 50,000 copies or more on the surface of the antigen-presenting tumor cell (APTC) herein, e.g., about 100 copies to about 20,000 copies, about 100 copies to about 10,000 copies, about 100 copies to about 8,000 copies, about 100 copies to about 6,000 copies, about 100 copies to about 4,000 copies, about 100 copies to about 2,000 copies, about 100 copies to about 1,000 copies, about 100 copies to about 500 copies, about 100 copies to about 250 copies, about 250 copies to about 50,000 copies, about 250 copies to about 20,000 copies, about250 copies to about 10,000 copies, about250 copies to about 8,000 copies, about 250 copies to about 6,000 copies, about 250 copies to about 4,000 copies, about 250 copies to about 2,000 copies, about250 copies to about 1,000 copies, about 250 copies to about 500 copies, about 500 copies to about 50,000 copies, about 500 copies to about 20,000 copies, about 500 copies to about 10,000 copies, about 500 copies to about 8,000 copies, about 500 copies to about 6,000 copies, about 500 copies to about4,000 copies, about 500 copies to about 2,000 copies, about 500 copies to about 1,000 copies, about 1,000 copies to about 50,000 copies, about 1000 copies to about 20,000 copies, about 1000 copies to about 10,000 copies, about 1000 copies to about 8,000 copies, about 1000 copies to about 6,000 copies, about 1000 copies to about 4,000 copies, about 1000 copies to about 2,000 copies, about 2,000 copies to about 50, 000 copies, about 2000 copies to about 20,000 copies, about 2000 copies to about 10,000 copies, about 2000 copies to about 8,000 copies, about 2000 copies to about 6,000 copies, about 2000 copies to about 4,000 copies, about 4000 copies to about 50,000 copies, about 4000 copies to about 20,000 copies, about 4000 copies to about 10,000 copies, about 4000 copies to about 8,000 copies, about 4000 copies to about 6,000 copies, about 6,000 copies to about 50,000 copies, about 6000 copies to about 20,000 copies, about 6000 copies to about 10,000 copies, about 6000 copies to about 8,000 copies, about 8,000 copies to about 50,000 copies, about 8000 copies to about 20,000 copies, about 8000 copies to about 10,000 copies, about 10,000 copies to about 50,000 copies, about 10,000 copies to about 20,000 copies, about 20,000 copies to about 50,000 copies.

[0220] In some embodiments, the tumor-APV is derived from its parental APTC comprising the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I wherein the endogenous pMHC-I comprises a natural pMHC-I.WSGR Docket No. 48295-719.601

[0221] In some embodiments, the parental APTC comprises the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I wherein the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules comprises an amino acid sequence with at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 94-111. In some embodiments, the parental APTC comprises the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC- I wherein the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules comprises an amino acid sequence set forth in any one of SEQ ID NO: 94 to SEQ ID NO: 111 in Table 5, or an amino acid sequence that is substantially identical to an amino acid sequence set forth in any one of SEQ ID NO: 94 to SEQ ID NO: 111 (e.g. 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity). In some embodiments, the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules comprises an amino acid sequence with at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 94-111.

[0222] In some embodiments, the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules comprises an amino acid sequence of at least 70% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 94-111, e.g., at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, or any percentage therebetween. In some embodiments, the first, the second, or the third fusion protein of the displayed costimulatory ligand molecules comprises an amino acid sequence of at least 80% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 94-111. In some embodiments, the costimulatory ligand molecule of the fusion protein comprises an amino acid sequence of at least 85% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 94-111 . In some embodiments, the costimulatory ligand molecule of the fusion protein comprises an amino acid sequence of at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 94-111. In some embodiments, the costimulatory ligand molecule of the fusion protein comprises an amino acid sequence of at least 95% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 94-111. In some embodiments, the costimulatory ligand molecule of the fusion protein comprises an amino acid sequence of at least 96% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 94-111 . In some embodiments, the costimulatory molecule of the fusion protein comprises an amino acid sequence of at least 97% sequence identity to an amino acid sequence according toWSGR Docket No. 48295-719.601 any one of SEQ ID NOs: 94-111. In some embodiments, the costimulatory ligand molecule of the fusion protein comprises an amino acid sequence of at least 98% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 94-111. In some embodiments, the costimulatory ligand molecule of the fusion protein comprises an amino acid sequence of at least 99% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 94-111.

[0223] In some embodiments, the tumor-derived antigen-presenting vesicle (tumor-APV) comprising a first fusion protein that comprises the costimulatory ligand molecule CD80 or CD86, and a second fusion protein that comprises the costimulatory ligand molecule GITRL, and a recombinant peptide major histocompatibility complex class I (pMHC-I), wherein the tumor-APV is derived from its parental antigen-presenting tumor cell (APTC) comprising the first fusion protein, the second fusion protein, and the recombinant pMHC-I, wherein the tumor-APV or its parental APTC lacks an endogenous pMHC-I or expresses a dysfunctional endogenous pMHC-I, wherein first fusion protein, the second fusion protein, or the recombinant pMHC-I is expressed at a valency of at least about 100 copies on a surface of the tumor-APV. In some embodiments, a fusion protein comprising a costimulatory ligand molecule that binds specifically to a T cell surface costimulatory receptor, an oligomerization domain, and a transmembrane domain, wherein the fusion protein is expressed at a valency of at least about 100 copies on a surface of a tumor-APV, wherein the tumor-APV is derived from its parental APTC. In some embodiments, the fusion protein comprises at least two separate fusion proteins, wherein the at least two separate fusion proteins each comprises a different costimulatory ligand molecule. In some embodiments, the costimulatory molecule comprises one or more selected from the group comprising GITR Ligand, CD80, CD86, 0X40 Ligand, CD48, CD30 Ligand, ICOS Ligand, CD70, CD40, CD40 Ligand, 4- BBL, TLIA, TIM4, SLAM, CD48, CD58, CD155, CD112, LIGHT, and ICAM1.

[0224] In some embodiments, the costimulatory ligand molecule comprises an amino acid sequence setforth in any one of SEQ ID NO: 23 to SEQ ID NO: 58 in Table 2, or an amino acid sequence that is substantially identical to an amino acid sequence set forth in any one of SEQ ID NO: 23 to SEQ ID NO: 58 (e.g. 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity). Methods of Use

[0225] Described herein, in some embodiments, is a tumor-APV capable of activating or stimulating an antigen-specific T cell wherein the tumor-APV is derived from its parental APTC comprising a first fusion protein, a second fusion protein, a third fusion protein of displayed costimulatory ligand molecules wherein any one of the fusion proteins is expressed at least about 100 copies on a surface of the tumor-APV.WSGR Docket No. 48295-719.601

[0226] Described herein, in some embodiments, are methods of preventing or treating cancer in a subject in need thereof comprising administering to the subject an antigen-presenting vesicle (APV) wherein the APV is derived from the APTC that is obtained from a subject. Described herein, in some embodiments, are methods of inducing protective immunity against cancer in a subject in need thereof comprising administering to the subject the tumor-derived antigen- presenting vesicle (tumor- APV), wherein the tumor- APV is derived from its parental APTC that is derived from the subject.

[0227] In some embodiments, the method of preventing or treating cancer in a subject in need thereof comprising administering to the subject a T cell or a TIL, wherein the T cell or the TIL is activated or stimulated by the tumor- APV before the administering to the subject. In some embodiments, the method of preventing cancer relapse in a subject in need thereof comprising administering to the subject tumor-APVs. In some embodiments, the method of preventing or treating established cancer in a subject in need thereof comprising administering to the subject autologous tumor-APVs. In some embodiments, the method of inducing protective immunity against cancer in a subject in need thereof comprising administering to the subject autologous tumor-APVs. In some embodiments, the autologous tumor-APVs is derived from a cancer cell produced in culture.

[0228] In some embodiments, the subject undergoes cancer treatment before the administering and does not have cancer after the treatment.

[0229] In some embodiments, the methods of preventing or treating cancer in a subject or inducing protective immunity against cancer in a subject comprise administering of tumor-APVs wherein the administering of tumor-APVs comprises administering a Toll-like receptor agonist in the subject.

[0230] In some embodiments, the methods of preventing or treating cancer in a subject or inducing protective immunity against cancer in a subject comprise administering of tumor-APVs wherein the administering of tumor-APVs comprises administering an anti-PDl, anti-PDLl, an anti-CTLA4 immune checkpoint blockade therapy, a targeted therapy, an antibody -drug therapy, a tyrosine kinase inhibitor, a chemotherapy, surgery, or a therapy of standard care.

[0231] Described herein, in some embodiments, are methods of enhancing anti-tumor T cell immunity in a subject, comprising administering to a subject in need thereof the tumor- APV or its parental APTC.

[0232] Many state-of-art cancer treatments include surgery, chemotherapy, targeted therapies (i.e., tyrosine kinase inhibitors), antibody -drug conjugates (ADC), CAR-T cells, and immune checkpoint blockade (ICB) therapies, either as monotherapies or in combination can lead to short -WSGR Docket No. 48295-719.601 term remission among many treated patients. In some embodiments, it is maybe preferred to administer the therapeutic agent with tumor-APVs, or its parental APTC.

[0233] In some embodiments, the methods of preventing or treating cancer in a subject, inducing protective immunity against cancer in a subject, or enhancing anti-tumor T cell immunity in a subject comprise administering a therapeutic agent wherein the therapeutic agent comprises an inflammatory cytokine, a dendritic cell targeting signal, or an innate modulating signal.

[0234] In some embodiments, administration of the tumor-APVs reduces or eliminates the cancer. In some embodiments, administration of the tumor-APVs increase cancer cell death, decrease tumor size, decrease cancer metastasis, or combinations thereof. In some embodiments, cell death is increased by about 1 -fold to about 2.5-fold, about 1-fold to about 5 -fold, about 2-fold to about 10-fold. In some embodiments, cell death is increased by at least 5 -fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 95-fold, 100-fold, or greater than 100-fold. In some embodiments, tumor size is decreased by about 1 -fold to about 2.5-fold, about 1-fold to about 5- fold, about 2-fold to about 10-fold. In some embodiments, tumor size is decreased by at least 5 - fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60- fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 95-fold, 100-fold, or greater than 100- fold. In some embodiments, cancer metastasis is decreased by about 1 -fold to about 2.5 -fold, about 1 -fold to about 5 -fold, about 2-fold to about 10-fold. In some embodiments, cancer metastasis is decreased by at least 5 -fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60 -fold, at least 70-fold, at least 80-fold, at least 90 -fold, at least 95 -fold, 100- fold, or greater than 100-fold.

[0235] In some embodiments, administration of the tumor-APVs reduces or eliminates the cancer as compared to a level prior to administration of the multivalent particles in the subject. In some embodiments, administration of the tumor-APVs reduces or eliminates the cancer as compared to a level if the subject had not received the multivalent particles. In some embodiments, administration of the tumor-APVs reduces or eliminates the cancer as compared to a level if the subject had received a different cancer treatment including but not limited to, radiation, surgery, and chemotherapy.

[0236] In some embodiments, the tumor APVs induce T cell mediated cytotoxicity against tumor cells.

[0237] In some instances, the subject is a mammal. In some instances, the subject is a mouse, rabbit, dog, pig, cattle, or human. Subjects treated by methods described herein may be infants, adults, or children.WSGR Docket No. 48295-719.601Compositions for generation of tumor-derived antigen-presenting vesicles (APVs)

[0238] Described herein, in some embodiments, are compositions comprising a tumor-APV that is derived from a parental APTC expressing fusion proteins of the displayed costimulatory ligand molecules and an endogenous peptide major histocompatibility complex class I (pMHC-I) on a surface of the tumor-APV. In some embodiments, the composition of the tumor-APV comprises a first fusion protein that comprises the costimulatory ligand molecule CD80 or CD86, a second fusion protein that comprises the costimulatory ligand molecule GITRL, and a recombinant peptide major histocompatibility complex class I (pMHC-I) on the surface of the tumor-APV.

[0239] The methods of producing the tumor-APV, in some embodiments, comprise administering to a subject a nucleic acid encoding a fusion protein comprising a costimulatory ligand molecule that binds specifically to a T cell surface costimulatory receptor, an oligomerization domain, and a transmembrane domain, wherein the administering results in the expression of the fusion protein on a surface of a cancer cell in the subject.

[0240] Alternatively, the methods of producing the tumor-APV comprise (A) Generating an antigen-presenting tumor cell (APTC) by introducing ex vivo into the cancer cell a nucleic acid encoding a fusion protein comprising a costimulatory ligand molecule that binds specifically to a T cell surface costimulatory receptor, an oligomerization domain, and a transmembrane domain to express the fusion protein on a surface of the cancer cell. (B) Culturing and expanding APTCs in culture dishes as a suspension, adherent, or organoid cell culture. (C) Generating tumor-derived antigen-presenting vesicles (tumor- APVs) by disrupting the antigen-presenting tumor cells (APTCs) into tumor-APVs using physical processes, including nitrogen cavitation, freeze and thaw, sonication, or size-exclusion filtration.

[0241] Compositions for generating the tumor-APV, in some embodiments, further comprise a nucleic acid sequence that each encodes one or more viral proteins. In some embodiments, the one or more viral proteins is a lentiviral protein, a retroviral protein, an adenoviral protein, or combinations thereof. In some embodiments, the one or more viral proteins comprises gag, pol, pre, tat, rev, or combinations thereof.

[0242] In some embodiments, the tumor-APV is a viral-like particle. In some embodiments, the multivalent particle is an extracellular vesicle. In some embodiments, the multivalent particle is an exosome. In some embodiments, the multivalent particle is an ectosome.

[0243] Compositions for generating the tumor-APV, in some embodiments, further comprise a nucleic acid sequence that encodes an expression construct for specifically targeting the fusion protein of the costimulatory molecules to the surface of an extracellular vesicle. In someWSGR Docket No. 48295-719.601 embodiments, the nucleic acid sequence encodes an expression construct for specifically targeting the fusion protein of the costimulatory molecules to the surface of an exosome.

[0244] Compositions for generating the tumor-APV, in some embodiments, further comprise a nucleic acid sequence that encodes a replication incompetent viral genome, a reporter, a therapeutic molecule, or combinations thereof. In some embodiments, the viral genome is derived from vesicular stomatitis virus, measles virus, Hepatitis virus, influenza virus, or combinations thereof.

[0245] In some embodiments, the method of producing the tumor-APV comprises disrupting the antigen-presenting tumor cell (APTC) expressing the fusion protein into the tumor-APV wherein the disrupting of APTCs is carried out using a physical method. In some embodiments, to disrupt APTCs, the physical method comprises nitrogen cavitation, sonication, freeze and thaw, or membrane exclusion. In some embodiments, the method of producing the tumor-APV further comprise purifying the tumor-APV to remove cellular debris, nucleic acid impurities, or protein impurities.

[0246] In some embodiments, the tumor-APV described herein comprises a size from about lOOnm to about 1 pm, e.g., about 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, 350 nm, 360 nm, 370 nm, 380 nm, 390 nm, 400 nm, 410 nm, 420 nm, 430 nm, 440 nm, 450 nm, 460 nm, 470 nm, 480 nm, 490 nm, 500 nm, 510 nm, 520 nm, 530 nm, 540 nm, 550 nm, 560 nm, 570 nm, 580 nm, 590 nm, 600 nm, 610 nm, 620 nm, 630 nm, 640 nm, 650 nm, 660 nm, 670 nm, 680 nm, 690 nm, 700 nm, 710 nm, 720 nm, 730 nm, 740 nm, 750 nm, 760 nm, 770 nm, 780 nm, 790 nm, 800 nm, 810 nm, 820 nm, 830 nm, 840 nm, 850 nm, 860 nm, 870 nm, 880 nm, 890 nm, 900 nm, 910 nm, 920 nm, 930 nm, 940 nm, 950 nm, 960 nm, 970 nm, 980 nm, 990 nm, or about 1 m, or any size therebetween. In some embodiments, the size of the tumor-APV is about lOOnm to about 200nm.Compositions and Pharmaceutical Compositions

[0247] Described herein, in some embodiments, are compositions comprising a tumor-APV derived from its parental APTC that expresses fusion proteins of co-stimulatory molecules and an endogenous peptide major histocompatibility complex class I (pMHC-I) on a surface of the tumor- APV. In some embodiments, the composition of the tumor-APV comprises a first fusion protein that comprises the costimulatory ligand molecule CD80 or CD86, a second fusion protein that comprises the costimulatory ligand molecule GITRL, and a recombinant peptide major histocompatibility complex class I (pMHC-I).

[0248] Described herein, in some embodiments, are compositions comprising a nucleic acid molecule encoding a first fusion protein, a second fusion protein, or a third fusion protein ofWSGR Docket No. 48295-719.601 displayed costimulatory ligand molecules. In some embodiments, the composition comprises the nucleic acid molecule encoding a first fusion protein of a displayed costimulatory ligand molecule CD80 or CD86, a second fusion protein of a displayed costimulatory ligand molecule GITRL.

[0249] Described herein, in some embodiments, are pharmaceutical compositions comprising a nucleic acid molecule encoding a first fusion protein, a second fusion protein, or a third fusion protein of displayed costimulatory ligand molecules. The method of producing the tumor-APV comprises administering to a subject a nucleic acid encoding a fusion protein comprising a costimulatory ligand molecule that binds specifically to a T cell surface costimulatory receptor, an oligomerization domain, and a transmembrane domain, wherein the administering results in the expression of the fusion protein on a surface of a cancer cell in the subject.

[0250] Described herein, in some embodiments, are pharmaceutical compositions comprising a tumor-APV derived from its parental APTC expressing fusion proteins of displayed costimulatory ligand molecules and an endogenous peptide major histocompatibility complex class I (pMHC-I) on a surface of the tumor-APV. The method of producing the tumor-derived antigen-presenting vesicle (tumor-APV), comprising: (A) Generating an antigen-presenting tumor cell (APTC) by introducing ex vivo into the cancer cell a nucleic acid encoding a fusion protein comprising a costimulatory ligand molecule that binds specifically to a T cell surface costimulatory receptor, an oligomerization domain, and a transmembrane domain to express the fusion protein on a surface of the cancer cell. (B) Culturing and expanding APTCs in culture dishes as a suspension, adherent, or organoid cell culture. (C) Generating tumor-derived antigen-presenting vesicles (tumor-APVs) by disrupting the antigen-presenting tumor cells (APTCs) into tumor-APVs using physical processes, including nitrogen cavitation, freeze and thaw, sonication, or size -exclusion filtration. In some embodiments, the method of producing the tumor APPV further comprises further comprising purifying the tumor-APV to remove cellular debris, nucleic acid impurities, or protein impurities.

[0251] In some embodiments, a pharmaceutical composition comprises the tumor-APV and a pharmaceutically acceptable excipient.

[0252] For administration to a subject, the tumor-APVs as disclosed herein, may be provided in a pharmaceutical composition together with one or more pharmaceutically acceptable carriers or excipients. In some embodiments, the tumor-APVs as disclosed herein, may be provided in a composition together with one or more carriers or excipients. The term "pharmaceutically acceptable carrier" includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the ingredients and that is not toxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil / water emulsions, variousWSGR Docket No. 48295-719.601 types of wetting agents, sterile solutions etc. Such carriers can be formulated by conventional methods and can be administered to the subject at a suitable dose. Preferably, the compositions are sterile. These compositions may also contain adjuvants such as preservative, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents.

[0253] The pharmaceutical composition may be in any suitable form, (depending upon the desired method of administration). It may be provided in unit dosage form, may be provided in a sealed container and may be provided as part of a kit. Such a kit may include in structions for use. It may include a plurality of said unit dosage forms.

[0254] The pharmaceutical composition maybe adapted for administration by any appropriate route, including a parenteral (e.g., subcutaneous, intramuscular, intravenous, or inhalation) route. Such compositions may be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.

[0255] Dosages of the substances of the present disclosure can vary between wide limits, depending upon the disease or disorder to be treated, the age and condition of the individual to be treated, etc. and a physician will ultimately determine appropriate dosages to be used.

[0256] The following examples are set forth to illustrate more clearly the principle and practice of embodiments disclosed herein to those skilled in the art and are not to be construed as limiting the scope of any claimed embodiments. Unless otherwise stated, all parts and percentages are on a weight basis.EXAMPLES

[0257] The following examples are given for the purpose of illustrating various embodiments of the disclosure and are not meant to limit the present disclosure in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the disclosure. Changes therein and other uses which are encompassed within the spirit of the disclosure as defined by the scope of the claims will occur to those skilled in the art.Example 1: Antigen-presenting Tumor Cells (APTCs) and Tumor- derived Antigen- presenting Vesicles from APTCs

[0258] This Example describes the development of antigen-presenting tumor cell (APTC) and its derivative: tumor-derived antigen-presenting vesicle (tumor-APV). APTCs and tumor-APVs both present all tumor antigens and can effectively activate anti-tumor antigen-specific T-cells in vitro and in vivo. APTCs and tumor-APVs represent the next-generation antigen-presenting T-cell vaccines that can be used to induce robust anti -tumor immunity for prevention and therapy.WSGR Docket No. 48295-719.601

[0259] Rationale, Design, and Method Development

[0260] Rationales

[0261] Effective activation of antigen-specific T cells by antigen-presenting cells (APCs), including B cells and dendritic cells (DCs), requires signal 1 from pMHC and TCR engagement and signal 2 from the interaction of costimulatory molecules (Figure 1A, 1C). In contrast, cancer cells, as well as normal somatic cells, generally lack the expression of costimulatory molecules. Thus, despite producing and presenting many mutant antigens and neoantigens, tumor cells cannot activate anti-tumor T cells properly. In fact, pMHC and TCR engagement without costimulatory signals induces tolerance and T cell anergy. It renders them unresponsive to further stimulation, resulting in tolerance of tumors by our immune systems (Figure IB, 1C).

[0262] Dearth of T cell co -stimulatory molecule expression in murine and human tumors

[0263] The expression of class I MHC molecules and a panel of T cell co -stimulatory molecules in murine tumor cell lines and human tumor samples were analyzed. Murine tumor cell lines from a variety of origins, including melanoma, B cell lymphoma, prostate, pancreatic, lung, renal, hepatic, and colorectal adenocarcinoma, were stained with a panel of MHC I and customspecific antibodies to determine the proportion of tumor cells and the relative expression levels of these molecules in tumors. The majority of murine tumor cell lines queried exhibited prevalent class I MHC expression, with over 90% of cells MHC I+(Figure 2A). Only 4T1 mammary carcinoma and B16F10 melanoma cell lines were below 90% MHC I+. Using conjugated primary monoclonal antibodies (mAb) and Quantibrite quantification beads, the copy number of MHC I molecules on various murine tumor cell lines was then determined. MHC I copy numbers ranged from 2.9E+05 molecules per cell forRenca renal adenocarcinoma cells to as low as 3.7E+03 MHC I molecules per cell for B16F10 melanoma cells. (Figure 2B). However, despite variations in relative MHC I expression level and cell valence, MHC I molecule expression was generally ubiquitous among tumor cells.

[0264] By contrast, expression of T cell co-stimulatory molecules was largely absent from murine tumor cell lines (Figure 2A). T cell costimulatory ligand molecules such as CD80, CD86, and OX40L were not detected in a meaningful proportion of cells or at significant expression levels in the murine tumor cell lines tested (Figure 2A). Scattered expression of a few co-stimulatory molecules, including CD48, ICAM-1, HVEM, and 4-1BBL, was detected in different tumor cell lines. 4-1BBL, in particular, was detected in more than 50% of cells in 4T1, MC38 (66%), and CT26 colon carcinoma (64%) and RM-1 prostate carcinoma (88%) cells, however, its expression was still more than 10-fold lower than that of MHC I, as determined by median fluorescence intensity (MFI). Expression of T cell costimulatory ligand CD80 and CD86 was muted acrossWSGR Docket No. 48295-719.601 almost all tumor cell lines surveyed. CD80 and CD86, also known as B7-1 and B7-2, respectively, are for proper antigen-specific CD8+ T cell activation by antigen-presenting cells. The expression of MHC I and T cell costimulatory ligands in human tumors was then analyzed. Data taken from 1020 human cancer cell lines provided by the Cancer Cell Line Encyclopedia (Novartis / Broad, Nature 2012) illustrates a similar phenomenon in human cancer cell lines. T cell costimulatory ligand molecules are not detected or are expressed at significantly lower levels than the MHC I genes HLA-A, HLA-B, and HLA-C across all tumor origins queried, as determined by mRNA microarray (Figure 2C). This analysis, coupled with the data surveying murine tumor cell lines, indicates that by and large, both human and murine tumor cell lines do not express T cell co- stimulatory molecules. Lack of proper anti-tumor T cell co-stimulation may present a significant barrier to T cell-based cancer immunotherapies, including checkpoint blockade and tumor- infiltrated lymphocyte (TIL) therapies.

[0265] Moreover, a universal set of costimulatory signals that may be used to reprogram the majority of the tumor types is yet to be defined. If such costimulatory signals are known, it is possible to reprogram tumor cells into antigen-presenting cells, dubbed antigen-presenting tumor cells (APTCs), by introducing the costimulatory ligands required into the tumor cells. Since cancer cells produce and present all tumor-specific antigens, APTCs may, in principle, be used to selectively activate tumor-specific T cells in vivo and ex vivo to function as anti-tumor T cell vaccines. Notably, some early studies showed that ectopic expression of costimulatory ligand CD80 or CD86 in few tumor cell lines caused delayed tumor growth in mice as the results of induction of T cell immunity. However, in those studies, ectopic expression of costimulatory ligand CD80 / CD86 only had such effects on few among all the tumor cell lines tested and often did not lead to the rejection of those tumor cell lines. Similarly, syngeneic mouse tumors generated using MC38 tumor cells expressing wild-type CD80 or wild-type GITRL grew significantly slower than those from the wild-type MC38 tumor cells and were not fully rejected (Figure 3). Furthermore, syngeneic mouse tumors generated from the Lewis lung cancer cells (LLC) expressing wild-type CD80 or wild-type GITRL grew at a similar rate compared to those from the wild-type LLC tumor cells (Figure 4). Together, these observations demonstrated that some tumor cells may not activate anti -tumor T cells properly because they lack proper costimulatory signals. However, it was also clear that ectopic expression of wild -type CD80 / CD86 or wild-type GITRL did not induce robust immunity against many of the tumor cell lines tested. So far, there has been no reported attempts to induce anti -tumor T cell immunity by programming tumor cells into antigen-presenting cells to effectively.WSGR Docket No. 48295-719.601

[0266] The challenge to define the costimulatory signals required for reprogramming tumor cells into APTCs

[0267] APCs express pMHCs and many activating costimulatory molecules that participate in TCR engagement (Figure 5). During antigen recognition by T cells, pMHC and TCR engagement leads to the formation of immunological synapses, which include highly condensed aggregates of the TCR subunits and signaling proteins, adhesion molecules, costimulatory receptors, and inhibitor receptors on the T cell membrane. Since there are many costimulatory molecules to choose from (Figure 5), we can reprogram tumor cells into potent and specific APTCs for antigen -specific T- cell activation.

[0268] However, there are many challenges to systematically examining the function of various costimulatory ligand molecules in reprogramming tumor cells into APTCs. First, T cells employ multiple co-stimulatory molecules to modulate T cell activation, proliferation, differentiation, and function (Figure 5). First, tumor cells are heterogeneous and may require different costimulatory signals to reprogram them into APTCs. Given the number of costimulatory ligands available, testing the function of individual and / or combinations of costimulatory molecules to turn diverse cancer cell types into APTCs may represent a daunting task technically and operationally. Beyond the complexity, tumor cells express various inhibitory immune checkpoints that may actively suppress activated T cells, and tumor cells may respond to T cell engagement to up-regulate additional signals to suppress T cell activation. These factors further confound the dissection of costimulatory signals required to help tumor cells overcome these negative regulatory events when trying to reprogram tumor cells into APTCs that can effectively activate and expand anti-tumor T cells in culture or in vivo.

[0269] Define the costimulatory signals required to reprogram vesicles into antigen- presenting vesicles

[0270] Instead of testing the function of individual or combination of costimulatory molecules in diverse tumor cells, an indirect approach was adopted to define the costimulatory signals required to program tumor cells into APTCs. Specifically, vesicles, such as exosomes (EVs) and virus-like particles (VLPs), were reprogrammed into antigen-presenting vesicles (APVs), which can effectively activate antigen -specific T cells. If the combination of costimulatory signals and an optimized display strategy can be determined to be strong enough to turn EVs and VLPs into robust antigen-presenting vesicles, the same combination of costimulatory signals and an optimized display strategy could be used to turn tumor cells into antigen-presenting tumor cells. The sizes of EVs and VLPs are around 100-200 nm, whereas APCs are around -10-20 microns in size. Larger particle sizes, which permit the display of a higher number of TCR and costimulatoryWSGR Docket No. 48295-719.601 engaging molecules (i.e. anti-CD3 and anti-CD28), are useful for producing scaffolding T-cell activation beads. For example, the optimal sizes of anti-CD3 / CD28 beads, which are commonly used for activating all T cells (not suitable for antigen-specific T cell activation), are in the range of 5 microns. Many attempts to build nanoparticle sized artificial antigen-presenting cells have not achieved efficient T cell activation. Moreover, EVs and VLPs from the right cell types may be devoid of negative signals that would compromise the screening.

[0271] Through the process of developing specific and potent APVs for in vitro and in vivo antigen-specific T-cell activation, the methods for displaying high copies of oligomerized proteins on the surface of exosomes or viral -like particles were established. Moreover, we defined the minimal combination of costimulatory ligand molecules, including CD80 / CD86 and GITRL, that are required for effective T-cell activation by the APVs (Figure 6A). Notably, APVs also display recombinant pMHC-I designated as single-chain trimers pMHC-I (SCT-pMHC-I), consisting of an ordered linked polypeptide with antigenic peptide, beta-2M, MHC-I alpha chain ectodomain, an oligomerization domain, and membrane anchoring domain (Figure 6A). So, the potent and specific APVs for antigen-specific T-cell activation on exosomes and VLPs were generated, demonstrating that high-efficiency surface display on vesicles has been optimized and the costimulatory signals has been defined. Such insights may be directly applied to reprogram tumor cells into APTCs.

[0272] Reprogramming tumor cells into APTCs

[0273] With the methods and insights from building APVs on EVs and VLPs, it is postulated that that the same set of co -stimulatory molecules, such as CD80 / CD86 and GITRL, can be displayed on tumor cells to turn them into antigen-presenting tumor cells (APTCs) by using the same display technology (Figure 6B). Similarly, high copy numbers of oligomerized CD80 / CD86 and GITRL on tumor cells will provide robust costimulatory signals for APTCs to activate all tumor-associated antigens and neoantigens from tumor cells following pMHC engagement with cognate TCRs. Notably, APVs display SCT-pMHC-I and can only be generated for defined antigenic peptides. In contrast, endogenous pMHC-I from APTCs will provide the signal 1 for pMHC-I and TCR engagement. Thus, APTCs can present a cocktail of tumor-specific antigens, tumor-associated antigens, and neoantigens form tumor cells and selectively activate antigen-specific T-cells that recognize all those tumor antigens. It is contemplated that there are different ways to apply APTCs in cancer treatment and prevention of cancer relapses after treatment, including autologous APTCs, in situ APTCs, and autologous T cell therapy with APTC-expanded anti-tumor T cells. Here the conceptual level rationales and designs of these APTC applications were provided.WSGR Docket No. 48295-719.601

[0274] Induction of antitumor immunity with autologous APTCs

[0275] It will first be demonstrated whether ex vivo reprogrammed APTCs may induce robust anti -tumor T cell immunity that lead to the rejection of APTCs, and whether the rejection of APTCs will result in anti-tumor T cell memory against cancer rechallenges. It will then be tested whether APTCs, which can activate anti-tumor T cells in vivo, may be used as T-cell vaccines for cancer prevention or treatment. It is contemplated that there are many advantages to APTC-based T-cell vaccines for cancer. First, APTCs can directly activate anti-tumor T cells. So, the process is much more direct and effective than the DC vaccines or mRNA / protein / DNA-based cancer vaccines, which rely on the highly complex and unpredictable process of transferring selected tumor antigens to dendritic cells in vitro or in vivo. Moreover, APTCs can directly activate T cells recognizing all tumor antigens presented, including tumor-specific antigens, tumor- associated antigens, and fast-evolving neoantigens, without manual searching or selecting antigens. In comparison, current cancer vaccine approaches mostly rely on manually selected cancer antigens. The selection process is, unfortunately, biased and limiting because of the everevolving cancer antigen landscape. Finally, recognition of APTCs by anti -tumor T cells may lead to the killing of APTCs, which will release more tumor antigens to prime DCs and other APCs. This approach should have tremendous advantages over existing cancer vaccine technology, including DC vaccine and mRNA / DNA / protein -based cancer vaccines.

[0276] In situ antigen-presenting tumor cells (in situ APTCs)

[0277] Once the ex vivo reprogrammed APTCs are demonstrated to function as potent T-cell vaccines in vivo, it will be logical to use similar costimulatory to reprogram tumor cells into antigen-presenting cells in situ — within the tumors. Specifically, costimulatory (i.e., CD80 and GITRL) signals are delivered to tumors in situ by using DNA vectors, viral vectors, or mRNAs encoding these molecules. Such an approach will generate APTCs within the tumor microenvironment and provide off-the-shelf in situ therapeutic vaccines for solid tumor types with accessible tumor masses without the need for culture and manipulation ex vivo. In situ, APTCs can be used as therapeutics vaccines for established tumors, either as a monotherapy or combination therapy with ICB, targeted therapies, ADC, TLR agonists, or chemo and standard care for cancer.

[0278] Autologous T cell therapy with ex vivo APTC-expanded anti-tumor T cells

[0279] Beyond the in vivo use of APTCs, APTCs may be used to expand anti -tumor T cells in culture for autologous T cell therapy. There are many advantages of using APTCs to expand autologous anti-tumor T cells. APTCs can be used to generate anti-tumor T cells from a patient's peripheral blood T cells or tumor-infiltrated T cells. Because the source of T cells is qualified for safety and effectiveness by positive and negative selection, APTC-expanded anti-tumor T cellsWSGR Docket No. 48295-719.601 are likely to be safe and effective. Moreover, these T cells are likely to recognize multiple tumor antigens, including tumor-specific antigens, tumor-associated antigens, and fast-evolving neoantigens, and, therefore, APTC-expanded T cells are more effective because of their polyclonal nature. Finally, using APTC-expanded T cells for autologous T cell therapy also helps to bypass the search of CAR-T cells for various tumor indications or the need to engineer TCRs, which often have unpredictable potency and safety.

[0280] Tumor-derived antigen-presenting vesicles (tumor-APVs) from APTCs

[0281] The results have demonstrated the use of tumor cell reprogramming into APTCs as a prophylactic and therapeutic cancer vaccine strategy capable of stimulating antigen -specific antitumor T cells and inducing robust anti -tumor immunity in vivo. However, using autologous cancer cells in any human cancer therapy poses significant safety and feasibility challenges. To circumvent this, APTCs were converted into tumor-derived antigen-presenting vesicles (tumor-APVs). Tumor- APVs may be generated from APTCs using various physical methods, including nitrogen cavitation, sonication, extrusion, freeze and thaw, or collected from the APTC culture as secreted EVs. Tumor- APVs maintain the function of APTCs in the activation of antigen-specific T cells in culture and in vivo as preventative and therapeutic cancer vaccines. Converting APTCs into tumor-APVs may abolish some of the negative signals in APTCs and permit robust expansion of activated antigenspecific T cells and better tumor control as therapeutic vaccines for established cancer.

[0282] High-density and oligomerized protein display on vesicles

[0283] Design of multivalent oligomeric protein display on enveloped vesicles

[0284] Given the relatively small size of exosomes and VLPs (-100 nm), the display strategy was optimized first to maximize the density and oligomerization of the displayed molecules on vesicles. The aim was to display pMHC and costimulatory ligands at high density and in oligomerized format on vesicles. It is postulated that high density and oligomerized display of pMHC and costimulatory ligands on these small vesicles may help them to engage a large number of TCR and costimulatory molecules on T cells and lead to productive engagement that mimics the formation of immunological synapses during DC and T cell engagement. To this end, the vesicle display systems were engineered by mimicking enveloped viruses, which often display their surface proteins in multivalent oligomeric format and high density on virions. Viruses such as Influenza or Hepatitis B display hundreds of copies of viral spikes per virion, enabling a single virion to simultaneously interact with multiple copies of host cell receptors and attachment proteins. In the case of coronaviruses, host cell receptors Angiotensin-converting enzyme 2 (ACE2) and dipeptidyl peptidase 4 (DPP4) serve as entry receptors for SARS Co V- 1 / 2 and MERS CoV, respectively. The densely packed spike proteins displayed on virions enable them to interact with multiple copies ofWSGR Docket No. 48295-719.601 entry receptors on the host cell surface with both high local avidity — spike to receptor — and global avidity — virion to host cell membrane. Thisevolutionarily conserved feature is shared by almost all enveloped viruses and underlies viruses’ ability to efficiently infect target cells by forming multivalent, Velcro-like interactions with cognate entry receptors on target cells. Many cellular membrane proteins, including TCR and costim complexes, are analogously clustered on the cell surface in oligomeric forms. It is reasoned that vesicles displaying pMHC and costims in oligomeric display formats could demonstrate both enhanced display density and oligomeric clustering, thus improving their local and global avidity to their corresponding oligomeric binding partners on the T cell membrane.

[0285] To mimic multivalent oligomeric protein expression on the cell surface, “display vectors” to express and efficiently target displayed peptides to the surface of enveloped vesicles, such as VLPs and exosomes, were designed (Figure 7). Display vectors are designed to express a fusion protein consisting of a signal peptide, a displayed peptide, an oligomerization domain, and a transmembrane and cytosolic tail for membrane anchoring and targeting. Furthermore, since viral surface proteins are highly efficient in targeting budding viral particles, these proteins may contain unique sequences required for vesicle display (Figure 7A). Based on this rationale, the transmembrane and cytosolic region of VSV-G (Vesicular Stomatitis virus glycoprotein) were used as a vesicle membrane anchoring protein. VSV-G can be used to effectively pseudotype lentivirus. Further, various well-defined oligomerization domains were selected to test their effects on display efficiency and oligomerization format on vesicles (Figure 7B). Finally, the display strategy was functionally tested and optimized by generating the “decoy-antivirus” — multivalent particles (MVPs) displaying oligomeric multivalent virus entry receptors as spike-capturing molecules. Specifically, viral entry receptors for SARS CoV, such as ACE2 and DDP4, were displayed on vesicles. Such design allowed to quantitatively correlate the copy number and oligomerization format of the displayed proteins to biological function in rapid virus neutralization assays. The neutralization activity of decoy-antivirus is tightly linked to their binding affinity to virus particles .

[0286] Generation of trimeric MVPs

[0287] Several trimeric display constructs were designed by fusing the displayed peptide to the D4 post-fusion trimerization domain of VSV-G, the Dengue E protein post-fusion trimerization domain, or the T4 Phage Fibritin Foldon domain, followed by the transmembrane and intracellular domains of VSV-G. By design, using a trimeric display vector, multivalent proteins can be displayed as trimers on the surface of the viral -like particles (VLP) and extracellular vesicles (EV), such as exosomes and exosomes. To produce trimeric VLP-based MVPs with viral RNA genomes, cells were co-transfected with a trimeric peptide fusion construct utilizing one of severalWSGR Docket No. 48295-719.601 trimerization domains — D4, DE, or Foldon — alongside a lentiviral packaging construct expressing packaging components, such as Gag-Pol and Rev proteins, and a viral genome transfer vector encoding a GFP / lucif erase reporter (Figure 8A). Alternatively, trimeric VLP -based MVPs without RNA genome were produced by co-transfecting display vector together with only a lentiviral packaging construct but not the viral genome transfer vector (Figure 8B). Finally, trimeric EV- based MVPs, including exosome-MVPs or ectosome-MVPs, were produced by transfecting only the trimeric peptide display vector (Figure 8C).

[0288] Trimeric ACE2-MVPs were generated and characterized using trimeric fusion constructs derived from several possible trimerization domains, including the D4 post -fusion trimerization domain of VSV-G, the Dengue E post-fusion trimerization domain, or the T4 Phage Foldon domain. ACE2-MVPs displaying each of these trimeric ACE2 fusion constructs were termed ACE2-D4, ACE2-DE, and ACE2-Fold-MVPs, respectively. Purified ACE2-D4, ACE2-DE, and ACE2-Fold-MVPs were quantified via P24 ELISA. Quantitative western blot analysis of each type of trimeric MVP revealed that ACE2-D4, ACE2-DE, and ACE2-Fold-MVPs display 3600 ± 1300, 2000 ±600, and 1450 ±150 copies of ACE2 per particle, respectively (Figure 9A), demonstrating that inclusion of trimerization domains in fusion constructs significantly increases display copy numbers as compared to monomeric fusion constructs. Notably, monomeric ACE2- VM-MVPs display only 1200 ±400 copies of ACE2 per particle. To verify that trimerization constructs indeed displayed ACE2 in trimers, monomeric and trimeric ACE2 MVPs were analyzed in western blots under reducing and non -reducing conditions (Figure 9B). While ACE2-VM-MVPs demonstrate only a single, monomeric band under non-reducing conditions, whereas ACE2 / D4, ACE2 / DE, and ACE2 / Fold-MVPs show larger, higher-order oligomeric bands, including trimeric bands (Figure 9B). Of these three, ACE2 / D4 and ACE2 / DE-MVPs but not the ACE2 / Fold-MVPs demonstrate a larger proportion of oligomerized fusion peptides at much higher copies / particle.Finally, trimeric ACE2-MVPs were tested in pseudovirus neutralization assays against SARS CoV- 2 to confirm functional ACE2 display. It was found that ACE2-D4, ACE2-DE, and ACE2-Fold- MVPs neutralize SARS CoV-2 pseudovirus at IC50s of 0.3, 1.2, and 1.1 pM (Figure 9C), respectively, and are upto 10 -fold more potent than monomeric ACE2-VMMVPs. This increase in neutralizing potency can be attributed to both the increase in overall ACE2 valency on trimeric MVPs, as well as the boosting of local multivalence between viral spike trimers and trimeric ACE2 fusion peptides, allowing trimeric ACE2 MVPs to bind and neutralize virions more effectively. These results demonstrate that ACE2 MVPs displaying trimeric fusion peptides display thousands of copies of functional, trimerized ACE2 per particle. Trimeric fusion display constructs incorporating trimerization domains such as D4, DE, or Foldon can be used to display thousands ofWSGR Docket No. 48295-719.601 functional trimeric peptides on MVPs. ACE2-D4-MVPs have the highest copies of trimeric / oligomeric ACE2 displayed per particle and are most potent in virus neutralization, demonstrating that D4 oligomerization domain maybe most effective in displaying high copies of trimeric peptides on the VLP surface. These results also demonstrate that optimized valency and oligomerization format of ACE2 on MVPs are key to developing more potent neutralizing antivirals against SARS CoV-2.

[0289] Comparison of decoy-MVPs with distinct oligomerized ACE2 display

[0290] Interestingly, ACE2-MVPs with distinct oligomerized ACE2 display have varied copies ACE2 molecules / particle (Figure 10A-D). For example, trimeric ACE2-D4 MVP has the highest level of ACE2 protein displayed at- 3600 ±1300 copies / particle, whereas monomeric ACE2-VM MVP and dimeric ACE2-LZ MVP display about -1000 copies ACE2 / particle. Among the ACE2- MVPs with D4, or DE, or Fold trimeric domains, ACE2-D4 and ACE2-DEMVPs utilizing trimeric domains from viral surface proteins display over 2000 copies ACE2 / particle, whereas ACE2-Fold MVPs utilizing a trimeric domain from bacteria phage protein. These results clearly illustrated that ACE2 surface display levels on VLPs can be enhanced by incorporating trimerization domains from viral surface proteins into the display vector, such as D4 of VSVG and DE of Dengue E surface protein (Figure 10A). Moreover, D4 and DE promote more robust trimerization / oligomerization of ACE2 display on VLPs (Figures 9B, 10B), suggesting that these trimeric domains may enhance the surface presentation of displayed peptides. Notably, the trimeric configuration is one of the most common features many viral surface / spike proteins share. Finally, the neutralizing potencies of ACE2-MVPs are also impacted by the oligomerization format of displayed ACE2 (Figure 10C). Notably, ACE2-D4 MVP has the highest neutralizing potency, which correlates with its high ACE2 valency and trimeric / oligomeric configuration. Nevertheless, all ACE2-MVPs have IC50S in the low picomolar range, demonstrating that they are all potent neutralizing molecules against SARS CoV-2 in pseudovirus neutralization assays.

[0291] Generation of tetrameric MVPs

[0292] A tetrameric display construct was designed by fusing the stem domain of Influenza Neuraminidase to the desired peptide (Figure 11). By design, multivalent proteins can be displayed as tetramers on the surface of VLPs and EVs, such as exosomes and ectosomes, using a tetrameric display vector. To produce tetrameric, VLP -based MVPs with viral RNA genomes, we cotransfected the tetrameric, NA-based Type II peptide fusion construct with a lentiviral packaging construct expressing packaging components, such as Gag-Pol and Rev proteins and a viral genome transfer vector encoding a GFP / lucif erase reporter (Figure 12A). Alternatively, tetrameric, VLP- based MVPs without RNA genomes were produced by co-transfecting the tetrameric peptideWSGR Docket No. 48295-719.601 display vector together with a lentiviral packaging construct but no viral genome transfer vector (Figure 12B). Finally, tetrameric, EV -based MVPs, including exosome-MVPs and ectosome- MVPs, were produced by transfecting only the tetrameric peptide displaying vector in 293T cells (Figure 12C).

[0293] Tetrameric MVPs displaying Dipeptidyl Peptidase 4 (DPP4) — the entry receptor for MERS CoV — termed DPP4-NA MVPs were generated and characterized. Two variations of the tetrameric fusion peptide derived from the Influenza Neuraminidase stem domain were tested (Figure 13 A). The concentration of purified DPP4-NA MVPs was determined via P24 ELISA. Quantitative western blot analysis revealed that DPP4 / NA MVPs displaying version 1 and 2 of the DPP4 / NA display constructs displayed approximately 390 ±220 and 150 ±70 copies of DPP4 per particle, respectively (Figure 13B). In addition, western blot analysis of both versions of DPP4 / NA MVPs under reducing and non-reducing conditions demonstrated that both versions result in multimeric DPP4 display on MVPs (Figure 13B). Finally, to determine whether our fusion peptides effectively display functional DPP4 on MVPs, both versions of DPP4 / NA MVPs against MERS CoV were tested in pseudovirus neutralization assays. Version 1 and 2 of DPP4 / NA MVPs neutralized MERS CoV pseudovirus atIC50s of 0.91 and 0.87 pM, respectively, more than 5 -logs more potent than soluble DPP4 protein (Figure 13C). These results demonstrate that DPP4-NA MVPs display hundreds of copies of multimeric, functional DPP4. Moreover, Neuraminidase stembased fusion peptides are effective, Type II transmembrane proteins capable of displaying numerous tetrameric peptide copies on MVP surfaces. This vector may be uniquely suited to display type II peptide on VLPs and EVs.

[0294] Generation MVPs displaying mixed oligomeric proteins

[0295] With the abovementioned display vectors, MVPs displaying multiple peptides in mixed oligomeric formats were also generated by co-transfecting distinct peptide display constructs with differing oligomerization domains. Such a design can be used to increase the display density of a displayed peptide or to create combinatorial display patterns of distinct display peptides. Again, mixed oligomeric MVPs can be built with VLPs and EVs, such as exosomes and ectosomes, by cotransfecting monomeric and oligomeric display vectors. To produce mixed VLP -based MVPs with viral RNA genomes, the mixed oligomeric peptide fusion constructs were co-transfected with a lentiviral packaging construct expressing packaging components, such as Gag-Pol and Rev proteins, and a viral genome transfer vector encoding a GFP / lucif erase reporter (Figure 14A). Alternatively, mixed VLP-based MVPs without RNA genome were produced by co-transfecting the mixed oligomeric display vectors together with only a lentiviral packaging construct but not the viral genome transfer vector (Figure 14B). Finally, mixed EV-based MVPs, including mixedWSGR Docket No. 48295-719.601 exosome-MVPs and ectosome-MVPs, were produced by transfecting the mixed oligomeric fusion constructs alone (Figure 14C). Mixed MVPs can be generated by co -transfecting display vectors utilizing distinct oligomerization domains.

[0296] Here it is being explored whether the neutralizing potency of MVPs displaying spike - recognizing antibodies can be further enhanced with multi-specificity. For example, MVPs can be genetically programmed to display combinations of distinct scFv antibodies recognizing multiple binding sites on the SARS CoV-2 spike protein. This design, if successful, would further enhance the neutralizing potency of such MVPs against pandemic viruses and mitigate the effects of typical spike escape mutagenesis on neutralizing antibodies. To this end, (aRBD:C18 / VM)(aNTD:CV26 / D4) bi-specific MVPs (bi-MVPs) that co-display two scFv fusion peptides — monomeric aRBD:C18 / VM and trimeric aNTD:CV26 / D4 — recognizing the RBD and the NTD regions of CoV-2 spike protein were designed and generated. The expression level and oligomerization of monomeric aRBD:C18 / VM and trimeric aNTD:CV26 / D4 on MVPs were confirmed by quantitative Western -blot analyses (Figure 15A). These biAb-antiviruses against their respective target viruses were then tested in pseudovirus neutralization assays. It showed that mixed (aRBD:C018 / VM)(aNTD:CV26 / D4) biAb-antiviruses have an IC50 of 3.06 ±0.23 pM in pseudovirus neutralization assays, a more than 2 -fold increase in potency as compared to mono- specific Ab -antiviruses displaying either aNTD:CV26 / D4 or aRBD:C018 / VM alone (Figure 15B). Furthermore, these mixed biAb -MVPs suppress pseudovirus infection by approximately 10,000- fold as determined by the decrease in pseudovirus infection luciferase signal, a 100 -fold increase in suppression compared to each respective mono-specific Ab-antivirus (Figure 15C). These results illustrate the potential to genetically program MVPs to display distinct peptides to create bi-specific functionalities.

[0297] EV-based ACE2-MVPs are highly potent inhibitors against live CoV-2 viruses

[0298] By transfecting only the trimeric ACE2-D4 displaying vector to 293 T cells (Figure 16A), EVs displaying multiple copies of oligomerized ACE2 were generated and designated as ACE2-D4 EVs. The sizes of ACE2-D4-EVs are in the range of 131 ±29 nm as determined by tunable resistive pulse sensing analysis (TRPS) using qNano (Figure 16B). Each ACE2-D4 EV particle displays approximately 14000 ±6000 copies of ACE2-D4VG fusion molecules (Figure 16C). Further, it showed that ACE2-D4 EVs displaying trimeric H2A / ACE2, a mutant ACE2 with no enzymatic activity, are highly potent inhibitors, neutralizing CoV -2 pseudovirus at IC50S of 26 ± 12 fM (Figure 16D). Thus, oligomerized ACE2-D4-EVs are highly potent against live CoV-2 vims infection (Figure 16D). Notably, the level of displayed ACE2 on EVs is at 14000 ±6000 copies / EV, whereas the level of displayed ACE2 on VLPs is 3600 ± 1300 copies / VLP,WSGR Docket No. 48295-719.601 demonstrating that the display vector with D4 oligomerization domain can display significantly higher copies of ACE2 on EVs than on VLPs.

[0299] Coiled-coil human peptides as oligomerization domains for vesicle display

[0300] To further optimize surface display on vesicles, several common structural motifs found across oligomeric human proteins were investigated. Coiled-coil domains, which are a series of identical 2 to 7 u-helices that snake around each other like the strand of rope, may make ideal oligomerization domains for vesicle display. These structurally repetitive motifs are found across numerous human proteins and can induce a range of oligomerization states from dimers to heptamers. It was postulated that coiled-coil domains from a variety of human proteins could be used to create various oligomeric patterns of peptide display on MVPs. For example, the LINE-1 (LI) retrotransposon (Figure 17), which accounts for roughly seventeen percent of the human genome, contains two open reading frames (orf), the first of which encodes a highly unique, 500 amino acid protein (orflp) including a LINE-1 retrotransposon orflp coiled-coil trimerization domain (Llcc). The prevalence of the LI retrotransposon in the human genome makes the Llcc an ideal non-immunogenic domain for trimeric peptide display on vesicles. In addition to the Llcc trimerization domain, the coiled coil domains from human collagen XV, lung surfactant protein D, tetranectin, mannose bidning protein, Factor X, and Fibrinogen can also be used to create oligomerized display patterns on the surface of VLPs and EVs. Such designs enable multivalent display of fusion peptides on the surface of VLPs and EVs in varied oligomeric configurations and at copy numbers comparable to or greater than their viral display counterparts. Since these coiled - coil oligomerization peptides originated from highly conserved human proteins, multivalent therapeutics produced using these MVP display constructs are less likely to be immunogenic.

[0301] To this end, a display vector was designed to present fusion peptides on VLPs or EVs by using Llcc as an oligomerization domain (Figure 17). Specifically, the vector includes a mammalian promoter driving the expression of a fusion peptide, which consists of a linked signal peptide(SP), a displayed peptide, an Llcc trimeric peptide, and the transmembrane and cytoplasmic tail domains of the VSV-G protein or to the transmembrane and cytoplasmic tail domains of the LINE1 orfl protein. MVPs displaying various types of Llcc fusion peptides were produced as VLPs both with and without genomes and as extracellular vesicles (EVs) such as exosomes and ectosomes. To produce MVPs displaying Llcc fusion peptides on VLPs containing RNA genomes, HEK 293T cells were co-transfected with Llcc-trimeric peptide display constructs along with a lentiviral packaging construct expressing packaging components, such as Gag-Pol and Rev proteins, and a viral genome transfer vector encoding a GFP / lucif erase reporter (Figure 18A). To produce MVPs displaying Llcc fusion peptides on VLPs without genomes, HEK 293T cells wereWSGR Docket No. 48295-719.601 co-transfected with Llcc-trimeric peptide display vectors and a lentiviral packaging construct without a viral genome transfer vector (Figure 18B). Finally, to produce MVPs displaying Llcc fusion peptides on EVs, 293T cells were transfected with Llcc-trimeric peptide display constructs alone (Figure 18C). Concentrations of VLP or EV -based humanized ACE2-MVPs were determined via P24 or tunable resistive pulse sensing (TRPS, qNano), respectively. Based on these concentrations, quantitative western blot analysis determined the copy numbers of displayed humanized fusion peptides on MVPs. The oligomerization patterns of displayed fusion peptides were determined via non-reducing PAGE analysis. In particular, MVPs displaying Llcc- ACE2 were found to display 7200 ±3600 copies of Llcc-ACE2 per particle, more than twice as many copies as ACE2-D4-MVPs (Figure 19A). Moreover, a significant fraction of displayed Llcc- ACE2 on VLPs is in the oligomerized format as indicated by western -blot analyses under nonreducing conditions (Figure 19B). These findings demonstrate that Llcc-trimeric domains derived from human LINE1 orf 1 protein can be used to increase the density and oligomerization of the displayed peptides on vesicles. Finally, ACE2-Llcc-MVPs were able to neutralize three different SARS CoV-2 spike variants at low or sub-picomolar IC50s in pseudovirus neutralization assays (Figure 19C), further supporting that increased peptide display density and oligomerization on vesicles correlates increased function potency of displayed vesicles.

[0302] Summary of high-density oligomerized protein display on VLPs and EVs

[0303] The sizes of EVs and VLPs are in the range of 100 to 200 nm. In comparison, the sizes of DCs or other natural APCs are more than 20 micrometers, and thus are more than 100 times larger than EVs and VLPs in diameters. So, DCs and APCs provide much larger surface area and hence provide more interacting molecules for T cell enegagement and the formation of immunological synapses. To overcome the size limitations of EVs and VLPs, efficient display technology was developed to target the high density and oligomerized displayed molecules to the surface of EVs and VLPs. These designs were functionally validated by testing their efficacy in pseudovirus neutralization. It showed the increase of the local and global avidity of the displayed molecules with their interaction partners. These findings laid the foundation to build antigen- presenting vesicles based onEVs and VLPs through targeted oligomerized display of the signaling proteins involved in immunological synapse formation during the engagement of antigen- presenting cells and T cells.

[0304] The compositions of antigen-presenting vesicles for T cell activation

[0305] Design and production of single-chain trimer pMHC-I APVs (SCT-APVs)

[0306] APCs express pMHCs and many activating costimulatory molecules that participate in TCR engagement (Figure 20A). During antigen recognition by T cells, TCR and pMHCWSGR Docket No. 48295-719.601 engagement leads to the formation of immunological synapses, which include highly condensed aggregates of the TCR subunits and signaling proteins, adhesion molecules, costimulatory receptors, and inhibitor receptors on the T cell membrane. With the display toolboxes described in section 1, we contemplated recreating such condensed multivalent interactions using engineered vesicles displaying high copies of oligomerized molecular machinery required for TCR engagement. We postulated that high-density, oligomerized expression of these TCR-engaging proteins may engage equivalently densities of cognate receptors on T cells and mimic the formation of immunological synapses. Furthermore, APVs may be designed to hardwire pro -inflammatory T- cell stimulation without sensitivity to negative environmental cues. In contrast, the outcomes of natural APC and T cell engagement under physiological conditions depend on the state of APCs and the presence or absence of activating and inhibitory receptors on the APCs, leading to either pro-inflammatory or tolerogenic outcomes.

[0307] To create single-chain trimer pMHC-I APVs (SCT-APVs), we used our established vesicle-display strategies to target the TCR-engaging proteins, including pMHC and various costimulatory molecules, onto VLPs or EVs (Figure 20B). Since the class I pMHCs consist of three polypeptides: antigenic peptide, MHC-I alpha chain, and P2m, we adopted the well- established single-chain trimer (SCT) to link the antigenic peptide, P2m, and MHC-I into a single polypeptide for pMHC-I display. We then connect the SCT to a type I transmembrane protein membrane anchor and specified oligomerization domain. Similarly, the extracellular domains of type I or type II costimulatory molecules are linked to corresponding type I or type II transmembrane protein membrane anchors and oligomerization domains. As demonstrated in Section 1, such constructions can lead to the high -copy surface display of SCT and costimulatory molecules at high copies and in oligomerized format on the surface of VLP and EVs (Figure 20C).

[0308] SCT-APVs may be produced as EVs or VLPs. We first developed S293 -HLA null cells by inactivating all three HLA alleles, HLA-A, HLA-B, and HLA-C, from a suspension S293 cell line. This cell line will enable the production of SCT-APVs without any endogenous human class I MHC molecules. Moreover, S293 -HLA null cells also enable scale-up production of SCT-APVs in industrial-scale bioreactors. To produce EV -based SCT-APVs, we transfected DNA constructs encoding SCT-pMHC-I / and costimulatory molecules (X, Y, Z) into S293-HLA null cells without a lentiviral transfer and packaging vector (Figure 21). Alternatively, to produce VLP -based SCT- APVs, we transfected DNA constructs encoding SCT-pMHC-E and costimulatory molecules (X, Y, Z) into S293-HLA null cells together with a lentiviral transfer and packaging vector (Figure 21). EV- or VLP-based SCT-APVs are harvested from the supernatant, purified through a multi-stepWSGR Docket No. 48295-719.601 process to remove all protein and nucleic acid contaminants, and reconstituted in a stabilizing buffer for in vitro and in vivo usage.

[0309] Define the optimal costimulatory signals required for SCT-APVs

[0310] Some early studies have attempted to build T cell activation particles by displaying SCT and CD80 on the surface of lentiviral VLPs. However, these T cell activation VLP particles had nominal functions in T cell activation in culture compared to anti-CD3 and anti-CD28 activation beads and were not suitable for in vivo T cell activation. Furthermore, the copies of SCT and CD80 displayed on these VLPs are unknown. There have been no efforts made to increase the density and / or oligomerization format of the displayed SCT and CD80 to maximize the effects of avidity during their engagement with TCR and target CD28 costimulatory molecules. Finally, no published studies have optimized the combinatorial code of costimulatory molecules required to reconstitute the T cell activation machinery on VLPs.

[0311] To this end, we first carried out a costim subtraction study to evaluate the contribution of a panel of costimulatory molecules on the T cell activation function of SCT-APVs (Figure 22). Specifically, we generated SCT-APVs displaying ova-SCT and a combination of costimulatory molecules, including CD80, CD86, GITRL, OX40L, CD48, CD30L, and ICAM-1, designated as all-costim APVs (Figure 22A). We then subtracted one costimulatory molecule from the all - costim APVs to create a list of costim subtracted APVs, including AGITRL, ACD80, ACD86, AOX40L, and ACD48-APVs. We also created SCT-APVs displaying only ova-SCT and GITRL only. All SCT-APVs are EV-based and display multivalent ICAM-1, and all Type I displayed molecules incorporate the D4 oligomerization domain. All Type II displayed molecules utilize the neuraminidase stem oligomerization domain and transmembrane anchor. We then examined the function of these SCT-APVs in activating naive OT-1 T cells (Figure 22B, 22C). Naive OT-1 T cells were isolated from mice and stimulated all-costim ova- APVs or various all-costim ova-APVs with one costimulatory molecule subtracted. We found that all-costim ova-APVs can effectively induce the expression of CD69, an early T cell activation marker, in naive OT-1 T cells, as indicated by FACS analysis at day 1 post-T cell activation. About 48% of the T cells stimulated by the all-costim ova-APVs are CD69+, with ~3% being CD69 and CD25 double-positive (Figure 22B, 22C). Interestingly, all costimulatory molecules contribute to T cell activation, as subtraction of each of the costimulatory molecules results in a significant decrease of the percent of total CD69+ cells at day 1 post-T cell activation by ova-APVs. The contribution by each of the costimulatory molecules is quantitatively distinct. Among the costimulatory molecules tested, GITRL has a dominant contribution to the function of all-costim ova-APVs in naive mouse OT-1 T cell activation. Subtracting GITRL from all-custom ova-APVs causes a reduction of the percent ofWSGR Docket No. 48295-719.601 total CD69 positive cells from -48% to ~ 9.7%, a nearly 80% decrease. Also, subtracting CD48 results in a reduction from -48% to x~28%. In contrast, subtracting other costimulatory molecules results in a less dramatic reduction in the percentage of total CD69 positive cells from -48% to -38%, -32%, -36%, and -33% for CD80, CD86, OX40L, and CD30L subtraction, respectively.

[0312] To further corroborate the above findings, we carried out a costimulatory addition study to evaluate the contribution of various function of GITRL in T cell activation by SCT-APVs, we created base ova-APVs displaying only ova-SCT, ICAM-1, and GITRL. We then created “+costim ova-APVs” by adding individual costimulatory molecules, including CD80, CD86, OX40L, and CD48, one at a time (Figure 23A). We then tested the function of +costim ova-APVs in activating splenic OT-1 T cells, including naive and antigen-experience OT-1 T cells, and control Pmel T cells, which do not recognize the ova peptide antigen (Figure 23B,23C). We found that base ova- APVs modestly increased the percent of CD69 and CD25 double -positive T cells among the activated OT-1 cells compared to that of stimulated Pmel T cells. Interestingly, the addition of CD80, CD86, XO40L, and CD48 increased the percent of CD69 and CD25 double-positive T cells among the activated OT-1 cells to 56%, 55%, 49%, and 44%, respectively. By contrast, Pmel T cells stimulated with these +costim ova-APVs have about -20% CD69 and CD25 double-positive T cells and thus do not respond to the addition of costimulatory molecules.

[0313] The above studies demonstrate that producing highly potent EV -based APVs for antigen-specific T-cell activation is feasible by displaying SCT-MHC-I as pMHC and a codisplaying a combination of T cell costimulatory molecules. Interestingly, we found that the GITRL costimulatory signal has a dominant role for SCT-APVs in activating antigen-specific mouse T cells. Nevertheless, we also found that all costimulatory molecules tested, including CD80, CD86, CD48, OX40L, CD30L, and GITRL, contributed to naive antigen -specific T-cell activation by ova-APVs. Finally, ova-APVs selectively activate OT-1 T cells but not Pmel T cells, suggesting the potential of antigen -specific T cell activation by ova-APVs. We have only examined the early T cell activation markers in the above analysis. The effects of SCT-APVs and individual costimulatory molecules onT cell proliferation and differentiation in culture and in vivo are not addressed here but may be tested in the future. Also, all-costimulatory ova-APVs are produced through transient transfection. Although the transfection efficiency in S293HLA null cells is generally high (>60%), all-costimulatory ova-APVs produced by this method are likely to be heterogeneous. Nevertheless, we still observed consistent and robust contribution of costimulatory molecules to SCT-APV functions in T cell activation, suggesting additional room to further improve SCT-APV function by increasing the displayed molecules' homogeneity.

[0314] Defining the optimal oligomerization format for SCT-MHC-I display on SCT-APVsWSGR Docket No. 48295-719.601

[0315] By evolutionary design, pMHC and TCRs form weak interactions with affinity in the low micromolar range. Interestingly, the formation of immunological synapses, which engage hundreds, if not thousands, copies of pMHC and TCR and costimulatory molecules, may overcome the weak interaction between pMHC and TCR complexes by taking advantage of the avidity effects of multivalent interaction. Because EVs and VLPs are significantly smaller than APCs, we aimed to optimize the density and oligomerization of displayed SCT-MHC-I to maximize their local and global avidity on vesicles, overcoming their size limitations and forming effective engagements with antigen -specific T cells. To this end, we designed SCT-MHC-I display vectors with varied oligomerization domains (Figure 24A). We then generated EV -based APVs displaying CD86 / D4, GITRL / NA, ICAM1 / D4, and ova-SCT-MHC-I without an oligomerization domain (VGTM, monomer) or incorporating a distinct oligomerization domain (Figure 24B). The oligomerization domains tested here include D4 (post -fusion trimeric domain from VSVG), cc-hex (coiled-coil hexamer domain), L24D (coiled-coil heterohexamer domain), L24H (coiled-coil heterohexamer domain), cc-Hept (coiled-coil heptamer domain), and wazOct (coiled-coil octamer domain). We sought to identify the optimal oligomerization format for SCT-MHC-I display while examining how varying oligomerized display of SCT-MHC-I on APVs might impact the strength and specificity of antigen-specific T cell activation, as well as the consequences for T cell proliferation.

[0316] To this end, we mixed naive OT-1 T cells (ova-specific, Ly5.2 positive) with nonspecific naive T cells from Ly5.1 mice (Ly5.1 or CD45.1 positive) and then stimulated mixed T cells with ova-APVs displaying ova-SCT with distinct oligomerization domains (Figure 25 A). The effects of ova-APVs on T cell activation were determined by FACS analysis of early T cell activation marker CD69 and CD25 expression, and measurement of T cell proliferation. (Figure 25B, 25C, 25D). Such analyses allowed us to compare the observed effects of ova-APVs on the antigen-specific OT-1 (Ly5.2+) and non-specific T cells (Ly5.1), and helped to discern how oligomerization domains used for ova-SCT-MHC-I display affect antigen -specific T cell recognition and activation. We found that SCT-APVs displaying ova-SCT with various oligomerization domains can effectively stimulate expression of early T cell activation markers CD69 and CD25 in OT-1 T cells but with distinct potency and specificity (Figure 25B, 25C).

[0317] Notably, T cells stimulated with ova-APV / D4 (trimeric domain) induced CD69 expression in ~94% OT-1 T cells and both CD69 andCD25 expression in ~74% of OT-1 cells, 48 hours post-stimulation. By contrast, T cells stimulated with ova-APV with various coiled-coil oligomerization domains (Figure 25B, 25C) yielded significantly lower proportions of CD69+CD25+ OT-1 cells, ranging from ~7% to ~20%, and T cells stimulated with ova- APV / VGTM (no SCT display oligomerization domain) resulted in approximately 44% ofWSGR Docket No. 48295-719.601CD69+CD25+ OT-1 cells. These results demonstrate that ova-APV / D4 are not only the most potent ova-APVs, but also more potent than anti-CD3 and CD28 Dyna beads at activating OT-1 T cells. Dynabead stimulation resulted in about 63% of CD69+ OT-1 T cells and 52% of CD69+CD25+ OT- 1 cells. Moreover, ova-APV / D4 is significantly more specific than ova-APVs with other oligomerization domains, as indicated by the significantly lower percent of total CD69+Ly5.1 cells and of CD69+CD25+Ly5.1 cells after stimulation with ova-APV / D4 (Figure 25B, 25C). Stimulation with Dynabeads resulted in significant expression of CD69 and CD25 in both OT-1 and non-specific Ly5.1 T cells. Notably, OT-1 T cells activated by ova-APV / D4 proliferated over 20-fold, comparable to those stimulated with anti-CD3 and CD28 Dyna beads. In contrast, the nonspecific Ly5.1 T cells activated by ova -APV / D4 did not proliferate (Figure 25D). Finally, OT-1 T cells activated by ova-APVs with other oligomerization domains also proliferated more than 10- fold, whereas the non-specific Ly5.1 T cells did not proliferate. These results demonstrate that ova- APV / D4 is highly potent and specific at activating antigen-specific T cells based on the FACS analyses of activation markers and proliferation. Interestingly, ova-APVs with other oligomerization domains had varied effects on antigen -specific T cell proliferation, despite some having weaker effects on inducing T cell activation marker expression.

[0318] Functional and physical properties of optimized base-SCT-APVs

[0319] As previously described, we systematically screened for costimulatory molecules and optimal oligomerization domains required for building effective antigen -specific SCT-APVs. Based on these results, we determined the composition of optimal base-SCT-APVs consisting of displayed type I molecules, SCT / D4, CD86 / D4, ICAM1 / D4, and a type II molecule, GITRL / NA (Figure 26A, 26B). We designated this base-SCT-APV as APV(86IG). D4 trimeric domains and VGTM transmembrane anchors are used for the display of type I molecules, whereas NA tetrameric domain and its type II transmembrane anchor were used for the display of type II receptors. The composition of the APV(86IG) should simplify the production but also provide flexibility to add additional costimulatory molecules for new and improved functionality when required. Since CD80 and CD86 bind to CD28, we use displayed CD80 and CD86 interchangeably in producing SCT-APVs. To this end, we produced the optimized APV(86IG) on EVs or VLPs. To produce EV -based APV(86IG), we transfected DNA constructs encoding SCT / D4 and costimulatory molecules (CD80 / D4, ICAM1 / D4, GITRL / NA ) into S293-HLA null cells without lentiviral transfer and packaging vector (Figure 26C). Alternatively, to produce VLP -based APV(86IG), we transfected DNA constructs encoding SCT / D4 and costimulatory molecules (CD80 / D4, ICAM1 / D4, GITRL / NA ) into S293 -HLA null cells together with lentiviral transfer and packaging vector (Figure 26C). EV- or VLP-based APV(86IG) are harvested from theWSGR Docket No. 48295-719.601 supernatant, purified through a multi-step process to remove all protein and nucleic acid contaminants, and reconstituted in a stabilizing buffer for in vitro and in vivo usage.

[0320] We determined the sizes of EV-APV(86IG) and VLP-APV(86IG) by tunable resistive pulse sensing analysis (TRPS) using qNano (Figure 27A, 27B). Their sizes are in the range of 189 or 193 nm, respectively. We then characterized the density of the displayed ova-SCT-MHCI molecules on EV-APV(86IG) and VLP-APV(86IG) by quantitative western-blot analyses (Figure 27C). We found that each EV-ova-APV(86IG) or VLP-ova-APV(86IG) particle displays approximately 6200 ± 1400 copies or 6500 ± 1700 copies of ova-SCT / D4VG fusion molecules (Figure 27C). We then characterized the density of the displayed costimulatory molecules on EV- APV(86IG) and VLP-APV(86IG) by quantitative Western-blot analyses (Figure 27D, 27E). We found that displayed costimulatory molecules, including CD80 / D4, ICAM1 / D4, and GITRL / NA are highly expressed on EVs or VLPs. Each EV-ova-APV(86IG) particle displays approximately 1400 ± 1300 copies of CD86 / D4, 2700 ± 1000 copies of ICAM1 / D4, and 1100 ± 500 copies of GITRL / NA. Furthermore, Each VLP-ova-APV(86IG) particle displays approximately 1100 ± 600 copies of CD86 / D4, 3100 ± 1700 copies of ICAM1 / D4, and 500 ± 40 copies of GITRL / NA. Overall, except that VLP-APV(86IG) slightly lower level of GITRL / NA, EV-APV(86IG) and VLP-APV(86IG) display comparable levels of ova-SCT / D4, CD80 / D4, ICAM1 / D4, and GITRL / NA.

[0321] We then examined the potency and specificity of EV- and VLP-based ova-APV(86IG) in activating antigen-specific OT-1 T cells (Figure 28). First, we compared the activation of OT-1 T cells by anti-CD3 / CD28 Dyna bead, and EV- and VLP-based ova-APV(86IG) and found that they induced comparable levels of T cell activation at day 1 post T cell activation, as indicated by the levels of total CD69+ cells and CD69+CD25+ cells determined by FACS analyses (Figure 28A, 28B). Furthermore, we stimulated mixed T cells, consisting of naive OT-1 T cells (ova- specific, Ly5.2 positive) and non-specific naive T cells from Ly5.1 mice (Ly5.1 positive), with anti-CD3 / CD28 Dynabead and EV -based ova-APV(86IG). We then examined their effects on T cell activation by FACS analyses of T cell activation markers CD69 and CD25 among OT-1 (Ly5.2) and non-specific Ly5.1 T cells (Figure 28C, 28D, 28E). Such analyses helped to discern how oligomerization domains used for ova-SCT-MHC-I display affect the antigen -specific T cell recognition and activation. We found that over 55% of OT-1 T cells are CD69+CD25+ cells, whereas only ~3% of Anti-CD3 / CD28 Dynabeads. Thus, EV-based ova-APV(86IG) selectively activated OT-1 T cells but not non-specific Ly 5.1 T cells. In contrast, anti-CD3 / CD28 Dynabeads are equally potent at activating OT-1 (Ly5.2) and non-specific Ly5.1 T cells (Figure 32C). Finally,WSGR Docket No. 48295-719.601EV-based ova-APV(86IG) differentially activates OT-1 T cells but not non-specific Ly5.1 T cells at various time points after stimulation (Figure 28D).

[0322] Summary of the effective compositions of SCT-APVs for T cell activation

[0323] As aforementioned, we systematically explored the effective molecular compositions of SCT-APVs. All SCT-APVs displayed SCT-pMHC-I with an antigenic peptide, ICAM-1, and a varied combination of activating co-stimulatory molecules. For type I membrane proteins, the displayed proteins were fused to the post-fusion D4 trimeric domain from VSVG and then to the VSVG transmembrane and intracellular domain. For type II membrane proteins, the neuraminidase stem and tetrameric domain were fused to the extracellular domain of displayed type II proteins.

[0324] pMHC engagement with cognate TCR is the triggering event of the T cell activation process, representing the signal 1 of T cell activation. It dictates the specificity of antigen recognition by TCRs. Notably, the optimal affinity of pMHC and TCR complexes falls in the range of sub -micromolar, which permits pMHC to rapidly scan through billions of TCRs to find the antigen-specific T cells. However, proper pMHC and TCR engagement leads to the formation of the immunological synapses, which results in clustered and multivalent interaction of pMHC and TCR and is useful for T cell activation. Therefore, EV- or VLP-based APVs can provide multivalent pMHC and TCR engagement that functionally mimics immunological synapses. To this end, we systematically explored the oligomerized display of SCT-MHC-1 on APVs with various oligomerization domains. We found that the D4 trimeric domain is most suitable for SCT -MHC-I display on SCT-APVs among the oligomerization domains tested. Notably, SCT-p MHC-I with the D4 trimeric domain were displayed at over 6000 copies / particles on EVs and VLPs.

[0325] Further, we systematically explore the display of costimulatory molecules on SCT- APVs. We were able to display most of the type I and type II costimulatory molecules at over 1000 copies / particles on EVs and VLPs. We found that displaying GITRL on SCT-APVs display can enable robust and specific activation of antigen-specific T cells by SCT-APVs as indicated by FACS analyses of early T cell activation markers or by the stimulation of T cell proliferation. Notably, we also found that all costimulatory molecules tested contributed to some degree to the ability of SCT-APVs to stimulate T cells. However, we want to emphasize that the in vitro T cell activation assays usedin characterizing the function of SCT-APVs are limited and may not reveal the function of the costimulatory molecules in many aspects of in vivo T cell biology, including lineage differentiation, migration, and memory formation. Collectively, we have demonstrated that EV- and VLP-based APVs displaying high-density and oligomerized SCT-MHC-1 and costimulatory molecules, including SCT-MHC-I, CD80 / CD86, and GITRL, provide robust and specific signals for antigen -specific T-cell activation in culture.WSGR Docket No. 48295-719.601

[0326] Reprogram tumor cells into antigen-presenting tumor cells (APTCs)

[0327] Reprogram tumor cells into APTCs by displaying multivalent, oligomeric co -stimulatory molecules on tumor cells

[0328] We demonstrated the design and use of single-chain trimer pMHC-I APVs (SCT-APVs) co-displaying single chain peptide-MHC trimers (SCT) and multivalent, oligomeric T cell costimulatory molecules, including CD80 and GITRL. APVs display hundreds or thousands of SCT and oligomeric custom peptides on the surfaces of extracellular vesicles (EVs) or viral -like particles (VLPs) and successfully activate antigen -specific T cells by mimicking the multivalent, velcro-like interactions atthe immunological synapse between T cells and antigen-presenting cells. With the methods and insights from building APVs on EVs and VLPs, we displayed the same set of co-stimulatory molecules used on APVs, including CD80 / CD86 and GITRL, on tumor cells to turn them into antigen-presenting tumor cells (APTCs) by using the same display technology (Figure 29A).

[0329] Our analysis of MHC I vs costimulatory ligand molecule expression in mouse and human tumor cell lines reveals that a majority of tumor cells likely still express MHC I but do not express costimulatory ligand molecules (Figure 2). While other barriers to proper antigen presentation may exist in tumors, including mutations in proper antigen processing, we posited that for tumors with functional class I MHC and antigen presentation, co -expression of the multivalent co-stimulatory ligands used in APVs would enable the tumor cells themselves to serve as antigen- presenting cells (APCs). Tumor cells themselves would then be able to directly activate a pool of polyclonal, anti-tumor T cells targeting various TAAs and neoantigens, bypassing the need for neoantigen discovery.

[0330] To this end, we designed antigen-presenting tumor cells (APTCs) by displaying multivalent, oligomerized costimulatory ligand molecules CD80 and GITRL alongside endogenous MHC I (Figure 29A). To induce stable expression of our fusion display peptides in tumor cells, we produced lentivirus packaging transfer vectors encoding trimeric murine CD80 / D4 and oligomeric murine GITRL / NA fusion display constructs. We then infected a panel of wild -type murine tumor cell lines and sorted a pool of CD8011|811 / GITRL11|811APTCs from the infected cell lines. We further characterized the expression levels of CD80 / D4 and GITRL / NA on each APTC cell line. In brief, APTCs were trypsinized, rested, stained with anti -murine GITRL and CD80 antibodies, and analyzed via fluorescence-activated cell sorting (FACS). Lentivirus infection efficiency varied between tumor cell lines, as did the expression level of multivalent CD80 and GITRL for each sorted APTC cell line (Figure 29B). We further quantified the exact number of CD80 and GITRL peptides per cell for each APTC cell line using PE-conjugated antibodies and QuantibriteWSGR Docket No. 48295-719.601 quantification beads. PancO2-APTCs were most efficiently infected, displaying 1.6 x 105± 8 x 104copies of CD80 / D4 and 1.6 x 105± 8 x 104copies of GITRL / NA per cell, while MC38-APTCs expressed as few as 5.8 x 103± 2.1 x 103copies of CD80 / D4 and 5.1 x 103± 1.7 x 103copies of GITRL / NA per cell (Figure 29C).

[0331] APTCs induce robust activation of antigen-specific T cells in vitro

[0332] We used chicken ovalbumin and OT-1 transgenic mouse T cells recognizing the ova peptide antigen as a model antigen recognition system to determine whether APTCs could selectively activate antigen -specific T cells. We began by generating APTC cell lines expressing chicken ovalbumin in addition to multivalent costimulatory s CD80 / D4 and GITRL / NA and coculturing ova-APTC cell lines in the presence of both antigen -specific OT-1 T cells and nonspecific Ly5.1 T cells. We then measured the specificity of APTC -induced T cell activation as a function of early T cell activation marker CD69 and CD25 expression in OT-1 vs Ly5.1 T cells. Ova MC38-APTCs strongly activated OT-1 T cells while stimulating relatively low non-specific Ly5.1 T cell activation. OT-1 T cells co-cultured with ova MC38-APTCs were more than 70% CD69+ / CD25+ 24 hours after co-culture, while Ly5. 1 T cells in the same culture remained less than 2.2% CD69+ / CD25+ (Figure 30A, 30B). Similar results were observed when OT-1 and Ly5.1 T cells were co-cultured with ova PancO2-APTCs (Figure 31A, 31B). By contrast, anti-CD3 / CD28 Mouse T Activator Dy nab eads activated both OT-1 and Ly5.1 T cells equally (Figures 30, 31). Notably, ova-APTCs were able to activate antigen-specific OT-1 T cells to a similar level as Dynabeads after 24-48 hours of co-culture. These results demonstrate that combinatorial expression of multivalent CD80 and GITRL on tumor cells enables potent, neoantigen-specific CD8+ T cell activation.

[0333] Effective rejection ofMC38-APTCs in mice

[0334] Based on the robust in vitro T-cell activation we observed, we tested whether APTCs would induce immune recognition and rejection in vivo. 7.5 x 105MC38-APTCs were implanted subcutaneously on the right flanks of female C57BL / 6 mice and monitored for tumor growth (Figure 32A). While wild-type MC38 tumors grew rapidly, MC38-APTC tumors were completely rejected in all mice challenged with APTCs (Figure 32B, 32C). Rejected tumors did not reappear after extended periods of observation, indicating complete immune rejection of implanted MC38- APTCs (Figure 32D). To determine whether the rejection of MC38-APTCs was indeed T cell- mediated, we implanted MC38-APTCs subcutaneously in the right flanks of athymic nude mice and compared their tumor growth to wild-type MC38 tumors (Figure 33 A). In athymic nude mice, wild-type MC38 and MC38-APTC tumors were equally tumorigenic (Figure 33B), demonstrating that rejection of MC38-APTCs is T cell-dependent.WSGR Docket No. 48295-719.601

[0335] MC38-APTCs induce the rejection of co-injected wild-type MC38 tumor cells

[0336] To determine whether MC38-APTCs were inducing broad anti -tumor T cell immunity against MC38 antigens, we challenged mice with a mixture of MC38-APTCs and an increasing number of wild-type MC38 cells implanted subcutaneously on the right flank (Figure 34A). We found that tumor mixtures were fully rejected in 4 / 5 mice at ratios of 10:1, 5:1, and 2:1 APTCs to wild-type tumor cells (Figure 34B, 34C). At higher ratios of 1 : 1 and 1 :2 APTCs to wild-type tumor cells, full or partial rejection is still induced in a subset of challenged mice (Figure 34C). This result demonstrates that MC38-APTCs activate anti -tumor T cells in vivo that recognize and kill wild-type MC38 tumor cells. Additionally, it demonstrates that a large enough population of wild- type tumor cells can subvert APTC-induced T-cell immunity via immune escape or suppression.

[0337] Rejection ofMC38-APTCs immunized mice from tumor rechallenge

[0338] Next, we investigated whether mice challenged with MC38-APTCs developed lasting anti-tumor immune memory against wild-type tumor cells. To this end, we challenged naive mice alongside mice that fully rejected MC38-APTCs with 7.5 x 105wild-type MC38 tumor cells implanted subcutaneously and monitored their tumor growth (Figure 35A). We found that MC38- APTC-vaccinated mice completely rejected wild-type MC38 tumor cells upon re-challenge, while naive mice were unable to reject implanted tumors and rapidly succumbed to tumor growth (Figure 35B, 35C, 35D). To determine the relative strength of anti -tumor immune recall imbued by MC38- APTC vaccination, we re-challenged MC38-APTC vaxxedmice with increasing doses of wild-type MC38 tumor cells (Figure 36 A). We found that 4 / 5 mice challenged with 1.5 x 106to 6 x 106wildtype tumor cells were able to completely reject or control re-challenged tumor growth compared to naive mice (Figure 36B, 36C). In other words, increasing the number of implanted tumor cells was not sufficient to overwhelm the anti -tumor T cell memory response induced by MC38-APTC vaccination.

[0339] Irradiated MC 38 -APTCs immunized mice from tumor rechallenge

[0340] We then assessed the vaccination potential of irradiated MC38-APTCs in vivo. Mice that received either one or two doses of 7.5 x 106gamma irradiated MC38-APTCs were rechallenged with 7.5 x 106wild-type MC38 tumor cells and monitored for tumor growth (Figure 37A). We found that after one dose, only 3 / 5 IR MC38-APTC vaccinated mice were able to reject wild-type tumor cells, while all mice that received two doses were able to fully reject wild -type tumor cells (Figure 37B, 37C). This result demonstrates that irradiated APTCs can also induce robust immune memory against the challenge of wild-type MC38 tumor cells, albeit slightly weaker than live MC38-APTCs. Taken together, these results demonstrate that MC38-APTCs areWSGR Docket No. 48295-719.601 capable of inducing strong, tumor-specific T cell-mediated primary immunity and immune memory.

[0341] Autologous APTCs induced rejection and robust protective immunity

[0342] We further developed APTC cell lines from a panel of murine cancer cells of various tissue origins and tested whether these APTCs were rejected in mice and induced anti -tumor immune memory. For Renca metastatic renal cell adenocarcinoma, 1 .0 x 106 Renca -APTCs were implanted subcutaneously in the right flanks of BALB / c mice and monitored for tumor growth (Figure 38 A). We found that Renca-APTCs were fully rejected in host mice, unlike wild-type Renca tumors (Figure 38B, 38C, 38D). Furthermore, Renca-APTC vaccinated mice that were rechallenged subcutaneously with 1.0 x 106 wild-type Renca cells (Figure 39A) fully rejected wildtype tumors, demonstrating induction of strong tumor-specific immune memory post-vaccination (Figure 39B, 39C, 39D).

[0343] For Panc02 pancreatic ductal adenocarcinoma, 1.0 x 106 PancO2-APTCs were implanted subcutaneously in the right flanks of C57BL / 6 mice and monitored for tumor growth (Figure 40A). We found that PancO2-APTCs were fully rejected in host mice, unlike wild-type Panc02 tumors (Figure 40B, 40C, 40D). Furthermore, PancO2-APTC vaccinated mice that were rechallenged subcutaneously with 1 .0 x 106 wild-type Panc02 cells (Figure 41 A) fully rejected wildtype tumors, demonstrating induction of strong tumor-specific immune memory post-vaccination (Figure 41B, 41C, 41D). Panc02 cells express relatively low numbers of MHC I compared to other fully rejected APTC cell lines (Figure 2 A). Complete rejection and robust immune memory induced by PancO2-APTC vaccination suggest that boosting anti -tumor T cell co-stimulation via high expression of displayed CD80 / D4 and GITRL / NA may compensate for lower MHC I expression.

[0344] For GL261 glioblastoma, 5.0 x 105 GL261-APTCs were implanted subcutaneously in the right flanks of C57BL / 6 mice and monitored for tumor growth (Figure 42A). We found that GL261 -APTCs were fully rejected in host mice, unlike wild-type GL261 tumors (Figure 42B, 42C, 42D) Furthermore, GL261-APTC vaccinated mice that were re-challenged subcutaneously with 5.0 x 105 wild-type GL261 cells (Figure 43A) fully rejected wild-type tumors, demonstrating induction of strong tumor-specific immune memory post-vaccination (Figure 43B, 43C, 43D).

[0345] Collectively, these results demonstrate that Renca metastatic renal cell adenocarcinoma, Panc02 pancreatic ductal adenocarcinoma, and GL261 glioblastoma cells can effectively be programmed into APTCs. Moreover, vaccination with these APTCs effectively induces robust protective immunity against rechallenge by wild-type tumor cells. These resultsWSGR Docket No. 48295-719.601 demonstrate that autologous APTCs represent a broader strategy for developing autologous cancer vaccines for various cancer types.

[0346] Summary of anti-tumor immunity induced by autologous APTCs

[0347] In summary, we have tried to reprogram ten tumor cell lines of different tissue origins into APTCs by displaying high copies, oligomerized CD80, and GITRL. We found that APTC lines derived from MC38-, Renca-, PancO2-, and GL-261-APTCs were fully rejected in mice and induced complete immune protection upon rechallenge with the parental tumor cell lines. These results demonstrate that reprogramming tumor cells into APTCs by displaying high copies and oligomerized costimulatory ligand molecules on tumor cells can elicit anti-tumor immunity in a broad spectrum of tumors in mice, albeit inducing varied degrees of anti -tumor immunity and memory.

[0348] Notably, we also found some APTC cell lines only conferred partial immunity in the rejection of certain APTC lines, the corresponding parental tumor cell lines, or both (Data not shown), suggesting that tumors may utilize diverse mechanisms to subvert the anti-tumor immunity induced by APTC-induced activation of tumor-specific T cells. Thus, it is possible that some APTCs may need additional signals beyond the costimulatory signals provided by the displayed CD80 and GITRL to further enhance the anti -tumor T cell immunity. Further studies are required to delineate what signals are required for APTCs to become broad -spectrum cancer vaccines for the prevention of cancer relapse and treatment of established tumors for all cancer.

[0349] Autologous APTCs as therapeutic T-cell vaccines for established tumors

[0350] The above results provide strong support that APTCs can effectively activate tumorspecific T cells in culture and in vivo. Activation of anti-tumor T cells often results in robust antitumor T cell immune response to prevent cancer development and the induction of robust memory response against rechallenges. These findings may lead to developing therapeutic applications through reprogramming tumor cells into APTCs.

[0351] Autologous APTCs as therapeutic vaccines to treat established cancer

[0352] Here we sought to determine whether the anti-tumor T cells induced by APTC vaccination would be able to exert tumor control over already established, wild -type tumors. Such activity would enable the use of APTCs as autologous therapeutic vaccines for established tumors. To this end, we designed studies treating established MC38 tumors in mice with MC38 -APTCs, in conjunction with innate immune adjuvants. Innate immune adjuvants are pathogen -associated molecular patterns (PAMPs) that activate various conserved pattern recognition receptors (PRRs) in dendritic cells, monocytes, and macrophages. It is well understood that induction of antigen - specific T cell immunity requires danger signals elicited from PAMPs. Thus, antigen -specific TWSGR Docket No. 48295-719.601 cells may be tolerated if they have seen the presented tumor antigens in the absence of danger signals. We tested the anti -tumor activity of MC38-APTCs in conjunction with bacterial lipopolysaccharides (LPS) and two forms of Polyinosinic-poly cytidylic acid (Poly(I:C)), synthetic double-stranded RNA analogs.

[0353] To test the effects of LPS and APTC treatment on established tumors, C57BL / 6 mice were implanted subcutaneously with 7.5 x 105 wild-type MC38 tumor cells. 7 days postimplantation, mice were randomized and treated with either 10 pg of LPS injected intratum orally, 7.5 x 105 MC38-APTCs injected peritumorally, or a combination of both LPS and APTCs (Figure 44 A). Subsequent LPS doses were administered on a weekly basis, while MC38-APTCs were delivered biweekly. We found that mice treated with MC38-APTCs alone demonstrated minimal tumor control, while mice treated with intratumoral LPS alone demonstrated partial rejection and initial tumor control, but eventual tumor escape in several mice (Figure 44B, 44C). Notably, treatment of established MC38 tumors with a combination of LPS and MC38-APTCs resulted in complete tumor rejection or long-term tumor control in all mice (Figure 44B, 44C).

[0354] We designed an analogous study to test the effects of APTC therapeutic vaccination in conjunction with NexaVantpoly(I:C), a synthetic double -stranded RNA sequence derived from the Chinese sac brood virus genome. Mice with established wild-type MC38 tumors were treated with either 10 g of NexaVant poly(LC) injected intratumo rally, MC38-APTCs as previously described, or a combination of the two (Figure 45A). Treatment with MC38-APTCs or NexaVant poly(I:C) alone had a negligible effect on established MC38 tumor growth (Figure 45B, 45C). Interestingly, combined NexaVant and APTC treatment resulted in significant synergistic tumor control of established tumors and prolonged survival in treated mice (Figure 45B, 45C, 45D).

[0355] We designed an analogous study to test the effects of APTC therapeutic vaccination in conjunction with high molecular weight (HMW) poly(LC), comprised of 1.5-1 ,8kb strands of inosine poly(I) homopolymers annealed to cytidine poly(C) homopolymers. Mice with established wild-type MC38 tumors were treated with either lOpg o...

Claims

1. WSGR Docket No. 48295-719.601CLAIMSWHAT IS CLAIMED IS:

1. An antigen-presenting tumor cell (APTC) comprising, a first fusion protein that comprises the displayed costimulatory ligand molecule CD80 or CD86, a second fusion protein that comprises the displayed costimulatory ligand molecule GITRL, and an endogenous protein that comprises an endogenous peptide major histocompatibility complex class I (pMHC-I), wherein the first fusion protein, the second fusion protein, or the endogenous protein, is expressed at a valency of at least about 1000 copies on a surface of the APTC.

2. The antigen-presenting tumor cell (APTC) of claim 1, wherein the first or second fusion protein binds specifically to the cognate costimulatory receptors on a T-cell surface.

3. The antigen-presenting tumor cell (APTC) of any one of the preceding claims, further comprising a third fusion protein comprising at least one, two, three, four, five, six, seven, eight, nine, ten, or more than ten separate fusion proteins, wherein the at least one, two, three, four, five, six, seven, eight, nine, ten, or more than ten separate fusion proteins each comprises 0X40 Ligand, CD48, CD30 Ligand, ICOS Ligand, CD70, CD40, CD40 Ligand, 4-BBL, TLIA, TIM4, SLAM, CD48, CD58, CD155, CD112, LIGHT, or ICAM-1.

4. The antigen-presenting tumor cell (APTC) of claim 1, wherein:(A) the endogenous pMHC-I binds specifically to an antigen-specific T cell receptor (TCR) that recognizes an antigenic peptide derived from an endogenous antigen, a viral antigen, a mutated tumor antigen, a tumor-specific antigen, a differentiated tumor antigen, an over-expressed tumor antigen, or a neoantigen within tumor cells; or(B) the endogenous pMHC-I binds specifically to a CD8+ T cell bearing an antigenspecific T cell receptor (TCR) recognizing an antigenic peptide derived from an endogenous antigen, a viral antigen, a mutated tumor antigen, a tumor-specific antigen, a differentiated tumor antigen, an over-expressed tumor antigen, or a neoantigen within tumor cells.

5. The antigen-presenting tumor cell (APTC) of any one of the preceding claims, wherein the endogenous pMHC-I comprises an endogenous antigenic peptide, an endogenous P-2- microglobulin (P2m), and an endogenous MHC-I alpha chain.

6. The antigen-presenting tumor cell (APTC) of any one of the preceding claims, wherein the endogenous antigenic peptide binds to the endogenous MHC-I alpha chain.

7. The antigen-presenting tumor cell (APTC) of any one of the preceding claims, wherein the endogenous antigenic peptide in the endogenous pMHC-I is derived from anWSGR Docket No. 48295-719.601 endogenous antigen, a viral antigen, a mutated tumor antigen, a tumor-specific antigen, a differentiated tumor antigen, an over-expressed tumor antigen, or a neoantigen within tumor cells.

8. The antigen-presenting tumor cell (APTC) of any one of the preceding claims, wherein the APTC is derived from pancreatic cancer, rectal cancer, colorectal cancer, lung cancer, endometrial cancer, liver cancer, testicular cancer, prostate cancer, cervical cancer, breast cancer, bladder cancer, head and neck cancer, skin cancer, brain cancer, ovarian cancer, salivary gland cancer, appendiceal cancer, esophagogastric cancer, thyroid cancer, soft tissue sarcoma, kidney cancer, leukemia, myeloma, lymphoma, melanoma, non-small cell lung cancer, Hodgkin’s lymphoma, or non-Hodgkin’s lymphoma.

9. The antigen-presenting tumor cell (APTC) of any one of the preceding claims, wherein the antigenic peptide is derived from pancreatic cancer, rectal cancer, colorectal cancer, lung cancer, endometrial cancer, liver cancer, testicular cancer, prostate cancer, cervical cancer, breast cancer, bladder cancer, head and neck cancer, skin cancer, brain cancer, ovarian cancer, salivary gland cancer, appendiceal cancer, esophagogastric cancer, thyroid cancer, soft tissue sarcoma, kidney cancer, leukemia, myeloma, lymphoma, melanoma, non-small cell lung cancer, Hodgkin’s lymphoma, or non-Hodgkin’s lymphoma.

10. The antigen-presenting tumor cell (APTC) of any one of the preceding claims, wherein the endogenous antigenic peptide comprises an endogenous antigenic peptide from a tumor cell.

11. The antigen-presenting tumor cell (APTC) of any one of the preceding claims, wherein the costimulatory ligand molecule comprises an amino acid sequence with at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 23 -58.

12. The antigen-presenting tumor cell (APTC) of any one of the preceding claims, wherein each of the first, second, or third fusion protein of the displayed costimulatory ligand molecules further comprises a transmembrane domain.

13. The antigen-presenting tumor cell (APTC) of any one of the preceding claims, wherein the transmembrane domain of the displayed costimulatory ligand molecules comprises a transmembrane domain derived from VSV-G, Dengue E protein, influenza hemagglutinin, influenza neuraminidase, spike protein SI, spike protein S2, Sindbis virus envelope (SINDBIS) protein, hemagglutinin envelope protein from measles virus, envelope glycoprotein of measles virus fusion (F) protein, RD114, BaEV, GP41, GP120, or the wild-type transmembrane domain of costimulatory ligand molecules.

14. The antigen-presenting tumor cell (APTC) of any one of the preceding claims, wherein the VSV-G comprises a transmembrane domain and cytoplasmic tail.WSGR Docket No. 48295-719.60115. The antigen-presenting tumor cell (APTC) of any one of the preceding claims, wherein the transmembrane domain comprises an amino acid sequence with at least 90% sequence identity to an amino acid sequence of SEQ ID NOs: 59-78.

16. The antigen-presenting tumor cell (APTC) of any one of the preceding claims, wherein each of the first, second, or third fusion protein further comprises an oligomerization domain.

17. The antigen-presenting tumor cell (APTC) of any one of the preceding claims, wherein the oligomerization domain of the displayed costimulatory ligand molecules is a dimerization domain, a trimerization domain, a tetramerization domain, a hexamerization domain, a heterohexamerization domain, a heptamerization domain, or an octamerization domain .

18. The antigen-presenting tumor cell (APTC) of any one of the preceding claims, wherein the oligomerization domain of the displayed costimulatory ligand molecules comprises a leucine zipper dimerization domain, a post-fusion oligomerization domain of viral surface protein, a D4 post-fusion trimerization domain of VSV-G protein, a Dengue E protein post-fusion trimerization domain, a foldon trimerization domain, a coiled coil domain, a LINE1 retrotransposon coiled coil domain (Llcc), a collagen XV coiled coil domain (CXVcc), a tetranectin coiled coil domain (TNTNcc), a cc-hex domain, a L24D domain, a L24DH domain, a cc-Hept domain, a wazOct domain, or an influenza neuraminidase stem domain.

19. The antigen-presenting tumor cell (APTC) of any one of the preceding claims, wherein the oligomerization domain of the displayed costimulatory ligand molecules comprises an amino acid sequence that has at least 90% sequence identity to an amino acid sequence according to SEQ ID NOs: 79-93.

20. The antigen-presenting tumor cell (APTC) of any one of the preceding claims, wherein the APTC is capable of activating or stimulating an antigen -specific T cell.21 . The antigen-presenting tumor cell (APTC) of any one of the preceding claims, wherein when the first fusion protein, the second fusion protein, or the third fusion protein of the costimulatory ligand molecules is expressed on the surface of the antigen-presenting tumor cell (APTC), the oligomerization domain is outside of the antigen-presenting tumor cell (APTC).

22. The antigen-presenting tumor cell (APTC) of any one of the preceding claims, wherein when the first fusion protein, the second fusion protein, or third fusion protein of the displayed costimulatory ligand molecules is expressed on the surface of the antigen-presenting tumor cell (APTC), the oligomerization domain is inside of the antigen-presenting tumor cell (APTC).WSGR Docket No. 48295-719.60123. The antigen-presenting tumor cell (APTC) of any one of the preceding claims, wherein the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 1000 copies or more on a surface of the antigen-presenting tumor cell (APTC).

24. The antigen-presenting tumor cell (APTC) of any one of the preceding claims, wherein the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 2500 copies on a surface of the antigen-presenting tumor cell (APTC).

25. The antigen-presenting tumor cell (APTC) of any one of the preceding claims, wherein the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 5000 copies on a surface of the antigen-presenting tumor cell (APTC).

26. The antigen-presenting tumor cell (APTC) of any one of the preceding claims, wherein the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 10,000 copies on a surface of the antigen-presenting tumor cell (APTC).

27. The antigen-presenting tumor cell (APTC) of any one of the preceding claims, wherein the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 20,000 copies on a surface of the antigen-presenting tumor cell (APTC).

28. The antigen-presenting tumor cell (APTC) of any one of the preceding claims, wherein the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 40,000 copies on a surface of the antigen-presenting tumor cell (APTC).

29. The antigen-presenting tumor cell (APTC) of any one of the preceding claims, wherein the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 60,000 copies on a surface of the antigen-presenting tumor cell (APTC).

30. The antigen-presenting tumor cell (APTC) of any one of the preceding claims, wherein the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 80,000 copies on a surface of the antigen-presenting tumor cell (APTC).

31. The antigen-presenting tumor cell (APTC) of any one of the preceding claims, wherein the first fusion protein, the second fusion protein, or the third fusion protein of theWSGR Docket No. 48295-719.601 displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 120,000 copies on a surface of the antigen-presenting tumor cell (APTC).

32. The antigen-presenting tumor cell (APTC) of any one of the preceding claims, wherein the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 200,000 copies on a surface of the antigen-presenting tumor cell (APTC).

33. The antigen-presenting tumor cell (APTC) of any one of the preceding claims, wherein the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 250,000 copies or more on a surface of the antigen-presenting tumor cell (APTC).

34. The antigen-presenting tumor cell (APTC) of any one of the preceding claims, wherein the endogenous pMHC-I comprises a natural pMHC-I.

35. The antigen-presenting tumor cell (APTC) of any one of the preceding claims, wherein the first, second, or third fusion protein of the displayed costimulatory ligand molecules comprises an amino acid sequence with at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 94-111 .

36. An antigen-presenting tumor cell (APTC) comprising a first fusion protein that comprises the costimulatory ligand molecule CD80 or CD86, and a second fusion protein that comprises the costimulatory ligand molecule GITRL, and a recombinant peptide major histocompatibility complex class I (pMHC-I), wherein the APTC lacks an endogenous pMHC-I or expresses a dysfunctional endogenous pMHC-I, wherein first fusion protein, the second fusion protein, or the recombinant pMHC-I is expressed at a valency of at least about 1000 copies on a surface of the APTC.

37. A pharmaceutical composition comprising the APTC of any one of the preceding claims, and a pharmaceutically acceptable excipient.

38. A fusion protein comprising a costimulatory ligand molecule that binds specifically to a T cell surface costimulatory receptor, an oligomerization domain, and a transmembrane domain, wherein the fusion protein is expressed at a valency of at least about 1000 copies on a surface of an antigen-presenting tumor cell (APTC).

39. The fusion protein of claim 38, wherein the fusion protein comprises at least two separate fusion proteins, wherein the at least two separate fusion proteins each comprises a different costimulatory ligand molecule.

40. The fusion protein of claim 38 or 39, wherein the costimulatory molecule comprises one or more selected from the group comprising GITR Ligand, CD80, CD86, 0X40WSGR Docket No. 48295-719.601Ligand, CD48, CD30 Ligand, ICOS Ligand, CD70, CD40, CD40 Ligand, 4-BBL, TLIA, TIM4, SLAM, CD48, CD58, CD155, CD112, LIGHT, and ICAM1.

41. An antigen-presenting tumor cell (APTC) comprising the fusion protein of any one of claims 38-40, wherein the APTC is capable of activating or stimulating an antigen -specific T cell and wherein the fusion protein is expressed at least about 1000 copies on a surface of the APTC.

42. A method of preventing or treating cancer in a subject in need thereof comprising administering to the subject an autologous cell comprising the APTC of any one of claims 1-36.

43. A method of preventing or treating established cancer in a subject in need thereof comprising administering to the subject an irradiated autologous cell comprising the APTC of any one of claims 1-36.

44. A method of inducing protective immunity against cancer in a subject in need thereof comprising administering to the subject an autologous cell comprising the APTC of any one of claims 1-36.

45. A method of inducing protective immunity against cancer in a subject in need thereof comprising administering to the subject an irradiated autologous cell comprising the APTC of any one of claims 1-36.

46. A method of preventing or treating established cancer in a subject in need thereof comprising administering to the subject a nucleic acid encoding the fusion protein of any one of claims 38-40, wherein the administering results in the expression of the fusion protein on a surface of a cancer cell in the subject.

47. A method of inducing protective immunity against cancer in a subject in need thereof comprising administering to the subject a nucleic acid encoding the fusion protein of any one of claims 38-40, wherein the administering results in expression of the fusion protein on a surface of a cancer cell in the subject.

48. A method of preventing cancer relapse in a subject in need thereof comprising administering to the subject an autologous cell comprising the APTC of any one of claims 1-36.

49. The method of claim 48, wherein the subject undergoes cancer treatment before the administering and does not have cancer after the treatment.

50. The method of claim 48 or 49, wherein the autologous cell comprisingthe APTC is produced in culture.

51. The method of claim 48 or 49, wherein the autologous cell comprisingthe APTC is a live cell, an irradiated cell, or an inactivated cell.WSGR Docket No. 48295-719.60152. The method of claim 48 or 49, wherein the administering comprises administering a Toll-like receptor agonist.

53. The method of any one of claims 48-52, wherein the administering comprises administering an anti-PDl, anti-PDLl, an anti-CTLA4 immune checkpoint blockade therapy, a targeted therapy, an antibody -drug therapy, a tyrosine kinase inhibitor, a chemotherapy, surgery, or a therapy of standard care.

54. The method of any one of claims 48-53, comprising introducing, before the administering, into the autologous cell a nucleic acid molecule encoding the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecule, wherein the autologous cell is cultured ex vivo.

55. The method of claim 46 or 47, wherein the nucleic acid comprises mRNA, DNA, or a viral vector.

56. The method of claim 55, wherein the viral vector comprises a lentiviral vector, a retroviral vector, an adeno-associated viral (AAV) vector, an adenoviral vector, a vector derived from herpes simplex virus (HSV), a vector derived from parvoviruses, or a vector derived from poxviruses.

57. The method of claim 56, wherein the poxviruses comprise vaccinia virus (VACV) or myxoma virus.

58. The method of claim 46 or 47, wherein the introducing comprises intratumoral delivery, intravenous delivery, intramuscular delivery, intraperitoneal delivery, or transdermal delivery.

59. The method of claim 46 or 47, wherein the administering comprises administering a Toll-like receptor agonist.

60. The method of claim 46 or 47, wherein the administering comprises administering an anti-PDl , anti-PDLl , an anti-CTLA4 immune checkpoint blockade therapy, a targeted therapy, an antibody-drug therapy, a tyrosine kinase inhibitor, a chemotherapy, surgery, or a therapy of standard care.

61. A method of inducing protective immunity against cancer in a subject in need thereof comprising administering to the subject an irradiated autologous cell comprising the APTC of any one of claims 1-36.

62. The method of claim 46 or 47, wherein the cancer cell is reprogrammed into an APTC that activates or stimulates a T cell in the subject.

63. A method of preventing or treating cancer in a subject in need thereof comprising:WSGR Docket No. 48295-719.601(A) obtaining a T cell, a tumor-infiltrated lymphocyte (TIL), or a cancer cell from the subject;(B) introducing ex vivo into the cancer cell a nucleic acid encoding the fusion protein of any one of claims 38-40 to express the fusion protein on a surface of the cancer cell to generate antigen-presenting tumor cells (APTCs);(C) contacting ex vivo the T cell or TIL with the antigen-presenting tumor cells (APTCs) to activate or stimulate the T cell or TIL; and(D) administering the T cell or TIL from step (C) to the subject.

64. The method of any one of claims 42-49, further comprising administering the subject a second therapy.

65. The method of claim 64, wherein the second therapy comprises an adjuvant.

66. The method of claim 64, wherein the second therapy comprises Poly (LC) or lipopolysaccharide (LPS).

67. The method of claim 64, wherein the second therapy comprises an immune checkpoint blocker therapy.

68. The method of claim 64, wherein the second therapy comprises an anti-PDl antibody, anti-PDLl antibody, or an anti-CTLA-4 antibody.

69. The method of claim 64, wherein the second therapy comprises a Toll-like receptor agonist for TLR3, TLR4, TLR7, TLR8, TLR9, CpG, or a STING agonist.

70. The method of claim 64, wherein the second therapy comprises a cytokine.

71. The method of claim 64, wherein the cytokine comprises IL-2, IL-7, IL-12, IL-15, IL-21, or interferon-gamma (IFN-gamma).

72. The method of claim 64, wherein the second therapy comprises a targeted therapy, and optionally a tyrosine kinase inhibitor or PARP inhibitor or RAS inhibitor.

73. The method of claim 64, wherein the second therapy comprises T cell therapy, and optionally a chimeric antigen -receptor T cell therapy or tumor-infiltrated lymphocyte therapy.

74. The method of claim 64, wherein the second therapy comprises standard care therapy, and optionally surgery or chemotherapy or radiation therapy.

75. The method of any one of claims 42-60, wherein the nucleic acid comprises mRNA, a viral vector, a DNA vector.

76. A method of producing an antigen-presenting tumor cell (APTC) comprising the APTC of any one of the preceding claims, comprising introducing into a tumor cell a nucleic acid molecule encoding the first fusion protein, the second fusion protein, or the third fusion protein of the displayed costimulatory ligand molecule.WSGR Docket No. 48295-719.60177. The method of claim 76, wherein the introduction is carried out ex vivo after the tumor cell is obtained from a subject.

78. The method of claim 76, wherein the introduction is carried out in vivo or intratumorally in a subject.

79. The method of any one of claims 76-78, wherein the nucleic acid comprises mRNA or DNA.

80. The method of any one of claims 76-79, wherein the nucleic acid comprises a DNA vector, an mRNA vector, or a viral vector.81 . The method of claim 76 or 77, wherein the tumor cell comprises an ex vivo cultured cell.

82. The method of claim 81, wherein the cultured cell comprises an adherent cell, a suspension cell, or an organoid.

83. The method of claim 80, wherein the viral vector comprises a lentiviral vector, a retroviral vector, an adeno-associated viral (AAV) vector, an adenoviral vector, a vector derived from herpes simplex virus (HSV), a vector derived from parvoviruses, or a vector derived from poxviruses.

84. The method of claim 83, wherein the poxviruses comprise vaccinia virus (VACV) or myxoma virus.

85. The method of claim 76 or 78, wherein the introducing comprises intratumoral delivery, intravenous delivery, intramuscular delivery, intraperitoneal delivery, or transdermal delivery.

86. The method of any of claims 76-85, wherein the nuclei acid molecule comprises an ectodomain of the costimulatory ligand molecule, an oligomerization domain, a transmembrane domain and a cytosolic domain.

87. A tumor-derived antigen-presenting vesicle (tumor- APV) comprising, a first fusion protein that comprises the displayed costimulatory ligand molecule CD80 or CD86, a second fusion protein that comprises the displayed costimulatory ligand molecule GITRL, and an endogenous protein that comprises an endogenous peptide major histocompatibility complex class I (pMHC-I), wherein the tumor-APV is derived from an antigen-presenting tumor cell (APTC) comprising the first fusion protein, the second fusion protein, and the endogenous protein, wherein the first fusion protein, the second fusion protein, or the endogenous protein, is expressed at a valency of at least about 1000 copies on a surface of the APTC or at 100 copies on a surface of the APV.WSGR Docket No. 48295-719.60188. The tumor-derived antigen-presenting vesicle (tumor-APV) of any one of the preceding claims, wherein the first or second fusion protein binds specifically to the cognate costimulatory receptors on a T-cell surface.

89. The tumor-derived antigen-presenting vesicle (tumor-APV) of any one of the preceding claims, wherein the tumor-APV or APTC further comprises a third fusion protein comprising at least one, two, three, four, five, six, seven, eight, nine, ten, or more than ten separate fusion proteins, wherein the at least one, two, three, four, five, six, seven, eight, nine, ten, or more than ten separate fusion proteins each comprises 0X40 Ligand, CD48, CD30 Ligand, ICOS Ligand, CD70, CD40, CD40 Ligand, 4-BB Ligand, TLIA, TIM4, SLAM, CD48, CD58, CD155, CD112, LIGHT, or ICAM-1.

90. The tumor-derived antigen-presenting vesicle (tumor-APV) of any one of the preceding claims, wherein:(A) the endogenous pMHC-I binds specifically to an antigen-specific T cell receptor (TCR) that recognizes an antigenic peptide derived from an endogenous antigen, a viral antigen, a mutated tumor antigen, a tumor-specific antigen, a differentiated tumor antigen, an over-expressed tumor antigen, or a neoantigen within tumor cells, or a self-antigen of normal protein within tumor cells; or(B) the endogenous pMHC-I binds specifically to a CD8+ T cell bearing an antigenspecific T cell receptor (TCR) recognizing an antigenic peptide derived from an endogenous antigen, a viral antigen, a mutated tumor antigen, a tumor-specific antigen, a differentiated tumor antigen, an over-expressed tumor antigen, a neoantigen, or a self- antigen of normal protein within tumor cells.91 . The tumor-derived antigen-presenting vesicle (tumor-APV) of any one of the preceding claims, wherein the endogenous pMHC-I comprises an endogenous antigenic peptide, an endogenous P-2 -microglobulin (P2m), and an endogenous MHC-I alpha chain.

92. The tumor-derived antigen-presenting vesicle (tumor-APV) of any one of the preceding claims, wherein the endogenous antigenic peptide binds to the endogenous MHC-I alpha chain.

93. The tumor-derived antigen-presenting vesicle (tumor-APV) of any one of the preceding claims, wherein the endogenous antigenic peptide in the endogenous pMHC-I is derived from an endogenous antigen, a viral antigen, a mutated tumor antigen, a tumor-specific antigen, a differentiated tumor antigen, an over-expressed tumor antigen, a neoantigen, or a self -antigen of normal protein within tumor cells.WSGR Docket No. 48295-719.60194. The tumor-derived antigen-presenting vesicle (tumor- APV) of any one of the preceding claims, wherein the tumor-APV or its parental APTC is derived from pancreatic cancer, rectal cancer, colorectal cancer, lung cancer, endometrial cancer, liver cancer, testicular cancer, prostate cancer, cervical cancer, breast cancer, bladder cancer, head and neck cancer, skin cancer, brain cancer, ovarian cancer, salivary gland cancer, appendiceal cancer, esophagogastric cancer, thyroid cancer, soft tissue sarcoma, kidney cancer, leukemia, myeloma, lymphoma, melanoma, non-small cell lung cancer, Hodgkin’s lymphoma, or non-Hodgkin’s lymphoma.

95. The tumor-derived antigen-presenting vesicle (tumor-APV) of any one of the preceding claims, wherein the antigenic peptide is derived from pancreatic cancer, rectal cancer, colorectal cancer, lung cancer, endometrial cancer, liver cancer, testicular cancer, prostate cancer, cervical cancer, breast cancer, bladder cancer, head and neck cancer, skin cancer, brain cancer, ovarian cancer, salivary gland cancer, appendiceal cancer, esophagogastric cancer, thyroid cancer, soft tissue sarcoma, kidney cancer, leukemia, myeloma, lymphoma, melanoma, non-small cell lung cancer, Hodgkin’s lymphoma, or non-Hodgkin’s lymphoma.

96. The tumor-derived antigen-presenting vesicle (tumor-APV) of any one of the preceding claims, wherein the endogenous antigenic peptide comprises an endogenous antigenic peptide from a tumor cell.

97. The tumor-derived antigen-presenting vesicle (tumor-APV) of any one of the preceding claims, wherein the costimulatory ligand molecule comprises an amino acid sequence with at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 23-58.

98. The tumor-derived antigen-presenting vesicle (tumor-APV) of any one of the preceding claims, wherein each of the first, second, or third fusion protein of the displayed costimulatory ligand molecules further comprises a transmembrane domain.

99. The tumor-derived antigen-presenting vesicle (tumor-APV) of any one of the preceding claims, wherein the transmembrane domain of the displayed costimulatory ligand molecules comprises a transmembrane domain derived from VSV-G, Dengue E protein, influenza hemagglutinin, influenza neuraminidase, spike protein SI, spike protein S2, Sindbis virus envelope (SINDBIS) protein, hemagglutinin envelope protein from measles virus, envelope glycoprotein of measles virus fusion (F) protein, RD114, BaEV, GP41, GP120, or the wild-type transmembrane domain of costimulatory ligand molecules.

100. The tumor-derived antigen-presenting vesicle (tumor-APV) of any one of the preceding claims, wherein the VSV-G comprises a transmembrane domain and cytoplasmic tail.WSGR Docket No. 48295-719.601101. The tumor-derived antigen-presenting vesicle (tumor- APV) of any one of the preceding claims, wherein the transmembrane domain comprises an amino acid sequence with at least 90% sequence identity to an amino acid sequence of SEQ ID NOs: 59-78.

102. The tumor-derived antigen-presenting vesicle (tumor- APV) of any one of the preceding claims, wherein each of the first, second, or third fusion protein further comprises an oligomerization domain.

103. The tumor-derived antigen-presenting vesicle (tumor-APV) of any one of the preceding claims, wherein the oligomerization domain of the displayed costimulatory ligand molecules is a dimerization domain, a trimerization domain, a tetramerization domain, a hexamerization domain, a heterohexamerization domain, a heptamerization domain, or an octamerization domain.

104. The tumor-derived antigen-presenting vesicle (tumor-APV) of any one of the preceding claims, wherein the oligomerization domain of the displayed costimulatory ligand molecules comprises a leucine zipper dimerization domain, a post-fusion oligomerization domain of viral surface protein, a D4 post-fusion trimerization domain of VSV-G protein, a Dengue E protein post-fusion trimerization domain, a foldon trimerization domain, a coiled coil domain, a LINE1 retrotransposon coiled coil domain (Llcc), a collagen XV coiled coil domain (CXVcc), a tetranectin coiled coil domain (TNTNcc), a cc-hex domain, a L24D domain, a L24DH domain, a cc-Hept domain, a wazOct domain, or an influenza neuraminidase stem domain.

105. The tumor-derived antigen-presenting vesicle (tumor-APV) of any one of the preceding claims, wherein the oligomerization domain of the displayed costimulatory ligand molecules comprises an amino acid sequence that has at least 90% sequence identity to an amino acid sequence according to SEQ ID NOs: 79-93.

106. The tumor-derived antigen-presenting vesicle (tumor-APV) of any one of the preceding claims, wherein the tumor-APV is capable of activating or stimulating an antigenspecific T cell.

107. The tumor-derived antigen-presenting vesicle (tumor-APV) of any one of the preceding claims, wherein when the first fusion protein, the second fusion protein, or the third fusion protein of the costimulatory ligand molecules is expressed on the surface of the tumor- APV, the oligomerization domain is outside of the tumor-APV.

108. The tumor-derived antigen-presenting vesicle (tumor-APV) of any one of the preceding claims, wherein when the first fusion protein, the second fusion protein, or third fusion protein of the displayed costimulatory ligand molecules is expressed on the surface of the tumor- APV, the oligomerization domain is inside of the tumor-APV.WSGR Docket No. 48295-719.601109. The tumor-derived antigen-presenting vesicle (tumor- APV) of any one of the preceding claims, wherein the first fusion protein, the second fusion protein, the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 100 copies or more on a surface of the tumor-APV.

110. The tumor-derived antigen-presenting vesicle (tumor APV) of any one of the preceding claims, wherein the first fusion protein, the second fusion protein, the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 250 copies on a surface of the tumor-APV.

111. The tumor-derived antigen-presenting vesicle (tumor-APV) of any one of the preceding claims, wherein the first fusion protein, the second fusion protein, the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 5,00 copies on a surface of the tumor-APV.

112. The tumor-derived antigen-presenting vesicle (tumor-APV) of any one of the preceding claims, wherein the first fusion protein, the second fusion protein, the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 1,000 copies on a surface of the tumor-APV.

113. The tumor-derived antigen-presenting vesicle (tumor-APV) of any one of the preceding claims, wherein the first fusion protein, the second fusion protein, the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 2,000 copies on a surface of the tumor-APV.

114. The tumor-derived antigen-presenting vesicle (tumor-APV) of any one of the preceding claims, wherein the first fusion protein, the second fusion protein, the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 4,000 copies on a surface of the tumor-APV.

115. The tumor-derived antigen-presenting vesicle (tumor-APV) of any one of the preceding claims, wherein the first fusion protein, the second fusion protein, the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 6,000 copies on a surface of the tumor-APV.

116. The tumor-derived antigen-presenting vesicle (tumor-APV) of any one of the preceding claims, wherein the first fusion protein, the second fusion protein, the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 8,000 copies on a surface of the tumor-APV.

117. The tumor-derived antigen-presenting vesicle (tumor-APV) of any one of the preceding claims, wherein the first fusion protein, the second fusion protein, the third fusionWSGR Docket No. 48295-719.601 protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 10,000 copies on a surface of the tumor-APV.

118. The tumor-derived antigen-presenting vesicle (tumor-APV) of any one of the preceding claims, wherein the first fusion protein, the second fusion protein, the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 20,000 copies on a surface of the tumor-APV.

119. The tumor-derived antigen-presenting vesicle (APV) of any one of the preceding claims, wherein the first fusion protein, the second fusion protein, the third fusion protein of the displayed costimulatory ligand molecules, or the endogenous pMHC-I is expressed at a valency of about 50,000 copies or more on a surface of the tumor-APV.

120. The tumor-derived antigen-presenting vesicle (tumor-APV) of any one of the preceding claims, wherein the endogenous pMHC-I comprises a natural pMHC-I.

121. The tumor-derived antigen-presenting vesicle (tumor-APV) of any one of the preceding claims, wherein the first, second, or third fusion protein of the displayed costimulatory ligand molecules comprises an amino acid sequence with at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 94-111.

122. A tumor-derived antigen-presenting vesicle (tumor-APV) comprising a first fusion protein that comprises the costimulatory ligand molecule CD80 or CD86, and a second fusion protein that comprises the costimulatory ligand molecule GITRL, and a recombinant peptide major histocompatibility complex class I (pMHC-I), wherein the tumor-APV is derived from its parental antigen-presenting tumor cell (APTC) comprising the first fusion protein, the second fusion protein, and the recombinant pMHC-I, wherein the tumor-APV or its parental APTC lacks an endogenous pMHC-I or expresses a dysfunctional endogenous pMHC-I, wherein first fusion protein, the second fusion protein, or the recombinant pMHC-I is expressed at a valency of at least about 100 copies on a surface of the tumor-APV.

123. A pharmaceutical composition comprising the tumor-APV of any one of the preceding claims, and a pharmaceutically acceptable excipient.

124. A fusion protein comprising a costimulatory ligand molecule that binds specifically to a T cell surface costimulatory receptor, an oligomerization domain, and a transmembrane domain, wherein the fusion protein is expressed at a valency of at least about 100 copies on a surface of a tumor-derived antigen-presenting vesicle (tumor-APV), wherein the tumor-APV is derived from its parental APTC.WSGR Docket No. 48295-719.601125. The fusion protein of any one of the preceding claims, wherein the fusion protein comprises at least two separate fusion proteins, wherein the at least two separate fusion proteins each comprises a different costimulatory ligand molecule.

126. The fusion protein of any one of the preceding claims, wherein the costimulatory molecule comprises one or more selected from the group comprising GITRL, CD80, CD86, 0X40 Ligand, CD48, CD30 Ligand, ICOS Ligand, CD70, CD40, CD40 Ligand, 4-BB Ligand, TLIA, TIM4, SLAM, CD48, CD58, CD155, CD112, LIGHT, and ICAM1.

127. A tumor-derived antigen-presenting vesicle (tumor- APV) comprising the fusion protein of any one of the preceding claims, wherein the tumor-APV is derived from its parental APTC and capable of activating or stimulating an antigen -specific T cell, wherein the fusion protein is expressed at least about 100 copies on a surface of the tumor-APV.

128. A method of preventing or treating cancer in a subject in need thereof comprising administering to the subject the antigen-presenting vesicle (APV) of any one of the preceding claims, wherein the APV is derived from the APTC that is derived from the subject.

129. A method of inducing protective immunity against cancer in a subject in need thereof comprising administering to the subject the tumor-derived antigen-presenting vesicle (tumor-APV) of any one of the preceding claims, wherein the tumor-APV is derived from its parental APTC that is derived from the subject.

130. A method of producing the tumor-derived antigen-presenting vesicle (APV) of any one of the preceding claims, comprising administering to a subject a nucleic acid encoding a fusion protein comprising a costimulatory ligand molecule that binds specifically to a T cell surface costimulatory receptor, an oligomerization domain, and a transmembrane domain, wherein the administering results in the expression of the fusion protein on a surface of a cancer cell in the subject.

131. A method of producing the tumor-derived antigen-presenting vesicle (tumor-APV) of any one of the preceding claims, comprising:(A) generating an antigen-presenting tumor cell (APTC) by introducing ex vivo into the cancer cell a nucleic acid encoding a fusion protein comprising a costimulatory ligand molecule that binds specifically to a T cell surface costimulatory receptor, an oligomerization domain, and a transmembrane domain to express the fusion protein on a surface of the cancer cell;(B) culturing and expanding APTCs in culture dishes as a suspension, adherent, or organoid cell culture; andWSGR Docket No. 48295-719.601(C) generating tumor-derived antigen-presenting vesicles (tumor-APVs) from APTC- derived exosomes (EVs) or by disruptingthe antigen-presenting tumor cells (APTCs) into tumor-APVs using physical processes, including nitrogen cavitation, freeze and thaw, sonication, or size-exclusion filtration.

132. The method of any one of the preceding claims, comprising deriving tumor- APV from the antigen-presenting tumor cell (APTC) expressing the fusion protein.

133. The method of any one of the preceding claims, comprising disrupting the antigen- presenting tumor cell (APTC) expressing the fusion protein into the tumor -APV.

134. The method of any one of the preceding claims, wherein the tumor-APV comprises a size from about lOOnm to about 1 pm.

135. The method of any one of the preceding claims, wherein the disrupting of APTCs is carried out using a physical method.

136. The method of any one of the preceding claims, wherein the physical method comprises nitrogen cavitation, sonication, freeze and thaw, or membrane exclusion.

137. The method of any one of the preceding claims, further comprising purifying the tumor-APV to remove cellular debris, nucleic acid impurities, or protein impurities.

138. The method of any one of the preceding claims, further comprising contacting ex vivo the T cell or TIL with the tumor-APV to activate or stimulate the T cell or TIL.

139. A method of preventing or treating cancer in a subj ect in need thereof comprising g administering to the subject the T cell or TIL of any one of the preceding claims, wherein the T cell or TIL is activated or stimulated by the tumor-APV before the administering.

140. A method of preventing cancer relapse in a subject in need thereof comprising administering to the subject tumor-APVs of any one of the preceding claims.

141. A method of preventing or treating established cancer in a subject in need thereof comprising administering to the subject autologous tumor-APVs of any one of the preceding claims.

142. A method of inducing protective immunity against cancer in a subject in need thereof comprising administering to the subject autologous tumor-APVs of any one of the preceding claims.

143. The method of any one of the preceding claims, wherein the subject undergoes cancer treatment before the administering and does not have cancer after the treatment.

144. The method of any one of the preceding claims, wherein the autologous tumor- APVs is derived from a cancer cell produced in culture.WSGR Docket No. 48295-719.601145. The method of any one of the preceding claims, wherein the administering of tumor-APVs comprises administering a Toll -like receptor agonist.

146. The method of any one of the preceding claims, wherein the administering of tumor-APVs comprises administering an anti-PDl, anti-PDLl, an anti-CTLA4 immune checkpoint blockade therapy, a targeted therapy, an antibody -drug therapy, a tyrosine kinase inhibitor, a chemotherapy, surgery, or a therapy of standard care.

147. A method of enhancing anti -turn or T cell immunity in a subject, comprising administering to a subject in need thereof the APTC of any one of the preceding claims or the APV of any one of the preceding claims.

148. The method of any one of the preceding claims, further comprising administering a therapeutic agent.

149. The method of any one of the preceding claims, wherein the therapeutic agent comprises an inflammatory cytokine, a dendritic cell targeting signal, or an innate modulating signal.

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