Allogeneic low immune biomimetic nanovesicles for treatment of cancer

Abstract

Disclosed herein are compositions comprising allogeneic, low immunogenic chimeric antigen receptor (CAR)-targeting biomimetic nanovesicles (BioNVs), and methods of treating, preventing, and / or ameliorating cancer using the same.

Description

Technical Field

[0001] The present disclosure provides, in part, compositions and methods comprising allogeneic, low-immunogenic biomimetic nanovesicles, and methods of using the same to treat or prevent cancer in, for example, mammalian subjects, such as humans.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 345,664, filed on May 25, 2022, the contents of which are incorporated herein by reference in their entirety. Background Art

[0004] Chimeric antigen receptor (CAR) whole cell therapy (T cells, NK cells, macrophages, tumor infiltrating lymphocytes (TIL), etc.) has been used to target cells to treat cancer. CAR whole cell therapy is a cancer therapy that usually requires collecting a patient's own immune cells (T cells) to treat their cancer, or developing allogeneic cell lines from stem cells to treat a wider patient population at a lower cost. Lymphocytes usually attack invasive microorganisms, but in CAR whole cell therapy, the cells are engineered to target cancer cells.

[0005] First, cells are isolated from the patient's blood and genetically engineered to express CAR on their surface, enabling the cells to bind to specific antigens. Alternatively, the cells are genetically engineered from stem cells (e.g., iPSC, MSC, or embryonic stem cells) to incorporate the allogeneic and stability properties of the immune system (e.g., incorporation of a molecular CD47 transmembrane tag to prevent clearance by macrophages), and then stably integrate the desired CAR. CARs are non-natural fusion proteins containing synthetic antibody fragments. CARs can recognize target cancer biomarkers through several binding parts: 1) the antibody fragment scFV region, or a bifunctional or BiTE antibody format; 2) through a viral epitope recognition receptor (VERR) derived from an oncolytic virus receptor; 3) through a camel-derived variable heavy chain IgG fragment, called V H H single domain nanobody (V H H nanobodies); 4) through cartilaginous fish-derived variable heavy chain IgG fragments, called variable neoantigen receptors (V NAR ); 5) through an engineered T cell receptor (TCR); 6) through any single heavy chain IgG fragment, from which the variable region can be engineered into a CAR structure to recognize any biomarker; 7) through an afilin (artificial binding domain with antigen selectivity); or 8) through a chimeric endocrine receptor (CER). CAR constructs in whole cell therapies rely on engineered signaling and costimulatory domains inside the cell to function.

[0006] Once CAR whole cells are produced, they are expanded to produce large numbers of cells that can then be infused back into the patient (autologous) or, if from an engineered stem cell source, into a wide range of patients (allogeneic). CAR whole cells recognize specific tumor antigens on cancer cells and are designed to kill cancer cells that have those specific antigens.

[0007] CAR-T cell therapy has been approved for the treatment of childhood acute lymphoblastic leukemia (ALL) and adult advanced lymphoma. For example, CAR T cells targeting CD-19 (tisangenlecleucel, Novartis) has been approved for the treatment of ALL. (axicabtagene ciloleucel, Gilead / Kite Pharmaceuticals) is approved for the treatment of lymphoma. Studies have also been conducted targeting CD-22 in cells that have lost CD-19 expression. Dual targeting of CD-19 and CD-123 in leukemias has also been studied. CAR T cells targeting B cell maturation antigen (BCMA) have recently been approved for the treatment of multiple myeloma (MM) (idecabtagene vicleucel, Bristol MyersSquibb). It is unclear whether CAR T cells can treat solid tumors because of the microenvironment surrounding them.

[0008] Despite recent advances, CAR whole-cell therapy still has some disadvantages. 1) It can cause cytokine release syndrome (CRS), which causes a systemic inflammatory response to immunotherapy, leading to high fever and hypotension. This may require additional treatment, such as blocking IL-6 activity. It also causes B cell death (B cell hypoplasia), requiring further treatment with immunoglobulin. Other side effects may include brain edema and neurotoxicity. 2) Autologous sources require that the patient receiving treatment has a sufficient number of T cells (or NK or macrophages) to harvest and engineer, which is sometimes not feasible. 3) Multiple rounds of treatment are usually required, and laborious re-engineering may also be required, especially when tumor cells lose antigen expression. 4) Whole-cell stability in the tumor microenvironment is poor, which is a major challenge because the hypoxic and acidic environment can trigger cell apoptosis. 5) Once a cell enters the tumor environment, it may become 'overactive', with increased metabolism, leading to exhaustion. 6) T cell responses can be shut down, for example, through the PD-1 pathway, by upregulating PD-L1 on tumor cells.

[0009] The PD-1 signaling axis blocks a key mechanism that kills normal healthy cells and is particularly important in whole-cell therapies. (pembrolizumab, Merck) or (nivolumab, Bristol-Myers Squibb) Potential side effects due to off-target effects occur in whole-cell therapies when PD-1 is blocked on T cells, exposing healthy cells containing PD-L1 to immune clearance, even when PD-L1 is upregulated on tumor cells. Drugs that block PD-1 on T cells prevent it from interacting with PD-L1 and PD-L2 on cancer cells (upregulated) or healthy cells and allow the immune system to clear cancer cells that express PD-L1. However, off-target effects on healthy cells may occur. (Pembrolizumab, Merck) There are other adverse side effects, such as blurred vision, body aches, confusion, constipation, abnormal stool or urine, depression, headache, nausea, etc.

[0010] PD-1 (Programmed Cell Death Protein 1) is a protein found on the surface of T cells that helps regulate the immune system's response to cells in the body by downregulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. PD-1 on T cells binds to PD-L1 and PD-L2 on normal healthy cells. The PD-1-PD-L1 interaction inactivates cell-mediated responses against normal cells to prevent T cell-mediated killing of healthy cells. This action also prevents the immune system from targeting and killing cancer cells, which express upregulated PD-L1. As T cell PD-1 binds cancer cells to PD-L1, this enhances the no-kill signal to the T cells, hiding the cancer cells from the immune system.

[0011] Although PD-1 blockade strategies have some efficacy against various cancers, standalone treatment is relatively ineffective for most solid tumor cancers. Several combination / combination therapy strategies have been developed to improve clinical outcomes targeting this signaling pathway. Some of these strategies include conjugated / combination therapy approaches with anti-PD-1 monoclonal antibodies, potent small molecules, monoclonal antibodies, gene editing, and combination with a variety of delivery systems including AAV and lipid nanoparticles (LNPs). Recently, LNPs have been used to target PD-L1 on cancer cells while delivering inhibitory / toxic nucleic acids, or to deliver anti-PD-L1 nucleic acids (e.g., mRNA). For example, Moderna has a checkpoint cancer vaccine (mRNA-4359) that expresses indoleamine 2,3-dioxygenase and PD-L1 antigen to stimulate effector T cells to target and kill immune and tumor cells expressing the target antigen. The latter approach may improve efficacy, but the accessibility of LNPs to solid tumor masses within the tumor microenvironment (TME) may be limited, and the delivery of toxic or anti-PD-L1 nucleic acids may be further limited due to the inability to penetrate into tumor cells.

[0012] There remains a need for cancer treatments that address the PD-L1 / PD-1 signaling problem and overcome the shortcomings of standard whole-cell therapies in treating cancer while taking advantage of the targeting and killing efficacy of whole-cell therapies. Summary of the invention

[0013] In an embodiment, a method for treating or preventing cancer comprises administering to a subject in need thereof (i) a therapeutically effective amount of a biomimetic nanovesicle (BioNV) comprising (a) a membrane-embedded chimeric antigen receptor (CAR) targeting a cell surface marker, and (b) a PD-1, PD-L1 and / or PD-L2 inhibitor; or (ii) a therapeutically effective amount of a BioNV comprising a membrane-embedded CAR targeting a cell surface marker, wherein the subject is being treated with a PD-1, PD-L1 and / or PD-L2 inhibitor.

[0014] In an embodiment, a method for treating or preventing cancer comprises administering to a subject in need thereof (i) a therapeutically effective amount of a biomimetic nanovesicle (BioNV) comprising (a) a membrane-embedded chimeric antigen receptor (CAR) targeting a cell surface marker, and a conjugated and / or membrane-anchored PD-L1 and / or PD-L2 inhibitor; or (ii) a therapeutically effective amount of a BioNV comprising (a) a membrane-embedded bispecific CAR targeting a first cell surface marker and a second cell surface marker, and a conjugated and / or membrane-anchored PD-L1 and / or PD-L2 inhibitor.

[0015] In an embodiment, a method for treating or preventing cancer comprises administering to a subject in need thereof (i) a therapeutically effective amount of a biomimetic nanovesicle (BioNV) comprising a bispecific chimeric antigen receptor (CAR) targeting a first cell surface marker and PD-L1 or PD-L2, wherein the first cell surface marker is not PD-1, PD-L1, or PD-L2; or (ii) a therapeutically effective amount of BioNV comprising a bispecific CAR targeting a first cell surface marker and PD-L1 or PD-L2, wherein the first cell surface marker is not PD-1, PD-L1, or PD-L2, and a PD-1, PD-L1, and / or PD-L2 inhibitor; or (iii) a therapeutically effective amount of BioNV comprising a bispecific CAR targeting PD-L1 or PD-L2 and a first cell surface marker, wherein the subject is being treated with a PD-1, PD-L1, and / or PD-L2 inhibitor, and wherein the first cell surface marker is not PD-1, PD-L1, or PD-L2.

[0016] In an embodiment, a method for treating or preventing cancer comprises administering to a subject in need thereof a therapeutically effective amount of a biomimetic nanovesicle (BioNV) comprising a bispecific chimeric antigen receptor (CAR) targeting a first cell surface marker and a second cell surface marker, and a PD-1, PD-L1 and / or PD-L2 inhibitor.

[0017] In an embodiment, a method for treating or preventing cancer comprises administering a therapeutically effective amount of a biomimetic nanovesicle (BioNV) comprising a bispecific chimeric antigen receptor (CAR) targeting a first cell surface marker and a second cell surface marker to a subject in need thereof, wherein the subject is receiving treatment with a PD-1, PD-L1 and / or PD-L2 inhibitor.

[0018] In embodiments, the first cell surface marker and the second cell marker are selected from Table 1 and / or Table 2. In embodiments, the first cell surface marker is selected from Table 1 and / or Table 2. In embodiments, BioNV binds to at least a first cell and at least a second cell to initiate an anti-cancer response. In embodiments, the first cell is a cancer cell and the second cell is an immune cell. In embodiments, the PD-1 inhibitor is an antibody targeting PD-1, optionally pembrolizumab, nivolumab, or cemiplizumab. In embodiments, the PD-L1 and / or PD-L2 inhibitor is an antibody targeting PD-L1 and / or PD-L2, optionally atezolizumab, avelumab, or durvalumab.

[0019] In embodiments, BioNV is derived from modified cells. In embodiments, modified cells are stem cells, induced pluripotent stem cells (iPSC), reprogrammed pluripotent or multipotent cells, embryonic stem cells, mesenchymal stem cells, or differentiated cells derived from any modified cells thereof. In embodiments, modified cells are iPSC. In embodiments, modified cells are T cells, helper T cells, T memory cells, or NK cells. In embodiments, modified cells are macrophages. In embodiments, modified cells are monocytes. In embodiments, low immunogenic cells may be any terminally differentiated cells, such as, but not limited to, muscle cells (satellite cells), adipocytes, bone cells, cardiomyocytes, hepatocytes, blood cells (including erythrocytes, thrombocytes, and all immune cell types), glial cells (and other neuronal cell types), epithelial cells, epidermal cells, interstitial cells (e.g., respiratory interstitial cells), fibroblasts (e.g., dermal fibroblasts), endothelial cells (e.g., bronchial endothelial cells), oral cells, stromal cells, or germ cells. In an embodiment, the low immunogenic cells can be any function-specific cell type, such as but not limited to exocrine secretory epithelial cells, hormone-secreting cells (e.g., enteroendocrine cells, thyroid cells, pancreatic islet cells, etc.), sensory transduction cells, autonomic nerve cells, sensory organ cells (e.g., columnar cells, olfactory cells, Schwann cells, satellite glial cells, etc.), barrier cells (e.g., lung cells, ductal cells, kidney cells, podocytes, etc.), extracellular matrix cells (e.g., tendon fibroblasts, osteoblasts, connective tissue cells, etc.) or contractile cells (e.g., skeletal muscle cells, cardiomyocytes, myoepithelial cells, etc.).

[0020] In embodiments, the modified cells are substantially lacking one or more MHC class I proteins, MHC class II proteins, T cell receptor (TCR) proteins and / or cytokine release syndrome (CRS) proteins. In embodiments, the modified cells have reduced or eliminated β2-macroglobulin (B2M) gene expression and / or reduced or eliminated MHC class I protein expression and / or activity. In embodiments, the modified cells have reduced or eliminated CIITA gene expression and / or reduced or eliminated MHC class II protein expression and / or activity.

[0021] In embodiments, the modified cells have reduced or eliminated HLA-A gene expression and / or reduced or eliminated HLA-A protein expression and / or activity. In embodiments, the modified cells have reduced or eliminated HLA-B gene expression and / or reduced or eliminated HLA-B protein expression and / or activity. In embodiments, the modified cells have reduced or eliminated HLA-C gene expression and / or reduced or eliminated HLA-C protein expression and / or activity. In embodiments, the modified cells have reduced or eliminated HLA-E or HLA-G gene expression and / or reduced or eliminated HLA-E or HLA-G protein expression and / or activity. In embodiments, the modified cells have reduced or eliminated HLA-F gene expression and / or reduced or eliminated HLA-F protein expression and / or activity.

[0022] In embodiments, the modified cells have reduced or eliminated T cell alpha constant (TRAC) gene expression and / or reduced or eliminated TRAC protein expression and / or activity. In embodiments, the modified cells have reduced or eliminated T cell beta constant (TRBC) gene expression and / or reduced or eliminated TRBC protein expression and / or activity.

[0023] In embodiments, the modified cell has reduced or eliminated PD-1 gene expression and / or reduced or eliminated PD-1 protein expression and / or activity.

[0024] In embodiments, the modified cells have reduced or eliminated IL-4 gene expression and / or reduced or eliminated IL-4 protein expression and / or activity. In embodiments, the modified cells have reduced or eliminated IL-6 gene expression and / or reduced or eliminated IL-6 protein expression and / or activity. In embodiments, the modified cells have reduced or eliminated IL-10 gene expression and / or reduced or eliminated IL-10 protein expression and / or activity. In embodiments, the modified cells have reduced or eliminated IL-16 gene expression and / or reduced or eliminated IL-16 protein expression and / or activity.

[0025] In embodiments, the modified cell has reduced or eliminated SerpinB9 gene expression and / or reduced or eliminated SerpinB9 protein expression and / or activity.

[0026] In embodiments, the modified cells express CD34 genes and / or gene products, or increase their expression. In embodiments, the modified cells express CCL2 genes and / or gene products, or increase their expression. In embodiments, the modified cells express PD-L1 genes and / or gene products, or increase their expression, and wherein the modified cells are not activated. In embodiments, the modified cells have reduced or eliminated PD-L1 gene and / or gene product expression, and wherein the modified cells are activated. In embodiments, the modified cells overexpress FasL. In embodiments, the modified cells overexpress SerpinB9. In embodiments, the modified cells express H2-M3 genes and / or gene products, or increase their expression.

[0027] In an embodiment, the modified cell expresses the CD47 gene and / or gene product, or increases its expression. In an embodiment, the modified cell expresses the CD24 gene and / or gene product, or increases its expression. In an embodiment, the modified cell expresses a chimeric CD24 / CD47 gene and / or gene product, or increases its expression.

[0028] In an embodiment, the modified cell overexpresses CTLA-4. In an embodiment, the modified cell expresses a chimeric CD200 gene and / or gene product, or increases its expression. In an embodiment, the modified cell expresses a chimeric CD24 / CD200 gene and / or gene product or a chimeric CD47 / CD200 gene and / or gene product, or increases its expression.

[0029] In an embodiment, the modified cell expresses a chimeric MFG-E8 gene and / or gene product, or increases its expression. In an embodiment, the modified cell expresses a NCAM gene and / or gene product, or increases its expression. In an embodiment, the modified cell expresses a chimeric α-phagocytic integrin gene and / or gene product, or increases its expression. In an embodiment, the modified cell expresses an antibody or antibody-like molecule targeting an IL-6 surface receptor (anti-IL-6R), or increases its expression.

[0030] In embodiments, the modified cell expresses, or increases expression of, the FasL gene and / or gene product. In embodiments, the modified cell does not overexpress the FasL gene and / or gene product.

[0031] In embodiments, the expression and / or activity of 3 or more immunogenic proteins, 4 or more immunogenic proteins, 5 or more immunogenic proteins, 6 or more immunogenic proteins, 7 or more immunogenic proteins, 8 or more immunogenic proteins, 9 or more immunogenic proteins, 10 or more immunogenic proteins, 11 or more immunogenic proteins, or 12 or more immunogenic proteins of the modified cells are reduced or eliminated. In embodiments, the modified cells express 3 or more immunoprotective proteins, 4 or more immunoprotective proteins, 5 or more immunoprotective proteins, 6 or more immunoprotective proteins, 7 or more immunoprotective proteins, 8 or more immunoprotective proteins, 9 or more immunoprotective proteins, or 10 or more immunoprotective proteins, or increase their expression.

[0032] In embodiments, the modified cells are allogeneic.In embodiments, the modified cells do not elicit an immune response in a patient to whom they or a BioNV derived therefrom is administered.

[0033] In embodiments, the modified cells are differentiated prior to BioNV formation.

[0034] In embodiments, the modified cell expresses CAR under the control of an adjustable expression element. In embodiments, CAR is activated before BioNV is formed. In embodiments, CAR is activated via its target, by another receptor and / or virus.

[0035] In embodiments, BioNVs are formed from modified cells or differentiated cells thereof by sonication, adaptive focused acoustic technology, French press, extrusion, continuous extrusion, detergent cell lysis and / or electroporation. In embodiments, BioNVs are formed from modified cells or differentiated cells thereof by continuous extrusion.

[0036] In embodiments, the size of the BioNV is about 10 nm to about 1200 nm. In embodiments, the size of the BioNV is about 10 nm to about 100 nm. In embodiments, the size of the BioNV is about 100 nm to about 200 nm. In embodiments, the size of the BioNV is about 200 nm to about 500 nm. In embodiments, wherein the size of the BioNV is about 500 nm to about 1200 nm.

[0037] In embodiments, the BioNV substantially lacks proteins and / or activities of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, and one of HLA-E or HLA-G.

[0038] In embodiments, the BioNV substantially lacks proteins and / or activities of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, SerpinB9, and one of HLA-E or HLA-G.

[0039] In embodiments, the BioNV substantially lacks proteins and / or activities of one of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, SerpinB9, CD200, HLA-E, or HLA-G, and one or more of IL-4, IL-10, and IL-16.

[0040] In embodiments, the BioNV has membrane embedded α-phagocytic integrin, CCL2, H2-M3, FasL, MFG-E8, anti-IL-6R antibody or antibody form, and PD-L1 (in BioNV derived from an unactivated cell source) and / or CTLA-4, and one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or two of CD24, CD47, and CD200.

[0041] In embodiments, the BioNV has membrane embedded α-phagocytic integrin, CCL2, H2-M3, FasL, MFG-E8, anti-IL-6R antibody or antibody form, SerpinB9, and PD-L1 (in BioNV derived from an unactivated cell source) and / or CTLA-4, and one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or two of CD24, CD47, and CD200.

[0042] In embodiments, the BioNV has a membrane embedded CD200 protein and substantially lacks CD24 or CD47 protein. In embodiments, the BioNV substantially lacks SerpinB9 and CD200 protein and / or activity.

[0043] In an embodiment, the CAR comprises an antibody or antibody form selected from one or more of the following: a monoclonal antibody, a polyclonal antibody, an antibody fragment, a V NAR 、V HH, Fab, Fab', Fab'-SH, F(ab')2, Fv, single-chain Fv (scFv), double antibody, nano antibody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody or a fusion protein comprising an antigen-binding portion of an antibody. In embodiments, the antibody form is scFv. In embodiments, CAR comprises a transmembrane domain derived from CD28, CD3ζ, CD4, CD8α, ICOS or a fragment and / or combination thereof. In embodiments, CAR comprises an intracellular domain, which further comprises an intracellular signaling domain and / or one or more co-stimulatory molecules of a CD3ζ chain, optionally selected from CD28, 4-1BB, ICOS, CD27 and OX40. In embodiments, CAR targets cancer-specific antigens.

[0044] In embodiments, BioNV or modified cells derived therefrom comprise nucleic acids encoding green fluorescent protein (GFP) and / or GFP protein. In embodiments, nucleic acids encoding GFP are operably linked to promoters from one or more of the following: IL-2, perforin, granzyme, alarmin, TNF, INF, and / or combinations thereof.

[0045] In embodiments, BioNV encapsulates a payload. In embodiments, the payload is one or more of a gene editor, a cytotoxic protein, a biologic, a nucleic acid, a fusion protein, a fluorescent protein, a tracer dye, a radionuclide, and / or a small molecule. In embodiments, the payload is a therapeutic payload for the type of cancer targeted by CAR. In embodiments, the payload comprises an alkylating agent. In embodiments, the payload comprises an anthracycline. In embodiments, the payload comprises an antimetabolite. In embodiments, the payload comprises an antitumor antibiotic. In embodiments, the payload comprises an antitumor antibody or antibody form. In embodiments, the payload comprises a corticosteroid. In embodiments, the payload comprises a plant alkaloid. In embodiments, the payload comprises a topoisomerase inhibitor. In embodiments, the payload comprises a checkpoint inhibitor.

[0046] In an embodiment, the payload is selected from Abecma, Abemaciclib, Abiraterone Acetate, Abraxane, ABVD, ABVE, ABVE-PC, AC, Acalabrutinib, AC-T, Actemra, Adcetris, ADE, Ado-Trastuzumab Emtansine, Doxorubicin, Afatinib Dimaleate, Afinitor, Akynzeo, Aldara, Aldesleukin, Alecensa, Alectinib, Alemtuzumab, Alimta, Aliqopa, Alkeran for injection, Alkeran tablets, Aloxi, Alpelisib, Amber Alunbrig, Ameluz, Amifostine, Aminolevulinic Acid Hydrochloride, Amivantamab-vmjw, Anastrozole, Apalutamide, Aprepitant, Aranesp, Aredia, Arimidex, Aromasin, Arranon, Arsenic Trioxide, Arzerra, Asciminib Hydrochloride Hydrochloride, Asparaginase Erwinia Chrysanthemi, Asparlas, Atezolizumab, Avapritinib, Avastin, Avelumab, Axicabtagene Ciloleucel, Axitinib, Ayvakit, Azacitidine, Azedra, Balversa, Bavencio, BEACOPP, BelantamabMafodotin-blmf, Beleodaq, Belinostat, Belzutifan, Bendamustine Hydrochloride, Bendeka, BEP, Besponsa, Besremi, Bevacizumab, Bexarotene, Bicalutamide, BiCNU, Binimetinib, Blenrep, Bleomycin Sulfate, Blinatumomab, Blincyto, Bortezomib, Bosulif, Bosutinib, Braftovi, Brentuximab Vedotin), Brexucabtagene Autoleucel, Breyanzi, Brigatinib, Brukinsa, BuMel, Busulfan, Busulfex, Cabazitaxel, Cablivi, Cabometyx, Cabozantinib-S-Malate, CAF, Calaspargase Pegol-mknl, Calquence, Campath, Camptosar, Capecitabine, Caplacizumab-yhdp, CapmatinibHydrochloride), CAPOX, Carac, Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, Carmustine, Carmustine Implant, Casodex, CEM, Cemiplimab-rwlc, Ceritinib, Cerubidine, Cervarix (recombinant HPV bivalent vaccine), Cetuximab, CEV, Chlorambucil, CHLORAMBUCIL-PREDNISONE, CHOP, Cisplatin, Cladribine, Clofarabine, Clolar, CMF, Cobimetinib fumarate Fumarate, Cometriq, Copanlisib Hydrochloride, COPDAC, Copiktra, COPP, COPP-ABV, Cosmegen, Cotellic, Crizotinib, CVP, Cyclophosphamide, Cyramza, Cytarabine, Dabrafenib Mesylate, Dacarbazine, Dacogen, Dacomitinib, Dactinomycin, Danyelza, Daratumumab, Daratumumab and Hyaluronidase-fihj, Darbepoetin alfa Alfa, Darolutamide, Darzalex, DarzalexFaspro, Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposomal, Daurismo, Decitabine, Decitabine and Cedazuridine, Defibrotide Sodium, Defitelio, Degarelix, DenileukinDiftitox, Denosumab, Dexamethasone, Dexrazoxane Hydrochloride, Dinutuximab, Docetaxel, Dostarlimab-gxly, Doxil, Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposomal, Durvalumab, Duvelisib, Efudex, Eligard, Elitek, Ellence, Elotuzumab, Eloxatin, Eltrombopag Olamine、Elzonris、Emapalumab-lzsg、Emend、Empliciti、Enasidenib Mesylate、Encorafenib、Enfortumab Vedotin-ejfv、Enhertu、Entrectinib、Enzalutamide、Epirubicin Hydrochloride、EPOCH、Epoetin Alfa、Epogen、Erbitux、Erdafitinib、Eribulin Mesylate、Erivedge、Erleada、Erlotinib Hydrochloride Hydrochloride), Erwinaze, Ethyol, Etopophos, Etoposide, Etoposide phosphate, Everolimus, Evista, Evomela, Exemestane, Exkivity, 5-FU, 5-FU, Fam-Trastuzumab Deruxtecan-nxki, Fareston, Faslodex, FEC, Fedratinib hydrochlorideHydrochloride), Femara, Filgrastim, Firmagon, Fludarabine Phosphate, Fluoroplex, Fluorouracil Injection, Fluorouracil Topical, Flutamide, FOLFIRI, FOLFIRI-bevacizumab, FOLFIRI-cetuximab, FOLFIRINOX, FOLFOX, Folotyn, Fostamatinib Disodium, Fotivda, Fulphila, FU-LV, Fulvestrant, Fyarro, Gamifant, Gardasil (Recombinant HPV Quadrivalent Vaccine), Gardasil 9 (Recombinant HPV Nine-Valent Vaccine), Gavreto, Gazyva, Gefitinib, Gemcitabine Hydrochloride, Gemcitabine-Cisplatin, Gemcitabine-Oxaliplatin, Gemtuzumab Ozogamicin, Gemzar, Gilotri, Gilteritinib Fumarate, Glasdegib Maleate, Gleevec, Gliadel Tablets Wafer, Glucarpidase, Goserelin Acetate, Granisetron, Granisetron Hydrochloride, Granix, Halaven, Hemangeol, Herceptin Hylecta, Herceptin, Recombinant HPV Bivalent Vaccine, Recombinant HPV Nine-Valent Vaccine, Recombinant HPV Quadrivalent Vaccine, Hycamtin, Hydrea, Hydroxyurea, Hyper-CVAD, Ibrance, Ibritumomab Tiuxetan, Ibrutinib, ICE, Iclusig, Idamycin PFS, Idarubicin Hydrochloride, IdecabtageneVicleucel, Idelalisib, Idhifa, Ifex, Ifosfamide, Interleukin-2 (recombinant), Imatinib Mesylate, Imbruvica, Imfinzi, Imiquimod, Imlygic, Infigratinib Phosphate, Infugem, Inlyta, Inotuzumab Ozogamicin, Inqovi, Inrebic, Interferon alpha-2b, Intron A, Iobenguane I131, Ipilimumab, Iressa, Irinotecan hydrochloride Hydrochloride), Isatuximab-irfc, Istodax, Ivosidenib, Ixabepilone, Ixazomib Citrate, Ixempra, Jakafi, JEB, Jelmyto, Jemperli, Jevtana, Kadcyla, Kepivance, Keytruda, Kimmtrak, Kisqali, Koselugo, Kymriah, Kyprolis, Lanreotide Acetate, Lapatinib Dimesylate Ditosylate、Larotrectinib Sulfate、Lenalidomide、Lenvatinib Mesylate、Lenvima、Letrozole、Calcium Folinate、Leukeran、Leuprolide Acetate、Levulan Kerastik、Libtayo、Lisocabtagene Maraleucel、Lomustine、LoncastuximabTesirine-lpyl, Lonsurf, Lorbrena, Lorlatinib, Lumakras, Lumoxiti, LupronDepot, Lubinectedin, Luspatercept-aamt, Lutathera, Lu 177-Dotatate), Lynparza, Margenza, Margetuximab-cmkb, Marqibo, Matulane, Dichloroethylamine Hydrochloride, Megestrol Acetate, Mekinist, Mektovi, Melphalan, Melphalan Hydrochloride, Mercaptopurine, Mesna, Mesnex, Methotrexate Sodium, Methylnaltrexone Bromide, Midostaurin, Mitomycin, Mitoxantrone Hydrochloride, Mobocertinib Succinate Succinate), Mogamulizumab-kpkc, Monjuvi, MOPP, Moxetumomab Pasudotox-tdfk, Mozobil, MVAC, Mvasi, Myleran, Mylotarg, Nanoparticle Paclitaxel, Naxitamab-gqgk, Necitumumab, Nelarabine, Neratinib Maleate Maleate, Nerlynx, Netupitant, Neulasta, Neupogen, Nexavar, Nilandron, Nilotinib, Nilutamide, Ninlaro, Niraparib TosylateMonoHydrate, Nivestym, Nivolumab, Nplate, Nubeqa, Nyvepria, Obinutuzumab, Odomzo, OEPA, Ofatumumab, OFF, Olaparib, Omacetaxine Mepesuccinate, Oncaspar, Ondansetron Hydrochloride, Onivyde, Ontak, Onureg, Opdivo, OPPA, Orgovyx, Osimertinib Mesylate Mesylate, Oxaliplatin, Paclitaxel, Paclitaxel albumin-stabilized nanoparticle formulation, PAD, Padcev, Palbociclib, Palifermin, Palonosetron Hydrochloride, Pamidronate Disodium, Panitumumab, Paraplatin, Pazopanib Hydrochloride, PCV, PEB, Pegaspargase, Pegfilgrastim, Pegylated interferon alpha-2b, PEG-Intron, Pemazyre, Pembrolizumab, Pemetrexed Disodium, Pemigatinib, Perjeta, Pertuzumab, Pexidartinib Hydrochloride, Phesgo, Piqray, Plerixafor, Polatuzumab Vedotin-piiq, Polivy, Pomalidomide, Pomalyst, Ponatinib HydrochlorideHydrochloride, Portrazza, Poteligeo, Portrazza, Pralsetinib, Prednisone, Procarbazine Hydrochloride, Procrit, Proleukin, Prolia, Promacta, Propranolol Hydrochloride, Provenge, Purinethol, Purixan, Qinlock, Radium 223 Dichloride, Raloxifene Hydrochloride Hydrochloride), Ramucirumab, Rasburicase, Ravulizumab-cwvz, Reblozyl, R-CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nine-Valent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Interferon α-2b, Regorafenib, Relistor, Relugolix, R-EPOCH, Retacrit, Retevmo, Revlimid, Ribociclib, R-ICE, Ripretinib, Rituxan, Rituxan Hycela, Rituximab, Rolapitant Hydrochloride, Romidepsin, Romiplostim, Ropeginterferon Alfa-2b-njft, Rozlytrek, Rubidomycin, Rubraca, Rucaparib Camsylate, Ruxolitinib Phosphate, Rybrevant, Rydapt, Rylaze, SacituzumabGovitecan-hziy, Sancuso, Sarclisa, Sclerosol intrapleural aerosol, Selinexor, Selpercatinib, Selumetinib Sulfate, Scemblix, Siltuximab, Sipuleucel-T, Sirolimus protein-bound particles, Soltamox, Somatuline Depot, Sonidegib, Sorafenib Tosylate, Sotorasib, Sprycel, STANFORD sterile talc, Steritalc, Stivarga, Sunitinib malate Malate, Sustol, Sutent, Sylatron, Sylvant, Synribo, Tabloid, Tabecta, TAC, Tafasitamab-cxix, Tafinlar, Tagraxofusp-erzs, Tagrisso, Talazoparib Tosylate, Talimogene Laherparepvec, Talzenna, Tamoxifen Citrate, Tarceva, Targretin, Tasigna, Tavalisse, Taxotere, Tazemetostat Hydrobromide, Tazverik, Tebentafusp-tebn, Tecartus, Tecentriq, Temodar, Temozolomide, Temsirolimus, Tepadina, Tepmetko, Tepotinib hydrochlorideHydrochloride、Thalidomide、Thalomid、Thioguanine、Thiotepa、Tibsovo、Tisagenlecleucel、Tisotumab Vedotin-tftv、Tivdak、Tivozanib Hydrochloride、Tocilizumab、Tolak、Topotecan Hydrochloride、Toremifene、Torisel、Totect、TPF、Trabectedin、Trametinib Dimethyl Sulfoxide Sulfoxide, Trastuzumab, Trastuzumab and Hyaluronidase-Oysk, Treanda, Trexall, Trifluridine and Tipiracil Hydrochloride, Trisenox, Trodelvy, Truseltiq, Truxima, Tucatinib, Tukysa, Turalio, Tykerb, Ukoniq, Ultomiris, Umbralisib Mesylate Tosylate, Undencyca, Unituxin, Uridine triacetate, VAC, Valrubicin, Valstar, Vandetanib, VAMP, Varubi, Vectibix, VeIP, Velcade, Vemurafenib, Venclexta, Venetoclax, Verzenio, Vidaza, Vinblastine Sulfate, Vincristine SulfateSulfate, Vincristine Sulfate Liposomal, Vinorelbine Tartrate, VIP, Vismodegib, Vistogard, Vitrakvi, Vizimpro, Voraxaze, Vorinostat, Votrient, Vyxeos, Welireg, Xalkori, Xatmep, Xeloda, XELIRI, XELOX, Xgeva, Xofigo, Xospata, Xpovio, Xtandi, Yervoy, Yescarta , Yondelis, Yonsa, Zaltrap, Zanubrutinib, Zarxio, Zejula, Zelboraf, Zepzelca, Zevalin, Ziextenzo, Zinecard, Zirabev (bevacizumab), Ziv-Aflibercept, Zofran, Zoladex, Zoledronic Acid, Zolinza, Zometa, Zyclara, Zydelig, Zykadia, Zynlonta, and Zytiga.

[0047] In embodiments, the payload is one or more of: pidilizumab, BMS-936559, tremelimumab, AGEN1884, and / or RG2077.

[0048] In an embodiment, the nucleic acid molecule encodes one or more of the following: a CRISPR / Cas component, a guide RNA (gRNA), a tracer RNA (tracrRNA), a microRNA (miRNA), RNA interference (RNAi), a small interfering RNA (siRNA), a double-stranded RNA, a Piwi-interacting RNA (piRNA), a small nuclear RNA (snRNA), a small nucleolar RNA (snoRNA), an antisense oligonucleotide (ASO), a locked nucleic acid (LNA), a splice-switching oligonucleotide (SSO), a tRNA, a complementary messenger RNA, a repeat-associated small interfering RNA (rasiRNA) and / or a small non-coding RNA. In an embodiment, the gene editing payload is one or more of: TALEN, ZFN, ribonuclease P RNA, C2c1, C2c2, C2c3, Cas9, Cpf1, TevCas9, Archaea Cas9, CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, CasX Cas omega, transposase, and / or any ortholog or homolog thereof.

[0049] In embodiments, BioNV encapsulates one or more perforin molecules. In embodiments, BioNV encapsulates one or more granzyme molecules. In embodiments, the granzyme molecules are selected from granzymes A, B, H, K, and M. In embodiments, BioNV encapsulates one or more perforin molecules and / or one or more granzyme molecules derived from a cell from which the BioNV is derived. In embodiments, BioNV encapsulates one or more perforin molecules and one and / or more granzyme molecules exogenously added to the BioNV.

[0050] In embodiments, the method of treatment further comprises co-administering a whole cell therapy. In embodiments, the method of treatment further comprises administering an additional therapeutic agent.

[0051] In embodiments, the BioNV is stored at or suitable for storage at about -80° C. In embodiments, the BioNV is lyophilized.

[0052] In embodiments, the PD-1 inhibitor is an antibody targeting PD-1, or pembrolizumab, nivolumab, or cemiplimab. In embodiments, the PD-L1 or PD-L2 inhibitor is an antibody targeting PD-L1 or PD-L2, optionally atezolizumab, avelumab, or durvalumab, or an antigen binding domain thereof, and is bound to the surface of BioNV. In embodiments, the PD-1 inhibitor is an antibody targeting PD-1, or pembrolizumab, nivolumab, or cemiplimab. In embodiments, the PD-L1 or PD-L2 inhibitor is an antibody targeting PD-L1 or PD-L2, optionally atezolizumab, avelumab, or durvalumab, or an antigen binding domain thereof, and is membrane anchored by transmembrane domain fusion.

[0053] In embodiments, the cancer is a carcinoma. In embodiments, the cancer is a sarcoma. In embodiments, the cancer is a myeloma. In embodiments, the cancer is a leukemia. In embodiments, the cancer is a lymphoma. In embodiments, the cancer is a mixed cancer. In embodiments, the cancer is metastatic.

[0054] In embodiments, the cancer is selected from acute biphenotypic leukemia, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid dendritic cell leukemia, acute myeloid leukemia, lung adenocarcinoma, adrenal tumors, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphomas, alveolar soft tissue and cardiac sarcomas, amyloidosis, anal cancer, anaplastic large cell lymphoma, angioimmunoblastic T-cell lymphoma, appendix cancer, astrocytoma, ataxia telangiectasia, attenuated familial adenomatous polyposis, B-cell prolymphocytic leukemia, basal cell carcinoma, Beckwith-Wiedemann Syndrome, bile duct cancer, Birt-Hogg-Dubé Syndrome, bladder cancer, bone cancer, brain and nervous system cancers, brain stem glioma, brainstem glioma, Glioma), Brain Tumor, Breast Cancer, Bronchial Adenoma / Carcinoid, Burkitt's Lymphoma, Carcinoid Tumor, Carney Complex, Central Nervous System Tumor, Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Childhood Fibroproliferative Ganglioglioma, Bile Duct Cancer, Chondrosarcoma, Chronic Lymphocytic Leukemia, Chronic Myeloid Leukemia, Chronic Myeloid Leukemia, Chronic T-Cell Lymphocytic Leukemia, Colon Cancer, Colorectal Cancer, Cowden Syndrome, Craniopharyngioma, Cutaneous T-Cell Lymphoma, Dermatofibrosarcoma Protuberans, Fibroproliferative Small Round Cell Tumor, Diffuse Gastric Cancer, Diffuse Large B-Cell Lymphoma, Endocrine System Cancer, Endocrine Tumor, Endometrial Cancer, Eosinophilic Leukemia, Ependymoma, Epithelioid Hemangioendothelioma (EHE), Esophageal Cancer, Ewing Sarcoma Sarcoma), extragonadal germ cell tumors, extrahepatic bile duct cancer, eye cancer, eyelid cancer, fallopian tube cancer, familial adenomatous polyposis, familial malignant melanoma, familial clear cell renal cell carcinoma (RCC), follicular lymphoma, gallbladder cancer, Gardner Syndrome, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gastrointestinal stromal tumor (GIST), genitourinary and gynecological cancers, germ cell tumors, gestational trophoblastic disease, gestational trophoblastic tumor, glioblastoma, glioma, hairy cell leukemia, head and neck cancer, hematopoietic cancer, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, HIV-related cancers, Hodgkin's lymphoma Lymphoma, hypopharyngeal cancer, inflammatory breast cancer, intravascular large B-cell lymphoma, invasive cribriform carcinoma, invasive lobular carcinoma, islet cell carcinoma (endocrine pancreas), islet cell tumors, juvenile polyposis syndrome, Kaposi sarcomaSarcoma), keratoacanthoma, renal cancer, lacrimal gland tumors, large granular lymphocytic leukemia, laryngeal and hypopharyngeal cancer, leiomyomatosis and renal cell carcinoma, leiomyosarcoma, Li-Fraumeni Syndrome (LFS), liposarcoma, liver cancer, lung cancer, primary cutaneous lymphoma, lymphomatoid granuloma, lymphoplasmacytic lymphoma, Lynch Syndrome, malignant fibrous histiocytoma of bone, mantle cell lymphoma, marginal zone B-cell lymphoma, mast cell leukemia, mastocytosis, mediastinal large B-cell lymphoma, medullary carcinoma, medulloblastoma, melanoma, meningioma, Merkel Cell Carcinoma, mesothelioma, mixed polyposis syndrome, mucosa-associated lymphoid tissue lymphoma, Muir-Torre syndrome Syndrome; MTS), Multiple Endocrine Neoplasia Syndrome, Multiple Endocrine Neoplasia Type 1, Multiple Endocrine Neoplasia Type 2, Multiple Myeloma, Multiple Myeloma / Plasma Cell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndrome, MYH-Associated Polyposis, Myxosarcoma, Nasal and Paranasal Sinus Cancer, Wilms Tumor, Nasopharyngeal Carcinoma, Nasopharyngeal Carcinoma, Neuroblastoma, Neuroendocrine Tumors, Neurofibromatosis Type 1, Neurofibromatosis Type 2, Nevoid Basal Cell Carcinoma Syndrome, Nodal Marginal Zone B-Cell Lymphoma, Non-Hodgkin Lymphoma, Non-Small Cell Lung Cancer, Non-Small Cell Lung Cancer, Oligodendroglioma, Optic Pathway Glioma, Oral and / or Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Pancreatic Cancer, Papillary Renal Cell Carcinoma, Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Cancer, Pelvic Cancer, Penile Cancer, Peutz-Jeghers Syndrome Syndrome), pharyngeal cancer, pheochromocytoma, phyllodes tumor, pilocytic astrocytoma, pineal astrocytoma, pituitary adenoma, pituitary tumor, plasmablastic lymphoma, pleuropulmonary blastoma, precursor B-lymphoblastic leukemia, primary central nervous system lymphoma, primary cutaneous follicular lymphoma, primary cutaneous immunocytoma, primary effusion lymphoma, primitive neuroectodermal tumor, prostate cancer, rectal cancer, kidney cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, primary cutaneous sarcoma, sebaceous carcinoma, Sézary Syndrome, skin appendage tumor, skin cancer, small bowel cancer, small cell lung cancer, small intestinal cancerCancer), splenic marginal zone lymphoma, squamous cell carcinoma of the lung, squamous cell carcinoma, squamous cell skin cancer, gastric cancer, surface epithelial-stromal tumors, T-cell prolymphocytic leukemia, testicular cancer, thoracic and respiratory tract cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell carcinoma, transitional cell carcinoma adenoid cystic carcinoma, tuberous sclerosis complex, tubular carcinoma, Turcot syndrome, carcinoma of unknown primary, unclassified carcinoma, ureteral cancer, urethral cancer, uterine cancer, uterine sarcoma, uveal melanoma, vaginal cancer, visual pathway and hypothalamic glioma, von Hippel-Lindau (VHL) syndrome, vulvar cancer, Wilms tumor, and xeroderma pigmentosum.

[0055] In an embodiment, an allogeneic biomimetic nanovesicle (BioNV) is provided, comprising (a) a membrane-embedded chimeric antigen receptor (CAR) targeting a cell surface marker, and (b) a binding and / or membrane-anchored targeting agent targeting PD-L1 or PD-L2, wherein the agent targeting PD-L1 or PD-L2 is atezolizumab, avelumab, or durvalumab, and wherein the CAR does not target PD-1, PD-L1, or PD-L2.

[0056] In an embodiment, an allogeneic biomimetic nanovesicle (BioNV) is provided, comprising (a) a first membrane-embedded chimeric antigen receptor (CAR) targeting a cell surface, wherein the first membrane-embedded CAR does not target PD-L1 or PD-L2, and (b) at least a second membrane-embedded CAR targeting PD-L1 or PD-L2.

[0057] In an embodiment, an allogeneic biomimetic nanovesicle (BioNV) is provided, comprising a bispecific, membrane-embedded chimeric antigen receptor (CAR), wherein the chimeric antigen receptor targets (a) a cell surface marker of a cancer cell; and (b) PD-L1 or PD-L2, and wherein the cell surface marker of the cancer cell is not PD-1, PD-L1 or PD-L2.

[0058] Description of the drawings

[0059] Figures 1A-1C A non-limiting illustration of T cells binding to normal cells is depicted. Figure 1A Depicted is the interaction of PD-1 on T cells (left) and PD-L1 / 2 expressed on healthy cells (right) to prevent an immune response. Figure 1B Depicted is the blocking of PD-1 on T cells by pembrolizumab or nivolumab, thereby preventing its interaction with PD-L1 / 2 on cancer cells (upregulated) and healthy cells. An immune response can then be initiated and the cancer cells can be eliminated; however, there may be off-target effects on healthy cells. Figure 1CDepicting overexpression of PD-L1 on cancer cells compared to healthy cells. Overexpression of PD-1 on cells protects them from immune attack, leading to T cell exhaustion.

[0060] Figure 2A-2G Non-limiting illustrations of various BioNVs with tumor-targeting CARs and anti-PD-L1 / 2 integral membrane antibodies and / or bispecific CARs are depicted. Figure 2A Depicted is a BioNV with a tumor-targeting CAR and an anti-PD-L1 / 2 integral membrane antibody that targets tumor cells. Figure 2B BioNVs with tumor-targeted anti-PD-L1-specific CAR or anti-PD-L1 / 2 bispecific CAR are depicted. Figure 2C Combination treatment of BioNV with tumor-targeted anti-PD-L1-specific CAR or anti-PD-L1 / 2 bispecific CAR with pembrolizumab or nivolumab is depicted. Figure 2D Combination treatment of BioNV with tumor-targeting CAR with pembrolizumab or nivolumab is depicted. Figure 2E Combination therapy of BioNV with tumor-targeting bispecific CAR with pembrolizumab or nivolumab is depicted. Figure 2F Combination treatment of BioNV with tumor-targeting CAR with pembrolizumab or nivolumab is depicted. Figure 2G Combination therapy of BioNV with tumor-targeting bispecific CAR with pembrolizumab or nivolumab is depicted.

[0061] Figure 3 A non-limiting schematic diagram of continuous extrusion to produce BioNV is depicted. DETAILED DESCRIPTION

[0062] This disclosure relates in part to allogeneic, low immunogenic cell-derived biomimetic nanovesicles (BioNV), which can be used to treat cancer by one or more surface-oriented chimeric antigen receptors (CARs), which recognize a single target or multiple targets (i.e., bispecific) by the binding portion of the desired biomarker / ligand. In an embodiment, the size of the designed BioNV is about 20-1200nm, which is much smaller than the size of traditional cell-based CAR-T / NK cell therapy. BioNV can be derived from cells, such as stem cells, iPSCs, reprogrammed pluripotent or multipotent cells, embryonic stem cells, mesenchymal stem cells, or differentiated cells from any stem cells. BioNV can also be derived from T cells, NK cells, macrophages, monocytes, etc. The plasma membrane-derived BioNV retains the low immunogenicity properties of the modified cells from which it is derived. The modified cells can be genetically engineered with a focus on reducing or eliminating (e.g., knocking out) immunogenic cell surface markers or immunogenic molecules (e.g., MHC class I / II, HLA, T cell receptor (TCR), cytokine release syndrome (CRS), etc.) and / or expressing or overexpressing immune protective cell surface markers (e.g., CD47, CD34, CD24, CD200, α-phagocytes, etc.). The binding portion of the CAR may include all variants of the antibody construct, including, for example, Fab, Fab', Fab'-SH, F(ab')2, scFv, diabodies, nanobodies, linear antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, and fusion proteins comprising an antibody antigen binding portion, V H H nanobody, V NARS and other antibody formats, thereby allowing BioNV to target any cancer cell of interest (targeting any type of cell surface biomarker). BioNV can also encapsulate any selected cytotoxic protein, small molecule, biologic, nucleic acid, gene editing therapeutic payload, etc. and deliver it to the intended cellular target for treating cancer. BioNV can include membrane-anchored and / or bound anti-cancer antibodies (e.g., pembrolizumab or nivolumab).

[0063] In addition, the present disclosure relates in part to methods of treating and / or preventing cancer by administering a combination of BioNV and a monospecific and / or bispecific CAR (e.g., a biomarker and anti-PD-L1 or anti-PD-L2), such as Figure 2B In embodiments, the treatment method involves targeting cancer cells and providing more specific PD-L1 blockade. In embodiments, BioNV can be administered with pembrolizumab or nivolumab, such as Figure 2C-2G In an embodiment, BioNV may include a tumor-targeted anti-PD-L1 specific or anti-PD-L1 / 2 bispecific CAR ( Figure 2B-2CIn embodiments, the therapeutic methods utilize BioNV ( ) having a monospecific and / or bispecific CAR and a binding and / or membrane-anchored antibody (e.g., pembrolizumab or nivolumab). Figure 2A In the embodiment, the method adopts Figure 2A , Figure 2B , Figure 2C , Figure 2D , Figure 2E , Figure 2F and / or Figure 2G The constructs or combination therapies indicated.

[0064] In the embodiments, anti-PD-1 molecules are not desired on BioNV because this may transfer CAR from cancer cells to T cells and / or potentially damage or kill T cells. However, in the embodiments, anti-PD-1 may be included as a whole protein construct or a minimal PD-L1 / PD-L2 interaction domain of PD-1. In the embodiments, it is designed to have a transmembrane domain so as to be integrated into the BioNV lipid layer. In the embodiments, CAR (monospecific or bispecific) can be rapidly changed to target mutants when mutants appear. The variability of biomarkers may be due to DNA repair mechanisms and drug selection and / or immune selection pressure in the host. In the embodiments, the treatment method uses BioNV derived from cytotoxic cells, thereby allowing BioNV to deliver anti-cancer molecules (e.g., cytokines, perforins, granzymes, etc.) to tumors in a more controllable manner than whole cells. In the embodiments, these molecules can be released from BioNV and show greatly improved penetration into TME compared to traditional LNPs or whole cells. In the embodiment, the scFV region of the CAR targets PD-L1 or PD-L2, while another scFV region (on the same CAR) recognizes a specific marker unique to cancer, allowing targeted cells to release perforin / granzymes to kill cells and block PD-L1 / PD-L2 of the PD-1 receptor on host T cells, thereby preventing cancer cells from escaping immunity. In the embodiment, the co-administration of pembrolizumab or nivolumab can bind to PD-1 on T cells, further enhancing the effect.

[0065] Methods for treating, preventing and / or ameliorating cancer

[0066] In some aspects, the present disclosure includes a method for treating, preventing and / or ameliorating cancer, comprising administering to a subject in need thereof (i) a therapeutically effective amount of a biomimetic nanovesicle (BioNV) comprising a membrane-embedded chimeric antigen receptor (CAR) targeting a cell surface marker, and (b) a PD-1, PD-L1 and / or PD-L2 inhibitor; or (ii) a therapeutically effective amount of a BioNV comprising a membrane-embedded CAR targeting a cell surface marker, wherein the subject is being treated with a PD-1, PD-L1 and / or PD-L2 inhibitor.

[0067] In some aspects, the present disclosure includes methods for treating, preventing and / or ameliorating cancer, comprising administering to a subject in need thereof (i) a therapeutically effective amount of BioNV comprising (a) a membrane-embedded CAR targeting a cell surface marker, and (b) a conjugated and / or membrane-anchored PD-L1 and / or PD-L2 inhibitor, or (ii) a therapeutically effective amount of BioNV comprising (a) a membrane-embedded bispecific CAR targeting a first cell surface marker and a second cell surface marker, and (b) a conjugated and / or membrane-anchored PD-L1 and / or PD-L2 inhibitor.

[0068] In some aspects, the present disclosure includes a method for treating, preventing and / or ameliorating cancer, the method comprising administering to a subject in need thereof (i) a therapeutically effective amount of BioNV comprising a bispecific CAR targeting a first cell surface marker and PD-L1 or PD-L2, wherein the first cell surface marker is not PD-1, PD-L1 or PD-L2; or (ii) a therapeutically effective amount of BioNV comprising (a) a bispecific CAR targeting a first cell surface marker and PD-L1 or PD-L2, wherein the first cell surface marker is not PD-1, PD-L1 or PD-L2, and (b) a PD-1, PD-L1 and / or PD-L2 inhibitor; or (iii) a therapeutically effective amount of BioNV comprising a bispecific CAR targeting PD-L1 or PD-L2 and a first cell surface marker, wherein the subject is receiving treatment with a PD-1, PD-L1 and / or PD-L2 inhibitor, and wherein the first cell surface marker is not PD-1, PD-L1 or PD-L2.

[0069] In some aspects, the present disclosure includes a method for treating, preventing and / or ameliorating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of BioNV comprising (a) a bispecific CAR targeting a first cell surface marker and a second cell surface marker, and (b) a PD-1, PD-L1 and / or PD-L2 inhibitor.

[0070] In some aspects, the present disclosure includes a method for treating, preventing and / or ameliorating cancer, comprising administering a therapeutically effective amount of BioNV comprising a bispecific CAR targeting a first cell surface marker and a second cell surface marker to a subject in need thereof, wherein the subject is receiving treatment with a PD-1, PD-L1 and / or PD-L2 inhibitor.

[0071] In embodiments, the CAR (monospecific or bispecific) targets a first cell surface marker and / or a second cell marker selected from Table 1 and / or Table 2. In embodiments, the bispecific CAR targets a first cell surface marker selected from Table 1 or Table 2 and PD-L1 or PD-L2.

[0072] Table 1. Illustrative checkpoint biomarkers and checkpoint biomarker ligands.

[0073]

[0074]

[0075] Table 2. Illustrative cancer cell biomarkers.

[0076]

[0077]

[0078]

[0079] In an embodiment, two or more biomarkers (or two or more epitopes of a single biomarker) can be targeted by BioNV with bispecific CAR or multiple monospecific CAR. In an embodiment, some biomarkers are present in various types of cancers, and BioNV can target various cancer types. In an embodiment, if resistance is produced to the main biomarker target on the cancer cell, a mutation biomarker can be selected, for example, because the biomarker on the cancer cell has a tendency to mutate or be suppressed. In an embodiment, the CAR construct (all its variants) can be changed to match the mutation. In an embodiment, the mutation present in cancer is predictable, so the construct can be prepared in advance, for example, SCN4A mutation can be used as a predictor of the treatment regimen of checkpoint inhibitors (Lin et al., "Potential Predictive Value of SCN4A Mutation Status for Immune Checkpoint Inhibitors in Melanoma." Biomedicine & Pharmacotherapy. 2020). In an embodiment, secondary biomarkers can be targeted in subsequent treatments, for example, a study of CD-22 in cells that have lost CD-19 expression has also been conducted.

[0080] In embodiments, BioNV binds to at least a first cell and at least a second cell to initiate an anti-cancer response. In embodiments, the first cell is a cancer cell and the second cell is an immune cell. In embodiments, the first cell and the second cell can both be the same or similar cell type (e.g., both are tumor cells).

[0081] In an embodiment, the PD-1 inhibitor is an antibody targeting PD-1, or pembrolizumab, nivolumab, or cemiplizumab. In an embodiment, the PD-L1 and / or PD-L2 inhibitor is atezolizumab, avelumab, or durvalumab.

[0082] In embodiments, BioNV is derived from modified cells, such as, but not limited to, stem cells, induced pluripotent stem cells (iPSCs), reprogrammed pluripotent or multipotent cells, embryonic stem cells, mesenchymal stem cells, or differentiated cells derived from any modified cells thereof. In embodiments, the modified cells are iPSCs. In embodiments, the modified cells are T cells, helper T cells, T memory cells, or NK cells. In embodiments, the modified cells are macrophages. In embodiments, the modified cells are monocytes.

[0083] In embodiments, the allogeneic and low immunogenic properties of modified cells (e.g., derived from iPSC) are produced by knocking out, silencing, inactivating, blocking or otherwise negating the expression, transcription efficiency and / or activity of one or more immunogenic molecules. In embodiments, the modified cells are substantially lacking one or more MHC class I proteins, MHC class II proteins, HLA proteins, TCR proteins and / or CRS proteins.

[0084] In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes β2-macroglobulin (B2M) gene disruption and / or reducing or eliminating the destruction of MHC I class protein expression and / or activity, such as in the case of CD8+T cell lineage. In embodiments, reducing or eliminating the expression and / or activity of one or more immunogenic proteins includes CIITA gene disruption and / or reducing or eliminating the destruction of MHC II class protein expression and / or activity, such as in the case of CD4+T cell lineage. Without wishing to be bound by theory, these proteins contribute to human leukocyte antigen (HLA) immunogenicity, which requires HLA allele matching in donor-recipient to be treated by cell-based therapy. In embodiments, allogeneic and / or low immunogenic properties are achieved by reducing or eliminating the expression and / or activity of genes encoding T cell receptor (TCR) proteins, including, for example, α and β chains (such as αβT cells) or γ and δ chains (such as γδT cells) that form ligand binding sites, and signaling modules CD3δ, CD3γ, CD3ε, and CD3ζ. In embodiments, this is done to reduce foreign T cell receptor types other than those of the CAR box, and further improve the homogeneity of the CAR of interest and reduce off-target effects in BioNV formation.

[0085] In embodiments, the modified cells include a β2-macroglobulin (B2M) gene disruption, or a gene disruption that disrupts MHC class I expression. In embodiments, knocking out the B2M gene reduces the number of potential doses that need to be administered because there is a risk of preventing the recipient from receiving BioNV for a long time, such as that observed in the above-mentioned whole cell-based methods. To address this problem, in embodiments, the HLA-E or HLA-G gene remains intact, so that the immune system can adapt to the produced BioNV. In embodiments, HLA-A, HLA-B, HLA-C, HLA-F, HLA-E, or HLA-G (but not both) are knocked out in sequence.

[0086] In embodiments, the modified cells have reduced or eliminated HLA-A gene expression and / or reduced or eliminated HLA-A protein expression and / or activity. In embodiments, the modified cells have reduced or eliminated HLA-B gene expression and / or reduced or eliminated HLA-B protein expression and / or activity. In embodiments, the modified cells have reduced or eliminated HLA-C gene expression and / or reduced or eliminated HLA-C protein expression and / or activity. In embodiments, wherein the modified cells have reduced or eliminated HLA-E or HLA-G gene expression and / or reduced or eliminated HLA-E or HLA-G protein expression and / or activity. In embodiments, the modified cells have reduced or eliminated HLA-F gene expression and / or reduced or eliminated HLA-F protein expression and / or activity.

[0087] In embodiments, modified cells have reduced or eliminated CIITA gene expression and / or reduced or eliminated MHC II class protein expression and / or activity.In embodiments, the MHC class I and MHC class II complexes of allogeneic iPSC are destroyed by knocking out the key proteins involved in its expression, such as β2-macroglobulin (B2M), which is a serum protein, found to be related to the MHC class I heavy chains on the surface of almost all nucleated cells, involved in presenting peptide antigens to the immune system.In embodiments, the CIITA gene of allogeneic iPSC is destroyed, and the gene is the main control transcription factor regulating the expression of all MHC II genes.In embodiments, the CIITA gene of allogeneic iPSC is destroyed, and the gene is the main control transcription factor regulating the expression of all MHC II genes, so that the resulting differentiated cell line (e.g., DC, mononuclear phagocytes, endothelial cells, thymic epithelial cells, B cells, etc.) does not express MHC II class proteins or its expression is reduced.

[0088] In embodiments, the modified cells have reduced or eliminated T cell alpha constant (TRAC) gene expression and / or reduced or eliminated TRAC protein expression and / or activity. In embodiments, the modified cells have reduced or eliminated T cell beta constant (TRBC) gene expression and / or reduced or eliminated TRBC protein expression and / or activity.

[0089] In embodiments, the modified cell has reduced or eliminated PD-1 gene expression and / or reduced or eliminated PD-1 protein expression and / or activity.

[0090] CRS is a major problem with whole cell therapies, and despite the presence of engineered low immunogenicity, the effector functions and other consequences of interaction with cells after infusion may lead to the release of biomolecules, resulting in systemic inflammatory syndrome characterized by fever, multiple organ dysfunction, etc. In embodiments, modified cells are engineered to destroy one or more proteins that cause CRS. In embodiments, modified cells have reduced or eliminated expression and / or activity (e.g., knockout or silencing) of CRS-related cytokines.

[0091] In embodiments, modified cells have reduced or eliminated IL-4 gene expression and / or reduced or eliminated IL-4 protein expression and / or activity. In embodiments, modified cells have reduced or eliminated IL-6 gene expression and / or reduced or eliminated IL-6 protein expression and / or activity. In embodiments, IL-6 knockouts prevent undesirable IL-6 from being packaged into BioNV, and reduce the contribution of BioNV to local (and concentrated due to biomarker targeting) and / or potential systemic CRS events. In embodiments, modified cells have reduced or eliminated IL-10 gene expression and / or reduced or eliminated IL-10 protein expression and / or activity. In embodiments, modified cells have reduced or eliminated IL-16 gene expression and / or reduced or eliminated IL-16 protein expression and / or activity. In embodiments, the reduction or elimination of interleukins reduces the possibility of CRS.

[0092] SerpinB9 is a member of the serine protease inhibitor superfamily. SerpinB9 is reported to protect cells from the immunological killing effect of granzyme B. In embodiments, for example, when BioNV is not designed to deliver granzymes, the modified cells from which BioNV is derived express SerpinB9 or increase its expression. In embodiments, for example, when BioNV is designed to deliver granzymes, the modified cells from which BioNV is derived knock out and / or silence SerpinB9. In embodiments, the modified cells have reduced or eliminated SerpinB9 gene expression and / or activity, and / or reduced or eliminated SerpinB9 protein expression and / or activity.

[0093] In embodiments, overexpression of SerpinB9 isolates the function of granzyme B associated with immune stimulation responses (e.g., apoptosis of target cells and / or diseased cells). In embodiments, granzyme B is inhibited in activated lymphocytes, NK cells, macrophages, and follicular DCs, as well as other cell types. In embodiments, for example, when BioNV is intended to deliver a non-granzyme payload (e.g., a gene editing payload), the modified cells may express SerpinB9 and / or increase its expression.

[0094] In embodiments, methods for treating, preventing and / or ameliorating cancer use BioNVs derived from cells modified to be low immunogenic due to expression of one or more immunoprotective proteins or increased expression thereof. In embodiments, modified cells express CD34 genes and / or gene products, or increase their expression. In embodiments, modified cells express CCL2 genes and / or gene products, or increase their expression. In embodiments, modified cells express PD-L1 genes and / or gene products, or increase their expression, wherein the modified cells are not activated, for example, and wherein the resulting BioNVs do not encapsulate perforin and / or granzymes. In embodiments, modified cells have reduced or eliminated expression of PD-L1 genes and / or gene products, and wherein the modified cells are activated, for example, and wherein the resulting BioNVs encapsulate perforin and / or granzymes. In embodiments, modified cells express H2-M3 genes and / or gene products, or increase their expression.

[0095] In an embodiment, the modified cells express CD47 genes and / or gene products, or increase their expression. Exosomes and cell-derived vesicles (CDV) are easily removed from the body by macrophages through phagocytosis. Phagocytosis greatly affects the therapeutic value and efficacy of CDV. In order to prevent the consumption of macrophages of BioNV, but not wishing to be bound by theory, in an embodiment, BioNV carries a CD47 tag on the surface. CD47tg (tag) sends a "don't eat me" signal, which, in an embodiment, increases the half-life and serum stability of BioNV in the subject. In an embodiment, the molecule CD47 isoform 2 (an isoform that interacts with SIRPα receptors on macrophages) is engineered into modified cells (e.g., iPSC cell lines). Without CD47tg, the half-life of BioNV is shortened due to phagocytosis inhibition, resulting in the need for higher and / or more frequent doses. In embodiments, the potential inhibitory phenotype of CD47 expression across cells is prevented by interfering with the inhibitory action mechanism of a series of microRNAs on the 3'UTR of the CD47 gene, either by deleting this region in a stable construct or by eliminating / inhibiting the expression of microRNAs. In embodiments, this can address the problem of inhibition caused by microRNAs across differentiated cell subsets.

[0096] In an embodiment, the modified cell overexpresses CD24. CD24 is a sialoglycoprotein expressed on mature granulocytes and B cells, and is also an antiphagocytic protein. CD24 prevents phagocytosis by interacting with Siglec-G / 10 on macrophages. In an embodiment, the modified cell expresses the CD24 gene and / or gene product, or increases its expression. In an embodiment, the modified cell expresses a chimeric CD24 / CD47 gene and / or gene product, or increases its expression. In an embodiment, the modified cell expresses a chimeric CD24 / CD47 with a tethered transmembrane domain. In an embodiment, the domains of CD47 isoform 2 and CD24 can be expressed separately or tethered to form a bilobed chimeric protein. In an embodiment, the modified cell is an iPSC from fibroblasts rather than ABO cells.

[0097] In an embodiment, the modified cell expresses a chimeric CD200 gene and / or gene product, or increases its expression. In an embodiment, the CD200 tag minimizes phagocytosis of macrophages and prevents activation of granulocytes. In an embodiment, when it is not desired to prevent granulocytes, such as in the TME, the modified cell does not express CD200, because the activation of granulocytes will supplement the mechanism of action of BioNV intended to release granzymes and perforins. However, in an embodiment, if a CD47 or CD24 tag is used, or a combination of a CD24 / CD47 chimeric bilobed protein tag (each preventing phagocytosis) and overexpressed H2-M3 (inhibiting NK reactions) is used, stability can be achieved without CD200 while allowing sufficient BioNV clearance. In embodiments where granzymes and perforins are not preferred therapeutic biomolecules, CD200 can be expressed to prevent activation of granulocytes while eliminating the CD47 tag or the CD24 tag, but not both tags. In embodiments, the modified cells express, or increase expression of, chimeric CD24 / CD200 genes and / or gene products or chimeric CD47 / CD200 genes and / or gene products.

[0098] In an embodiment, the modified cell (or a cell differentiated therefrom) does not express all three of CD47, CD24, and CD200. In an embodiment, the modified cell from which the BioNV is derived is engineered so that the BioNV is stable, but not to the extent that the BioNV is difficult to clear from the body. An overly stable BioNV may eventually trigger a humoral response, thereby limiting the dose or number of treatments that can be administered.

[0099] In an embodiment, the modified cell expresses a chimeric CTLA-4 gene and / or gene product, or increases its expression. In an embodiment, the modified cell expresses a chimeric MFG-E8 gene and / or gene product, or increases its expression. In an embodiment, the modified cell expresses a NCAM gene and / or gene product, or increases its expression. In an embodiment, the modified cell expresses a chimeric α-phagocytic integrin gene and / or gene product, or increases its expression.

[0100] In an embodiment, the modified cell expresses an antibody or antibody-like molecule (anti-IL-6R) targeting an IL-6 surface receptor, or increases its expression. In an embodiment, the iPSC cell line used to generate the anti-cancer BioNV has an anti-IL-6R antibody engineered into the cell line. In an embodiment, the BioNV carries α-IL-6R, thereby preventing the signaling pathway on local immune cells in the tumor environment from being activated.

[0101] In embodiments, the modified cells express the FasL gene and / or gene product, or increase its expression. In embodiments, the modified cells do not overexpress the FasL gene and / or gene product. In embodiments, overexpression of FasL in the modified cells is avoided because enrichment of the natural expression level of FasL is observed in the membrane of BioNV after processing (e.g., by continuous extrusion). Too high a concentration of FasL may be counterproductive and prevent T cell recruitment to solid tumors and / or cause premature death of T cells.

[0102] In embodiments, the modified cells may express one or more fusion proteins of one or more portions of any of the immunoprotective proteins described herein. For example, in embodiments, constructs may be prepared in which appropriate portions of a selected ligand are tethered to a transmembrane domain, such that the entire protein (e.g., an intracellular signaling domain) is not necessary. In embodiments, constructs may be prepared in which biologically relevant portions of two or more proteins are tethered together and / or tethered to a transmembrane domain.

[0103] In embodiments, the expression and / or activity of one or more immunogenic proteins of the modified cells is reduced or eliminated, e.g., proteins that cause an immune response in a subject, donor-recipient mismatch, HLA alloimmunization, inflammation, CRS, etc., e.g., MHC class I proteins, MHC class II proteins, HLA proteins, TCR proteins, CRS proteins, etc. In embodiments, the expression and / or activity of 3 or more immunogenic proteins, 4 or more immunogenic proteins, 5 or more immunogenic proteins, 6 or more immunogenic proteins, 7 or more immunogenic proteins, 8 or more immunogenic proteins, 9 or more immunogenic proteins, 10 or more immunogenic proteins, 11 or more immunogenic proteins, or 12 or more immunogenic proteins of the modified cells is reduced or eliminated.

[0104] In embodiments, the modified cells have expression or increased expression and / or activity of one or more immunoprotective proteins, e.g., proteins that result in preventing or reducing an immune response in a subject, preventing or reducing premature clearance of a subject's BioNV, preventing or reducing phagocytosis, conferring barrier crossing function, etc., e.g., CD47, CD24, CD200, CD34, CCL2, H2-M3, MFG-E8, PD-L1 (for non-activated cells), CTLA-4, etc. In embodiments, the modified cells express 3 or more immunoprotective proteins, 4 or more immunoprotective proteins, 5 or more immunoprotective proteins, 6 or more immunoprotective proteins, 7 or more immunoprotective proteins, 8 or more immunoprotective proteins, 9 or more immunoprotective proteins, or 10 or more immunoprotective proteins, or increase their expression.

[0105] In embodiments, "increased expression" and "increased expression and / or activity" as used herein refers to increased expression and / or activity in low immunogenic cells and / or resultant BioNVs compared to native or wild-type homologous cells. For example, in embodiments, increased expression and / or activity of one or more biomolecules described herein can confer low immunogenicity to iPSCs relative to iPSCs that do not have the same protein expression pattern or expression level. In embodiments, "increased expression" is due to a genetic modification, such as a knock-in.

[0106] In embodiments, the modified cells are allogeneic.In embodiments, the modified cells do not elicit an immune response in a patient to whom they or a BioNV derived therefrom is administered.

[0107] In embodiments, before making BioNV, the modified cells undergo specific processing, such as activation of modified cells (e.g., by TCR / CD3, CD28, etc. to increase the expression of granzymes, perforins, etc.), differentiation of modified cells (e.g., differentiation from iPSC into T cells, NK cells, macrophages, etc.) and / or activation of CAR. In embodiments, the modified cells differentiate before BioNV is formed. In embodiments, the modified cells express CAR through adjustable expression elements. In embodiments, CAR is activated before BioNV is formed. In embodiments, CAR is activated via its target, by another receptor and / or virus. In embodiments, the modified cells are amplified after engineering; any small-scale amplification or large-scale feeding system amplification method known in the art can be used.

[0108] In embodiments, the type of cancer to be targeted drives differentiation into a given cell line. In embodiments, if brain cancer is targeted, the BioNV may be derived from activated macrophages / monocytes. In embodiments, the BioNV inherits some phenotypic properties from the cells from which it is derived (its parent cells). For example, in embodiments, macrophages / monocytes easily cross the BBB, and this property (which may be membrane receptor driven) may be transferred to the BioNV. In embodiments, the BioNV may be generated from tumor infiltrating lymphocytes (TILs) of dense tumors.

[0109] In embodiments, BioNVs are formed from modified cells or differentiated cells thereof (e.g., after activation) by sonication, adaptive focused acoustic technology, French press, extrusion, continuous extrusion, detergent cell lysis, enzymatic disruption of cells (e.g., trypsinization), and / or electroporation. In embodiments, BioNVs are formed from modified cells or differentiated cells thereof by continuous extrusion.

[0110] In embodiments, the size of the BioNV is about 10 nm to 1200 nm. In embodiments, the size of the BioNV is about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 120 nm, about 140 nm, about 160 nm, about 180 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, about 1000 nm, about 1100 nm, or about 1200 nm. In embodiments, the size range of the BioNV is about 10 nm to 100 nm, about 100 nm to 200 nm, about 200 nm to 500 nm, or about 500 nm to 1200 nm.

[0111] In embodiments, in the methods of treating, preventing and / or ameliorating cancer described herein, the size of the BioNVs is matched to the cancer type, tumor type, disease progression (e.g., metastasis, spread, pathology, etc.), tissue type, etc. In embodiments, methods of treating very dense / sticky tumor types (e.g., retinoblastoma) utilize BioNVs of small size (e.g., less than about 500 nm in size, less than about 400 nm in size, less than about 300 nm in size, less than about 200 nm in size, less than about 100 nm in size, or less than about 50 nm in size) to achieve higher permeability. In embodiments, methods of treating loose tumor types (e.g., lung cancer) utilize larger BioNVs (e.g., greater than about 500 nm in size, greater than about 600 nm in size, greater than about 700 nm in size, greater than about 800 nm in size, greater than about 900 nm in size, or greater than about 1000 nm in size).

[0112] In embodiments, the BioNV substantially lacks proteins and / or activities of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, and one of HLA-E or HLA-G.

[0113] In embodiments, the BioNV substantially lacks proteins and / or activities of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, SerpinB9, and one of HLA-E or HLA-G.

[0114] In embodiments, the BioNV substantially lacks proteins and / or activities of one of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, SerpinB9, CD200, HLA-E, or HLA-G, and one or more of IL-4, IL-10, and IL-16.

[0115] In embodiments, the BioNV has membrane embedded α-phagocytic integrin, CCL2, H2-M3, FasL, MFG-E8, anti-IL-6R antibody or antibody form, and PD-L1 (in BioNV from non-activated cells) and / or CTLA-4, and one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or two of CD24, CD47, and CD200.

[0116] In embodiments, the BioNV has membrane embedded α-phagocytic integrin, CCL2, H2-M3, FasL, MFG-E8, anti-IL-6R antibody or antibody form, SerpinB9, and PD-L1 (in BioNV from non-activated cells) and / or CTLA-4, and one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or two of CD24, CD47, and CD200.

[0117] In embodiments, the BioNV has a membrane embedded CD200 protein and substantially lacks CD24 or CD47 protein. In embodiments, the BioNV substantially lacks SerpinB9 and CD200 protein and / or activity.

[0118] In embodiments, BioNV CAR comprises an antibody or antibody form selected from one or more of the following: monoclonal antibody, polyclonal antibody, antibody fragment, VNAR, VHH, Fab, Fab', Fab'-SH, F(ab')2, Fv, single-chain Fv (scFv), double antibody, nano antibody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody or a fusion protein comprising an antigen binding portion of an antibody. In embodiments, the antibody form is scFv. In embodiments, CAR comprises a viral ligand.

[0119] In embodiments, CAR comprises a transmembrane domain derived from CD28, CD3ζ, CD4, CD8α, ICOS or a fragment and / or combination thereof. In embodiments, CAR comprises an intracellular domain, which further comprises an intracellular signaling domain and / or one or more co-stimulatory molecules of a CD3ζ chain, optionally selected from CD28, 4-1BB, ICOS, CD27 and OX40. In embodiments, CAR lacks an intracellular domain and / or a co-stimulatory domain.

[0120] In embodiments, CAR targets cancer-specific antigens, for example, but not limited to, in a method of treating hepatocellular carcinoma (HCC), CAR targets AFP (alpha-ferritin); in a method of treating mesothelioma, CAR targets mesothelin; in a method of treating breast cancer, CAR targets ER (estrogen receptor), PR (progesterone receptor) or HER-2 / neu; in a method of treating non-small cell lung cancer, CAR targets EGFR (epidermal growth factor receptor); in a method of treating non-small cell lung cancer and / or colorectal cancer, CAR targets KRAS; in a method of treating colorectal cancer, CAR targets UG T1A1; in methods of treating blood cancer, gastric cancer and / or skin cancer, the CAR targets c-KIT; in methods of treating lymphoma, the CAR targets CD30; in methods of treating gastrointestinal cancer, the CAR targets PDGFR (platelet-derived growth factor receptor); in methods of treating lung cancer, breast cancer and colorectal cancer, the CAR targets TEM8 (tumor endothelial marker 8); in methods of treating various solid tumors, the CAR targets EIIIB; in methods of treating ovarian cancer, the CAR targets CA-125; in methods of treating renal cell carcinoma, the CAR targets CD9; in methods of treating various tumors In the method of treating lymphoma, the CAR targets CD11b and / or CD18; in the method of treating lymphoma, the CAR targets CD19, CD20 and / or CD21; in the method of treating endometrial cancer, colon cancer, skin cancer and / or leukemia, the CAR targets CD22; in the method of treating B cell cancer, the CAR targets CD24; in the method of treating lymphoma, the CAR targets CD30; in the method of treating cancer driven by dormant T cells carrying viruses, the CAR targets CD32a; in the method of treating ovarian cancer, the CAR targets CD41; in the method of treating lymphoma, the CAR targets CD42b; in the method of treating In the method of treating breast cancer, colon cancer, gastric cancer and / or lung cancer, CAR targets CD43; in the method of treating colorectal cancer, CAR targets CD51 and / or CD54; in the method of treating breast cancer and / or ovarian cancer, CAR targets CD61; in the method of treating lung cancer, CAR targets CD63; in the method of treating cancer metastasis, CAR targets CD66a / b / c / e; in the method of treating hepatocellular carcinoma and / or gastric cancer, CAR targets CLEC-2; in the method of treating bladder cancer, CAR targets CD147; in the method of treating thrombosis caused by cancer, CAR targets GPVI. In the embodiment, CAR targets cancer-specific antigens of one or more targets in Table 1 and / or Table 2.

[0121] In embodiments, BioNV encapsulates a payload; for example, "lumen load", or the ability of BioNV to load a payload into a lumen (space in a biomimetic nanovesicle). In embodiments, the payload is one or more of a gene editor, a cytotoxic protein, a biologic, a nucleic acid, a fusion protein, a fluorescent protein, a tracer dye, a radionuclide, and / or a small molecule. In embodiments, the payload is a therapeutic payload for the type of cancer targeted by CAR. In embodiments, the payload includes one or more of the following: an alkylating agent, an anthracycline, an antimetabolite, an antitumor antibiotic, an antitumor antibody or antibody form, a corticosteroid, a plant alkaloid, a topoisomerase inhibitor, and / or a checkpoint inhibitor.

[0122] In embodiments, the payload is selected from one or more of the following: abemaciclib, abiraterone acetate, abraxan, ABVD, ABVE, ABVE-PC, AC, acalabrutinib, AC-T, Antenol, Ansili, ADE, ado-trastuzumab emtansine, doxorubicin, afatinib dimaleate, afinitor, Oconze, idela, aldesleukin, Ansentsa, alectinib, alemtuzumab, limita, elezo, Alkran for injection, Alkran tablets, aloxi, apelis, Amberi, Ameruz, amifostine, aminolevulinic acid hydrochloride, ervantumab-vmjw, anastrozole, apalutamide, aprepitant, espu, Acorda, Arimidex, Arnoxin, Anruijin, trioxygen Arsenicum arsenicum, Asera, Ascimini hydrochloride, Asparaginase Erwinia, Asparagus, Atezolizumab, Afatinib, Avastin, Avelumab, Akilencel, Axitinib, Tajiva, Azacitidine, Azedra, Balvesa, Bavencio, BEACOPP, Belantamab Mofortin-blmf, Belyodak, Belinostat, Beztifan, Bendamustine hydrochloride, Bendeka, BEP, Bebossa, Bevacizumab, Bexarotene, Bicalutamide, BiCNU, Bimetinib, Brenrep, Bleomycin sulfate, Belinto, Belyto, Bortezomib, Bosuliv, Bosutinib, Bravtovi, Vebutin, Brecatuximab, Brekacin, Breyanzi, Brigatinib, Brukinsa, BuMel, busulfan, busulfan, cabazitaxel, cabizumab, cabozantinib, cabozantinib-S-malate, CAF, carbaspagas peg-mknl, Concordia, Campas, Camptosa, capecitabine, capecitabine-yhdp, capmatinib hydrochloride, CAPOX, Caracol, carboplatin, carboplatin-paclitaxel, carfilzomib, carmustine, carmustine implant, Concordia, CEM, cemiprilimab-rwlc, ceritinib, ceribid, Cerax (recombinant HPV bivalent vaccine), cetuximab, CEV, chlorambucil, chlorambucil-prednisone, CHOP, cisplatin, cladribine, clofarabine, Corolla, CMF, cobimetinib fumarate, Comtril, hydrochloride Copanisib, COPDAC, Kebituo, COPP, COPP-ABV, Cosmegen, Cotelici, Crizotinib, CVP, Cyclophosphamide, Ciranze, Cytarabine, Dabrafenib Mesylate, Dacarbazine, Dacotinib, Actinomycin D, Dayouze, Daratumumab, Daratumumab and Hyaluronidase-fihj, Darbepoetin alfa, Darofem, Zakata, Zakata Express, Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposomal, Dorismo, Decitabine, Decitabine and Cidaruci, Defibrotide Sodium, Defitelio, Degarelix, Denileukin, Denosumab, Dexamethasone, Dexazolidinone Hydrochloride, Denutoximab, Docetaxel, Dotalimumab-GXLY, Doxitaxel,Doxorubicin hydrochloride, liposomal doxorubicin hydrochloride, durvalumab, duvelith, efdex, eligad, elitek, ellens, elotuzumab, eloxadine, eltrombopag, elzolis, epavazumab-lzsg, emend, empliximab, ensidipine mesylate, connefenib, enrofloxacin vedotin-ejfv, euhede, entrectinib, enzalutamide, epirubicin hydrochloride, EPOCH, epoetin alfa, epogen, erbitux, erdatinib, eribulin mesylate, erici, erida, erlotinib hydrochloride, elvinaze, isol, vanbifu, etoposide, etoposide phosphate, everolimus, ivert, evomera, exemestane, anvili, 5-FU, 5-FU, Fa m-trastuzumab drutilecan-nxki, falotin, fusodex, FEC, fedratinib hydrochloride, fulon, filgrastim, fermoneg, fludarabine phosphate, fluprednisone, fluorouracil injection, fluorouracil topical, flutamide, forfuri, forfuri-bevacizumab, forfuri-cetuximab, forfino, fulvestrant, fulvestrant, fostamicin, fostamicin disodium, fortivar, forfiltrates, FU-LV, fulvestrant, filaro, gamivant, Gardasil (recombinant HPV quadrivalent vaccine), Gardasil 9 (recombinant HPV nine-valent vaccine), Gavreto, Gaziva, gefitinib, gemcitabine hydrochloride, gemcitabine-cisplatin, gemcitabine-oxaliplatin, gemtuzumab ozogamicin, gemcitabine, gillotoxifene, fumarole Gliardinib, granisetron maleate, Gleevec, Gliadal powder tablets, Glucarpivirine, Goserelin acetate, granisetron, granisetron hydrochloride, Granix, Haraven, Hermangio, Herceptin Helekta, Herceptin, recombinant HPV bivalent vaccine, recombinant HPV nine-valent vaccine, recombinant HPV four-valent vaccine, Hecantin, Aizhi, hydroxyurea, Hyper-CVAD, Ibrance, Ibrutinib, ICE, Icluzig, Idarubicin PFS, Idarubicin hydrochloride, Idicatargen, Vicruse, Idelalis, Idifa, Ifex, Ifosfamide, Interleukin-2 (recombinant), Imatinib mesylate, Ibvecar, Imfenzi, Imiquimod, Imlijik, Indole phosphate gretinib, infgem, inlita, ituzumab ozogamicin, incovi, inrebic, interferon alfa-2b, intron A, iobenguanidine I131, ipilimumab, ressa, irinotecan hydrochloride, isatuximab-irfc, istodex, ivosideb, ixabepilone, ixazomib citrate, ixampura, jacafi, jeb, germitor, janperli, jeftana, cadsila, capivoxil, kepivans, kymida, kimstrak, kiskali, coselugol, kimria, caprolis, lanreotide acetate, lapatinib dimesylate, larotrectinib sulfate, lenalidomide, lenvatinib mesylate, lenvima, letrozole, leucovorin, rocloma, leuprolide acetate, leupromide, leuprolide, leucomitin, leuprolide, leuprolide,Lisocartogenmal, lomustine, lantuximab, tecillin-lpyl, lansef, lorbrena, lorlatinib, lumaclas, lumositide, lipan depot, lumenectin, ruspactin-aamt, lutetase, lutetium (Lu 177-dotat), leupro, meganle, magetuximab-cmkb, marcibo, maturaner, dichloroethylamine hydrochloride, megestrol acetate, meginister, mektovi, melphalan, melphalan hydrochloride, mercaptopurine, mesna, mesnex, methotrexate sodium, methylnaltrexone bromide, mistolin, mitomycin, mitoxantrone hydrochloride, mobotinib succinate, moglizumab-kpkc, monjuvi, mopp, mosetuximab pasudotox-tdfk, siprelin, mvac, mvasi, myleran, mylotarg, nanoparticle paclitaxel, nacitozumab-gqgk, nacitozumab, nelarabine, nelarabine maleate Latinib, Helian, Netupitant, Neurastat, Nupregin, Nexavar, Nilandron, Nilotinib, Nilutamide, Enlari, Niraparib tosylate monohydrate, Nivestim, Nivolumab, Napre, Nubeka, Nivipriya, Obinutuzumab, Odomzo, OEPA, Ofatumumab, OFF, Olaparib, Omacitabine mesylate, Oncaspar, Ondansetron hydrochloride, Onived, Ontaq, Onureg, Odivo, OPPA, Orgovix, Osimertinib mesylate, Oxaliplatin, Paclitaxel, Paclitaxel albumin-stabilized nanoparticle formulation, PAD, Padchev, Palbociclib, Palifermin, Palofermin hydrochloride nosetron, pamidronate disodium, panitumumab, paraplatin, pazopanib hydrochloride, PCV, PEB, pegaspargase, pegfilgrastim, peginterferon alfa-2b, PEG-Intron, pemaziret, pembrolizumab, pemetrexed disodium, pemigatinib, perjeta, pertuzumab, pesidatinib hydrochloride, fisgo, pixar, plerixafor, polatuzumab vedotin-piiq, plavixet, pomalidomide, pomalistatin, ponatinib hydrochloride, portraz, portligio, pralatrexate, pralatrexate, prednisone, procarbazine hydrochloride, procrete, prolegin, prolia, promactor, propranolol hydrochloride, provange, Mercaptopurine, Prixan, Qinlock, radium 223 dichloride, raloxifene hydrochloride, ramucirumab, rasburicase, ravulizumab-cwvz, libloze, R-CHOP, R-CVP, recombinant human papillomavirus (HPV) bivalent vaccine, recombinant human papillomavirus (HPV) nine-valent vaccine, recombinant human papillomavirus (HPV) quadrivalent vaccine, recombinant interferon α-2b, regorafenib, restesta, relugolix, R-EPOCH, retacli, retefumo, relimide, ribociclib, R-ICE, ripretinib, rituximab, rituximab, rolapitant hydrochloride, romidepsin, romiprosin,Rope interferon alpha-2b-NJFT, Rosinquan, rubracin, rucaparib camphorsulfonate, ruxolitinib phosphate, rebuvant, redpath, relaze, gosatuzumab-hziy, shanqi, saklisa, sloso intrapleural aerosol, selinexor, selpatinib, selumetinib sulfate, senblix, selutuximab, siprexa-t, sirolimus protein-bound particles, sotamox, somadulin reservoir, solidegi, sorafenib tosylate, sotolasib, shidasai, stanford sterile talc, stritaq, baiwango, sunitinib malate, soto, sotent, seratron, sawinco, simrib, tabloid, Tabexin, TAC, Tancitumomab-CXIX, Tafele, Taglax, Tarisa, Tarazopanib Tosylate, Talimoginlaheparevek, Tazena, Tamoxifen Citrate, Tarceva, Tagretin, Dasina, Tavaris, Taxotere, Tazemetostat Hydrobromide, Davic, Terbentafussp, Tecatus, Tesanqi, Temoda, Temozolomide, Temsirolimus, Tepadina, Todocan, Tepotinib Hydrochloride, Thalidomide, Salomede, Thioguanine, Thiotepa, Tosuvo, Tisagan Leclercel, Tisomab Vedotin-TFTV, Tivodak, Tivozanib Hydrochloride, Tocilizumab, Tolac, Topotecan Hydrochloride, Tocilizumab remifene, torcet, torcet, tpf, trabectin, trametinib dimethyl sulfoxide, trastuzumab, trastuzumab and hyaluronidase-oysk, cunda, terensu, trofluridine and tipiracil hydrochloride, tresenoside, todavi, truseltic, trusima, tucatinib, tuxa, tularo, tykerb, ukonix, utomis, erblisab mesylate, vindensica, unitusin, uridine triacetate, vac, valrubicin, valstatin, vandetanib, vamp, valubi, victivib, veip, vemurafenib, vecorapil, venetoclax, vezenio, vedaza, vinblastine sulfate, vinblastine sulfate Vincristine, liposomal vincristine sulfate, vinorelbine tartrate, VIP, Vimodegi, Vistoga, Vitec, Dozin, Volacoze, Vorinostat, Viquant, Vexos, Velirege, Xelcore, Setmep, Xeloda, Cialis, Cielox, Angawi, Sofigo, Sigatan, Civio, Ancotan, Yiwo, Ikeda, Yoderlis, Yongsha, Zatrap, Zabutinib, Zarcio, Zele, Zorbov, Zepzeka, Zevalin, Zietenzo, Zinecad, Zilabev (bevacizumab), Ziv-Aflibercept, Zofran, Norad, Zoledronic Acid, Zolinza, Zometa, Zikalala, Zvib, Zancorta, Sinlonta and Zeco.

[0123] In embodiments, the payload is one or more of: pidilizumab, BMS-936559, tremelimumab, AGEN1884, and / or RG2077.

[0124] In an embodiment, the nucleic acid molecule encodes one or more of the following: a CRISPR / Cas component, a guide RNA (gRNA), a tracer RNA (tracrRNA), a microRNA (miRNA), RNA interference (RNAi), a small interfering RNA (siRNA), a double-stranded RNA, a Piwi-interacting RNA (piRNA), a small nuclear RNA (snRNA), a small nucleolar RNA (snoRNA), an antisense oligonucleotide (ASO), a locked nucleic acid (LNA), a splice-switching oligonucleotide (SSO), a tRNA, a ribosomal RNA (rRNA), a short hairpin (shRNA) complementary messenger RNA, a repeat-associated small interfering RNA (rasiRNA) and / or a small non-coding RNA.

[0125] In an embodiment, the gene editing payload includes a gene editing nucleic acid and / or protein, such as TALEN, ZFN, RNase P RNA, C2c1, C2c2, C2c3, Cas9, Cpf1, TevCas9, Archaea Cas9, CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, CasX Cas omega, transposase, and / or any ortholog or homolog of any of these editing agents. In an embodiment, the gene editing agent may also include a gRNA, which refers to a guide RNA as used herein. In an embodiment, the gRNA may be a sequence complementary to a coding or non-coding sequence and may be customized according to a specific sequence to be targeted. In an embodiment, the gRNA sequence may be a sense sequence or an antisense sequence. In an embodiment, when a gene editing agent composition is applied herein, it is preferably, but not limited to, including two or more gRNAs; however, a single gRNA may also be used.

[0126] In embodiments, BioNV delivers a gene editing payload comprising a transcription activation response region (TAR) loop system. In embodiments, BioNV encapsulates a plasmid expressing a gene editor and contains a TAR loop sequence between the 5' end of the promoter and the gene editor / guide box and acts as a barrier to block transcription. In embodiments, transcription will only be triggered in cells that are infected and contain the HIV Tat protein. In embodiments, the Tat protein binds to the TAR loop, relaxes it, and releases the promoter for transcription, thereby expressing the editor and its guide.

[0127] In embodiments, BioNV encapsulates one or more perforin molecules. In embodiments, BioNV encapsulates one or more granzyme molecules. In embodiments, granzyme molecules are selected from granzymes A, B, H, K and M. In embodiments, BioNV encapsulates one or more perforin molecules and / or one or more granzyme molecules derived from cells from which BioNV is derived. In embodiments, BioNV encapsulates one or more perforin molecules and one and / or more granzyme molecules exogenously added to BioNV. In the cell processing stage (e.g., continuous extrusion), in embodiments, the chamber between the extrusion filters may be filled with a concentration gradient of extracellular, purified perforin, granzymes, etc., which may be encapsulated in BioNV during the extrusion process. In embodiments, BioNV expressing PD-L1 is derived from unactivated cells and BioNV is substantially lacking perforin and / or granzymes.

[0128] In embodiments, methods for treating, preventing and / or ameliorating cancer include co-administering whole cell therapy (e.g., T cells, NK cells, TIL, macrophage therapy). In embodiments, supplementing whole cell therapy with BioNV can be used to reduce the effective dose of whole cell therapy required, reduce CRS, teratoma potential, off-target effects, etc.

[0129] In embodiments, the method of treating or preventing cancer comprises administering an additional therapeutic agent. In embodiments, the additional therapeutic agent may be any additional anti-cancer agent, analgesic agent and / or non-steroidal anti-inflammatory agent (NSAID).

[0130] In embodiments, the BioNV can be frozen at -80°C, or suitable for storage and / or lyophilization (e.g., for reconstitution in a buffer) at about -80°C. In embodiments, the BioNV can be stable at about ambient temperature, about -20°C, about 4°C, about 25°C, or about 37°C for at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, at least about a week, or at least about a month or more.

[0131] In embodiments, the PD-1 inhibitor is an antibody targeting PD-1, or pembrolizumab, nivolumab or cemiplimab. In embodiments, the PD-L1 or PD-L2 inhibitor is an antibody targeting PD-L1 or PD-L2, optionally atezolizumab, avelumab or durvalumab, or its antigen binding domain, and is bound to the surface of BioNV. In embodiments, the PD-1 inhibitor is an antibody targeting PD-1, or pembrolizumab, nivolumab or cemiplimab. In embodiments, the PD-L1 or PD-L2 inhibitor is an antibody targeting PD-L1 or PD-L2, optionally atezolizumab, avelumab or durvalumab, or its antigen binding domain, and is membrane anchored by transmembrane domain fusion. In embodiments, the antibody can be membrane anchored by any transmembrane domain suitable for CAR. In embodiments, "binding" may refer to chemical association of the antibody with the lipid layer of BioNV, such as covalent binding via a linker; linkers for antibodies and antibody-drug conjugates are well known in the art.

[0132] In embodiments, treatment methods include BioNVs with integrated antibodies or antibody forms (e.g., pembrolizumab or nivolumab). In embodiments, "integration" may refer to integration of the antibody into the lipid layer of the BioNV. In embodiments, the antibody may be expressed on the surface of the modified cell from which the BioNV is derived. In embodiments, the antibody is engineered to have a transmembrane domain to allow the antibody or antibody form to be surface exposed on the cell prior to BioNV formation.

[0133] In embodiments, the cancer is one or more of carcinoma, sarcoma, myeloma, leukemia, lymphoma, mixed cancer, and / or metastatic cancer.

[0134] In embodiments, the cancer is selected from acute biphenotypic leukemia, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid dendritic cell leukemia, acute myeloid leukemia, lung adenocarcinoma, adrenal tumors, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphomas, alveolar soft tissue and cardiac sarcomas, amyloidosis, anal cancer, anaplastic large cell lymphoma, angioimmunoblastic T-cell lymphoma, appendix cancer, astrocytoma, ataxia telangiectasia, attenuated familial adenomatous polyposis, B-cell prolymphocytic leukemia, basal cell carcinoma, Beckwith-Wiedemann syndrome, bile duct cancer, Bert-Hoag-Dub syndrome, bladder cancer, bone cancer, brain and nervous system cancers, brain stem glioma, brain stem glioma, Glioma), brain tumors, breast cancer, bronchial adenoma / carcinoid, Burkitt's lymphoma, carcinoid tumor, Carney syndrome, central nervous system tumors, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, childhood fibroproliferative ganglioglioma, bile duct cancer, chondrosarcoma, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloid leukemia, chronic T-cell lymphocytic leukemia, colon cancer, colorectal cancer, Cowden syndrome, craniopharyngioma, cutaneous T-cell lymphoma, dermatofibrosarcoma protuberans, fibroproliferative Small round cell tumor, diffuse gastric cancer, diffuse large B-cell lymphoma, endocrine system cancer, endocrine tumors, endometrial cancer, eosinophilic leukemia, ependymoma, epithelioid hemangioendothelioma (EHE), esophageal cancer, Ewing's sarcoma, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, eyelid cancer, fallopian tube cancer, familial adenomatous polyposis, familial malignant melanoma, familial clear cell renal cell carcinoma (RCC), follicular lymphoma, gallbladder cancer, Gardner syndrome, gastric cancer, gastrointestinal cancer, gastrointestinal Carcinoma, Gastrointestinal Stromal Tumor, Gastrointestinal Stromal Tumor (GIST), Genitourinary and Gynecological Cancers, Germ Cell Tumors, Gestational Trophoblastic Disease, Gestational Trophoblastic Tumors, Glioblastoma, Glioma, Hairy Cell Leukemia, Head and Neck Cancer, Hematopoietic Cancers, Hepatocellular Carcinoma, Hepatosplenic T-Cell Lymphoma, HIV-Related Cancers, Hodgkin Lymphoma, Hypopharyngeal Cancer, Inflammatory Breast Cancer, Intravascular Large B-Cell Lymphoma, Aggressive Cribiform Carcinoma, Aggressive Lobular Carcinoma, Islet Cell Carcinoma (Endocrine Pancreas), Islet Cell Tumors, Juvenile juvenile polyposis syndrome, Kaposi's sarcoma, keratoacanthoma, renal cancer, lacrimal gland tumors, large granular lymphocytic leukemia, laryngeal and hypopharyngeal cancer, leiomyomatosis and renal cell carcinoma, leiomyosarcoma, Li-Fraumeni syndrome (LFS), liposarcoma, liver cancer, lung cancer, primary cutaneous lymphoma, lymphomatoid granuloma, lymphoplasmacytic lymphoma, Lynch syndrome, malignant fibrous histiocytoma of bone, mantle cell lymphoma, marginal zone B-cell lymphoma, mast cell leukemia, mastocytosis, mediastinal large B-cell lymphoma,Medullary carcinoma, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, mixed polyposis syndrome, mucosa-associated lymphoid tissue lymphoma, Muir-Tore syndrome (MTS), multiple endocrine neoplasia syndrome, multiple endocrine neoplasia type 1, multiple endocrine neoplasia type 2, multiple myeloma, multiple myeloma / plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, MYH-associated polyposis, myxosarcoma, nasal cavity and paranasal sinus cancer, Wilms tumor, nasopharyngeal carcinoma, nasopharyngeal carcinoma, neuroblastoma, neuroendocrine tumor, type 1 Neurofibromatosis, neurofibromatosis type 2, nevoid basal cell carcinoma syndrome, nodal marginal zone B-cell lymphoma, non-Hodgkin lymphoma, non-small cell lung cancer, non-small cell lung cancer, oligodendroglioma, optic nerve glioma, oral cavity and / or oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, papillary renal cell carcinoma, paranasal sinus and nasal cavity cancer, parathyroid cancer, pelvic cancer, penile cancer, Peutz-Jeghers syndrome, pharyngeal cancer, pheochromocytoma, phyllodes tumor, pilocytic astrocytoma, pineal astrocytoma, pituitary adenoma, pituitary Tumors, plasmablastic lymphoma, pleuropulmonary blastoma, precursor B lymphoblastic leukemia, primary central nervous system lymphoma, primary cutaneous follicular lymphoma, primary cutaneous immunocytoma, primary effusion lymphoma, primitive neuroectodermal tumor, prostate cancer, rectal cancer, kidney cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, primary cutaneous sarcoma, sebaceous carcinoma, Sezary syndrome, skin appendage tumors, skin cancer, small intestine cancer, small cell lung cancer, small intestine cancer, splenic marginal zone lymphoma, lung squamous cell carcinoma, squamous Cell carcinoma, squamous cell skin cancer, gastric cancer, surface epithelial-stromal tumors, T-cell prolymphocytic leukemia, testicular cancer, thoracic and respiratory tract cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell carcinoma, transitional cell carcinoma adenoid cystic carcinoma, tuberous sclerosis complex syndrome, tubular carcinoma, Turker's syndrome, carcinoma of unknown primary, unclassified carcinoma, ureteral cancer, urethral cancer, uterine cancer, uterine sarcoma, uveal melanoma, vaginal cancer, visual pathway and hypothalamic glioma, von Hippel-Lindau (VHL) syndrome, vulvar cancer, Wilms tumor, and xeroderma pigmentosum.

[0135] In embodiments, BioNV can target cancer cells associated with adenoid cystic carcinoma, adrenal tumors, amyloidosis, anal cancer, appendix cancer, astrocytoma, ataxia telangiectasia, familial adenomatous polyposis, Beckwith-Wiedemann syndrome, bile duct cancer, Bert-Hoag-Dub syndrome, bladder cancer, bone cancer, brain stem glioma, brain tumor, breast cancer, carcinoid tumor, Carney syndrome, central nervous system tumor, cervical cancer, colorectal cancer, Cowden syndrome, craniopharyngioma, desmoplastic infantile ganglioglioma, endocrine tumor, ependymoma, esophageal cancer, Ewing's sarcoma tumors, eye cancer, eyelid cancer, fallopian tube cancer, familial adenomatous polyposis, familial malignant melanoma, familial non-VHL clear cell renal cell carcinoma, gallbladder cancer, Gardner syndrome, gastrointestinal stromal tumors, germ cell tumors, gestational trophoblastic disease, head and neck cancer, diffuse gastric cancer, leiomyomatosis and renal cell carcinoma, mixed polyposis syndrome, pancreatitis, papillary renal cell carcinoma, HIV and AIDS-related cancers, islet cell tumors, juvenile polyposis syndrome, kidney cancer, lacrimal gland tumors, laryngeal and hypopharyngeal cancers, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, B cell Proliferative lymphocytic leukemia, hairy cell leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic T-cell lymphocytic leukemia, eosinophilic leukemia, Li-Fraumeni syndrome, liver cancer, lung cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, Lynch syndrome, mastocytosis, medulloblastoma, melanoma, meningioma, mesothelioma, Muir-Tore syndrome, multiple endocrine neoplasia type 1, multiple endocrine neoplasia type 2, multiple myeloma, myelodysplastic syndrome, MYH-associated polyposis, nasal and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, neuroendocrine tumors, Neurofibromatosis type 1, neurofibromatosis type 2, nevoid basal cell carcinoma syndrome, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, Peutz-Jeghers syndrome, pituitary tumors, pleuropulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, alveolar soft tissue and cardiac sarcoma, Kaposi's sarcoma, skin cancer, small intestine cancer, stomach cancer, testicular cancer, thymoma, thyroid cancer, tuberous sclerosis complex syndrome, Tork's syndrome, primary unknown, uterine cancer, vaginal cancer, von Hippel-Lindau syndrome, Wilms tumor, and xeroderma pigmentosum.

[0136] In embodiments, treating, preventing and / or ameliorating cancer includes one or more of the following: clinical remission, reduction in tumor volume, reduction in angiogenesis, reduction in metastasis size and number, increased tumor infiltration, reduction in tumor volume, or improvement in cancer-related clinical symptoms within about 2 weeks, within about 4 weeks, within about 6 weeks, within about 12 weeks, within about 18 weeks, within about 24 weeks, within about 6 months, within about 1 year, or about 2 years or longer from administration of the compositions and methods of using such compositions.

[0137] Dosage and Administration

[0138] The dosage and dosing schedule of any BioNV disclosed herein may depend on various parameters and factors, including, but not limited to, the specific BioNV, the cancer being treated, the severity of the condition, whether the condition is being treated or prevented, the age, weight and general health of the subject, and the judgment of the attending physician. In addition, pharmacogenomic (the effect of genotype on the pharmacokinetics, pharmacodynamics, or efficacy spectrum of the therapeutic agent) information about a particular subject may affect the dosage used. Furthermore, the exact individual dosage may be adjusted to some extent based on a variety of factors, including the specific combination of drugs being administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the disease being treated, the severity of the condition, and the anatomical location of the condition. Some variation in dosage may be expected.

[0139] In embodiments, delivery of BioNV can be similar to delivery of vesicles, particularly liposomes (see Langer, 1990, Science 249: 1527-1533; Treat et al., Liposomes in Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989)).

[0140] In embodiments, the methods of treating and / or preventing cancer using the BioNV described herein include a dosage range in units of the number concentration of BioNV per kilogram (kg) of subject body weight. In embodiments, a suitable dosage range for the methods described herein may include about 10 3 BioNV / kg to about 10 12 In one embodiment, the BioNV is present in the composition at a concentration of about 10 3 BioNV / mL to about 10 14 Alternatively, in embodiments, the BioNV composition is present in the composition at a weight / volume in the range of about 5 ng / mL to about 500 mg / mL. In embodiments, the BioNV dosage is based on the size of the BioNV used in the treatment, for example, at an equivalent dose, a 1000 nm BioNV is provided in an amount approximately 5-fold to 10-fold less than the amount of a 100 nm BioNV.

[0141] In an embodiment, the BioNV disclosed herein is administered by controlled or sustained release means or by delivery by devices well known to those of ordinary skill in the art. Examples include, but are not limited to, those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; and 5,733,556, each of which is incorporated herein by reference in its entirety. Such dosage forms may be suitable for providing controlled or sustained release of one or more active ingredients using, for example, hydroxypropyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, microspheres, or combinations thereof, thereby providing a desired release profile in varying proportions. Controlled or sustained release of an active ingredient can be stimulated by various conditions including, but not limited to, changes in pH, changes in temperature, stimulation by light of an appropriate wavelength, concentration or availability of an enzyme, concentration or availability of water, or other physiological conditions or compounds.

[0142] In an embodiment, a polymeric material is used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61; Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105).

[0143] In an embodiment, a controlled release system is placed adjacent to the target area to be treated, thereby requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release, supra, Vol. 2, pp. 115-138 (1984)). Other controlled release systems discussed in the review by Langer, 1990, Science 249: 1527-1533 may be used.

[0144] In embodiments, methods of using BioNV include applying BioNV to the surface of a device (e.g., a catheter) or containing it in a pump, patch, or other drug delivery device. The excipient or carrier can be selected based on the mode and route of administration. Suitable pharmaceutical carriers and pharmaceutical necessities for pharmaceutical formulations are described in the well-known reference books in the art, Remington's Pharmaceutical Sciences (EW Martin) and USP / NF (United States Pharmacopeia and National Formulary).

[0145] Pembrolizumab is a humanized anti-PD-1 monoclonal antibody used to treat cancers such as melanoma, lung cancer, head and neck cancer, Hodgkin's lymphoma, gastric cancer, cervical cancer, and breast cancer. It binds to PD-1 on cancer cells, blocking the interaction with lymphocyte receptors. In embodiments, this allows the immune system to target cancer cells that can no longer escape the cytotoxic response while preventing the immune system from attacking healthy cells. In embodiments, pembrolizumab can be administered at a dose of 200 mg to 400 mg or 2 mg / kg.

[0146] Nivolumab is a humanized anti-PD-1 monoclonal antibody used to treat cancers such as melanoma, lung cancer, malignant pleural mesothelioma, renal cell carcinoma, colon cancer, esophageal squamous cell carcinoma, liver cancer, gastric cancer, and esophageal cancer or gastroesophageal junction cancer. In embodiments, nivolumab works in the same manner as pembrolizumab. In embodiments, nivolumab can be administered at a dose of 240 mg to 480 mg or 3 mg / kg.

[0147] In embodiments, BioNV can be administered at a dose consistent with that of pembrolizumab and / or nivolumab. In embodiments, BioNV can be administered at a dose consistent with that of whole cells, for example, based on CAR concentration. In embodiments, the typical concentration range of CAR protein per microgram of T cell is between 0.20ng-0.70ng, and the total number of CARs in a single BioNV may be 5 to 10,000 times less than that of whole cells, resulting in the conversion of BioNV mass into CAR concentration, where it can be assumed that the CAR concentration is equal to the cell (e.g., T cell) from which it is derived (e.g., the case in exosomes) or increases (e.g., the case in BioNV). In embodiments, the concentration and / or surface density of the targeting agent (e.g., CAR) on BioNV increases compared to the whole cell from which it is derived. In embodiments, the concentration and / or surface density of the targeting agent (e.g., CAR) is enriched by continuous extrusion processing of whole cells. In embodiments, the concentration and / or surface density of the targeting agent (e.g., CAR) and other cell surface molecules on BioNV increases by 2 to 100 times relative to the whole cell.

[0148] The dosage regimen using any BioNV disclosed herein can be selected based on a variety of factors, including the cancer type, race, age, weight, sex, and medical condition of the subject; the severity of the condition to be treated; the route of administration; the renal or liver function of the subject; the pharmacogenomic composition of the individual; and the specific composition of the present disclosure employed. Any BioNV disclosed herein can be administered in a single daily dose, or the total daily dose can be administered in divided doses twice, three times, or four times a day. In addition, any BioNV disclosed herein can be administered continuously rather than intermittently throughout the dosing regimen.

[0149] In embodiments, BioNV is administered in continuous doses about once per hour, about once every 2 hours, about once every 6 hours, about once every 12 hours, about once every 24 hours, about once every 2 days, about once every 4 days, about once every 7 days, about once every 2 weeks, about once every 4 weeks, or about once a month.

[0150] In embodiments, comprehensive remission or clinical remission of cancer, reduction in tumor volume, reduction in angiogenesis, reduction in size and number of metastases, increase in tumor infiltration, reduction in primary tumor volume, or improvement in clinical symptoms is achieved within about 24 weeks, about 18 weeks, about 12 weeks, about 8 weeks, about 6 weeks, about 4 weeks, about 2 weeks, or about 1 week after administration of the compositions and methods of using such compositions.

[0151] Composition of BioNV

[0152] In some aspects, the present disclosure relates to a composition for treating and / or preventing cancer, the composition comprising an allogeneic BioNV comprising a membrane-embedded CAR targeting a cell surface marker, and a binding and / or membrane-anchored targeting agent targeting PD-L1 or PD-L2, wherein the agent targeting PD-L1 or PD-L2 is atezolizumab, avelumab, or durvalumab, and wherein the CAR does not target PD-1, PD-L1, or PD-L2.

[0153] In some aspects, the present disclosure relates to a composition for treating and / or preventing cancer, comprising a first membrane-embedded CAR that targets a cell surface, and at least a second membrane-embedded CAR that targets PD-L1 or PD-L2, wherein the first CAR does not target PD-1, PD-L1 or PD-L2.

[0154] In some aspects, the present disclosure relates to a composition for treating and / or preventing cancer, comprising a bispecific, membrane-embedded CAR targeting a cell surface marker of a cancer cell and PD-L1 or PD-L2, wherein the cell surface marker of the cancer cell is not PD-1, PD-L1 or PD-L2.

[0155] In embodiments, the composition comprises BioNV. In embodiments, the composition comprises BioNV and at least one anticancer therapeutic agent, as described herein. In embodiments, BioNV can adsorb therapeutic molecules onto the surface of NV and / or encapsulate the therapeutic payload in the aqueous compartment of NV. In embodiments, the composition comprises BioNV and at least one checkpoint inhibitor encapsulated in the aqueous core. In embodiments, the composition comprises a therapeutically effective amount of BioNV. In embodiments, BioNV and checkpoint inhibitors may be combined in solution or may be co-administered in a separate solution. In embodiments, the composition comprises BioNV loaded with cytotoxic molecules (e.g., perforin, granzymes, etc.).

[0156] In embodiments, the compositions are derived from iPSCs (as well as other cell types) that have been modified to reduce expression of immunogenic molecules and / or to increase expression of immunoprotective molecules.

[0157] In embodiments, the composition is allogeneic and / or has low immunogenicity. For example, in embodiments, the composition does not cause an inflammatory response and / or an immune response when administered. In embodiments, BioNV is allogeneic and / or has low immunogenicity. In embodiments, after administration to a subject, the composition (optionally the BioNV therein) causes less than about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 24%, about 23%, about 22%, about 21%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2% or about 1% of inflammation or immune response. An immune response is measured as a function of the concentration of a cytokine, chemokine, or immunomodulatory enzyme, such as IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-20, IFN-α / β / γ, TNFα / β, IDO, HLA-G, HGF, PGE2, etc., or any combination thereof, for example, compared to a homologous whole cell therapy counterpart.

[0158] In embodiments, BioNV is present in the composition at a concentration of about 10 3 BioNV / mL to about 10 14 Alternatively, in embodiments, the BioNV composition is present in the composition at a weight / volume range of about 5 ng / mL to about 500 mg / mL.

[0159] In embodiments, the composition is substantially free of one or more bacteria, viruses, fungi, spores, mycoplasmas, pyrogens, and in more specific embodiments, substantially free of all of the foregoing substances. In embodiments, the composition is substantially free of whole cells and intracellular cell components, including organelles, such as nuclei, mitochondria, Golgi bodies, etc., and / or substantially free of non-CAR-expressing NVs and / or substantially free of ruptured, damaged NVs. In embodiments, the composition is substantially free of cell chromatin, nucleosomes, genomic DNA, and other cellular genetic material and non-therapeutic nucleic acids.

[0160] BioNV's pharmaceutical compositions and formulations

[0161] In some aspects, the composition is a pharmaceutical composition. In an embodiment, the pharmaceutical composition of the present disclosure is formulated to provide a therapeutically effective amount of BioNV as an active ingredient. In an embodiment, the pharmaceutical composition of the present disclosure is formulated to provide a therapeutically effective amount of one or more anticancer therapeutic agents as a payload within BioNV as an active ingredient. Typically, the pharmaceutical composition also includes one or more pharmaceutically acceptable excipients, carriers (including inert solid diluents and fillers), diluents (including sterile aqueous solutions and various organic solvents), penetration enhancers, solubilizers, and adjuvants.

[0162] Pharmaceutical excipients can be liquids, such as water and oils, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Pharmaceutical excipients can be, for example, saline, gum arabic, gelatin, starch paste, talc, keratin, silica gel, urea, etc. In addition, adjuvants, stabilizers, thickeners, lubricants and colorants can be used. When applied to a subject, pharmaceutically acceptable excipients are usually sterile. When any agent disclosed herein is administered intravenously, water is a useful excipient. Saline solutions and dextrose aqueous solutions and glycerol solutions can also be used as liquid excipients, particularly for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, any composition disclosed herein can also be formulated with a wetting agent or emulsifier or pH buffer. Other examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro, ed., 19th ed. 1995), which is incorporated herein by reference.

[0163] In an embodiment, the composition comprises an excipient or carrier. In an embodiment, the diluent is a pharmaceutically acceptable excipient or carrier.

[0164] In embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable diluent. Non-limiting examples of diluents include liquid diluents such as water, ethanol, propylene glycol, glycerol, and various combinations thereof, and inert solid diluents such as calcium carbonate, calcium phosphate, or kaolin. In embodiments, the diluent includes one or more of the following: saline, phosphate buffered saline, Dulbecco's modified Eagle's medium (DMEM), α modified minimum essential medium (αMEM), Roswell Park Memorial Institute medium 1640 (RPMI medium 1640), HBSS, human albumin, Ringer's solution, etc., or any combination thereof.

[0165] In embodiments, the active ingredient is typically mixed with an excipient, diluted by an excipient, or encapsulated in a carrier in the form of, for example, a capsule, tablet, pouch, paper, or other container. When an excipient is used as a diluent, it may be a solid, semisolid, or liquid substance (e.g., saline) as a vehicle, carrier, or medium for the active ingredient. In embodiments, the composition may be in the form of a tablet, pill, powder, lozenge, sachet, cachet, elixir, suspension, emulsion, solution, syrup, aerosol (as a solid or in a liquid medium), lotion, cream, ointment, gel, soft and hard gelatin capsules, suppository, sterile injection solution, and sterile packaged powder. As known in the art, the type of diluent may vary according to the intended route of administration. In embodiments, the resulting composition may include additional agents, such as preservatives, cryopreservatives (e.g., DMSO), and / or lyoprotectants (e.g., polyols, salts). In embodiments, the carrier may be or may include a lipid-based or polymer-based colloid. In embodiments, the carrier material may be a colloid formulated as a liposome, a hydrogel, a microparticle, a nanoparticle, or a block copolymer micelle. In embodiments, the carrier material may form a capsule, and the material may be a polymer-based colloid.

[0166] In an embodiment, a pharmaceutical composition comprising BioNV includes a solubilizing agent. In an embodiment, a pharmaceutical composition comprising BioNV includes a cryoprotectant or an agent that improves thermal stability, such as DMSO or glycerol. In an embodiment, the pharmaceutical composition can be delivered with a suitable vehicle or delivery device known in the art.

[0167] In embodiments, the composition comprises a scaffold. In embodiments, the scaffold comprises a biomaterial. In a non-limiting example, the three-dimensional biomaterial comprises BioNV embedded in an extracellular matrix that is attached to, dispersed within, or captured within the scaffold. In embodiments, the biomaterial is biodegradable and / or synthetic.

[0168] In embodiments, the scaffold comprises a biodegradable biomaterial. Non-limiting examples of biodegradable biomaterials include fibrin, collagen, elastin, gelatin, vitronectin, fibronectin, laminin, reconstructed basement membrane matrix, starch, dextran, alginate, hyaluronic acid, chitin, chitosan, agarose, sugar, hyaluronic acid, poly (lactic acid), poly (glycolic acid), polyethylene glycol, decellularized tissue, self-assembling peptides, polypeptides, glycosaminoglycans, derivatives and mixtures thereof. Other useful biodegradable polymers or polymer types include, but are not limited to, polydioxanone, polycarbonate, polyoxalate, poly(α-ester), polyanhydride, polyacetate, polycaprolactone, poly(orthoester), polyamino acid, polyamide and mixtures and copolymers thereof, L-lactic acid and D-lactic acid stereopolymers, copolymers of bis(p-carboxyphenoxy) propionic acid and sebacic acid, sebacic acid copolymers, caprolactone copolymers, poly(lactic acid) / poly(glycolic acid) / polyethylene glycol copolymers, polyurethane and poly(lactic acid) copolymers, polyurethane and poly(lactic acid) copolymers, α-amino acid copolymers, α-amino acid and caproic acid copolymers, A-glutamic acid benzyl ester and polyethylene glycol copolymers, succinate and poly(ethylene glycol) copolymers, polyphosphazenes, polyhydroxyalkanoates and mixtures thereof. Binary and ternary systems are also contemplated. In embodiments, the scaffold includes one or more of collagen, various proteoglycans, alginate-based matrix and chitosan. In embodiments, the scaffold comprises one or more of a hydrogel, silk, matrigel, acellular and / or decellularized scaffolds, poly-ε-caprolactone scaffolds, absorbable scaffolds, and nanofiber-hydrogel composites.

[0169] In embodiments, the support comprises a synthetic biomaterial. Non-limiting examples of synthetic biomaterials include lactone-based polyesters or copolyesters, such as polylactide, polycaprolactone, polyorthoesters, polyanhydrides, polyamino acids, polysaccharides, polyphosphazenes, poly(ether-ester) copolymers (e.g., PEO-PLLA); polydimethylsiloxane, poly(ethylene-vinyl acetate), acrylate-based polymers or copolymers (e.g., polyhydroxyethyl methyl methacrylate, polyvinyl pyrrolidone), fluorinated polymers (e.g., polytetrafluoroethylene) and cellulose esters.

[0170] In embodiments, the composition can be prepared in any manner known in the pharmaceutical art, and can be administered by a variety of routes (e.g., subcutaneous, intravenous, etc.), depending on whether local or systemic treatment is required and the area to be treated. In embodiments, administration can be local (including ocular and mucosal, including intranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powder or aerosol, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), ocular, oral or parenteral. In embodiments, methods may include ocular delivery, topical administration (eye drops), subconjunctival, periocular or intravitreal injection or introduction by surgical placement of a balloon catheter or ophthalmic insert in the conjunctival sac. In embodiments, parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial administration, such as intrathecal or intraventricular administration. In embodiments, parenteral administration may be in the form of a single bolus dose, or may be, for example, by a continuous infusion pump.

[0171] In embodiments, pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, powders, etc. In embodiments, methods of treating and / or preventing cancer include the use of pharmaceutical carriers; aqueous, powder or oily bases; thickeners, etc.

[0172] In an embodiment, the pharmaceutical composition contains nucleic acids and vectors described herein as active ingredients and one or more pharmaceutically acceptable carriers. In an embodiment, the term "pharmaceutically acceptable" (or "pharmacologically acceptable") refers to molecular entities and compositions that do not produce side effects, allergies or other adverse reactions when applied to animals or humans, as appropriate. The methods and compositions disclosed herein can be applied to a variety of species, such as humans, non-human primates (e.g., monkeys), horses or other livestock, dogs, cats, ferrets, or other mammals, rats, mice or other experimental animals raised as pets. In an embodiment, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial agents, isotonic agents and absorption delaying agents, buffers, excipients, adhesives, lubricants, gels, surfactants, etc. that can be used as media for pharmaceutically acceptable substances.

[0173] In embodiments, the composition can be applied to the surface of a device (e.g., a catheter) or contained in a pump, patch or other drug delivery device. In embodiments, the composition can be applied alone or in the presence of a pharmaceutically acceptable excipient or carrier (e.g., normal saline) in the form of a mixture. Excipients or carriers are selected based on the mode of administration and approach. Suitable pharmaceutical carriers and pharmaceutical necessities for pharmaceutical formulations are described in the well-known reference books Remington's Pharmaceutical Sciences (EW Martin) and USP / NF (United States Pharmacopoeia and National Formulary) in the art.

[0174] In an embodiment, a composition disclosed herein, e.g., a pharmaceutical composition, is suspended in a saline buffer (including but not limited to TBS, PBS, etc.).

[0175] The present technology includes the disclosed BioNV in various formulations of pharmaceutical compositions. In embodiments, the BioNV disclosed herein can take the form of solutions, suspensions, emulsions, drops, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained release formulations, emulsions, aerosols, sprays, suspensions, or any other form suitable for use.

[0176] Pharmaceutical compositions comprising the BioNV described herein may conveniently be presented in unit dosage form and may be prepared by any method well known in the pharmaceutical art. Such methods generally include the step of combining the therapeutic agent with a carrier consisting of one or more additional ingredients. Typically, the pharmaceutical composition is prepared by uniformly and intimately combining the therapeutic agent with a liquid carrier, a finely divided solid carrier, or both, and then, if desired, molding the product into a dosage form of the desired formulation (e.g., wet or dry granulation, powder blends, etc., followed by tableting using conventional methods known in the art).

[0177] In an embodiment, any BioNV disclosed herein is formulated according to routine procedures into a pharmaceutical composition suitable for the modes of administration disclosed herein.

[0178] Additional therapeutic agents

[0179] In embodiments, the compositions or methods described herein further comprise a therapeutically effective amount of one or more additional therapeutic agents. In embodiments, the therapeutically effective amount of one or more additional therapeutic agents may be in solution with the BioNV, adsorbed onto the surface of the NV, or as a payload encapsulated within the BioNV. In embodiments, the additional therapeutic agent is one or more of a checkpoint inhibitor, an analgesic, and / or an anti-infective agent.

[0180] In embodiments, the compositions or methods of the invention contemplate other additional therapeutic agents, such as analgesics to help treat inflammation or pain at the site of application, or anti-infective agents to prevent infection at the site treated with the composition. Non-limiting examples of additional therapeutic agents include analgesics such as nonsteroidal anti-inflammatory drugs, opioid agonists, and salicylates; anti-infective agents such as anthelmintics, antianaerobics, antibiotics, aminoglycoside antibiotics, antifungal antibiotics, cephalosporin antibiotics, macrolide antibiotics, other beta-lactam antibiotics, penicillin antibiotics, quinolone antibiotics, sulfonamide antibiotics, tetracycline antibiotics, antimycobacterial drugs, antituberculosis antimycobacterial drugs, antiprotozoal drugs, antimalarial antiprotozoal drugs, antivirals, antiretrovirals, scabicides, anti-inflammatory drugs, corticosteroids, Steroidal anti-inflammatory drugs, antipruritic / local anesthetics, topical anti-infectives, antifungal topical anti-infectives, antiviral topical anti-infectives; electrolyte and renal agents, such as acidifiers, alkalizers, diuretics, carbonic anhydrase inhibitor diuretics, loop diuretics, osmotic diuretics, potassium sparing diuretics, thiazide diuretics, electrolyte substitutes, and uricosuric agents; enzymes, such as pancreatic enzymes and thrombolytic enzymes; gastrointestinal agents, such as antidiarrheals, antiemetics, gastrointestinal anti-inflammatory agents, salicylic acid gastrointestinal anti-inflammatory agents, antacids and anti-ulcer agents, acid pump inhibitor anti-ulcer agents, gastric mucosal anti-ulcer agents , H2-blockers, antiulcers, gallstone dissolving agents, digestives, emetics, laxatives and stool softeners and prokinetics; general anesthetics, such as inhalation anesthetics, halogenated inhalation anesthetics, intravenous anesthetics, barbiturate intravenous anesthetics, benzodiazepine intravenous anesthetics and opioid agonist intravenous anesthetics; hormones and hormone modulators, such as abortifacients, adrenal drugs, corticosteroid adrenal drugs, androgens, antiandrogens; immunobiological agents, such as immunoglobulins, immunosuppressants, toxoids and vaccines; local anesthetics, such as amide local anesthetics and ester local anesthetics Anesthetics; musculoskeletal agents, such as anti-gout anti-inflammatory agents, corticosteroid anti-inflammatory agents, gold compound anti-inflammatory agents, immunosuppressive anti-inflammatory agents, non-steroidal anti-inflammatory drugs (NSAIDs), salicylate anti-inflammatory agents; minerals; vitamins, such as water-soluble or fat-soluble vitamins, vitamin A, vitamin B, vitamin C, vitamin D, vitamin E and / or vitamin K; and radionuclides, such as yttrium-90, iodine-131, samarium-153, lutetium-177, astatine-211, lead-212 / bismuth-212, radium-223, actinium-225 and thorium-227.

[0181] Additional non-limiting examples of useful therapeutic agents in the above categories include: (1) general analgesics, such as lidocaine or its derivatives, and NSAID analgesics, including diclofenac, ibuprofen, ketoprofen, and naproxen; (2) opioid agonist analgesics, such as codeine, fentanyl, hydromorphone, and morphine; (3) salicylate analgesics, such as aspirin (ASA) (enteric-coated ASA); (4) Hi-blocker antihistamines, such as clemastine and terfenadine; (5) sedative analgesics, such as ... enadine); (5) anti-infectives, such as mupirocin; (6) anti-anaerobic anti-infectives, such as chloramphenicol and clindamycin; (7) anti-fungal antibiotic anti-infectives, such as amphotericin B, clotrimazole, fluconazole and ketoconazole; (8) macrolide antibiotic anti-infectives, such as azithromycin and erythromycin; (9) other β-lactam antibiotic anti-infectives, such as aztreonam and imipenem; (10) penicillin antibiotic anti-infectives, such as nafcillin, oxacillin, penicillin G and penicillin V; (11) quinolone antibiotic anti-infectives, such as ciprofloxacin ) and norfloxacin; (12) tetracycline antibiotics, such as doxycycline, minocycline and tetracycline; (13) anti-tuberculosis and anti-mycobacterial drugs, such as isoniazid (INH) and rifampin; (14) antiprotozoal drugs, such as atovaquone and dapsone; (15) antimalarial and antiprotozoal drugs, such as chloroquine and pyrimethamine; (16) antiretroviral drugs, such as ritonavir and zidovudine; (17) antiviral drugs, such as acyclovir, ganciclovir, interferon α , remdesivir and rimantadine; (18) antifungal topical anti-infective drugs, such as amphotericin B, clotrimazole, miconazole and nystatin; (19) antiviral topical anti-infective drugs, such as acyclovir; (20) electrolyte and renal drugs, such as lactulose; (21) loop diuretics, such as furosemide; (22) potassium-sparing diuretics, such as triamterene; (23) thiazide diuretics, such as hydrochlorothiazide (HCTZ); (24) uricosuric drugs, such as probenecid; (25) enzymes, such as RNAse and DNAse; (26) antiemetics, such as prochlorperazine; (27) salicylic acid gastrointestinal anti-inflammatory drugs, such as sulfasalazine; (28) gastric acid pump inhibitor anti-ulcer drugs, such as omeprazole;(29) H2-receptor blocker anti-ulcer drugs, such as cimetidine, famotidine, nizatidine and ranitidine; (30) digestive agents, such as pancreatic lipase; (31) prokinetic drugs, such as erythromycin; (32) ester local anesthetics, such as benzocaine and procaine; (33) musculoskeletal corticosteroid anti-inflammatory drugs, such as beclomethasone, betamethasone, cortisone, etc. sone), dexamethasone, hydrocortisone, and prednisone; (34) musculoskeletal anti-inflammatory immunosuppressants, such as azathioprine, cyclophosphamide, and methotrexate; (35) musculoskeletal nonsteroidal anti-inflammatory drugs (NSAIDs), such as diclofenac, ibuprofen, ketoprofen, ketoprofen, and naproxen; (36) minerals, such as iron, calcium, and magnesium; (37) vitamin B compounds, such as cyanocobalamin (vitamin B12) and niacin (vitamin B3); (38) vitamin C compounds, such as ascorbic acid; and (39) vitamin D compounds, such as calcitriol.

[0182] Anti-cancer BioNV

[0183] In some aspects, the present disclosure includes BioNV. BioNV is approximately 20-1200nm in size and contains an outward-facing, membrane-embedded CAR capable of binding to a target molecule. Without wishing to be bound by theory, the biomimetic quality is attributed to the nanovesicle composition derived from the plasma membrane of allogeneic, low immunogenic modified cells. In an embodiment, BioNV comprises a plasma membrane-derived lipid bilayer that completely encapsulates an aqueous core that can accommodate a variety of cell-derived molecules, including perforins, granzymes, cytokines, and the like. In an embodiment, the aqueous core of NV can further encapsulate exogenous biologics, fluorescent proteins, tracer dyes, radionuclides, and small molecules, as well as other therapeutic agents.

[0184] To ensure the correct directionality of the CAR and eliminate BioNVs lacking the CAR, in embodiments, HPLC-based affinity chromatography techniques can be used to select and concentrate only the BioNVs exposed to the solvent CAR with sufficient surface concentrations. HPLC-based affinity chromatography techniques can be used to reduce the concentration of contaminating cell material and NVs containing immunogenic cell surface markers by positive or negative selection.

[0185] CAR constructs may include a variety of structural molecules. The structural function of prototype CAR includes a fusion protein, which includes an extracellular (or outward-facing) binding portion (e.g., scFv), which is connected to a transmembrane domain (e.g., CD28, CD3ζ, CD4, CD8α, ICOS, etc.) by a hinge peptide (e.g., CH2 / CH3 domains from IgG Fc regions, Gly-Gly-Ser peptide bonds, CD28 peptides, CD8α peptides, etc.), followed by a variety of intracellular signaling domains (e.g., 4-1BB, CD3ζ, CD28, 4-1BB, ICOS, CD27, OX40, etc.). In embodiments, BioNV lacks the intracellular mechanism of whole cells, so CAR design does not require any intracellular signaling molecules (primary CAR constructs). In embodiments, CAR constructs include an extracellular scFV binding portion fused to a CD28 transmembrane domain with an IgG CH2 / CH3 joint, and substantially lack any intracellular domain or function. In an embodiment, the CAR construct has a prototypical intracellular domain exchanged or otherwise fused with an anchor protein, such as a PLA2 domain from AAV, a fusion protein, a radionuclide binding domain, a cytoskeletal element, a small molecule transport domain, etc., which may facilitate fusion with target cells and / or packaging and release of the therapeutic payload.

[0186] In embodiments, CAR antigen binding molecules include a variety of binding moieties, including binding domains based on antibodies or antibody forms. In embodiments, BioNV includes antibodies or antibody forms binding moieties selected from one or more of the following: monoclonal antibodies, polyclonal antibodies, antibody fragments, Fab, Fab', Fab'-SH, F(ab')2, Fv, single-chain Fv (scFv), double antibodies, nano antibodies, linear antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies and fusion proteins comprising the antigen binding moiety of antibodies. In embodiments, CAR constructs include binding moieties with bispecific T cell engagers (BiTE), variable heavy chain IgG fragments VHH, VNAR or by engineered T cell receptors (TCR). In embodiments, the binding moiety may be a viral epitope recognition receptor (VERR) derived from one or more oncolytic virus receptors and / or viral ligands.

[0187] In the embodiments, BioNV is formed by destroying the cell membrane of engineered iPSC. In the embodiments, hypo-iPSC is characterized by B2M- / -, CIITA- / -, CD47+ / +, PD1- / - plasma membrane spectrum, and can be used to produce BioNV of the present invention. Hypo-BioNV can be produced from parental iPSC cell lines by methods such as ultrasonic treatment, adaptive focused acoustic technology, French press, extrusion, continuous extrusion, detergent cell lysis and electroporation. In the embodiments, continuous extrusion is a method for producing Hypo-BioNV. Continuous extrusion of iPSC can produce BioNV with HLA1 / HLA2 negative (low immunogenicity) and tgCD47+, showing PD1 resistance elimination.

[0188] In embodiments, the size uniformity of BioNV can be analyzed by dynamic light scattering (DLS), flow cytometry, mass spectrometry, and other methods for determining particle size. In embodiments, BioNV can be filtered according to particle size or size range to optimize renal clearance and other clinically relevant NV characteristics. In embodiments, the size of BioNV is about 20nm to 1200nm. In embodiments, the size of BioNV is about 10nm, about 20nm, about 30nm, about 40nm, about 50nm, about 60nm, about 70nm, about 80nm, about 90nm, about 100nm, about 120nm, about 140nm, about 160nm, about 180nm, about 200nm, about 300nm, about 400nm, about 500nm, about 600nm, about 700nm, about 800nm, about 900nm, about 1000nm, about 1100nm, or about 1200nm. The size range of BioNV is about 10nm to 20nm, about 20nm to 30nm, about 30nm to 40nm, about 40nm to 50nm, about 50nm to 60nm, about 60nm to 70nm, about 70nm to 80nm, about 80nm to 90nm, about 90nm to 100nm, about 10nm to 100nm, about 100nm to 200nm, about 200nm to 400nm, about 400nm to 600nm, about 600nm to 800nm, about 800nm ​​to 1000nm, or about 1000nm to 1200nm.

[0189] In embodiments, iPSC-derived BioNVs include NVs with only an outer plasma membrane leaflet, only an inner plasma membrane leaflet, and / or both leaflets of the plasma membrane lipid bilayer intact. In embodiments, iPSC-derived NVs include additional lipid additives (e.g., phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, ceramide, lecithin, etc.), nonionic surfactants (e.g., sorbitol monostearate, octadecylamine, etc.), sterols (e.g., cholesterol, bile salt derivatives, etc.), polyols (e.g., maltodextrin, sorbitol, sucrose, mannitol, etc.), and proteins (e.g., serum albumin, Fc, etc.) to improve physicochemical properties such as thermal stability, clearance, and therapeutic payload packaging / release. The content of cholesterol and the length and saturation of the hydrocarbon chain of the phospholipids affect the rigidity and stability of the bilayer, and thus the ability of the NV to carry and release drugs, biomolecules, and other therapeutic payloads. In embodiments, BioNV also incorporates zwitterionic lipids and methods of using zwitterionic lipids, for example, as described in U.S. Patent Publication No. US20130216607, the contents of which are incorporated herein by reference in their entirety. Accordingly, functionalizing the hydrophilic head of the lipid with a polymer or biomolecule can provide additional features to the vesicle surface, thereby shaping its interaction with blood components, tissues, and the immune system in vivo.

[0190] In embodiments, the CAR targets include multiple cell surface markers, including markers associated with a particular cancer, wherein the BioNV is cancer specific.

[0191] In an embodiment, BioNV is configured to encapsulate a variety of therapeutic payloads. In an embodiment, the primary targeted BioNV can be used to deliver small molecule therapeutic payloads. In an embodiment, the second generation (or third or fourth generation) of BioNV containing CAR derived from activated lymphocytes may contain cytokines and other cytotoxic peptides. In an embodiment, BioNV can be formatted to encapsulate and deliver plasmid DNA, for example, to express gene editing nucleases and gRNA in target cells. Alternatively or additionally, in an embodiment, BioNV can encapsulate nucleases and gRNA. In an embodiment, the targeted second generation (or third or fourth generation) BioNV can be designed to encapsulate and deliver additional therapeutic proteins or peptides of interest.

[0192] In embodiments, BioNV delivers a gene editing payload comprising a transcription activation response region (TAR) loop system. In embodiments, BioNV encapsulates a plasmid expressing a gene editor and contains a TAR loop sequence between the 5' end of the promoter and the gene editor / guide box and acts as a barrier to block transcription. In embodiments, transcription will only be triggered in cells that are infected and contain the HIV Tat protein. In embodiments, the Tat protein binds to the TAR loop, relaxes it, and releases the promoter for transcription, thereby expressing the editor and its guide.

[0193] In the embodiments, BioNV is CD34+, or derived from CD34+ cells, such as human CD34+ umbilical cord blood. CD34+ umbilical cord blood-derived cell lines can be used as the basic cell lines for the development, production and manufacture of BioNV for the delivery of gene editing therapeutics. CD34+ umbilical cord blood-derived low immunogenic cell lines have been experimentally confirmed to have low expression of HLA 1 / 2 and overexpression of CD47 (Deuse T. et al. Hypoimmunogenic derivatives of induced pluripotent stem cells evade immune rejection in fully immunocompetent allogeneic recipients. Nat Biotechnol. 2019 March; 37(3): 252-258).

[0194] In embodiments, BioNV and / or compositions comprising BioNV are administered in combination with one or more additional compounds. In embodiments, BioNV is pre-treated with one or more additional compounds, for example, prior to administration to a subject.

[0195] In the embodiments, BioNV is modular and allogeneic (off-the-shelf) due to the lack of immunogenicity of engineered iPSC. In the embodiments, the lack of whole-cell signaling components enables BioNV to easily regulate target specificity and resistance to immunosuppressive signals. In the embodiments, BioNV lacks genetic elements that cause uncontrolled cytokine storms, thereby minimizing the risk of cytokine release syndrome (CRS) in patients. In the embodiments, the amount of active cytokines, perforins, granzymes, interferons, interleukins, etc. encapsulated in BioNV is regulated in the process upstream of the cell (before BioNV is derived). In the embodiments, BioNV is derived from cells that can pass through biological barriers and / or viral receptors known to promote penetration.

[0196] Without wishing to be bound by theory, the BioNV produced by the iPSC engineered allogeneic basal cell line represents an immune stealth BioNV with the potential for multiple doses. The neutralization mediated by the BioNV antibody is minimized, and immune cell-mediated clearance is avoided (T cells and macrophages). In embodiments, BioNV does not contain live genetic material from cells derived therefrom to cause CRS or teratoma. In embodiments, the expression of certain cytokines encapsulated in BioNV increases and natural T cells can be recruited. In embodiments, BioNV can be derived from modified cell types with or without barrier-penetrating ligands to further control activity after infusion.

[0197] BioNV formation method

[0198] In embodiments, modified cells are low immunogenic cells derived from iPSC, and the iPSC has been engineered to have reduced or eliminated immunogenic protein expression and / or activity, and / or express immune protective proteins or increase their expression. In embodiments, iPSC is recovered from somatic cell state using microRNA technology instead of small molecule transactivators. A tighter differentiation system can be provided using microRNA, thereby producing higher quality iPSC. It is not desirable to be bound by theory, and these high-quality iPSCs are less prone to expression inhibition (rear engineering proteins, such as CD47) and genetic drift, and have higher culture division quality / quantity (before cell integrity problems occur, cultures can be divided more times than other methods).

[0199] In embodiments, BioNVs derived from iPSC-derived low immunogenic cells retain the functions of low immunogenic cells, such as, but not limited to, the ability to cross the blood-brain barrier, such as in the case of macrophages / monocytes, or tissue-specific factors, such as in the case of cardiomyocytes, hepatocytes, etc.

[0200] In an embodiment, the MHC class I and MHC class II complexes of allogeneic iPSCs are disrupted by knocking out key proteins involved in their expression, such as B2M, which is a serum protein found associated with MHC class I heavy chains on the surface of nearly all nucleated cells and is involved in presenting peptide antigens to the immune system.

[0201] In the embodiments, once B2M knockout (KO), CIITA KO, IL-6KO and CD47tg knock-in (KI) are engineered into iPSC, TRAC and TRBC genes can be knocked out. In the embodiments, only one gene of each is knocked out, rather than two genes on a single allele. In the embodiments, TRAC and TRBC genes can be knocked out as described herein. The purpose of knocking out TRAC and TRBC genes is to eliminate T cell receptors. In the embodiments, modified cells are differentiated into T cell subsets lacking T cell receptors to derive BioNV. Cells are genetically modified so that they are substantially lacking TCR, reducing the chance of competing ligands for CAR constructs that can non-specifically target alternative tissues. Therefore, in the embodiments, TCR genes are knocked out as a strategy to reduce the off-target effects of BioNV. In the embodiments, TRAC / TRBC knockouts typically reduce the possibility of CRS and BioNV toxicity.

[0202] In embodiments, the modified cells are expanded following engineering; any small-scale expansion or large-scale feeder system expansion method known in the art may be used.

[0203] In embodiments, after constructing B2M KO, CIITA KO, IL-6KO, CD47tg KI and IL-2 promoter-driven green fluorescent protein (GFP) (IL-2p GFP) reporter genes, CAR constructs can be integrated / engineered into cells. In embodiments, CAR constructs can be knocked into TRAC / TRBC genes, while knocking out the remaining TRAC / TRBC genes, thereby producing CAR+ and TRAC / TRBC- / - cells. In embodiments, CAR constructs can knock into the TRAC / TRBC gene positions on two loci at the same time, thereby producing CAR+ / + and TRAC / TRBC- / - cells.

[0204] In the embodiments, once B2M KO, CIITA KO, IL-6KO, CD47tg KI, IL-2p GFP KI and CAR modified cells (e.g., iPSC) are engineered, the quality of the immune synapse (IS) is measured between the CAR recognition domain and the biomarker. In the embodiments, the IS quality of BioNV can be directly related to the efficacy of whole cell therapy.

[0205] In embodiments, BioNV or low immunogenic cells derived therefrom include nucleic acids encoding GFP (and other fluorescent proteins). In embodiments, once B2M KO, CIITA KO, CD47tg KI, IL-6KO, TRAC / TRBC single KO are engineered into iPSC, GFP molecules are engineered into modified cell lines. In embodiments, this serves as a control cell line. In embodiments, non-control cell lines (therapeutic cell lines) do not have GFP. In embodiments, nucleic acids encoding GFP are operably connected to promoters from one or more of the following: IL-2, perforin, granzyme, alarmin, TNF, INF, combinations thereof and / or any other cell-specific genes or reporter genes. When lymphocytes are widely / overall activated due to various stimuli, the IL-2 promoter will be constitutively activated. In embodiments, more concentrated activation / inhibition (regulation) is used. In embodiments, IL-2p GFP reporter gene serves as an indicator (as part of BioNV derivatization process) of the extensive / overall activation degree of cells. In embodiments, GFP signaling combined with immunoblot analysis of cytokine levels (e.g., perforin, granzymes, alarmins, TNF, and INF) can effectively modulate the extent of widespread / overall activation of lymphocytes upon exposure to activation antigens. In embodiments, GFP is used to compare activation levels between manufacturing batches and ensure consistency in therapeutic development.

[0206] In the embodiments, low immunogenic cells are CD34+, or derived from CD34+ cells, such as human CD34+ umbilical cord blood. In the embodiments, CD34+ umbilical cord blood-derived cell lines can be used as the basic cell lines developed, produced and manufactured by BioNV for delivery of gene editing therapeutics. In the embodiments, low immunogenic cell lines derived from CD34+ umbilical cord blood have been experimentally confirmed to have low expression of HLA 1 / 2 and overexpression of CD47 (Deuse et al. "Hypoimmunogenic derivatives of induced pluripotent stem cells evade immune rejection in fullyimmunocompetent allogeneic recipients." Nat. Biotechnol. Vol. 37, No. 3, 2019: pp. 252-258. doi: 10.1038 / s41587-019-0016-3).

[0207] In embodiments, hypoimmunogenic cells can be engineered using a variety of hypoimmunogenic engineering techniques, for example, as described in Deuse et al., Han et al., Xu et al., and Harding et al., and as described in published U.S. patent applications US20190376045, US20190376045, US20210308183, and US20210292715 (to Deuse), US20210161971 (to Nagy), US20180141992 (to Strominger), and published European patent application 3693384 (to Poirot), each of which is incorporated herein by reference in its entirety (Han X et al., "Generation of hypoimmunogenic human pluripotent stem cells"). cells.” PNAS. Vol. 116, No. 21, 2019: pp. 10441-10446. doi: 10.1073 / pnas.1902566116.), (Xu H et al. “Targeted Disruption of HLA Genes via CRISPR-Cas9 Generates iPSCs with Enhanced Immune Compatibility.” Cell Stem Cell. Vol. 24, No. 4, 2019: pp. 566-578. doi: 10.1016 / j.stem.2019.02.005.) and (Harding et al., “Induction of long-term allogeneic cell acceptance and formation of immune privileged tissue in immunocompetent hosts.” BioRxiv 716571 [preprint], July 30, 2019. doi: 10.1101 / 716571.).

[0208] In an embodiment, BioNV is derived from cells that have been eliminated from HLA genes that encode MHC membrane glycoproteins that confer immune responses associated with GVHD rejection. HLA gene clusters can be divided into three categories: 1) MHC class I pathway, 2) MHC class II pathway, and 3) MHC class III pathway. Only MHC class I and class II pathways express protein complexes that trigger immune responses in GVHD, while MHC class III complexes do not participate in immune activities.

[0209] The elimination of MHC class protein complexes can trigger NK cells and macrophages to enter active clearance mode, and then the cells are destroyed. In order to avoid this killing mechanism, in an embodiment, CD47 isotype 2 transmembrane molecule protein tags can be added to the cell membrane of the modified cell to avoid NK and macrophage-mediated killing reactions, for example, as described in Willingham et al., Deuse et al. and Han et al. (Willingham SB et al. "The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors." PNAS. Vol. 109, No. 17, 2012: 6662-7 pages. doi: 10.1073 / pnas.1121623109.). In embodiments, cells can be engineered to use additional mechanisms to prevent these responses, such as those described in the following literature: 1) CD24 transmembrane molecule protein tag (e.g., as performed in Zhao et al.) (Zhao W et al. "Strategies for Genetically Engineering Hypoimmunogenic Universal Pluripotent Stem Cells." iScience. Vol. 23, No. 6, 2020: 101162. doi: 10.1016 / j.isci.2020.101162.), 2) membrane-bound surfactant protein-D (SP-D) (e.g., as performed in Jiaravuthisan et al.) (Jiaravuthisan P et al. "Amembrane-type surfactant protein D (SP-D) suppresses macrophage-mediated cytotoxicity in swine endothelial cells." Transpl Immunol. Vol. 47, 2018: pp. 44-48. doi: 10.1016 / j.trim.2018.02.003.), and 3) a molecular PD-L1 tag for preventing T cell responses. In embodiments, BioNV derived from 'activated' cells will encapsulate and / or release perforin and / or granzymes, resulting in targeted cell death. In embodiments, activated cells will produce perforin and / or granzymes to be packaged into BioNV. In embodiments, low immunogenic cells to be activated will not express PD-L1 to avoid the resulting BioNV being targeted to PD-1 on T cells. In embodiments, this reduces the possibility of releasing perforin and / or granzymes, resulting in unwanted T cell death.In embodiments, PD-L1 is overexpressed in BioNVs derived from cells that have not been activated and not loaded with apoptotic cytokines. In embodiments, PD-L1 of low immunogenic cells to be activated is downregulated, knocked out, or otherwise silenced. In embodiments, low immunogenic cells that have not been activated have upregulated PD-L1, i.e., for BioNVs used for gene editing agent delivery. In an embodiment, CD47 can be used in genetically engineered iPSCs to achieve immune tolerance to innate immune cells, such as in studies by Chhabra et al., Han et al., and Jaiswal et al. (Chhabra A et al. “Hematopoietic stem cell transplantation in immunocompetent hosts without radiation or chemotherapy.” Sci Transl Med. Vol. 8, No. 351, 2016: 351ra105. doi: 10.1126 / scitranslmed.aae0501.) and (Jaiswal S et al. “CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis.” Cell. Vol. 138, No. 2, 2009: pp. 271-85. doi: 10.1016 / j.cell.2009.05.046.). In embodiments, cells may be modified as described in U.S. Patent No. 8,562,997 to Jaiswal et al., which is incorporated herein by reference in its entirety.

[0210] In embodiments, some methods that do not completely knock out all HLA genes can be used, for example, as done by Xu et al. and Han et al., only HLA genes that are highly associated with immune response are knocked out, while HLA genes that inhibit macrophage or NK response remain intact (e.g., HLA-E, HLA-F, and HLA-G). In embodiments, this method does not require the addition of a CD47 tag; modified cells can be engineered to produce BioNVs with or without CD47.

[0211] In an embodiment, the method improves upon the low immunogenicity method of Table 3.

[0212] Table 3: Three methods for modifying cells using HLA knockout combined with a CD47 isoform 2 tag and a PD-L1 transmembrane tag (Zhao et al. 2020) (Gornalusse GG et al. "HLA-E-expressing pluripotent stem cells "Escape allogeneic responses and lysis by NK cells." Nat Biotechnol. Vol. 35, No. 8 Journal of Clinical Oncology, 2017: pp. 765-772. doi:10.1038 / nbt.3860.).

[0213]

[0214]

[0215] In embodiments, developing allogeneic modified cells involves removing MHC class I and MHC class II protein complexes by destroying certain HLA genes or B2M knockouts, and then knocking out the CIITA gene. In embodiments, due to its rapid mechanism of action, CRISPR gene editing methods can be used for knockout. In embodiments, zinc finger nucleases (ZFNs) and / or TALENS are used for knockout. In embodiments, Cre / Lox recombinase system is used to produce modified cells. In embodiments, RNA silencing (RNAi, shRNA, microRNA, CRISPR Cas13a-d, etc.) is used to produce modified cells.

[0216] In the embodiments, the method for developing allogeneic, low immunogenic modified cells is different from the method of Harding et al. to produce allogeneicity. The method of Harding et al. includes an alternative method based on immune escape mechanisms in nature, rather than deleting MHC class I / II genes and risking long-term acceptance of receptors. The method relies on the biomimicry of Harding et al. based on horizontally transmitted cancer DFTD type 2 (dominant in Tasmanian devils). In the embodiments, the development of allogeneic modified cells may include expressing immunomodulatory proteins CCL21, PD-L1, FasL, SerpinB9, H2-M3, CD47, CD200 and / or MFG-E8 or increasing their expression to protect cell derivatives from long-term immune rejection in mice (and humans) without deleting MHC class I / II proteins. In the embodiments, the modified cells express one or more proteins shown in Table 4, including any splice variants and / or isoforms (e.g., CD200 splice variants) of any of the proteins shown. In the embodiments, this system can be used to interfere with the activity of APCs, macrophages, NK cells, and T lymphocytes. In an embodiment, the modified cell line may also contain a safe cell system developed by Liang et al., in which a cell division gene is linked to a suicide gene to prevent uncontrolled teratomas from causing cancer (Liang Q et al. "Linking a cell-division gene and a suicide gene to define and improve cell therapy safety." Nature. Vol. 563, No. 7733, 2018: pp. 701-704. doi: 10.1038 / s41586-018-0733-7.).

[0217] In an embodiment, the method improves upon the low immunogenicity method of Table 4.

[0218] Table 4: Expression or increased expression of illustrative proteins used to generate allogeneic modified cells.

[0219]

[0220] In an embodiment, the low immunogenic cells from which BioNV are derived are engineered to knock out one or more of the following: HLA-A, HLA-B, HLA-C, HLA-E, HLA-G, HLA-F, CIITA, IL-6, IL-4, IL-10, IL-16, TRAC, TRBC, SerpinB9 and / or any combination thereof; and knock in one or more of the following: CCL2, PD-L1 (in BioNV from unactivated cell sources), CTLA-4, H2-M3, CD24, CD47 (minus the 3'UTR region or an alternative 3'UTR region that does not contain inhibitory microRNA binding sites), MFG-E8, CD200 and / or any combination thereof.

[0221] In embodiments, BioNV is produced by low immunogenic cells having one or more modifications of Table 5.

[0222] Table 5: Illustrative engineered cell expression profiles for BioNV formation for human use (Fife BT and Bluestone JA."Control of peripheral T-cell tolerance and autoimmunity via the CTLA-4and PD-1pathways.” Immunol Rev. Vol. 224, 2008: pp. 166-82. doi:10.1111 / j.1600-065X.2008.00662.x.) and (Rong Z et al. "An effective approach to prevent Immune rejection of human ESC-derived allografts." Cell Stem Cell. Vol. 14, No. 1 2014: pp. 121-30. doi:10.1016 / j.stem.2013.11.014.).

[0223]

[0224] In embodiments, the inactivation / activation of genes is controlled by inducible promoters during the entire differentiation, activation and manufacturing process of BioNV. In embodiments, the destruction of MHC, TCR and CRS genes will produce - / -CRS and - / -TCR allogeneic iPSC, resulting in it having a plasma membrane showing low immunogenicity characteristics when infused into a subject. The CRS genes related to CRS pathogenesis include IL-6, IL-10, IFN-γ, monocyte chemotactic protein 1 (MCP-1), granulocyte macrophage colony stimulating factor (GM-CSF), and other cytokines, including tumor necrosis factor (TNF), IL-1, IL-2, IL-2-receptor-α and IL-8. In embodiments, one or more of these genes are inactivated, for example, in cells derived from BioNV.

[0225] In the embodiments, BioNV is formed by destroying the cell membrane of engineered iPSC. In the embodiments, hypo-iPSC is characterized by B2M- / -, CIITA- / -, CD47+ / +, PD1- / - plasma membrane spectrum, and can be used to produce BioNV. Low immunogenicity BioNV can be produced from parental iPSC cell lines by methods such as ultrasonic treatment, adaptive focused acoustic technology, French press, extrusion, continuous extrusion, detergent cell lysis and electroporation. In the embodiments, continuous extrusion is a method for producing low immunogenicity BioNV. In the embodiments, continuous extrusion of iPSC can produce HLA1 / HLA2 negative (low immunogenicity) and tgCD47+ BioNV, showing PD1 resistance elimination.

[0226] In embodiments, the genetic engineering of iPSC includes gene editing technology, such as CRISPR-based gene editing system, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), large range nuclease and other gene editing methods, with the purpose of producing allogeneic / low immunogenic iPSC and / or for CAR box integration. In embodiments, the genetic engineering of iPSC may refer to the reduction or elimination of transcription of any genetic element; similarly, the genetic engineering of iPSC may refer to the increase or knock-in of expression of any genetic element (including both endogenous and exogenous genetic elements).

[0227] For example, in an embodiment, stable cell integration (safe harbor genetic position) in iPSC can be controlled by implementing a Tet-regulated CRISPRa+ targeting 3x transcription factor targeting gRNA system. The activation of three upstream transcription factors by the CRISPR activation system can trigger a signal cascade event, enhancing the production of CAR that replaces endogenous antibody ORF at a specified site. This system can be 'tuned' by including a Tet-regulated promoter, so that the concentration of cell surface CAR can be changed. Next, stable cell replacement of CDR and heavy and light chain antibody regions with a CAR box can be achieved by Cpf-1 directed homology-directed repair (HDR). Finally, the stably integrated CAR box may contain flanking gRNA binding sites, which allow scFV (and other antibody forms) or VERR / viral ligands to be repeatedly exchanged or changed to quickly and consistently insert the desired sequence.

[0228] In embodiments, the allogeneic and low immunogenic properties of iPSCs can be further improved by inducing overexpression of immunoprotective molecules. For example, and without limitation, overexpression of CD47 (and other cell surface integrins) can reduce the kinetics of macrophages consuming BioNV products from the blood. In embodiments, in addition, the allogeneic and low immunogenic properties of iPSCs can be improved by the expression of α and β phagocytic integrins. In embodiments, overexpression of similar immunogenic protective cell surface markers that signal leukocytes can be used as a strategy to increase the half-life of BioNV after infusion.

[0229] In embodiments, iPSCs are genetically engineered for CAR cassette integration. CAR cassette integration may include both integrative and non-integrative transgenic insertions. Non-limiting examples of non-integrative transgenic insertions include mRNA, non-integrative lentivirus, and endonuclease targeting methods. Integrative CAR cassette insertion methods include stable retroviral vector insertion and transposase-based integration systems. For example, a retroviral vector can be used to achieve stable CAR cassette transduction, which can enable iPSCs to maintain genetic elements encoding CAR throughout differentiation, amplification, and activation. In embodiments, brexucabtagene autoleucel ( Kite Pharma Inc.) and axicabtagene ciloleucel ( Kite Pharma Inc.) CAR cassette into T cells, and tisagenlecleucel ( Novartis International AG) (Labbé, RP, et al. "Lentiviral Vectors for T Cell Engineering: Clinical Applications, Bioprocessing and Future Perspectives." Viruses 13 (2021); 1528. doi: 10.3390 / v13081528).

[0230] In embodiments, a variety of transcriptional control elements, such as tetracycline on / off promoters (or similar drug-regulated promoters) can be used to regulate the CAR concentration on the surface of iPSC basal cell lines or any downstream differentiated cells (and the resulting BioNV), to drive the expression of CRISPR activation / gRNA (CRISPRa) systems. Then, the CRISPRa system can activate antibody regulatory transcription factors, such as Drm2, Fr5, and Bxp2, which regulate the expression of engineered CAR boxes integrated into antibody gene loci (positions where antibody genes have been replaced). In addition, similar transcriptional control elements can be provided to control the overexpression of genes (such as CD47) at defined manufacturing stages, drive genes that control differentiation, etc.

[0231] Subjects and / or Animals

[0232] In embodiments, subject and / or animal are mammals, for example, people, mice, rats, guinea pigs, dogs, cats, horses, cattle, pigs, rabbits, sheep or non-human primates. In embodiments, subject and / or animal are non-mammals, for example zebrafish. In embodiments, subject and / or animal are transgenic animals, which include fluorescent cells, for example RPE cells and / or immune cells with GFP. In embodiments, subject and / or animal are people. In embodiments, BioNV is derived from fluorescently labeled cells and / or is packaged with fluorescently labeled proteins or labels (for example, GFP). In embodiments, mankind is pediatric mankind, adults, elderly, infants or children. In other embodiments, mankind is referred to as patient.

[0233] In an embodiment, the method of treatment comprises administering to a human whose age ranges are about 0 months to about 6 months, about 6 months to about 12 months, about 12 months to about 18 months, about 18 months to about 36 months, about 1 year to about 5 years, about 5 years to about 10 years, about 10 years to about 15 years, about 15 years to about 20 years, about 20 years to about 25 years, about 25 years to about 30 years, about 30 years to about 35 years, about 35 years to about 40 years, about 40 years to about 45 years, about 45 years to about 50 years, about 50 years to about 55 years, about 55 years to about 60 years, about 60 years to about 65 years, about 65 years to about 70 years, about 70 years to about 75 years, about 75 years to about 80 years, about 80 years to about 85 years, about 85 years to about 90 years, about 90 years to about 95 years, or about 95 years to about 100 years.

[0234] In embodiments, the subject is a non-human animal, and thus the disclosure relates to veterinary uses. In embodiments, the non-human animal is a domestic pet. In embodiments, the non-human animal is a livestock animal.

[0235] In embodiments, serum and / or immune cells and / or tumor cells are assessed and / or affected. In embodiments, immune cells include cells of the innate immune system of a subject and / or animal. In embodiments, such cells include but are not limited to NK cells, monocytes, DCs, B cells, macrophages, CD4+T cells, and CD8+T cells. In various embodiments, the disclosure provides for detecting the presence, absence, or measurement of tumor volume, tumor cells, metastasis, cDNA, or RNA in a sample derived from a subject.

[0236] Reagent test kit

[0237] In an embodiment, the present disclosure provides a kit that can simplify the administration of any agent described herein. An exemplary kit of the present disclosure includes any agent described herein in unit dosage form. In an embodiment, the unit dosage form is a container, such as a prefilled syringe, which can be sterile, containing any agent described herein and a pharmaceutically acceptable carrier, diluent, excipient or vehicle. In an embodiment, the kit also includes a label or printed instructions indicating the use of any agent described herein. In an embodiment, the kit also includes an eyelid speculum, a local anesthetic, and a cleaning agent for injection surface. In an embodiment, the kit further includes one or more additional agents described herein.

[0238] In aspects, the disclosure includes syringes comprising one or more compositions of the disclosure. In embodiments, the syringe is pre-filled with a certain volume of the composition. In embodiments, the volume of the syringe pre-filled is about 1 mL to about 10 mL. In embodiments, the syringe is pre-filled with a volume of about 10 mL, about 9 mL, about 8 mL, about 7 mL, about 6 mL, about 5 mL, about 4 mL, about 3 mL, about 2 mL, about 1.9 mL, about 1.8 mL, about 1.7 mL, about 1.6 mL, about 1.5 mL, about 1.4 mL, about 1.3 mL, about 1.2 mL, about 1.1 mL, or about 1.0 mL or less of the composition.

[0239] In an embodiment, the syringe comprises a composition having a shelf life in the range of about 1 hour to about 1 week. In an embodiment, the syringe comprises a composition having a shelf life of at least about 12 hours, about 24 hours, about 36 hours, about 48 hours, or about 72 hours when stored at a temperature in the range of about -85°C to about 25°C. In an embodiment, the syringe comprises a composition having a shelf life of at least about 12 hours, about 24 hours, about 36 hours, about 48 hours, or about 72 hours when stored at a temperature in the range of about 15°C to about 25°C.

[0240] In embodiments, the storage temperature is about -80°C. In embodiments, the storage temperature is about -20°C. In embodiments, the storage temperature is about 4°C. In embodiments, the storage temperature is about 21°C. In embodiments, the kit comprises lyophilized BioNV.

[0241] In one embodiment, the kit includes a container containing a composition comprising a BioNV of the disclosure, a therapeutically effective amount of an additional therapeutic agent (eg, a therapeutic agent described herein), and instructions for use.

[0242] Example

[0243] Example 1: Production of BioNV by continuous extrusion

[0244] Biomimetic nanovesicles (BioNV) can be produced from low immunogenic cell lines, as illustrated by the scheme depicted in Figure 17. The following scheme outlines the experiment of micro CAR BioNV. Micro CAR BioNV can be derived from any cell type. The following outlines BioNV derived from activated or unactivated natural killer lymphocytes (NK cells). BioNV can be designed to target any antigen receptor, as described herein.

[0245] A combination of flow cytometry and iodixanol density gradients can be used to measure CAR expression levels in poorly immunogenic cell lines (e.g., Figure 3 Step 1).

[0246] The differentiation of iPSCs expressing surface CAR into lymphocytes can be analyzed by identification of lymphocyte markers, including, for example, CD4 / CD8 (T cells) or CD56 / CD16 (natural killer cells) and other cell surface markers (e.g., Figure 3 Step 2). The expression profile can be determined by flow cytometry, RT-PCR and / or CRISPR-based analysis.

[0247] Next, lymphocyte activation can be achieved using low predetermined concentrations of biomarker antigen-coated beads in two phases over two weeks (e.g. Figure 3 This process also allows analysis of the quality of the immunological synapse (IS) between the CAR and the antigen-coated beads, using well-established protocols to measure i) quantification of F-actin accumulation at the site of synapse formation, ii) distribution of pZeta at the synapse, iii) clustering of antigen across the IS site, and / or iv) polarization of lytic granules (LGs) containing perforin and granzymes.

[0248] After lymphocyte activation, the cells are expanded using a defined protocol (e.g. Figure 3After expansion, each cell population is analyzed for levels of perforin and granzymes (or other luminal payload, if applicable) to ensure that concentration levels are consistent across batches. This is accomplished using a series of qPCR, immunoblotting, flow cytometry, and / or mass spectrometry. If a sufficiently large cell population is available in step 3, the expansion step may not be necessary.

[0249] Once the cells are activated and producing the desired therapeutic protein, they are expanded, harvested, washed several times, and then placed into a buffered extrusion medium. The cells are then fully processed (e.g., Figure 3 Step 5). In the initial extrusion step of the continuous extrusion process, the nucleus (along with nuclear components, including nuclear pores, genomic material and transcription factors) and mitochondria are eliminated. The sample is then treated with an endonuclease, such as BENZONASE. BENZONASE is a non-specific recombinant endonuclease that cuts all types of DNA and RNA variants into non-functional fragments of <8 soluble base pairs. This minimizes the amount of nucleic acid loaded per sample and on a scalable basis without interfering with the BioNV membrane chemistry. The lysis process also eliminates nucleic acid viscosity, allowing the material to be subsequently loaded and passed through the next set of extrusion filters.

[0250] The continuous extrusion process will avoid eliminating other organelles, such as the Golgi or endoplasmic reticulum. The membrane systems of these organelles are highly evolved to transport vesicles (release and absorption) between folded membranes. For example, the cis and trans faces of the Golgi contain unique lipid compositions that contribute to low energy barrier absorption and release in vesicle transport. These components are relatively low in the cytoplasmic membrane. Therefore, it is not so advantageous to separate the cytoplasmic membrane for BioNV derivatization. When BioNVs pass through polycarbonate filters during continuous extrusion, they are disrupted and spontaneously formed depending on the pore size. The BioNVs produced by this process contain membranes with a uniform mixture of cytoplasmic, Golgi and ER lipid content and protein components, which can significantly improve their affinity for cell and tissue delivery absorption compared to BioNVs that have been treated to eliminate these organelles. These features can be translated into better and more consistent absorption of BioNVs into target cells at much lower doses than systems that do not have these characteristics.

[0251] After the extrusion step, the BioNV was passed through an α-CD3 HPLC (FPLC under expansion) column to remove a low percentage (approximately 0.05%) of reversed BioNV (e.g. Figure 3This is done to ensure that the resulting BioNV has homogenous orientation relative to the membrane. Yield loss in this step is lower because it is a flow-through process that captures impurities. After the BioNV is collected after the HPLC / FPLC step, it will be tested through a standardized process.

[0252] The standardization process includes one or more of the following measurements:

[0253] BioNV Homogeneity: Nanoparticle flow cytometry (NanoFCM) can be used to confirm the BioNV concentration, homogeneity of size, density of BioNV, and / or homogeneity of the BioNV luminal composition.

[0254] Luminal payload concentration NanoFM technology can be used to determine the type and concentration of nucleic acids / proteins packaged into the BioNV lumen. These data can be confirmed in parallel by one or more methods including immunoblotting, mass spectrometry and BCA analysis to determine the nucleic acid and protein content of BioNV.

[0255] BioNV stability A combination of nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), and electron microscopy (EM) can be used in combination with immunoblotting and mass spectrometry to determine the physical and biochemical characteristics of BioNV over 8 to 10 months. Data from these assays can include protein expression profiles, the extent of intact BioNV membrane / packaging, and / or the extent of aggregation.

[0256] Membrane Integrity: The integrity of the BioNV membrane was assessed using a calcein release assay in combination with NanoFCM to evaluate membrane permeability. The results provided insight into the leaky properties of the BioNV relative to a standardized set of BioNVs.

[0257] Mass of lumen payload: Depending on the nature of the payload, a variety of analytical assays can be used to determine the quality of the lumen-encapsulated payload. If the delivery is a nucleic acid, qPCR and / or sequencing at 8 to 10 months can be used to check the integrity and quantity of the nucleic acid payload. For proteins, BioNV components can be analyzed using one or more of NanoFCM, mass spectrometry, and immunoblot analysis to analyze protein payloads.

[0258] CAR mass and surface density:NanoFCM, mass spectrometry and / or immunoblotting analysis can be used to determine the CAR surface density. Compared with the whole cell surface density, the CAR surface density in BioNV is expected to be at least about 5 times to at least about 10 times higher. Compared with whole cells, this can significantly enhance the targeting of antigens. CAR quality can be determined at the cell stage, as described above (e.g., as described in step 3). Mathematical models can be used to infer cell quality data and apply them to BioNV related to efficacy study data results.

[0259] BioNV features: BioNV can be tested for basic functionality, including multiple well-defined standardized assays, such as in vitro cellular uptake into target cells with or without expressed antigens, and its ability to cross dense tissues, such as those in a human retina model. These basic functional assays can be performed immediately after the continuous extrusion process, after which preclinical studies will address the remaining quality and functional properties of BioNV.

[0260] After the extrusion process, the mini-CAR BioNVs (as well as VERR and / or viral ligand BioNVs) will be classified into the following groups based on activation of immune synapse (IS) binding:

[0261] 1) Low-quality whole-cell IS pool (activated), where “low-quality” refers to the degree of IS binding;

[0262] 2) medium-quality whole-cell IS pool (activated), where “medium-quality” refers to the degree of IS binding relative to “low” and “high” masses;

[0263] 3) High-quality whole-cell IS pools (activated), where “high-quality” refers to the extent of IS binding; and

[0264] 4) High quality whole-cell IS pool (not activated).

[0265] BioNV can be cryopreserved at this stage.

[0266] Statistical analysis will be used to evaluate and address batch-to-batch consistency. Upper and lower control limits will be based on data from use controls (e.g., mean, 95% confidence interval, range, and / or percentile distribution).

[0267] Example 2: In vitro evaluation of BioNV

[0268] Three experiments will be performed using each of low-, medium-, and high-quality activated mini-CAR NK BioNVs and high-quality mini-CAR NK unactivated BioNVs. Experiments will be performed in combination with whole-cell CAR-NKs.

[0269] Cytotoxicity: will use Chromium-51( 51Cr) release assay, using multiple cell lines derived from various cancers as well as control cell lines to evaluate the cytotoxicity of the mini-CAR NK BioNV (and whole-cell mini-CAR NK cells). 51 Cr, 51Cr will be incorporated into the cells. After the cancer cell lines are treated with BioNV (and / or whole cells), 51 Cr is then released from the target cells during lysis. The supernatant can be collected, centrifuged to clarify the supernatant, and measured 51 The amount of Cr was used as a proxy for cell lysis. 51 Cr can be measured directly using a gamma counter, or mixed with scintillation cocktail for reading in a plate reader (eg, using LUMAPLATE, Perkin Elmer).

[0270] Nanoparticle flow cytometry (NanoFCM), ELISA and / or immunoblot analysis will be used to monitor the concentration and presence of lumen-loaded therapeutic payloads, such as perforin, granzymes, p53, alarmins, TNF and / or INF. The concentration and / or presence of these proteins can also be determined during cell killing assays. PCR will be used to monitor the activation state of apoptosis-related genes (e.g., BAD, BAX, and Caspase-3) during treatment. The uptake of each subpopulation of micro-CAR NK BioNV by cells will be measured by rhodamine staining. Control treatment using "high quality" unactivated micro-CAR NK BioNV will be used for comparison.

[0271] Evaluations will be performed in one or more of the following cell lines.

[0272] Lung cancer cell lines:

[0273] A549: A widely used non-small cell lung cancer (NSCLC) cell line derived from lung cancer.

[0274] H1975: A NSCLC cell line derived from a patient with acquired resistance to the epidermal growth factor receptor (EGFR) inhibitor gefitinib.

[0275] H1650: A NSCLC cell line carrying an EGFR exon 19 deletion mutation that is associated with sensitivity to EGFR tyrosine kinase inhibitors (TKIs).

[0276] HCC827: A NSCLC cell line harboring an EGFR exon 19 deletion mutation that is highly sensitive to EGFR TKIs.

[0277] H1299: A p53-deficient NSCLC cell line commonly used in lung cancer research.

[0278] Calu-3: a lung adenocarcinoma cell line derived from a metastatic site.

[0279] PC-9: A NSCLC cell line derived from a patient with lung adenocarcinoma harboring a deletion in EGFR exon 19. PC-9 cells are used to study EGFR-targeted therapies.

[0280] Hepatocellular carcinoma cell lines:

[0281] HepG2: Hepatocellular carcinoma cell line.

[0282] Huh7: Hepatocellular carcinoma cell line.

[0283] PLC / PRF / 5: A liver cancer cell line for studying viral hepatitis-associated hepatocellular carcinoma.

[0284] Hep3B: a hepatocellular carcinoma cell line lacking functional p53.

[0285] SK-Hep-1: A hepatocellular carcinoma cell line derived from lymph node metastases of a patient with hepatocellular carcinoma.

[0286] Breast Cancer Cell Lines:

[0287] MCF-7: Hormone receptor positive (estrogen receptor positive and progesterone receptor positive).

[0288] T47D: A hormone receptor-positive breast cancer cell line derived from a metastatic site of a patient with invasive ductal carcinoma.

[0289] MDA-MB-231: A triple-negative breast cancer cell line that lacks expression of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2).

[0290] BT-474: A breast cancer cell line overexpressing the HER2 / neu receptor.

[0291] SK-BR-3: A HER2-positive breast cancer cell line derived from the pleural effusion of a patient with metastatic breast adenocarcinoma.

[0292] ZR-75-1: an estrogen receptor-positive breast cancer cell line derived from a metastatic site of a patient with invasive ductal carcinoma.

[0293] Brain cancer cell lines:

[0294] U87MG: glioblastoma cell line.

[0295] U251: A glioma cell line derived from human glioblastoma multiforme.

[0296] LN-18: A glioma cell line derived from human glioblastoma used to study glioma invasion.

[0297] T98G, SF268, D54, A172: glioblastoma cell lines derived from human glioblastoma multiforme.

[0298] Renal Cell Carcinoma Cell Lines:

[0299] 786-O: A renal cell carcinoma cell line derived from a primary renal tumor from a patient with clear cell carcinoma.

[0300] ACHN: a renal cell carcinoma line derived from human renal adenocarcinoma.

[0301] Caki-1, 769-P: A renal cell carcinoma cell line derived from human renal clear cell carcinoma.

[0302] A498: A renal cell carcinoma cell line derived from a human renal cell carcinoma with clear cell histology.

[0303] RCC-MF: (Renal Cell Carcinoma Metastasized to the Fibula) A renal cell carcinoma cell line derived from a metastatic lesion in the fibula of a patient with renal cell carcinoma.

[0304] Lymphoma cell lines:

[0305] Raji, Daudi: Burkitt's lymphoma cell lines as models of aggressive B-cell lymphomas.

[0306] Jurkat: A T-cell lymphoma cell line derived from a patient with acute T-cell leukemia.

[0307] SU-DHL-4: diffuse large B-cell lymphoma (DLBCL) cell line.

[0308] HUT78: A cutaneous T-cell lymphoma (CTCL) cell line derived from a patient with Sézary syndrome.

[0309] OCI-LY10: A DLBCL cell line derived from a DLBCL patient.

[0310] Toledo: A mantle cell lymphoma cell line derived from a mantle cell lymphoma patient.

[0311] Leukemia cell lines:

[0312] K-562: A chronic myeloid leukemia (CML) cell line derived from a patient with blast crisis CML.

[0313] HL-60: A promyelocytic leukemia cell line derived from a patient with acute promyelocytic leukemia.

[0314] THP-1: A monocytic leukemia cell line derived from a patient with acute monocytic leukemia.

[0315] Jurkat: A T-cell leukemia cell line derived from a patient with acute T-cell leukemia.

[0316] MV4-11: a myelomonocytic leukemia cell line derived from an AML patient.

[0317] NALM-6: A B-cell precursor leukemia cell line derived from a patient with B-cell acute lymphoblastic leukemia (ALL).

[0318] REH: A B-cell precursor leukemia cell line derived from a patient with B-cell ALL.

[0319] Melanoma cell lines:

[0320] A375, SK-MEL-28, SK-MEL-2, WM-266-4, UACC-62: Melanoma cell lines derived from metastatic sites of patients with malignant melanoma.

[0321] M14, MeWo: melanoma cell lines derived from primary tumors of patients with malignant melanoma.

[0322] Example 3: In vivo evaluation of the efficacy of micro-CAR NK cells and BioNV

[0323] Experiments will be performed using mini-CAR NK whole cells and BioNV against GPC3. GPC3 CAR NK cells will be used to establish an immune synapse (IS) quality standard curve.

[0324] Mouse Model: BioNV and / or whole cells will be tested using one or more of the following mouse models: hGPC3TG-NSG: hepatocellular carcinoma (HCC) expressing human GP3 protein in immunodeficient mice lacking mature T cells, B cells, and NK cells; HCC NSG: hepatocellular carcinoma (HCC) immunodeficient mice lacking mature T cells, B cells, and NK cells; HCC PDX: hepatocellular carcinoma (HCC) patient-derived xenograft mice with tumor implants; and severe combined immunodeficient (SCID) mice. The mice may have a C57BL / 6J background.

[0325] Tumor implantation and visualization:Prior to administration to establish in vivo tumors, tumor cells will be fluorescently labeled for visualization and measurement using in vivo animal imaging devices, such as dual positron emission tomography (PET) scans and computed tomography (CT) scans (PET / CT), such as IVIS known dose, high-resolution micro-CT (Perkin Elmer). Tumor cells will be transduced with retrovirus encoding one or more luciferase genes, such as RLuc luciferase genes (sea pansy / Renilla reniformis), FLuc, and teLuc, under puromycin selection. To evaluate the efficacy and persistence of GPC3 CARNK in vivo, several subpopulations will be administered, including "low", "medium", and "high" IS quality GPC3 CARNK (e.g., as described above), in which cells will be transduced with plasmid eGFP (enhanced green fluorescent protein). 8-10 week old mice (e.g., hGPC3TG-NSG, HCC NSG, and / or HCC PDX mice) will be injected intraperitoneally (IP) with 5×10 6 After about 5-10 days, when palpable tumors (approximately 50 mm 2 ) were administered by IP injection. 6 GPC3 CAR NK and control GCP3-CAR-NK cells. In order to perform in vivo imaging of GPC3 CAR NK expressing eGFP and tumor cells expressing luciferase genes, mice will be IP injected with D-luciferin (about 150 mg / kg, eGFP-FLuc substrate) and coelenterazine (about 1 mg / kg, RLuc substrate), respectively. Since eGFP and RLuc use different substrates, they can be combined as dual reporters to monitor tumors and GPC3 CAR NK.

[0326] Tumors in hGPC3TG-NSG, HCC NSG, and / or HCC PDX mouse models will be visualized and measured using PET / CT, such as IVIS known dose, high resolution micro-CT (Perkin Elmer). Alternatively, tumor size can be measured using calipers at the maximum longitudinal diameter (length) and maximum transverse diameter (width).

[0327] Tumor Penetration:The tumor penetration properties of each IS quality microCAR NK GPC3BioNV will be evaluated at various stages of tumor growth and compared with whole cell infiltration of the tumor. BioNV tumor penetration can also be compared with other systems such as oncolytic viruses, AAV and / or exosomes. MicroCAR NK BioNV (and cells) from the three groups and activated and non-activated whole cells will be tested by tumor cross-sectioning and / or immunostaining at various stages and time courses of tumor growth.

[0328] In vivo antitumor activity ： The homing, persistence and anti-tumor activity (alone and in combination with whole cell CAR NK) of each mass of micro CAR NK BioNV will be evaluated in chip-based tumor models and animal models. Severe combined immunodeficiency (SCID) and patient-derived xenograft (PDX) mouse models will be used with in vivo imaging systems (e.g., PET / CT and / or IVIS, Perkin Elmer). Efficacy will be measured in a dose-dependent response manner over time using one or more of tumor volume, tumor weight, and tumor suppression. Micro CAR therapeutic cargoes for each tumor will be monitored, such as perforin, granzyme, and p53 release. PCR, ELISA, and / or immunoblotting will be used to evaluate apoptotic gene activation during treatment, such as BAD, BAX, and Caspase-3.

[0329] Example 4: In vitro and in vivo evaluation of CRS-related toxicity

[0330] Cytokine Activation: The extent of in vitro cytokine activation in one or more of the cell lines outlined herein will be assessed using each mass of micro-CAR NK GCP3 BioNV and CAR NK GCP3, as well as one or more control CAR cell lines (e.g., CAR-T-T1E, CAR-T cells with a pan-ErbB targeting CAR). Cancer cell lines will be co-cultured with CD8 T cells. Micro-CAR NK GCP3 BioNV and CAR NK GCP3 cells will be added in increasing concentrations and the extent of cytokine activation will be determined over time. Levels of molecules IL-6, IL-2 and / or INF-γ will be measured by PCR, immunoblotting, ELISA and / or flow cytometry. In addition, as described above, 51 Cr release assay was used to evaluate cytotoxicity.

[0331] CRS reduction:The in vivo model will be used to measure the extent of CRS caused by each mass of micro-CAR NK GCP3 BioNV, CAR NKGCP3 cells and their combination therapy compared to the standardized CAR-T-T1E cells and CAR-EXO-T1E BioNV that cause cytokine release syndrome (CRS). The extent of CRS (in vivo) will be determined by measuring serum levels of various cytokines (molecules IL-6, IL-2 and / or INF-γ) and comparing them to the extent of cytokine release of whole cell controls. The concentrations of micro-CAR NK GCP3 BioNV, CAR NK GCP3 cells and controls will be increased in a dose-dependent manner until CRS induction is observed.

[0332] Lymphocyte recruitment and PD-L1 blockade: BioNV uptake and cell / tumor killing of microCAR BioNVs targeting checkpoint inhibitors (CPIs) (e.g., PDL1 or CTLA4) (e.g., by single or bispecific CAR constructs or transmembrane antibodies) will be determined as described above. BioNVs with anti-CPI CARs will also be used for natural lymphocyte recruitment and / or lymphocyte activity. Lymphocyte recruitment and / or activity to the tumor microenvironment (TME) will be assessed by flow cytometry and / or by measuring cytokine concentrations associated with lymphocyte migration by PCR, ELIA, immunoblotting, and / or NanoFCM.

[0333] Animal Model: One or more of the following animal models will be used:

[0334] SCID (CB17) or SCID (beige) model: SCID mice with a CB17 or beige background are used. These mice have a non-functional immune system.

[0335] NOD-SCID model: NOD-SCID (non-obese diabetic SCID) mice have combined immunodeficiency and are commonly used in xenograft studies. They allow the implantation of human lung cancer cells or tumor tissue.

[0336] NSG (NOD-SCID IL-2Rγnull) model: NSG mice are highly immunodeficient and lack the IL-2Rγ receptor, making them amenable to human cell engraftment.

[0337] Rag2- / -γc- / - model: Rag2- / -γc- / - mice are double knockout mice lacking the Rag2 gene and the common γ chain (γc) gene.

[0338] NOG (NOD / Shi-scid / IL2Rγnull) model: NOG mice are severely immunodeficient and lack T cells and B cells.

[0339] The following xenograft models can be used (or adapted tumor models on a chip - 'tumor on a chip') to study the effects of the mini-CAR BioNV (alone or as a combination therapy with whole cell CAR therapy) in vivo:

[0340] Lung cancer tumors:

[0341] A549 xenograft model: A human lung adenocarcinoma cell line used to establish xenograft models in mice.

[0342] H460 Xenograft Model: A human lung cancer cell line used to establish xenograft models in mice.

[0343] HCC827 xenograft model: a human lung adenocarcinoma cell line harboring an epidermal growth factor receptor (EGFR) mutation.

[0344] NCI-H1975 xenograft model: A human lung adenocarcinoma cell line containing both EGFR and T790M mutations associated with resistance to EGFR targeted therapies.

[0345] Patient-derived xenograft (PDX) models: PDX models involve implanting patient-derived lung cancer tissue directly into mice. The models retain the characteristics and heterogeneity of the original patient tumor.

[0346] Hepatocellular Carcinoma Tumors:

[0347] HepG2 and Huh7 xenograft models: Human hepatocellular carcinoma cell lines used to establish xenograft models in mice.

[0348] PLC / PRF / 5 xenograft model: a human hepatocellular carcinoma cell line derived from a liver cancer patient.

[0349] SK-HEP-1 xenograft model: A human hepatocellular carcinoma cell line commonly used to study HCC biology and investigate therapeutic approaches.

[0350] Patient-derived xenograft (PDX) models: PDX models involve the direct implantation of patient-derived HCC tissue into mice. The models retain the characteristics and heterogeneity of the original patient tumor.

[0351] FRG (Fah- / -Rag2- / -Il2rg- / -) model: FRG mice are triple knockout mice lacking the fumaryl acetoacetate hydrolase (Fah) gene, the Rag2 gene, and the Il2rg gene. These mice have liver damage and are highly susceptible to implantation of human hepatoma cells.

[0352] Breast cancer tumors:

[0353] MCF-7, MDA-MB-231, and 4T1 xenograft models: Human breast cancer cell lines used in xenograft models. The cells are implanted into the mammary fat pad of nude mice, where they grow and form tumors.

[0354] Brain Cancer Tumors:

[0355] U87MG U251 xenograft models: human glioblastoma multiforme, an aggressive brain cancer.

[0356] GL261 Xenograft Model: Mouse glioblastoma used as a syngeneic mouse xenograft model.

[0357] D283 Med xenograft model: a childhood brain tumor called medulloblastoma.

[0358] BT-474 Xenograft Model: The BT-474 cell line was originally derived from a breast cancer patient and has also been used as a xenograft model for brain metastases colonizing the brain.

[0359] D283 Med: Medulloblastoma in children.

[0360] Patient-derived xenograft (PDX) models: PDX models involve implanting patient-derived lung cancer tissue directly into mice. The models retain the characteristics and heterogeneity of the original patient tumor.

[0361] Renal Cell Carcinoma:

[0362] Renca xenograft model: A murine renal adenocarcinoma cell (RCC) line derived from BALB / c mice.

[0363] ACHN, Caki-1, 786-O xenograft models: human RCC cell lines.

[0364] OS-RC-2 xenograft model: a human RCC cell line derived from a patient with clear cell RCC.

[0365] Lymphoma Tumors:

[0366] Raji and Ramos xenograft models: derived from Burkitt's lymphoma patients and used to study B-cell lymphomas.

[0367] SU-DHL-4 xenograft model: diffuse large B-cell lymphoma (DLBCL) cell line.

[0368] Granta-519 Xenograft Model: Mantle Cell Lymphoma (MCL) Cell Line for MCL Tumor Growth, Invasion, and Response to Therapy.

[0369] Pfeiffer xenograft models: Derived from patients with primary effusion lymphoma (PEL) with unique characteristics, such as association with human herpesvirus 8 (HHV-8).

[0370] Leukemia tumors:

[0371] Subcutaneous xenograft models: Human leukemia cells are injected subcutaneously into immunodeficient mice, usually SCID, NOD-SCID, NSG, or NOG mice.

[0372] Orthotopic xenograft models: Orthotopic models involve the injection of leukemia cells into an anatomically relevant site, such as the bone marrow or spleen, of immunodeficient mice.

[0373] Intravenous xenograft models: Human leukemia cells are injected directly into the bloodstream of immunodeficient mice.

[0374] Patient-derived xenograft (PDX) models: PDX models involve transplanting patient-derived leukemia cells into immunodeficient mice. These models recapitulate the heterogeneity and features of human leukemia in mice.

[0375] Genetically Manipulated Models: In addition to direct implantation of human leukemia cells, leukemias can also be developed using genetically engineered mouse models (GEMMs).

[0376] Melanoma Tumors:

[0377] Subcutaneous xenograft models: Human melanoma cells are injected subcutaneously into immunodeficient mice, usually SCID, NOD-SCID, NSG, or NOG mice.

[0378] Orthotopic xenograft models: Melanoma cells are injected into an anatomically relevant site, such as the skin or dermis, of immunodeficient mice. These models mimic the melanoma microenvironment and demonstrate tumor invasion, angiogenesis, and interactions with surrounding tissues.

[0379] Metastatic xenograft models: Melanoma cells are injected directly into the bloodstream or by organ-specific injection (eg, intravenous or intrasplenic) to promote metastatic spread.

[0380] Patient-derived xenograft (PDX) models: PDX models involve transplanting patient-derived melanoma cells or tumor tissue fragments into immunodeficient mice.

[0381] Genetically manipulated models: Genetically engineered mouse models (GEMMs) can be used to develop melanomas with specific genetic alterations, such as activating mutations in genes such as BRAF and / or NRAS, to initiate melanoma tumorigenesis.

[0382] Cell lines can be used in combination or as controls. For example, in a mini-CAR-EGFR BioNV therapy experiment targeting lung cancer, EGFR+ve and EGFR-ve models were used to test specificity.

[0383] Any of the cell lines and / or animal models described herein can be used to evaluate BioNV and cell lines used to produce BioNV, as well as to evaluate controls thereof.

[0384] definition

[0385] The following definitions are used in conjunction with the disclosure herein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0386] An "effective amount" or "therapeutically effective amount" is an amount effective to treat, prevent, or ameliorate cancer, such as the cancers described herein, or an amount intended to reduce the number of malignant cells, reduce the size of a primary tumor, reduce angiogenesis, reduce the size and / or number of metastases, or improve symptoms associated with cancer in a subject.

[0387] As used herein, "a", "an" or "the" may mean one or more than one.

[0388] As used herein, the word "include" and variations thereof are intended to be non-limiting, such that a recitation of items in a list does not exclude other similar items that may also be applicable to materials, compositions, devices, and methods of the present technology. Likewise, the terms "can" and "may" and variations thereof are intended to be non-limiting, such that a statement that an embodiment may or may include certain elements or features does not exclude other embodiments of the present technology that do not include those elements or features.

[0389] Although the open-ended term "comprising" as a synonym for terms such as including, containing, or having is used herein to describe and claim the present disclosure or embodiments thereof, and may alternatively be described using alternative terms such as "consisting of" or "consisting essentially of."

[0390] In embodiments, "BioNV" refers to an allogeneic, low immunogenic and biomimetic nanovesicle (NV) comprising at least one surface-oriented, membrane-embedded CAR. In embodiments, the "nanovesicle (NV)" mentioned herein is a lipid-bound vesicle having a size of about 10nm to about 1200nm, which encapsulates an aqueous core. In embodiments, lipid-bound NVs can be formed using a lipid monolayer, a lipid bilayer, or maintain a multilayer form. In embodiments, BioNV refers to a biologically derived nano-sized vesicle that can have a designed biological functionalization. In embodiments, BioNV is "biomimetic" because it is derived from endogenous cell material, more specifically, it substantially reproduces the plasma membrane material found in cells. In embodiments, the cells from which BioNV are derived may include any kind of stem cells, including cell types differentiated from the stem cells. In embodiments, BioNV is substantially free of encapsulated cell debris, including cell genomic nucleic acids, organelles, or organelle parts. In embodiments, BioNV is characterized by having one or more, 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more of the following:

[0391] a. A size of about 10 nm to about 1200 nm;

[0392] b. Total volume is about 500nm 3 to about 5 μm 3 (assuming a spherical shape);

[0393] c. having a content of at least one phospholipid and cholesterol;

[0394] d. having a surface membrane, the surface membrane having one or more of CD34, CCL21, PD-L1 (in BioNV derived from non-activated cell sources), FasL, SerpinB9, H2-M3, CD47, CTLA-4, CD24, CD200, MFG-E8, NCAM, α-phagocytic integrin, and / or anti-6R antibody or antibody form, or a chimera of one or more thereof; having a surface membrane, the surface membrane substantially lacking T cell receptor components (TRAC and / or TRBC), MHC class I components and / or MHC Class II components lacking proteins of one or more of HLA-A, HLA-B, HLA-C, HLA-E or HLA-G (but not both HLA-E and HLA-G), HLA-F and / or CIITA, SerpinB9, and substantially lacking proteins of one or more of IL-4, IL-6, IL-10 and / or IL-16 within the vesicles;

[0395] e. A membrane-embedded CAR comprising a target binding portion, wherein the target binding portion may include an antibody or antibody format selected from one or more of the following: a monoclonal antibody, a polyclonal antibody, an antibody fragment, Fab, Fab′, Fab′-SH, F(ab′)2, Fv, a single-chain Fv (scFv), a diabody, a nanobody, a linear antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, a humanized antibody, a human antibody, a fusion protein comprising an antigen binding portion of an antibody, a bispecific T cell engager (BiTE), a VERR / viral ligand, or a variable heavy chain IgG fragment V H H or V NAR or engineered T cell receptors (TCRs), where the CAR can target a single biomarker, multiple biomarkers, or multiple portions of a single biomarker;

[0396] f. payloads capable of adsorbing and / or encapsulating: one or more perforins, granzymes, cytokines, cytotoxic proteins, checkpoint inhibitors, gene editing payloads, antibodies or antibody fragments, fusion proteins, small molecules, biologics, radionuclides, tracers, dyes, fluorescent proteins, and other therapeutic payloads and / or any combination thereof; and

[0397] g. be capable of not causing harmful immune response in the subject.

[0398] In embodiments, "induced pluripotent stem cells" or "iPSCs" refer to stem cells that can be generated directly from adult cells. iPSCs can be derived from differentiated cells that are reprogrammed back to an embryonic-like pluripotent state. iPSCs can generally propagate indefinitely and become any cell type of the organism from which they originated.

[0399] In embodiments, "allogeneic" as used herein refers to biological material, tissue or cells that are genetically different and initially immunologically incompatible, although derived from the same species. For example, an allogeneic BioNV is material derived from a first subject (iPSC donor) and can be provided to any number of different genetically non-identical subjects.

[0400] In the embodiments, "low immunogenicity" or "low immunity" used herein for modified cells and / or BioNV refers to a reduced ability to produce an immune response. In the embodiments, iPSC and BioNV may have low immunogenicity due to reduced or eliminated expression and / or activity of one or more immunogenic cell surface proteins and / or secretory proteins (e.g., TCR proteins, CRS proteins, MHC class I or MHC class II proteins, etc.). In the embodiments, iPSC and BioNV may have low immunogenicity due to expression of one or more immune protective cell surface proteins (e.g., CD47, CD34, CD24, CD200, α-phagocytic integrin, etc.). In the embodiments, BioNV may have low immunogenicity due to not triggering CRS in the subject and / or not inducing HLA incompatibility.

[0401] In embodiments, "knockout", "silencing", "inactivation", "destruction" or "blocking" and equivalents thereof, with respect to transcription, gene expression or protein expression, refers to a reduction in the amount of transcription, gene or protein expression from a normal state or below the wild-type state in a particular cell subpopulation. The reduction may be significant, such that gene expression does not occur, or only a negligible amount of expression occurs.

[0402] In an embodiment, "overexpression" as used herein refers to an increase in the amount of transcription, or expression of a gene or protein from a normal state or exceeding the wild-type state in a specific cell subpopulation.

[0403] Equivalent solutions

[0404] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments specifically described herein. Such equivalents are intended to be encompassed within the scope of the appended claims.

[0405] As used herein, all headings are for organizational purposes only and are not intended to limit the disclosure in any way. The contents of any individual section may apply equally to all sections.

[0406] Incorporated by Reference

[0407] All patents and publications cited herein are hereby incorporated by reference in their entirety, including published PCT application WO 2020 / 227369, filed on May 6, 2020, entitled “Tailored Hypoimmune Nanovesicle Delivery Systems for Cancer Tumors,” and published U.S. non-provisional application US 20220040106 A1, filed on August 3, 2021, entitled “Tailored Hypoimmune Nanovesicle Delivery Systems for Cancer Tumors, Hereditary and Infectious Diseases.”

Claims

1. A method for treating or preventing cancer, the method comprising administering to a subject in need thereof: (i) a therapeutically effective amount of a biomimetic nanovesicle (BioNV) comprising: (a) a membrane-embedded chimeric antigen receptor (CAR) targeting a cell surface marker; and (b) PD-1, PD-L1 and / or PD-L2 inhibitors; or (ii) a therapeutically effective amount of BioNV comprising a membrane-embedded CAR targeting a cell surface marker, wherein the subject is being treated with a PD-1, PD-L1 and / or PD-L2 inhibitor.

2. A method for treating or preventing cancer, the method comprising administering to a subject in need thereof: (i) a therapeutically effective amount of a biomimetic nanovesicle (BioNV) comprising: (a) a membrane-embedded chimeric antigen receptor (CAR) targeting a cell surface marker; and (b) a bound and / or membrane-anchored PD-L1 and / or PD-L2 inhibitor; or (ii) a therapeutically effective amount of BioNV comprising: (a) a membrane-embedded bispecific CAR targeting a first cell surface marker and a second cell surface marker; and (b) Bound and / or membrane-anchored PD-L1 and / or PD-L2 inhibitors.

3. A method for treating or preventing cancer, the method comprising administering to a subject in need thereof: (i) a therapeutically effective amount of a biomimetic nanovesicle (BioNV) comprising a bispecific chimeric antigen receptor (CAR) targeting a first cell surface marker and PD-L1 or PD-L2, wherein the first cell surface marker is not PD-1, PD-L1 or PD-L2; or (ii) a therapeutically effective amount of BioNV comprising (a) a bispecific CAR targeting the first cell surface marker and PD-L1 or PD-L2, wherein the first cell surface marker is not PD-1, PD-L1 or PD-L2; and (b) PD-1, PD-L1 and / or PD-L2 inhibitors; or (iii) a therapeutically effective amount of BioNV comprising a bispecific CAR targeting PD-L1 or PD-L2 and a first cell surface marker, wherein the subject is being treated with a PD-1, PD-L1 and / or PD-L2 inhibitor, and wherein the first cell surface marker is not PD-1, PD-L1 or PD-L2.

4. A method for treating or preventing cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of a biomimetic nanovesicle (BioNV), comprising (a) a bispecific chimeric antigen receptor (CAR) targeting a first cell surface marker and a second cell surface marker; and (b) PD-1, PD-L1 and / or PD-L2 inhibitors.

5. A method for treating or preventing cancer, the method comprising administering a therapeutically effective amount of a biomimetic nanovesicle (BioNV) comprising a bispecific chimeric antigen receptor (CAR) targeting a first cell surface marker and a second cell surface marker to a subject in need thereof, wherein the subject is receiving treatment with a PD-1, PD-L1 and / or PD-L2 inhibitor.

6. The method of any one of claims 2, 4 and 5, wherein the first cell surface marker and the second cell marker are selected from Table 1 and / or Table 2.

7. The method of claim 3, wherein the first cell surface marker is selected from Table 1 and / or Table 2.

8. The method of any one of claims 2 to 7, wherein the BioNV binds to at least a first cell and at least a second cell to initiate an anti-cancer response.

9. The method of claim 8, wherein the first cell is a cancer cell and the second cell is an immune cell.

10. The method of any of the preceding claims, wherein the PD-1 inhibitor is an antibody targeting PD-1, optionally pembrolizumab, nivolumab or cemiplizumab.

11. The method of any one of claims 1 to 9, wherein the PD-L1 and / or PD-L2 inhibitor is an antibody targeting PD-L1 and / or PD-L2, optionally atezolizumab, avelumab or durvalumab.

12. The method of any one of the preceding claims, wherein the BioNV is derived from a modified cell.

13. The method of claim 12, wherein the modified cell is a stem cell, an induced pluripotent stem cell (iPSC), a reprogrammed pluripotent or multipotent cell, an embryonic stem cell, a mesenchymal stem cell, or a differentiated cell derived from any modified stem cell thereof. The method of claim 13 , wherein the modified cells are iPSCs.

15. The method of claim 12, wherein the modified cell is a T cell, a helper T cell, a T memory cell, or a NK cell.

16. The method of claim 12, wherein the modified cells are macrophages.

17. The method of claim 12, wherein the modified cells are monocytes.

18. The method of any one of claims 12 to 17, wherein the modified cells substantially lack one or more MHC class I proteins, MHC class II proteins, T cell receptor (TCR) proteins, and / or cytokine release syndrome (CRS) proteins.

19. The method of any one of claims 12 to 17, wherein the modified cells have reduced or eliminated β2-macroglobulin (B2M) gene expression and / or reduced or eliminated MHC class I protein expression and / or activity.

20. The method of any one of claims 12 to 19, wherein the modified cells have reduced or eliminated CIITA gene expression and / or reduced or eliminated MHC class II protein expression and / or activity.

21. The method of any one of claims 12 to 20, wherein the modified cells have reduced or eliminated HLA-A gene expression and / or reduced or eliminated HLA-A protein expression and / or activity.

22. The method of any one of claims 12 to 21, wherein the modified cells have reduced or eliminated HLA-B gene expression and / or reduced or eliminated HLA-B protein expression and / or activity.

23. The method of any one of claims 12 to 22, wherein the modified cells have reduced or eliminated HLA-C gene expression and / or reduced or eliminated HLA-C protein expression and / or activity.

24. The method of any one of claims 12 to 23, wherein the modified cells have reduced or eliminated HLA-E or HLA-G gene expression and / or reduced or eliminated HLA-E or HLA-G protein expression and / or activity.

25. The method of any one of claims 12 to 24, wherein the modified cells have reduced or eliminated HLA-F gene expression and / or reduced or eliminated HLA-F protein expression and / or activity.

26. The method of any one of claims 12 to 25, wherein the modified cells have reduced or eliminated T cell alpha constant (TRAC) gene expression and / or reduced or eliminated TRAC protein expression and / or activity.

27. The method of any one of claims 12 to 26, wherein the modified cells have reduced or eliminated T cell beta constant (TRBC) gene expression and / or reduced or eliminated TRBC protein expression and / or activity.

28. The method of any one of claims 12 to 27, wherein the modified cells have reduced or eliminated PD-1 gene expression and / or reduced or eliminated PD-1 protein expression and / or activity.

29. The method of any one of claims 12 to 28, wherein the modified cells have reduced or eliminated IL-4 gene expression and / or reduced or eliminated IL-4 protein expression and / or activity.

30. The method of any one of claims 12 to 29, wherein the modified cells have reduced or eliminated IL-6 gene expression and / or reduced or eliminated IL-6 protein expression and / or activity.

31. The method of any one of claims 12 to 30, wherein the modified cells have reduced or eliminated IL-10 gene expression and / or reduced or eliminated IL-10 protein expression and / or activity.

32. The method of any one of claims 12 to 31, wherein the modified cells have reduced or eliminated IL-16 gene expression and / or reduced or eliminated IL-16 protein expression and / or activity.

33. The method of any one of claims 12 to 32, wherein the modified cell has reduced or eliminated SerpinB9 gene expression and / or reduced or eliminated SerpinB9 protein expression and / or activity.

34. The method of any one of claims 12 to 33, wherein the modified cells express, or increase expression of, the CD34 gene and / or gene product.

35. The method of any one of claims 12 to 34, wherein the modified cells express, or increase expression of, the CCL2 gene and / or gene product.

36. The method of any one of claims 12 to 35, wherein the modified cell expresses the PD-L1 gene and / or gene product, or increases its expression, and wherein the modified cell is not activated; or wherein the modified cell has reduced or eliminated expression of the PD-L1 gene and / or gene product, and wherein the modified cell is activated.

37. The method of any one of claims 12 to 36, wherein the modified cell expresses, or increases expression of, the H2-M3 gene and / or gene product.

38. The method of any one of claims 12 to 37, wherein the modified cells express, or increase expression of, the CD47 gene and / or gene product.

39. The method of any one of claims 12 to 38, wherein the modified cells express, or increase expression of, the CD24 gene and / or gene product.

40. The method of any one of claims 12 to 39, wherein the modified cells express, or increase expression of, chimeric CD24 / CD47 genes and / or gene products.

41. The method of any one of claims 12 to 40, wherein the modified cells express a chimeric CD200 gene and / or gene product, or increase expression thereof.

42. The method of any one of claims 12 to 41, wherein the modified cells express, or increase expression of, chimeric CD24 / CD200 genes and / or gene products or chimeric CD47 / CD200 genes and / or gene products.

43. The method of any one of claims 12 to 42, wherein the modified cells express a chimeric CTLA-4 gene and / or gene product, or increase expression thereof.

44. The method of any one of claims 12 to 43, wherein the modified cell expresses, or increases expression of, a chimeric MFG-E8 gene and / or gene product.

45. The method of any one of claims 12 to 44, wherein the modified cell expresses, or increases expression of, the NCAM gene and / or gene product.

46. ​​The method of any one of claims 12 to 45, wherein the modified cell expresses, or increases expression of, a chimeric alpha-phagocytic integrin gene and / or gene product.

47. The method of any one of claims 12 to 46, wherein the modified cells express an antibody or antibody-like molecule targeting an IL-6 surface receptor (anti-IL-6R), or increase its expression.

48. The method of any one of claims 12 to 46, wherein the modified cell expresses, or increases expression of, the FasL gene and / or gene product.

49. The method of any one of claims 12 to 48, wherein the modified cell does not overexpress the FasL gene and / or gene product therein.

50. The method of any one of claims 12 to 49, wherein the expression and / or activity of 3 or more immunogenic proteins, 4 or more immunogenic proteins, 5 or more immunogenic proteins, 6 or more immunogenic proteins, 7 or more immunogenic proteins, 8 or more immunogenic proteins, 9 or more immunogenic proteins, 10 or more immunogenic proteins, 11 or more immunogenic proteins, or 12 or more immunogenic proteins of the modified cells is reduced or eliminated.

51. The method of any one of claims 12 to 50, wherein the modified cells express, or increase the expression of, 3 or more immunoprotective proteins, 4 or more immunoprotective proteins, 5 or more immunoprotective proteins, 6 or more immunoprotective proteins, 7 or more immunoprotective proteins, 8 or more immunoprotective proteins, 9 or more immunoprotective proteins, or 10 or more immunoprotective proteins.

52. The method of any one of claims 12 to 51, wherein the modified cells are allogeneic.

53. The method of any one of claims 12 to 52, wherein the modified cells do not elicit an immune response in a patient to which they or a BioNV derived therefrom is administered.

54. The method of any one of claims 12 to 53, wherein the modified cells are differentiated prior to BioNV formation.

55. The method of any one of claims 12 to 54, wherein the modified cell expresses the CAR under the control of a regulatable expression element.

56. The method of any of the preceding claims, wherein the CAR is activated prior to BioNV formation.

57. The method of claim 56, wherein the CAR is activated via its target, by another receptor and / or a virus.

58. The method of any one of claims 12 to 57, wherein the BioNV is formed from the modified cells or differentiated cells thereof by sonication, adaptive focused acoustic technology, French press, extrusion, continuous extrusion, detergent cell lysis and / or electroporation.

59. The method of claim 58, wherein the BioNV is formed from the modified cells by continuous extrusion.

60. The method of any of the preceding claims, wherein the BioNV has a size of about 10 nm to about 1200 nm.

61. The method of any of the preceding claims, wherein the BioNV has a size of about 10 nm to about 100 nm.

62. The method of any one of claims 1 to 60, wherein the BioNV has a size of about 100 nm to about 200 nm.

63. The method of any one of claims 1 to 60, wherein the BioNV has a size of about 200 nm to about 500 nm.

64. The method of any one of claims 1 to 60, wherein the BioNV has a size of about 500 nm to about 1200 nm.

65. The method of any of the preceding claims, wherein the BioNV substantially lacks expression and / or activity of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, and one of HLA-E or HLA-G.

66. The method of any of the preceding claims, wherein the BioNV substantially lacks expression and / or activity of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, SerpinB9, and one of HLA-E or HLA-G.

67. The method of any of the preceding claims, wherein the BioNV substantially lacks expression and / or activity of one of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, SerpinB9, CD200, HLA-E, or HLA-G, and one or more of IL-4, IL-10, and IL-16.

68. The method of any of the preceding claims, wherein the BioNV comprises membrane embedded α-phagocytic integrin, CCL2, H2-M3, FasL, MFG-E8, anti-IL-6R antibody or antibody form, and PD-L1 and / or CTLA-4; and One of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200 and chimeric CD47 / CD200, or two of CD24, CD47 and CD200.

69. The method of any one of claims 1 to 67, wherein the BioNV comprises membrane embedded α-phagocytic integrin, CCL2, H2-M3, FasL, MFG-E8, anti-IL-6R antibody or antibody form, SerpinB9, and PD-L1 and / or CTLA-4; and One of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200 and chimeric CD47 / CD200, or two of CD24, CD47 and CD200.

70. The method of any one of claims 1 to 67, wherein the BioNV comprises membrane-embedded CD200 protein and is substantially devoid of CD24 or CD47.

71. The method of any one of claims 1 to 67, wherein the BioNV substantially lacks SerpinB9 and CD200 protein and / or activity.

72. The method of any one of the preceding claims, wherein the CAR comprises an antibody or antibody form selected from one or more of the following: a monoclonal antibody, a polyclonal antibody, an antibody fragment, a V NAR 、V H H, Fab, Fab', Fab'-SH, F(ab')2, Fv, single-chain Fv (scFv), diabody, nanobody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, or a fusion protein comprising the antigen-binding portion of an antibody.

73. The method of claim 71, wherein the antibody format is a scFv.

74. A method as described in any of the preceding claims, wherein the CAR comprises a transmembrane domain derived from CD28, CD3ζ, CD4, CD8α, ICOS, or a fragment and / or combination thereof.

75. A method as described in any of the preceding claims, wherein the CAR comprises an intracellular domain, which further comprises an intracellular signaling domain of the CD3ζ chain and / or one or more co-stimulatory molecules, optionally selected from CD28, 4-1BB, ICOS, CD27 and OX40.

76. A method as described in any of the preceding claims, wherein the CAR targets a cancer-specific antigen.

77. The method of any of the preceding claims, wherein the BioNV or modified cells derived therefrom comprise nucleic acid encoding green fluorescent protein (GFP) and / or GFP protein.

78. The method of claim 77, wherein the nucleic acid encoding GFP is operably linked to a promoter from one or more of: IL-2, perforin, granzyme, alarmin, TNF, INF, and / or a combination thereof.

79. The method of any of the preceding claims, wherein the BioNV encapsulates a payload.

80. The method of claim 79, wherein the payload is one or more of: a gene editing agent, a cytotoxic protein, a biologic, a nucleic acid, a fusion protein, a fluorescent protein, a tracer dye, a radionuclide, and / or a small molecule.

81. The method of claim 79, wherein the payload is a therapeutic payload for the type of cancer targeted by the CAR.

82. The method of claim 79, wherein the payload comprises an alkylating agent.

83. The method of claim 79, wherein the payload comprises an anthracycline.

84. The method of claim 79, wherein the payload comprises an antimetabolite.

85. The method of claim 79, wherein the payload comprises an anti-tumor antibiotic.

86. The method of claim 79, wherein the payload comprises an anti-tumor antibody or antibody form.

87. The method of claim 79, wherein the payload comprises a corticosteroid.

88. The method of claim 79, wherein the payload comprises a plant alkaloid.

89. The method of claim 79, wherein the payload comprises a topoisomerase inhibitor.

90. The method of claim 79, wherein the payload comprises a checkpoint inhibitor.

91. The method of claim 79, wherein the payload is selected from abemaciclib, abiraterone acetate, abraxan, ABVD, ABVE, ABVE-PC, AC, acalabrutinib, AC-T, Antenol, Ansili, ADE, Ado-trastuzumab Entansin, doxorubicin, afatinib dimaleate, Afinitor, Oconze, Idala, Aldesleukin, Ansentsa, Alectinib, Alemtuzumab, Alimta, Alefil, Alkran for injection, Alkran tablets, Aloxi, Apelis, Amberi, Ameruz, Amifostine, Aminolevulinic acid hydrochloride, Evantumab-vmjw, Anastrozole, Apalutamide, Aprepitant, Espre, Acort, Arimidex, Arnoxin, Anrejin, Arsenic trioxide, Asera, Asciminib hydrochloride, Asparaginase Erwinia, Asparagus, Atezolizumab, Afatinib, Avastin, Avelumab, Akilencel, Axitinib, Tajivir, Azacitidine, Azedra, Balvesa, Bavencio, BEACOPP, Belantamab Mofortin-blmf, Belyodak, Belinostat, Beztifan, Bendamustine hydrochloride, Bendeka, BEP, Bebossa, Bevacizumab, Bevacizumab, Bexarotene, Bicalutamide, BiCNU, Bimetinib, Brenrep, Bleomycin sulfate, Belinto, Belyto, Bortezomib, Bosuliv, Bosutinib, Braftovir, Vebutin, Brecatuximab, Brecatuximab, Breyanzi, Brigatinib, Brukin SA, BuMel, busulfan, busulfan, cabazitaxel, cabizumab, cabozantinib, cabozantinib-S-malate, CAF, CARASA PEG-MKNL, CONCOX, CAMPAS, CAPTOS, capecitabine, capecitabine-yhdp, capmatinib hydrochloride, CAPOX, CARAQ, carboplatin, carboplatin-paclitaxel, carfilzomib, carmustine, carmustine implant, CANSTA, CEM, cemiprilimab-rwlc, ceritinib, ceribid, Cervarix (recombinant HPV bivalent vaccine), cetuximab, CEV, chlorambucil, chlorambucil-prednisone, CHOP, cisplatin, cladribine, clofarabine, Corolla, CMF, cobimetinib fumarate, Comtril, salt Copenhagen acid, COPDAC, Cerbitol, COPP, COPP-ABV, Cosmegen, Cotelici, Crizotinib, CVP, Cyclophosphamide, Ciranze, Cytarabine, Dabrafenib Mesylate, Dacarbazine, Dacotinib, Actinomycin D, Dayouze, Daratumumab, Daratumumab and Hyaluronidase-fihj, Darbepoetin alfa, Darofem, Zakata, Zakata Express, Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposomal, Dorismo, Decitabine, Decitabine and Cidaruci, Defibrotide Sodium, Defitelio, Degarelix, Denileukin, Denosumab, Dexamethasone, Dexazolidinone Hydrochloride, Denutoximab, Docetaxel, Dotalimumab-GXLY, Doxitaxel,Doxorubicin hydrochloride, liposomal doxorubicin hydrochloride, durvalumab, duvelith, efdex, eligad, elitek, ellens, elotuzumab, eloxadine, eltrombopag, elzolis, epavazumab-lzsg, emend, empliximab, ensidipine mesylate, connefenib, enrofloxacin vedotin-ejfv, euhede, entrectinib, enzalutamide, epirubicin hydrochloride, EPOCH, epoetin alfa, epogen, erbitux, erdatinib, eribulin mesylate, erici, erida, erlotinib hydrochloride, elvinaze, isol, vanbifu, etoposide, etoposide phosphate, everolimus, ivert, evomera, exemestane, anvili, 5-FU, 5-FU, Fa m-trastuzumab drutilecan-nxki, falotin, fusodex, FEC, fedratinib hydrochloride, fulon, filgrastim, fermoneg, fludarabine phosphate, fluprednisone, fluorouracil injection, fluorouracil topical, flutamide, forfuri, forfuri-bevacizumab, forfuri-cetuximab, forfino, fulvestrant, fulvestrant, fostamicin, fostamicin disodium, fortivar, forfiltrates, FU-LV, fulvestrant, filaro, gamivant, Gardasil (recombinant HPV quadrivalent vaccine), Gardasil 9 (recombinant HPV nine-valent vaccine), Gavreto, Gaziva, gefitinib, gemcitabine hydrochloride, gemcitabine-cisplatin, gemcitabine-oxaliplatin, gemtuzumab ozogamicin, gemcitabine, gillotoxifene, fumarole Gliardinib, granisetron maleate, Gleevec, Gliadal powder tablets, Glucarpivirine, Goserelin acetate, granisetron, granisetron hydrochloride, Granix, Haraven, Hermangio, Herceptin Helekta, Herceptin, recombinant HPV bivalent vaccine, recombinant HPV nine-valent vaccine, recombinant HPV four-valent vaccine, Hecantin, Aizhi, hydroxyurea, Hyper-CVAD, Ibrance, Ibrutinib, ICE, Icluzig, Idarubicin PFS, Idarubicin hydrochloride, Idicatargen, Vicruse, Idelalis, Idifa, Ifex, Ifosfamide, Interleukin-2 (recombinant), Imatinib mesylate, Ibvecar, Imfenzi, Imiquimod, Imlijik, Indole phosphate gretinib, infgem, inlita, ituzumab ozogamicin, incovi, inrebic, interferon alfa-2b, intron A, iobenguanidine I131, ipilimumab, ressa, irinotecan hydrochloride, isatuximab-irfc, istodex, ivosideb, ixabepilone, ixazomib citrate, ixampura, jacafi, jeb, germitor, janperli, jeftana, cadsila, capivoxil, kepivans, kymida, kimstrak, kiskali, coselugol, kimria, caprolis, lanreotide acetate, lapatinib dimesylate, larotrectinib sulfate, lenalidomide, lenvatinib mesylate, lenvima, letrozole, leucovorin, rocloma, leuprolide acetate, leupromide, leuprolide, leucomitin, leuprolide, leuprolide,Lisocartogenmal, lomustine, lantuximab, tecillin-lpyl, lansef, lorbrena, lorlatinib, lumaclas, lumositide, lipan depot, lumenectin, ruspactin-aamt, lutetase, lutetium (Lu 177-dotat), leupro, meganle, magetuximab-cmkb, marcibo, maturanet, dichloroethylamine hydrochloride, megestrol acetate, meginister, mektovi, melphalan, melphalan hydrochloride, mercaptopurine, mesna, mesnex, methotrexate sodium, methylnaltrexone bromide, mistolin, mitomycin, mitoxantrone hydrochloride, mobotinib succinate, moglizumab-kpkc, monjuvi, mopp, mosetuximab pasudotox-tdfk, siprelin, mvac, mvasi, myleran, mylotarg, nanoparticle paclitaxel, nacitozumab-gqgk, nacitozumab, nelarabine, nelarabine maleate Latinib, Helian, Netupitant, Neurastat, Nupregin, Nexavar, Nilandron, Nilotinib, Nilutamide, Enlari, Niraparib tosylate monohydrate, Nivestim, Nivolumab, Napre, Nubeka, Nivipriya, Obinutuzumab, Odomzo, OEPA, Ofatumumab, OFF, Olaparib, Omacitabine mesylate, Oncaspar, Ondansetron hydrochloride, Onived, Ontaq, Onureg, Odivo, OPPA, Orgovix, Osimertinib mesylate, Oxaliplatin, Paclitaxel, Paclitaxel albumin-stabilized nanoparticle formulation, PAD, Padchev, Palibociclib, Palifermin, Palofermin hydrochloride nosetron, pamidronate disodium, panitumumab, paraplatin, pazopanib hydrochloride, PCV, PEB, pegaspargase, pegfilgrastim, peginterferon alfa-2b, PEG-Intron, pemaziret, pembrolizumab, pemetrexed disodium, pemigatinib, perjeta, pertuzumab, pesidatinib hydrochloride, fisgo, pixar, plerixafor, polatuzumab vedotin-piiq, plavixet, pomalidomide, pomalistatin, ponatinib hydrochloride, portraz, portligio, pralatrexate, pralatrexate, prednisone, procarbazine hydrochloride, procrete, prolegin, prolia, promactor, propranolol hydrochloride, provange, Mercaptopurine, Prixan, Qinlock, radium 223 dichloride, raloxifene hydrochloride, ramucirumab, rasburicase, ravulizumab-cwvz, libloze, R-CHOP, R-CVP, recombinant human papillomavirus (HPV) bivalent vaccine, recombinant human papillomavirus (HPV) nine-valent vaccine, recombinant human papillomavirus (HPV) quadrivalent vaccine, recombinant interferon α-2b, regorafenib, restesta, relugolix, R-EPOCH, retacli, retefumo, relimide, ribociclib, R-ICE, ripretinib, rituximab, rituximab, rolapitant hydrochloride, romidepsin, romiprotinib,Rope interferon alpha-2b-NJFT, Rosinquan, rubracin, rucaparib camphorsulfonate, ruxolitinib phosphate, rebuvant, redpath, relaze, gosatuzumab-hziy, shanqi, saklisa, sloso intrapleural aerosol, selinexor, selpatinib, selumetinib sulfate, senblix, selutuximab, siprexa-t, sirolimus protein-bound particles, sotamox, somadulin reservoir, solidegi, sorafenib tosylate, sotolasib, shidasai, stanford sterile talc, stritaq, baiwango, sunitinib malate, soto, sotent, seratron, sawinco, simrib, tabloid, Tabexin, TAC, Tancitumomab-CXIX, Tafele, Taglax, Tarisa, Tarazopanib Tosylate, Talimoginlaheparevek, Tazena, Tamoxifen Citrate, Tarceva, Tagretin, Dasina, Tavaris, Taxotere, Tazemetostat Hydrobromide, Davic, Terbentafussp, Tecatus, Tesanqi, Temoda, Temozolomide, Temsirolimus, Tepadina, Todocan, Tepotinib Hydrochloride, Thalidomide, Salomede, Thioguanine, Thiotepa, Tosuvo, Tisagan Leclercel, Tisomab Vedotin-TFTV, Tivodak, Tivozanib Hydrochloride, Tocilizumab, Tolac, Topotecan Hydrochloride, Tocilizumab remifene, torcet, torcet, tpf, trabectin, trametinib dimethyl sulfoxide, trastuzumab, trastuzumab and hyaluronidase-oysk, cunda, terensu, trofluridine and tipiracil hydrochloride, tresenoside, todavi, truseltic, trusima, tucatinib, tuxa, tularo, tykerb, ukonix, utomis, erblisab mesylate, vindensica, unitusin, uridine triacetate, vac, valrubicin, valstatin, vandetanib, vamp, valubi, victivib, veip, vemurafenib, vecorapil, venetoclax, vezenio, vedaza, vinblastine sulfate, vinblastine sulfate Vincristine, liposomal vincristine sulfate, vinorelbine tartrate, VIP, Vimodegi, Vistoga, Vitec, Dozin, Volacoze, Vorinostat, Viquant, Vexos, Velirege, Xelcore, Setmep, Xeloda, Cialis, Cielox, Angawi, Sofigo, Sigatan, Civio, Ancotan, Yiwo, Ikeda, Yoderlis, Yongsha, Zatrap, Zabutinib, Zarcio, Zele, Zorbov, Zepzeka, Zevalin, Zietenzo, Zinecad, Zilabev (bevacizumab), Ziv-Aflibercept, Zofran, Norad, Zoledronic Acid, Zolinza, Zometa, Zikalala, Zvib, Zancorta, Sinlonta and Zeco.

92. The method of claim 79, wherein the payload is one or more of: pidilizumab, BMS-936559, tremelimumab, AGEN1884, and / or RG2077.

93. The method of claim 80, wherein the nucleic acid molecule encodes one or more of: a CRISPR / Cas component, a guide RNA (gRNA), a tracer RNA (tracrRNA), a microRNA (miRNA), RNA interference (RNAi), a small interfering RNA (siRNA), a double-stranded RNA, a Piwi-interacting RNA (piRNA), a small nuclear RNA (snRNA), a small nucleolar RNA (snoRNA), an antisense oligonucleotide (ASO), a locked nucleic acid (LNA), a splice-switching oligonucleotide (SSO), a tRNA, a ribosomal RNA (rRNA), a short hairpin (shRNA) complementary messenger RNA, a repeat-associated small interfering RNA (rasiRNA), and / or a small non-coding RNA.

94. The method of claim 80, wherein the gene editing payload is one or more of: TALEN, ZFN, ribonuclease P RNA, C2c1, C2c2, C2c3, Cas9, Cpf1, TevCas9, Archaea Cas9, CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, CasX Cas omega, transposase, and / or any ortholog or homolog thereof.

95. The method of any of the preceding claims, wherein the BioNV encapsulates one or more perforin molecules.

96. The method of any of the preceding claims, wherein the BioNV encapsulates one or more granzyme molecules.

97. The method of claim 96, wherein the granzyme molecule is selected from the group consisting of granzymes A, B, H, K and M.

98. The method of any of the preceding claims, wherein the BioNV encapsulates one or more perforin molecules and / or one or more granzyme molecules derived from a cell from which the BioNV is derived.

99. The method of any one of claims 1 to 97, wherein the BioNV encapsulates one or more perforin molecules and / or one or more granzyme molecules that are exogenously added to the BioNV.

100. The method of any of the preceding claims, further comprising co-administering whole cell therapy.

101. The method of any of the preceding claims, further comprising administering an additional therapeutic agent.

102. The method of any of the preceding claims, wherein the BioNV is stored or suitable for storage at about -80°C.

103. The method of any of the preceding claims, wherein the BioNV is lyophilized.

104. The method of any one of claims 2 or 6 to 103, wherein the PD-1 inhibitor is an antibody targeting PD-1, optionally pembrolizumab, nivolumab, or cemiplizumab.

105. The method of any one of claims 2 or 6 to 103, wherein the PD-L1 or PD-L2 inhibitor is an antibody targeting PD-L1 or PD-L2, optionally atezolizumab, avelumab or durvalumab, or an antigen binding domain thereof, and is bound to the surface of the BioNV.

106. The method of any one of claims 2 or 6 to 103, wherein the PD-1 inhibitor is an antibody targeting PD-1, optionally pembrolizumab or cemiplizumab.

107. The method of any one of claims 2 or 6 to 103, wherein the PD-L1 or PD-L2 inhibitor is atezolizumab, avelumab, or durvalumab, or an antigen binding domain thereof, and is membrane anchored by fusion of a transmembrane domain.

108. The method of any of the preceding claims, wherein the cancer is a carcinoma.

109. The method of any one of claims 1 to 107, wherein the cancer is a sarcoma.

110. The method of any one of claims 1 to 107, wherein the cancer is myeloma.

111. The method of any one of claims 1 to 107, wherein the cancer is leukemia.

112. The method of any one of claims 1 to 107, wherein the cancer is lymphoma.

113. The method of any one of claims 1 to 107, wherein the cancer is a mixed cancer.

114. The method of any of the preceding claims, wherein the cancer is metastatic.

115. The method of any one of claims 1 to 107, wherein the cancer is selected from the group consisting of acute biphenotypic leukemia, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid dendritic cell leukemia, acute myeloid leukemia, lung adenocarcinoma, adrenal tumors, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphomas, alveolar soft tissue and cardiac sarcomas, amyloidosis, anal cancer, anaplastic large cell lymphoma, angioimmunoblastic T-cell lymphoma, appendix cancer, astrocytoma, ataxia telangiectasia, attenuated familial adenomatous polyposis, B-cell prolymphocytic leukemia, basal cell carcinoma, Beckwith-Wiedemann syndrome, bile duct cancer, Bert-Hoag-Dub syndrome, bladder cancer, bone cancer, brain and nervous system cancers, brain stem glioma, brain stem glioma, Glioma), brain tumors, breast cancer, bronchial adenoma / carcinoid, Burkitt's lymphoma, carcinoid tumor, Carney syndrome, central nervous system tumors, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, fibroproliferative ganglioglioma of childhood, bile duct cancer, chondrosarcoma, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloid leukemia, chronic T-cell lymphocytic leukemia, colon cancer, colorectal cancer, Cowden syndrome, craniopharyngioma, cutaneous T-cell lymphoma, cutaneous fibrosis protuberans Ewing's sarcoma, fibroplastic small round cell tumor, diffuse gastric cancer, diffuse large B-cell lymphoma, endocrine system cancer, endocrine tumors, endometrial cancer, eosinophilic leukemia, ependymoma, epithelioid hemangioendothelioma (EHE), esophageal cancer, Ewing's sarcoma, extragonadal germ cell tumors, extrahepatic bile duct cancer, eye cancer, eyelid cancer, fallopian tube cancer, familial adenomatous polyposis, familial malignant melanoma, familial clear cell renal cell carcinoma (RCC), follicular lymphoma, gallbladder cancer, Gardner syndrome, Gastric cancer, Gastrointestinal cancer, Gastrointestinal carcinoid tumor, Gastrointestinal stromal tumor, GIST, Genitourinary and gynecological cancers, Germ cell tumors, Gestational trophoblastic disease, Gestational trophoblastic tumor, Glioblastoma, Glioma, Hairy cell leukemia, Head and neck cancer, Hematopoietic cancer, Hepatocellular carcinoma, Hepatosplenic T-cell lymphoma, HIV-related cancer, Hodgkin lymphoma, Hypopharyngeal cancer, Inflammatory breast cancer, Intravascular large B-cell lymphoma, Invasive cribriform carcinoma, Invasive lobular carcinoma, Pancreatic islet Cell carcinoma (endocrine pancreas), islet cell tumors, juvenile polyposis syndrome, Kaposi's sarcoma, keratoacanthoma, kidney cancer, lacrimal gland tumors, large granular lymphocytic leukemia, laryngeal and hypopharyngeal cancer, leiomyomatosis and renal cell carcinoma, leiomyosarcoma, Li-Fraumeni syndrome (LFS), liposarcoma, liver cancer, lung cancer, primary cutaneous lymphoma, lymphomatoid granuloma, lymphoplasmacytic lymphoma, Lynch syndrome, malignant fibrous histiocytoma of bone, mantle cell lymphoma, marginal zone B-cell lymphoma,Mast cell leukemia, mastocytosis, mediastinal large B-cell lymphoma, medullary carcinoma, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, mixed polyposis syndrome, mucosa-associated lymphoid tissue lymphoma, Muir-Tore syndrome (MTS), multiple endocrine neoplasia syndrome, multiple endocrine neoplasia type 1, multiple endocrine neoplasia type 2, multiple myeloma, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, MYH-associated polyposis, myxosarcoma, nasal and paranasal sinus cancer, Wilms tumor, nasopharyngeal carcinoma, nasopharyngeal carcinoma, neuroblastoma, neuroendocrine tumors, neurofibromatosis type 1, neurofibromatosis type 2, nevoid basal cell carcinoma syndrome, nodal marginal zone B-cell lymphoma, non-Hodgkin lymphoma, non-small cell lung cancer, non-small cell lung cancer, oligodendroglioma, optic nerve glioma, oral and / or oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, papillary renal cell carcinoma, paranasal sinus and nasal cavity cancer, parathyroid cancer, pelvic cancer, penile cancer, Peutz-Jeghers syndrome, pharyngeal cancer, pheochromocytoma, phyllodes tumor, pilocytic astrocytoma, pineal gland Astrocytoma, Pituitary adenoma, Pituitary tumor, Plasmablastic lymphoma, Pleuropulmonary blastoma, Precursor B lymphoblastic leukemia, Primary central nervous system lymphoma, Primary cutaneous follicular lymphoma, Primary cutaneous immunocytoma, Primary effusion lymphoma, Primitive neuroectodermal tumor, Prostate cancer, Rectal cancer, Kidney cancer, Renal cell carcinoma, Retinoblastoma, Rhabdomyosarcoma, Salivary gland cancer, Sarcoma, Primary cutaneous sarcoma, Sebaceous carcinoma, Sezary syndrome, Skin appendage tumor, Skin cancer, Small intestine cancer, Small cell lung cancer, Small intestine cancer, Splenic marginal zone lymphoma, Lung squamous cell carcinoma Cell carcinoma, squamous cell carcinoma, squamous cell skin cancer, gastric cancer, surface epithelial-stromal tumors, T-cell prolymphocytic leukemia, testicular cancer, thoracic and respiratory tract cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell carcinoma, transitional cell carcinoma adenoid cystic carcinoma, tuberous sclerosis complex syndrome, tubular carcinoma, Turker's syndrome, carcinoma of unknown primary, unclassified carcinoma, ureteral cancer, urethral cancer, uterine cancer, uterine sarcoma, uveal melanoma, vaginal cancer, visual pathway and hypothalamic glioma, von Hippel-Lindau (VHL) syndrome, vulvar cancer, Wilms tumor, and xeroderma pigmentosum.

116. An allogeneic biomimetic nanovesicle (BioNV), comprising: (a) a membrane-embedded chimeric antigen receptor (CAR) targeting a cell surface marker; and (b) binding and / or membrane-anchored targeting agents targeting PD-L1 or PD-L2, wherein the agent targeting PD-L1 or PD-L2 is atezolizumab, avelumab, or durvalumab, and wherein the CAR does not target PD-L1 or PD-L2.

117. An allogeneic biomimetic nanovesicle (BioNV), comprising: (a) a first membrane-embedded chimeric antigen receptor (CAR) that targets a cell surface, wherein the first membrane-embedded CAR does not target PD-L1 or PD-L2; and (b) at least a second membrane-embedded CAR targeting PD-L1 or PD-L2.

118. An allogeneic biomimetic nanovesicle (BioNV) comprising a bispecific, membrane-embedded chimeric antigen receptor (CAR) targeting: (a) cell surface markers of cancer cells; and (b) PD-L1 or PD-L2, and Wherein the cell surface marker of the cancer cell is not PD-L1 or PD-L2.

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