Allogeneic hypoimmunogenic biomimetic nanovesicles for cancer treatment

Abstract

Disclosed herein are compositions comprising allogeneic low-immunogenic chimeric antigen receptor (CAR)-targetable biomimetic nanovesicles (BioNVs), and methods of using the same for the treatment, prevention, and / or amelioration of cancer.

Description

Technical Field

[0001] The present disclosure provides, in part, compositions and methods comprising allogeneic hypoimmunogenic biomimetic nanovesicles, and methods of using them, for example, in mammalian subjects such as humans, for the treatment or prevention of cancer.

[0002] Cross - reference to related applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 345,664, filed May 25, 2022, the content of which is hereby incorporated by reference in its entirety.

Background Art

[0003] Chimeric antigen receptor (CAR) whole - cell therapies (T cells, NK cells, macrophages, tumor - infiltrating lymphocytes (TIL), etc.) are used to target cells for the treatment of cancer. CAR whole - cell therapies generally require collecting a patient's own immune cells (T cells) to treat their own cancer, or developing allogeneic cell lines from stem cells to treat a broader patient population at a lower cost. Lymphocytes normally attack invasive microorganisms, but in CAR whole - cell therapies, the cells are engineered to target cancer cells.

[0004] First, cells are isolated from a patient's blood and genetically engineered to express on their surface a chimeric antigen receptor (CAR) that enables the cells to bind to a specific antigen. Alternatively, the cells are genetically engineered from stem cells (e.g., iPSCs, MSCs, or embryonic stem cells) and incorporate allogeneic and stable properties from the immune system (e.g., incorporation of the transmembrane tag of the molecule CD47 to prevent clearance by macrophages), achieved by subsequent stable incorporation of the desired CAR. The CAR is a non - naturally occurring fusion protein containing fragments of synthesized antibodies. The CAR can recognize a target cancer biomarker through several binding moieties, namely: 1) the single - chain variable fragment (scFV) region of an antibody, or a bispecific or BiTE antibody format, 2) a virus epitope recognition receptor (VERR) derived from a tumor - lytic virus receptor, 3) by a variable heavy - chain IgG fragment from camelids called V H H single - domain nanobody (V H H nanobody), 4) by a variable heavy - chain IgG fragment from cartilaginous fish called variable new antigen receptor (V NAR ), 5) by an engineered T - cell receptor (TCR), 6) by any single - chain IgG fragment from which the variable region is engineered into a CAR structure to recognize any biomarker, 7) by an affilin (an artificial binding domain with antigen selectivity), or 8) by a chimeric endocrine receptor (CER). CAR constructs in whole - cell therapy rely on engineered signaling and co - stimulatory domains inside the cell to function.

[0005] Once CAR - whole cells are produced, they can be expanded for mass production and then reinjected into the patient (autologous), or if they are from an engineered stem - cell source, they can be injected into a wide range of patients (allogeneic). CAR - whole cells are designed to recognize a specific tumor antigen on the cancer cells they are designed to target and kill the cancer cells that have those specific antigens.

[0006] CAR T cell therapy has been approved for the treatment of pediatric acute lymphoblastic leukemia (ALL) and adult advanced lymphoma. For example, CD-19-targeted CAR T cells (tisangenlecleucel, KYMRIAH®, Novartis) are approved for treating ALL. YESCARTA® (axicabtagene ciloleucel, Gilead / Kite Pharmaceuticals) is approved for the treatment of lymphoma. Tests targeting CD-22 in cells that have lost CD-19 expression are also being conducted. Dual targeting of CD-19 and CD-123 in leukemia has also been studied. CAR T cells targeting B cell maturation antigen (BCMA) were recently approved as a treatment for multiple myeloma (MM) (idecabtagene vicleucel, ADECMA®, Bristol Myers Squibb). Whether CAR T cells can treat solid tumors remains unclear at present due to the microenvironment surrounding them.

[0007] Despite recent advances, CAR whole-cell therapy has several drawbacks. 1) It can cause cytokine release syndrome (CRS), which results in a systemic inflammatory response due to immunotherapy, leading to high fever and hypotension. This may require additional treatment, such as blocking IL-6 activity. It can also cause B cell death (B cell aplasia), requiring further treatment with immunoglobulins. Other side effects may include cerebral edema and neurotoxicity. 2) In autologous settings, the patient to be treated needs to have an appropriate number of T cells (or NK or macrophages) for harvesting and manipulation, which may not be feasible. 3) Multiple treatments are often required, and difficult re-engineering may also be needed, especially if tumor cells have lost antigen expression. 4) The stability of whole cells in the tumor microenvironment is low due to the ability of hypoxia and acidic environments to induce apoptosis, presenting a major challenge. 5) Once cells enter the tumor environment, they can become exhausted with increased metabolism and become "hyperactive." 6) T cell responses can be shut down, for example, via upregulation of PD-L1 on tumor cells through the PD-1 pathway.

[0008] The PD-1 signaling axis blocks an important mechanism for killing normal, healthy cells and is particularly important in the context of whole-cell therapies. Whole-cell therapies have potential side effects caused by off-target effects, where KEYTRUDA® (pembrolizumab, Merck) or OPDIVO® (nivolumab, Bristol-Myers Squibb) blocks PD-1 on T cells, thus exposing PD-L1-containing healthy cells to immune clearance despite PD-L1 being upregulated in tumor cells. Agents that block PD-1 on T cells prevent the interaction with (upregulated) cancer cells or PD-L1 and PD-L2 on healthy cells, enabling the removal of PD-L1-expressing cancer cells by the immune system. However, off-target effects targeting healthy cells can occur. KEYTRUDA® (pembrolizumab, Merck) also has other undesirable side effects, such as blurred vision, body aches and pains, confusion, constipation, fecal or urinary abnormalities, depression, headache, nausea, etc.

[0009] 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 the inflammatory activity of T cells. PD-1 on T cells binds to PD-L1 and PD-L2 on normal, healthy cells. The PD-1-PD-L1 interaction inactivates the cell-mediated response against normal cells and prevents the killing of healthy cells by T cells. Since cancer cells can express upregulated PD-L1, this also prevents the immune system from targeting and killing cancer cells. T cell PD-1 binds to cancer cells with PD-L1, enhancing the non-killing signal to T cells, making cancer cells invisible to the immune system.

[0010] The PD-1 blockade strategy has some efficacy in various cancers, but monotherapy for most solid tumor cancers is relatively ineffective. To improve the clinical outcomes regarding targeting of this signaling pathway, several combination / co-therapy strategies have been developed. Some of these strategies involve coupling / co-therapy approaches combined with a number of delivery systems including anti-PD-1 monoclonal antibodies, potent small molecules, monoclonal antibodies, gene editing, AAV, and lipid nanoparticles (LNP). More recently, LNP has been utilized to target PD-L1 on cancer cells while delivering inhibitory / toxic nucleic acids or to deliver anti-PD-L1 nucleic acids (such as mRNA). For example, Moderna has a checkpoint cancer vaccine (mRNA-4359) that expresses indoleamine 2,3-dioxygenase and the PD-L1 antigen to target immune and tumor cells expressing the target antigen and to stimulate effector T cells that kill. In this latter approach, improved efficacy can be achieved, but access of the LNP to solid tumor masses within the tumor microenvironment (TME) may be limited, and delivery of toxic nucleic acids or anti-PD-L1 nucleic acids may be further limited due to lack of penetration into tumor cells.

[0011] There is still a need for cancer therapies that overcome the challenges of addressing the PD-L1 / PD-1 signaling problem while leveraging the targeting and killing effects of whole cell therapies and overcoming the drawbacks of standard whole cell therapies in cancer treatment. SUMMARY OF THE INVENTION

[0012] In various embodiments, a method of treating or preventing cancer, the method comprising (i) administering to a subject in need thereof a therapeutically effective amount of a biomimetic nanovesicle (BioNV) comprising (a) a membrane-embedded chimeric antigen receptor (CAR) targeted to a cell surface marker and (b) a PD-1, PD-L1, and / or PD-L2 inhibitor, or (ii) administering to a subject in need thereof a therapeutically effective amount of a BioNV comprising a membrane-embedded CAR targeted to a cell surface marker, wherein the subject is undergoing treatment with a PD-1, PD-L1, and / or PD-L2 inhibitor.

[0013] In various embodiments, a method of treating or preventing cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of a biomimetic nanovesicle (BioNV) comprising (i) (a) a membrane-embedded chimeric antigen receptor (CAR) targeted to a cell surface marker, and a conjugate and / or a membrane anchor PD-L1 and / or PD-L2 inhibitor, or (ii) (a) a membrane-embedded bispecific CAR targeted to a first cell surface marker and a second cell surface marker, and a therapeutically effective amount of a BioNV comprising a conjugate and / or a membrane anchor PD-L1 and / or PD-L2 inhibitor.

[0014] In various embodiments, a method of treating or preventing cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of a biomimetic nanovesicle (BioNV) comprising (i) a bispecific chimeric antigen receptor (CAR) targeted to a first cell surface marker and either PD-L1 or PD-L2, wherein the first cell surface marker is not PD-1, PD-L1, or PD-L2, or (ii) a bispecific CAR targeted to a first cell surface marker and either PD-L1 or PD-L2, wherein the first cell surface marker is not PD-1, PD-L1, or PD-L2 and is not a PD-1, PD-L1, and / or PD-L2 inhibitor, or (iii) a therapeutically effective amount of a BioNV comprising a bispecific CAR targeted to either PD-L1 or PD-L2 and a first cell surface marker, the subject having been treated with a PD-1, PD-L1, and / or PD-L2 inhibitor, and the first cell surface marker not being PD-1, PD-L1, or PD-L2.

[0015] In various embodiments, a method of treating or preventing cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of biomimetic nanovesicles (BioNV) comprising a bispecific chimeric antigen receptor (CAR) targeted to a first cell surface marker and a second cell surface marker, and a PD-1, PD-L1, and / or PD-L2 inhibitor.

[0016] In various embodiments, a method of treating or preventing cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of biomimetic nanovesicles (BioNV) comprising a bispecific chimeric antigen receptor (CAR) targeted to a first cell surface marker and a second cell surface marker, wherein the subject is undergoing treatment with a PD-1, PD-L1, and / or PD-L2 inhibitor.

[0017] In various embodiments, the first cell surface marker and the second cell marker are selected from Table 1 and / or Table 2. In various embodiments, the first cell surface marker is selected from Table 1 and / or Table 2. In various embodiments, the BioNV binds to at least a first cell and at least a second cell and initiates an anti-cancer response. In various embodiments, the first cell is a cancer cell and the second cell is an immune cell. In various embodiments, the PD-1 inhibitor is an antibody targeted to PD-1, optionally pembrolizumab, nivolumab, or semaprilumab. In various embodiments, the PD-L1 and / or PD-L2 inhibitor is an antibody targeted to PD-L1 and / or PD-L2, optionally atezolizumab, avelumab, or durvalumab.

[0018] In various embodiments, BioNV is derived from a modified cell. In various embodiments, the modified cell is a stem cell, induced pluripotent stem cell (iPSC), reprogrammed pluripotent or multipotent cell, embryonic stem cell, mesenchymal stem cell, or a differentiated cell derived from any modified cell thereof. In various embodiments, the modified cell is an iPSC. In various embodiments, the modified cell is a T cell, helper T cell, T memory cell, or NK cell. In various embodiments, the modified cell is a macrophage. In various embodiments, the modified cell is a monocyte. In various embodiments, the low immunogenic cell can be any terminally differentiated cell, such as, but not limited to, muscle cells (satellite cells), adipocytes, osteocytes, cardiomyocytes, hepatocytes, blood cells (including erythrocytes, platelets, and all immune cell types), glial cells (such as other neuron cell types), epithelial cells, epidermal cells, stromal cells (such as respiratory stromal cells), fibroblasts (such as skin fibroblasts), endothelial cells (such as bronchial endothelial cells), oral cells, stromal cells, or germ cells. In various embodiments, the low immunogenic cell can be any functionally specific cell type, such as, but not limited to, exocrine gland secretory epithelial cells, hormone-secreting cells (such as enteroendocrine cells, thyroid cells, pancreatic islet cells, etc.), sensory transducer cells, autonomic nerve cells, sensory organ cells (such as pillar cells, olfactory cells, Schwann cells, satellite glial cells, etc.), barrier cells (such as lung cells, duct cells, kidney cells, podocytes, etc.), extracellular matrix cells (such as tendon fibroblasts, osteoblasts, connective tissue cells, etc.), or contractile cells (such as skeletal muscle cells, cardiomyocytes, myoepithelial cells, etc.).

[0019] In various embodiments, 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. In various embodiments, the modified cells have reduced or absent expression of the β2-microglobulin (B2M) gene, and / or reduced or absent expression and / or activity of MHC class I proteins. In various embodiments, the modified cells have reduced or absent expression of the CIITA gene, and / or reduced or absent expression and / or activity of MHC class II proteins.

[0020] In various embodiments, the modified cells have reduced or absent expression of the HLA-A gene, and / or reduced or absent expression and / or activity of HLA-A proteins. In various embodiments, the modified cells have reduced or absent expression of the HLA-B gene, and / or reduced or absent expression and / or activity of HLA-B proteins. In various embodiments, the modified cells have reduced or absent expression of the HLA-C gene, and / or reduced or absent expression and / or activity of HLA-C proteins. In various embodiments, the modified cells have reduced or absent expression of the HLA-E or HLA-G gene, and / or reduced or absent expression and / or activity of HLA-E or HLA-G proteins. In various embodiments, the modified cells have reduced or absent expression of the HLA-F gene, and / or reduced or absent expression and / or activity of HLA-F proteins.

[0021] In various embodiments, the modified cells have reduced or absent expression of the T cell alpha constant (TRAC) gene, and / or reduced or absent expression and / or activity of TRAC proteins. In various embodiments, the modified cells have reduced or absent expression of the T cell beta constant (TRBC) gene, and / or reduced or absent expression and / or activity of TRBC proteins.

[0022] In various embodiments, the modified cells have reduced or abolished expression of the PD-1 gene and / or reduced or abolished expression and / or activity of the PD-1 protein.

[0023] In various embodiments, the modified cells have reduced or abolished expression of the IL-4 gene and / or reduced or abolished expression and / or activity of the IL-4 protein. In various embodiments, the modified cells have reduced or abolished expression of the IL-6 gene and / or reduced or abolished expression and / or activity of the IL-6 protein. In various embodiments, the modified cells have reduced or abolished expression of the IL-10 gene and / or reduced or abolished expression and / or activity of the IL-10 protein. In various embodiments, the modified cells have reduced or abolished expression of the IL-16 gene and / or reduced or abolished expression and / or activity of the IL-16 protein.

[0024] In various embodiments, the modified cells have reduced or abolished expression of the SerpinB9 gene and / or reduced or abolished expression and / or activity of the SerpinB9 protein.

[0025] In various embodiments, the modified cells express the CD34 gene and / or gene product or have increased expression thereof. In various embodiments, the modified cells express the CCL2 gene and / or gene product or have increased expression thereof. In various embodiments, the modified cells express the PD-L1 gene and / or gene product or have increased expression thereof, and the modified cells are not activated. In various embodiments, the modified cells have reduced or abolished expression of the PD-L1 gene and / or gene product, and the modified cells are activated. In various embodiments, the modified cells overexpress FasL. In various embodiments, the modified cells overexpress SerpinB9. In various embodiments, the modified cells express the H2-M3 gene and / or gene product or have increased expression thereof.

[0026] In some embodiments, the modified cells express the CD47 gene and / or gene product, or have increased expression thereof. In some embodiments, the modified cells express the CD24 gene and / or gene product, or have increased expression thereof. In some embodiments, the modified cells express the chimeric CD24 / CD47 gene and / or gene product, or have increased expression thereof.

[0027] In some embodiments, the modified cells overexpress CTLA-4. In some embodiments, the modified cells express the chimeric CD200 gene and / or gene product, or have increased expression thereof. In some embodiments, the modified cells express the chimeric CD24 / CD200 gene and / or gene product, or the chimeric CD47 / CD200 gene and / or gene product, or have increased expression thereof.

[0028] In some embodiments, the modified cells express the chimeric MFG-E8 gene and / or gene product, or have increased expression thereof. In some embodiments, the modified cells express the NCAM gene and / or gene product, or have increased expression thereof. In some embodiments, the modified cells express the chimeric α-phagocytic integrin gene and / or gene product, or have increased expression thereof. In some embodiments, the modified cells express an antibody or antibody format molecule (anti-IL-6R) that targets the IL-6 surface receptor, or have increased expression and / or activity thereof.

[0029] In some embodiments, the modified cells express the FasL gene and / or gene product, or have increased expression thereof. In some embodiments, the modified cells do not overexpress the FasL gene and / or gene product.

[0030] In various embodiments, the modified cells have reduced or absent expression 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. In various 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 have increased expression thereof.

[0031] In various embodiments, the modified cells are allogeneic. In various embodiments, the modified cells do not elicit an immune response in a patient to whom the cells or BioNV derived therefrom are administered.

[0032] In various embodiments, the modified cells are differentiated prior to BioNV formation.

[0033] In various embodiments, the modified cells express CAR under the control of a controllable expression element. In various embodiments, the CAR is activated prior to BioNV formation. In various embodiments, the CAR is activated via its target, another receptor, and / or through a virus.

[0034] In various embodiments, BioNV is formed from the modified cells, or differentiated cells thereof, by sonication, adaptive focused acoustics technology, French press, extrusion, continuous extrusion, cell lysis with a detergent, and / or electroporation. In various embodiments, BioNV is formed from the modified cells or differentiated cells thereof by continuous extrusion.

[0035] In various embodiments, BioNV is sized from about 10 nm to about 1200 nm. In various embodiments, BioNV is sized from about 10 nm to about 100 nm. In various embodiments, BioNV is sized from about 100 nm to about 200 nm. In various embodiments, BioNV is sized from about 200 nm to about 500 nm. In various embodiments, BioNV is sized from about 500 nm to about 1200 nm.

[0036] In various embodiments, BioNV substantially lacks any one protein and / or activity of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, and HLA-E or HLA-G.

[0037] In various embodiments, BioNV substantially lacks any one protein and / or activity of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, SerpinB9, and HLA-E or HLA-G.

[0038] In various embodiments, BioNV substantially lacks any 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 proteins and / or activities of IL-4, IL-10, and IL-16.

[0039] In various embodiments, BioNV comprises membrane-embedded α-gulphagocytic integrin, CCL2, H2-M3, FasL, MFG-E8, an anti-IL-6R antibody or antibody format, and PD-L1 (in BioNVs derived from non-activated cell sources) and / or CTLA-4, and any one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or any two of CD24, CD47, and CD200.

[0040] In various embodiments, BioNV comprises a membrane-embedded α-phagocytic integrin, CCL2, H2-M3, FasL, MFG-E8, an anti-IL-6R antibody or antibody format, SerpinB9, and PD-L1 (in BioNVs derived from non-activated cell sources) and / or CTLA-4, and any one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or any two of CD24, CD47, and CD200.

[0041] In various embodiments, BioNV has a membrane-embedded CD200 protein and is substantially lacking in either the CD24 or CD47 protein. In various embodiments, BioNV is substantially lacking in the SerpinB9 and CD200 proteins and / or activities.

[0042] In various embodiments, the CAR comprises an antibody or antibody format selected from one or more of monoclonal antibodies, polyclonal antibodies, antibody fragments, 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 an antigen-binding portion of an antibody. In various embodiments, the antibody format is scFv. In various embodiments, the CAR comprises a transmembrane domain derived from CD28, CD3ζ, CD4, CD8α, ICOS, or fragments and / or combinations thereof. In various embodiments, the CAR further comprises an intracellular domain comprising the intracellular signaling domain of the CD3ζ chain and / or optionally one or more costimulatory molecules selected from CD28, 4-1BB, ICOS, CD27, and OX40. In various embodiments, the CAR is targeted to a cancer-specific antigen.

[0043] In some embodiments, BioNV, or modified cells derived therefrom, contain a nucleic acid encoding green fluorescent protein (GFP) and / or the GFP protein. In some embodiments, the nucleic acid encoding GFP is operably linked to a promoter derived from one or more of IL-2, perforin, granzyme, alarmin, TNF, INF, and / or combinations thereof.

[0044] In some embodiments, BioNV encapsulates a payload. In some 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 tracking dye, a radionuclide, and / or a small molecule. In some embodiments, the payload is a therapeutic payload for the cancer type targeted by the CAR. In some embodiments, the payload includes an alkylating agent. In some embodiments, the payload includes an anthracycline. In some embodiments, the payload includes an antimetabolite. In some embodiments, the payload includes an antitumor antibiotic. In some embodiments, the payload includes an antitumor antibody or antibody format. In some embodiments, the payload includes an adrenal corticosteroid. In some embodiments, the payload includes a plant alkaloid. In some embodiments, the payload includes a topoisomerase inhibitor. In some embodiments, the payload includes a checkpoint inhibitor.

[0045] In various embodiments, the payloads are Abecma, Abemaciclib, Abiraterone Acetate, Abraxane, ABVD, ABVE, ABVE-PC, AC, Acalabrutinib, AC-T, Actemra, Adcetris, ADE, Ado-Trastuzumab Emtansine, Adriamycin, Afatinib Dimaleate, Afinitor, Akynzeo, Aldara, Aldesleukin, Alecensa, Alectinib, Alemtuzumab, Alimta, Aliqopa, Alkeran for Injection, Alkeran Tablets, Aloxi, Alpelisib, Alunbrig, Ameluz, Amifostine, Aminolevulinic Acid Hydrochloride, Amivantamab-vmjw, Anastrozole, Apalutamide, Aprepitant, Aranesp, Aredia, Arimidex, Aromasin, Arranon, Arsenic Trioxide, Arzerra, Asciminib Hydrochloride, Asparaginase Erwinia Chrysanthemi, Asparlas, Atezolizumab, Avapritinib, Avastin, Avelumab, Axicabtagene Ciloleucel, Axitinib, Ayvakit, Azacitidine, Azedra, Balversa, Bavencio, BEACOPP, Belantamab Mafodotin-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, BrexucabtageneAutoleucel, Breyanzi, Brigatinib, Brukinsa, BuMel, Busulfan, Busulfex, Cabazitaxel, Cablivi, Cabometyx, Cabozantinib-S-Malate, CAF, Calaspargase Pegol-mknl, Calquence, Campath, Camptosar, Capecitabine, Caplacizumab-yhdp, Capmatinib Hydrochloride, 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, 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, Darolutamide, Darzalex, Darzalex Faspro, Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposome, Daurismo, Decitabine, Decitabine and Cedazuridine, DefibrotideSodium, Defitelio, Degarelix, Denileukin Diftitox, Denosumab, Dexamethasone, Dexrazoxane Hydrochloride, Dinutuximab, Docetaxel, Dostarlimab-gxly, Doxil, Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome, 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, Erwinaze, Ethyol, Etopophos, Etoposide, Etoposide Phosphate, Everolimus, Evista, Evomela, Exemestane, Exkivity, 5-FU, 5-FU, Fam-Trastuzumab Deruxtecan-nxki, Fareston, Faslodex, FEC, Fedratinib Hydrochloride, Femara, Filgrastim, Firmagon, Fludarabine Phosphate, Fluoroplex, Fluorouracil Injection, Fluorouracil-Topical, Flutamide, FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRI-CETUXIMAB, FOLFIRINOX, FOLFOX, Folotyn, FostamatinibDisodium、Fotivda、Fulphila、FU-LV、Fulvestrant、Fyarro、Gamifant、Gardasil(Recombinant HPV Quadrivalent Vaccine)、Gardasil 9(Recombinant HPV Nonavalent Vaccine)、Gavreto、Gazyva、Gefitinib、Gemcitabine Hydrochloride、GEMCITABINE-CISPLATIN、GEMCITABINE-OXALIPLATIN、Gemtuzumab Ozogamicin、Gemzar、Gilotrif、Gilteritinib Fumarate、Glasdegib Maleate、Gleevec、Gliadel Wafer、Glucarpidase、Goserelin Acetate、Granisetron、Granisetron Hydrochloride、Granix、Halaven、Hemangeol、Herceptin Hylecta、Herceptin、HPV Bivalent Vaccine、Recombinant、HPV Nonavalent Vaccine、Recombinant、HPV Quadrivalent Vaccine、Recombinant、Hycamtin、Hydrea、Hydroxyurea、Hyper-CVAD、Ibrance、Ibritumomab Tiuxetan、Ibrutinib、ICE、Iclusig、Idamycin PFS、Idarubicin Hydrochloride、Idecabtagene Vicleucel、Idelalisib、Idhifa、Ifex、Ifosfamide、Interleukin-2(recombinant)、Imatinib Mesylate、Imbruvica、Imfinzi、Imiquimod、Imlygic、Infigratinib Phosphate、Infugem、Inlyta、Inotuzumab Ozogamicin、Inqovi、Inrebic、Interferon Alfa-2b、Intron A、Iobenguane I131、Ipilimumab、Iressa、IrinotecanHydrochloride、Isatuximab-irfc、Istodax、Ivosidenib、Ixabepilone、Ixazomib Citrate、Ixempra、Jakafi、JEB、Jelmyto、Jemperli、Jevtana、Kadcyla、Kepivance、Keytruda、Kimmtrak、Kisqali、Koselugo、Kymriah、Kyprolis、Lanreotide Acetate、Lapatinib Ditosylate、Larotrectinib Sulfate、Lenalidomide、Lenvatinib Mesylate、Lenvima、Letrozole、Leucovorin Calcium、Leukeran、Leuprolide Acetate、Levulan Kerastik、Libtayo、Lisocabtagene Maraleucel、Lomustine、Loncastuximab Tesirine-lpyl、Lonsurf、Lorbrena、Lorlatinib、Lumakras、Lumoxiti、Lupron Depot、Lurbinectedin、Luspatercept-aamt、Lutathera、Lutetium(Lu 177-Dotatate)、Lynparza、Margenza、Margetuximab-cmkb、Marqibo、Matulane、Mechlorethamine Hydrochloride、Megestrol Acetate、Mekinist、Mektovi、Melphalan、Melphalan Hydrochloride、Mercaptopurine、Mesna、Mesnex、Methotrexate Sodium、Methylnaltrexone Bromide、Midostaurin、Mitomycin、Mitoxantrone Hydrochloride、Mobocertinib Succinate、Mogamulizumab-kpkc、Monjuvi、MOPP、Moxetumomab Pasudotox-tdfk、Mozobil、MVAC、Mvasi、Myleran、Mylotarg、NanoparticlePaclitaxel, Naxitamab-gqgk, Necitumumab, Nelarabine, Neratinib Maleate, Nerlynx, Netupitant, Neulasta, Neupogen, Nexavar, Nilandron, Nilotinib, Nilutamide, Ninlaro, Niraparib Tosylate Monohydrate, Nivestym, Nivolumab, Nplate, Nubeqa, Nyvepria, Obinutuzumab, Odomzo, OEPA, Ofa tumumab, OFF, Olaparib, Omacetaxine Mepesuccinate, Oncaspar, Ondansetron Hydrochloride, Onivyde, Ontak, Onureg, Opdivo, OPPA, Orgovyx, Osimertinib Mesylate, Oxaliplatin, Paclitaxel, Paclitaxel Albumin-stabilized Nanoparticle Formulation, PAD, Padcev, Palbociclib, Palifermin, Palonosetron Hydrochloride, Pamidronate Disodium, Panitumumab, Paraplatin, Pazopanib Hydrochloride, PCV, PEB, Pegaspargase, Pegfilgrastim, Peginterferon Alfa-2b, PEG-Intron, Pemazyre, Pembrolizumab, Pemetrexed Disodium, Pemigatinib, Perjeta, Pertuzumab, Pexidartinib Hydrochloride, Phesgo, Piqray, Plerixafor, Polatuzumab Vedotin-piiq, Polivy, Pomalidomide, Pomalyst, Ponatinib Hydrochloride, Portrazza, Poteligeo, Pralatrexate, Pralsetinib, Prednisone, Procarbazine Hydrochloride, Procrit, Proleukin, Prolia, Promacta, Propranolol Hydrochloride, Provenge, Purinethol, Purixan, Qinlock, Radium 223 Dichloride, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, Ravulizumab-cwvz, Reblozyl, R-CHOP, R-CVP, Recombinant Human Papillomavirus(HPV) Bivalent Vaccine, Recombinant HumanPapillomavirus(HPV)Nonavalent Vaccine、Recombinant Human Papillomavirus(HPV)Quadrivalent Vaccine、Recombinant Interferon Alfa-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、Sacituzumab Govitecan-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 V、Sterile Talc Powder、Steritalc、Stivarga、Sunitinib Malate、Sustol、Sutent、Sylatron、Sylvant、Synribo、Tabloid、Tabrecta、TAC、Tafasitamab-cxix、Tafinlar、Tagraxofusp-erzs、Tagrisso、Talazoparib Tosylate、Talimogene Laherparepvec、Talzenna、Tamoxifen Citrate、Tarceva、Targretin、Tasigna、Tavalisse、Taxotere、TazemetostatHydrobromide, Tazverik, Tebentafusp-tebn, Tecartus, Tecentriq, Temodar, Temozolomide, Temsirolimus, Tepadina, Tepmetko, Tepotinib Hydrochloride, Thalidomide, Thalomid, Thioguanine, Thiotepa, Tibsovo, Tisagenlecleucel, Tisotumab Vedotin-tftv, Tivdak, Tivozanib Hydrochloride, Tocilizumab, Tolak, Topotecan Hydrochloride, Toremifene, Torisel, Totect, TPF, Trabectedin, Trametinib Dimethyl Sulfoxide, Trastuzumab, Trastuzumab and Hyaluronidase-oysk, Treanda, Trexall, Trifluridine and Tipiracil Hydrochloride, Trisenox, Trodelvy, Truseltiq, Truxima, Tucatinib, Tukysa, Turalio, Tykerb, Ukoniq, Ultomiris, Umbralisib Tosylate, Undencyca, Unituxin, Uridine Triacetate, VAC, Valrubicin, Valstar, Vandetanib, VAMP, Varubi, Vectibix, VeIP, Velcade, Vemurafenib, Venclexta, Venetoclax, Verzenio, Vidaza, Vinblastine Sulfate, Vincristine Sulfate, Vincristine Sulfate Liposome, VinorelbineIt is selected from 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 (Bevcizumab), Ziv-Aflibercept, Zofran, Zoladex, Zoledronic Acid, Zolinza, Zometa, Zyclara, Zydelig, Zykadia, Zynlonta, and Zytiga.

[0046] In some embodiments, the payload is one or more of pidilizumab, BMS-936559, tremelimumab, AGEN1884, and / or RG2077.

[0047] In some embodiments, the nucleic acid molecule comprises 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), 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 some embodiments, the gene editing payload is one or more of a TALEN, a ZFN, an 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, a transposase, and / or any ortholog or homolog thereof.

[0048] In some embodiments, BioNV encapsulates one or more perforin molecules. In some embodiments, BioNV encapsulates one or more granzyme molecules. In some embodiments, the granzyme molecule is selected from granzyme A, B, H, K, and M. In some embodiments, BioNV encapsulates one or more perforin molecules and / or one or more granzyme molecules derived from the cells from which BioNV is derived. In some embodiments, BioNV encapsulates one or more perforin molecules and one or more granzyme molecules that are exogenously added to BioNV.

[0049] In some embodiments, the method of treatment further comprises co-administering a whole cell therapy. In some embodiments, the method of treatment further comprises administering an additional therapeutic agent.

[0050] In various embodiments, BioNV is stored at about -80°C or is suitable for storage at about -80°C. In various embodiments, BioNV is lyophilized.

[0051] In various embodiments, the PD-1 inhibitor is an antibody targeted against PD-1, or pembrolizumab, nivolumab, or semiprimab. In various embodiments, the PD-L1 or PD-L2 inhibitor is an antibody targeted against PD-L1 or PD-L2, optionally atezolizumab, avelumab, or durvalumab, or an antigen-binding domain thereof, and is conjugated to the surface of BioNV. In various embodiments, the PD-1 inhibitor is an antibody targeted against PD-1, or pembrolizumab, nivolumab, or semiprimab. In various embodiments, the PD-L1 or PD-L2 inhibitor is an antibody targeted against PD-L1 or PD-L2, optionally atezolizumab, avelumab, or durvalumab, or an antigen-binding domain thereof, and is membrane-anchored via transmembrane domain fusion.

[0052] In various embodiments, the cancer is a carcinoma. In various embodiments, the cancer is a sarcoma. In various embodiments, the cancer is a myeloma. In various embodiments, the cancer is a leukemia. In various embodiments, the cancer is a lymphoma. In various embodiments, the cancer is a mixed cancer. In various embodiments, the cancer is metastatic.

[0053] In various embodiments, the cancer is acute biphenotypic leukemia, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid dendritic cell leukemia, acute myeloid leukemia, lung adenocarcinoma, adrenal tumor, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, alveolar soft part sarcoma and cardiac sarcoma, amyloidosis, anal cancer, anaplastic large cell lymphoma, angioimmunoblastic T cell lymphoma, appendiceal cancer, astrocytoma, ataxia telangiectasia, attenuated familial adenomatosis, B-cell prolymphocytic leukemia, basal cell carcinoma, Beckwith-Wiedemann syndrome, bile duct cancer, Birt-Hogg-Dubé syndrome, bladder cancer, bone cancer, central nervous system cancer, brain stem glioma, brain stem glioma, brain tumor, breast cancer, bronchial adenoma / carcinoid, Burkitt lymphoma, carcinoid tumor, carcinoid tumors, Carney complex, central nervous system tumor, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, childhood desmoplastic ganglioglioma, cholangiocarcinoma, chondrosarcoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloid leukemia, chronic T-cell lymphocytic leukemia, colon cancer, colorectal cancer, Cowden syndrome, craniopharyngioma, cutaneous T-cell lymphoma, dermatofibrosarcoma protuberans, desmoplastic small round cell tumor, diffuse gastric cancer, diffuse large B-cell lymphoma, endocrine cancer, endocrine tumor, endometrial cancer, eosinophilic leukemia, epithelioma, epitheloid hemangioendothelioma (EHE), esophageal cancer, Ewing sarcoma, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, eyelid cancer, fallopian tube cancer, familial adenomatosis, 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 cancer and gynecological cancer, germ cell tumor, 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, islet cell carcinoma (pancreatic endocrine),Pancreatic islet cell tumor, juvenile polyposis syndrome, Kaposi sarcoma, keratoacanthoma, renal cancer, lacrimal gland tumor, large granular lymphocyte leukemia, laryngeal cancer and hypopharyngeal cancer, leiomyomatosis and renal cell carcinoma, leiomyosarcoma, Li-Fraumeni syndrome (LFS), liposarcoma, liver cancer, lung cancer, primary cutaneous lymphoma, lymphomatoid granulomatosis, lymphoplasmacytic lymphoma, Lynch syndrome, malignant fibrous histiocytoma of bone, mantle cell lymphoma, marginal zone B-cell lymphoma, mast cell leukemia, mastocytosis, mediastinal large cell B-cell lymphoma, medullary carcinoma, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, mixed polyposis syndrome, mucosa-associated lymphoid tissue lymphoma, Muir-Torre syndrome (MTS), multiple endocrine neoplasia syndrome, multiple endocrine neoplasia type 1, multiple endocrine neoplasia type 2, multiple myeloma, multiple myeloma / plasma cell tumor, fungating polypoid tumor, myelodysplastic syndrome, MYH-related polyposis, myxosarcoma, nasal and paranasal cavity cancer, nephroblastoma, nasopharyngeal cancer, nasopharyngeal carcinoma, neuroblastoma, neuroendocrine tumor, 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 carcinoma, anaplastic glioma, optic nerve glioma, oral cancer and / or oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, papillary renal cell carcinoma, paranasal cavity 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 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, renal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, primary cutaneous sarcoma, sebaceous adenocarcinoma, Sézary syndrome, skin appendage tumor, skin cancer, small bowel cancer, small cell lung cancer, small intestine cancer, splenic marginal zone lymphoma, squamous cell carcinoma of lung, squamous cell carcinoma, squamous cell skin cancer, stomach cancer, superficial epithelial stromal tumor, T-cell prolymphocytic leukemia, testicular cancer, thoracic and respiratory cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell carcinoma, transitional cell carcinoma adenoid cystic carcinoma, tuberous sclerosis syndrome, tubular carcinoma, Turcot syndrome, cancer of unknown primary origin, unclassified carcinoma, ureteral cancer, urethral cancer, uterine cancer, uterine sarcoma, uveal melanomaSelected from vaginal cancer, hypothalamic glioma, von Hippel-Lindau (VHL) syndrome, vulvar cancer, Wilms tumor, and xeroderma pigmentosum.,

[0054] In various embodiments, an allogeneic biomimetic nanovesicle (BioNV) comprising (a) a membrane-embedded chimeric antigen receptor (CAR) targeted to a cell surface marker and (b) a conjugate and / or a membrane anchor targeting agent targeted to PD-L1 or PD-L2, wherein the agent targeted to PD-L1 or PD-L2 is atezolizumab, avelumab, or durvalumab, and the CAR is not targeted to PD-1, PD-L1, or PD-L2.

[0055] In various embodiments, an allogeneic biomimetic nanovesicle (BioNV) comprising (a) a first membrane-embedded chimeric antigen receptor (CAR) targeted to a cell surface, wherein the first membrane-embedded CAR is not targeted to PD-L1 or PD-L2, and (b) at least a second membrane-embedded CAR targeted to PD-L1 or PD-L2.

[0056] In various embodiments, there is provided an allogeneic biomimetic nanovesicle (BioNV) comprising (a) a cell surface marker of a cancer cell and (b) a bispecific membrane-embedded chimeric antigen receptor (CAR) targeted to PD-L1 or PD-L2, wherein the cell surface marker of the cancer cell is not PD-1, PD-L1, or PD-L2. BRIEF DESCRIPTION OF THE DRAWINGS

[0057]

Figure 1A

Figure 1B

Figure 1C

Figure 2A

Figure 2B

Figure 2C

Figure 2D

Figure 2E

Figure 2F

Figure 2G

Figure 3

Mode for Carrying Out the Invention

[0058] The present disclosure relates, in part, to allogeneic, low-immunogenic cell-derived biomimetic nanovesicles (BioNVs) that are found to be useful in the treatment of cancer by one or more surface-oriented chimeric antigen receptors (CARs) that recognize a single target or multiple targets (i.e., bispecificity) via a binding moiety for a desired biomarker / ligand. In various embodiments, the designed BioNVs have a size of about 20 - 1200 nm, which is much smaller than conventional cell-based CAR-T / NK cell therapies. BioNVs 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 cell. BioNVs can also be derived from cells such as T cells, NK cells, macrophages, monocytes, etc. Plasma membrane-derived BioNVs retain the low-immunogenic properties of the modified cells from which they are derived. The modified cells can undergo genetic manipulation focused on the reduction or elimination (e.g., knockout) of 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 the expression or overexpression of immunoprotective cell surface markers (e.g., CD47, CD34, CD24, CD200, alpha-macrophage, etc.). The binding moiety of the CAR can be, for example, Fab, Fab’, Fab’-SH, F(ab’)2, scFv, diabody, nanobody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and antibody, V H H nanobody, V NARSIt can include any variant of an antibody construct that enables BioNV to target any cancer cell of interest (target any type of cell surface biomarker), including a fusion protein containing the antigen-binding portion of . BioNV can also encapsulate and deliver any selected cytotoxic protein, small molecule, biologic, nucleic acid, gene editing therapeutic payload, etc. to a cell target intended for cancer treatment. BioNV can include a membrane anchor and / or a conjugated anti-cancer antibody (e.g., pembrolizumab or nivolumab).

[0059] Furthermore, the present disclosure relates, in part, to a method of treating and / or preventing cancer by administering a composition of BioNV having a single-specificity and / or bispecific CAR (e.g., a biomarker and anti-PD-L1 or anti-PD-L2), as shown in FIG. 2B. In embodiments, the treatment method includes targeting cancer cells and providing a more specific block of PD-L1. In embodiments, BioNV can be administered with pembrolizumab or nivolumab, as shown in FIGS. 2C-2G. In embodiments, BioNV can include a tumor-targeting anti-PD-L1 specific or anti-PD-L1 / 2 bispecific CAR (FIGS. 2B-2C). In embodiments, the method of treatment utilizes a BioNV having a single-specificity and / or bispecific CAR, as well as a conjugated and / or membrane-anchored antibody (e.g., pembrolizumab or nivolumab) (FIG. 2A). In embodiments, the method uses constructs or combination therapies as shown in FIGS. 2A, 2B, 2C, 2D, 2E, 2F, and / or 2G.

[0060] In various embodiments, having an anti-PD-1 molecule on the BioNV is undesirable because it may convert the CAR from cancer cells to T cells and / or potentially damage or kill T cells. However, in various embodiments, the anti-PD-1 can be included as an entire protein construct or as the minimal PD-L1 / PD-L2 interaction domain of PD-1. In various embodiments, this is engineered with a transmembrane domain to be incorporated into the BioNV lipid layer. In various embodiments, the CAR (either single-specificity or bispecific) can rapidly change to these when target variants occur. Biomarker variability can result from DNA repair mechanisms within the host and drug selection and / or immunological selection pressures. In various embodiments, the method of treatment uses BioNVs derived from cytotoxic cells to enable the delivery of anti-cancer molecules (e.g., cytokines, perforin, granzyme, etc.) to the tumor in a more controlled manner than whole cells do. In various embodiments, these molecules can be released from the BioNVs and have significantly improved penetration into the TME compared to conventional LNPs or whole cells. In various embodiments, the scFV region of the CAR targets either PD-L1 or PD-L2, and the other scFV region (on the same CAR) recognizes a cancer-specific marker, enabling targeting of the cell for both the release of perforin / granzyme to kill the cell and the blocking of PD-L1 / PD-L2 from the PD-1 receptor on host T cells, thereby preventing immunological escape of cancer cells. In various embodiments, co-administration of pembrolizumab or nivolumab acts to bind to PD-1 on T cells and further enhance the effect.

[0061] Methods of treating, preventing, and / or ameliorating cancer In various aspects, the present disclosure includes methods of treating, preventing, and / or ameliorating cancer, the methods comprising administering to a subject in need thereof a therapeutically effective amount of a biomimetic nanovesicle (BioNV) comprising (i) a membrane-embedded chimeric antigen receptor (CAR) targeted to 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 targeted to a cell surface marker, wherein the subject is undergoing treatment with a PD-1, PD-L1, and / or PD-L2 inhibitor.

[0062] In various aspects, the present disclosure includes methods of treating, preventing, and / or ameliorating cancer, the methods comprising administering to a subject in need thereof a therapeutically effective amount of a BioNV comprising (i) (a) a membrane-embedded CAR targeted to a cell surface marker and (b) a conjugate and / or a membrane anchor PD-L1 and / or PD-L2 inhibitor, or (ii) (a) a membrane-embedded bispecific CAR targeted to a first cell surface marker and a second cell surface marker and (b) a therapeutically effective amount of a BioNV comprising a conjugate and / or a membrane anchor PD-L1 and / or PD-L2 inhibitor.

[0063] In various aspects, the present disclosure includes methods of treating, preventing, and / or ameliorating cancer, the methods comprising administering to a subject in need thereof a therapeutically effective amount of BioNV that (i) comprises a bispecific CAR targeted to a first cell surface marker and either PD-L1 or PD-L2, wherein the first cell surface marker is not PD-1, PD-L1, or PD-L2, or (ii) comprises a bispecific CAR targeted to a first cell surface marker and either PD-L1 or PD-L2, wherein the first cell surface marker is not PD-1, PD-L1, or PD-L2 and is not a PD-1, PD-L1, and / or PD-L2 inhibitor, or (iii) a therapeutically effective amount of BioNV comprising a bispecific CAR targeted to either PD-L1 or PD-L2 and a first cell surface marker, wherein the subject has been treated with a PD-1, PD-L1, and / or PD-L2 inhibitor and the first cell surface marker is not PD-1, PD-L1, or PD-L2.

[0064] In various aspects, the present disclosure includes methods of treating, preventing, and / or ameliorating cancer, the methods comprising administering to a subject in need thereof a therapeutically effective amount of BioNV comprising (a) a bispecific CAR targeted to a first cell surface marker and a second cell surface marker, and (b) a PD-1, PD-L1, and / or PD-L2 inhibitor.

[0065] In various aspects, the present disclosure includes methods of treating, preventing, and / or ameliorating cancer, the methods comprising administering to a subject in need thereof a therapeutically effective amount of BioNV comprising a bispecific CAR targeted to a first cell surface marker and a second cell surface marker, wherein the subject has been treated with a PD-1, PD-L1, and / or PD-L2 inhibitor.

[0066] In various embodiments, the CAR (single - specificity or bispecificity) is targeted to a first cell - surface marker and / or a second cell marker selected from Table 1 and / or 2. In various embodiments, the bispecific CAR is targeted to a first cell - surface marker selected from either Table 1 or 2 and PD - L1 or PD - L2. [Table 1] [Table 2] TIFF2025518126000004.tif230159TIFF2025518126000005.tif98159

[0067] In various embodiments, two or more biomarkers (or two or more epitopes of a single biomarker) can be targeted by a bispecific CAR, or a BioNV having multiple single - specificity CARs. In various embodiments, some biomarkers are found in multiple types of cancer, and the BioNV can be targeted to multiple cancer types. In various embodiments, for example, since biomarkers on cancer cells tend to mutate or be suppressed, if resistance occurs to the primary biomarker target on cancer cells, a mutated biomarker can be selected. In various embodiments, the CAR construct (all of its variants) can be modified to match the mutation. In various embodiments, since the mutations present in cancer are predictable, the construct can be made in advance. For example, the SCN4A mutation can be used as a predictor for checkpoint inhibitor treatment regimens (Lin et al., “Potential Predictive Value of SCN4A Mutation Status for Immune Checkpoint Inhibitors in Melanoma.” Biomedicine & Pharmacotherapy. 2020). In various embodiments, secondary biomarkers can be targeted in subsequent treatments. For example, tests are also being conducted to target CD - 22 in cells that have lost CD - 19 expression.

[0068] In various embodiments, BioNV binds to at least a first cell and at least a second cell to initiate an anti-cancer response. In various embodiments, the first cell is a cancer cell and the second cell is an immune cell. In various embodiments, both the first cell and the second cell can be of the same or a similar cell type (e.g., both are tumor cells).

[0069] In various embodiments, the PD-1 inhibitor is an antibody targeted to PD-1, or pembrolizumab, nivolumab, or cemiplimab. In various embodiments, the PD-L1 and / or PD-L2 inhibitor is atezolizumab, avelumab, or durvalumab.

[0070] In various embodiments, BioNV derived from a modified cell is, but not limited to, a differentiated cell derived from a stem cell, induced pluripotent stem cell (iPSC), reprogrammed pluripotent or multipotent cell, embryonic stem cell, mesenchymal stem cell, or any modified cell thereof. In various embodiments, the modified cell is an iPSC. In various embodiments, the modified cell is a T cell, helper T cell, T memory cell, or NK cell. In various embodiments, the modified cell is a macrophage. In various embodiments, the modified cell is a monocyte.

[0071] In various embodiments, the allogeneic and hypoimmunogenic properties of the modified cell (e.g., derived from iPSC) are created by knocking out, silencing, inactivating, blocking, or otherwise invalidating the expression, transcription efficiency, and / or activity of one or more immunogenic molecules. In various embodiments, the modified cell substantially lacks one or more MHC class I proteins, MHC class II proteins, HLA proteins, TCR proteins, and / or CRS proteins.

[0072] In some embodiments, reduction or elimination of the expression and / or activity of one or more immunogenic proteins includes, for example, disruption of the β2-microglobulin (B2M) gene and / or disruption that reduces or eliminates the expression and / or activity of MHC class I proteins, in the case of CD8+ T cell lines. In some embodiments, reduction or elimination of the expression and / or activity of one or more immunogenic proteins includes, for example, disruption of the CIITA gene and / or disruption that reduces or eliminates the expression and / or activity of MHC class II proteins, in the case of CD4+ T cell lines. Without wishing to be bound by theory, these proteins contribute to the immunogenicity of human leukocyte antigen (HLA), and HLA allele matching in donor-recipient is required for treatment by cell-based therapies. In some embodiments, allogeneic and / or hypoimmunogenic properties are achieved, for example, by reducing or eliminating the expression and / or activity of genes encoding T cell receptor (TCR) proteins, including the α and β chains (in the case of αβ T cells) or the γ and δ chains (in the case of γδ T cells) that form the ligand-binding site, as well as the signal transduction modules CD3δ, CD3γ, CD3ε, and CD3ζ. In some embodiments, this is performed to reduce foreign T cell receptor types other than those of the CAR cassette, further improve the uniformity of the CAR of interest, and reduce off-target effects in BioNV formation.

[0073] In some embodiments, the modified cells include β2-microglobulin (B2M) gene disruption or gene disruption that interferes with MHC class I expression. In some embodiments, knocking out the B2M gene reduces the number of possible doses that can be administered because there is a risk of preventing long-term acceptance of BioNV by the recipient, as observed in the all-cell-based approach described above. To overcome this problem, in some embodiments, the HLA-E or HLA-G genes are left intact to allow the immune system to adapt to the resulting BioNV. In some embodiments, HLA-A, HLA-B, HLA-C, HLA-F, and HLA-E or HLA-G (but not both) are sequentially knocked out.

[0074] In various embodiments, the modified cells have reduced or absent expression of the HLA-A gene and / or reduced or absent expression and / or activity of the HLA-A protein. In various embodiments, the modified cells have reduced or absent expression of the HLA-B gene and / or reduced or absent expression and / or activity of the HLA-B protein. In various embodiments, the modified cells have reduced or absent expression of the HLA-C gene and / or reduced or absent expression and / or activity of the HLA-C protein. In various embodiments, the modified cells have reduced or absent expression of the HLA-E or HLA-G gene and / or reduced or absent expression and / or activity of the HLA-E or HLA-G protein. In various embodiments, the modified cells have reduced or absent expression of the HLA-F gene and / or reduced or absent expression and / or activity of the HLA-F protein.

[0075] In various embodiments, the modified cells have reduced or absent expression of the CIITA gene and / or reduced or absent expression and / or activity of the MHC class II protein. In various embodiments, in allogeneic iPSCs, the MHC class I and MHC class II complexes are disrupted by knocking out β2-microglobulin (B2M), a serum protein found associated with the MHC class I heavy chain on the surface of almost all nucleated cells and involved in peptide antigen presentation to the immune system, which is an important protein involved in the expression of the MHC class I and MHC class II complexes. In various embodiments, in allogeneic iPSCs, the CIITA gene, which is the master regulatory transcription factor that controls the expression of all MHC II genes, is disrupted. In various embodiments, in allogeneic iPSCs, the CIITA gene, which is the master regulatory transcription factor that controls the expression of all MHC II genes, is disrupted, and as a result, the resulting differentiated cell lines (e.g., DCs, monocytes, endothelial cells, thymic epithelial cells, B cells, etc.) do not express the MHC class II protein or have reduced expression thereof.

[0076] In various embodiments, the modified cells have reduced or absent expression of the T cell alpha constant (TRAC) gene and / or reduced or absent expression and / or activity of the TRAC protein. In various embodiments, the modified cells have reduced or absent expression of the T cell beta constant (TRBC) gene and / or reduced or absent expression and / or activity of the TRBC protein.

[0077] In various embodiments, the modified cells have reduced or absent expression of the PD-1 gene and / or reduced or absent expression and / or activity of the PD-1 protein.

[0078] CRS is a major concern in whole cell therapies, and despite being engineered to be hypoimmunogenic, biomolecules can be released due to effector function and other consequences of the interaction of the cells after injection, which may result in a systemic inflammatory syndrome characterized by fever, multiple organ dysfunction, etc. In various embodiments, the modified cells are engineered to disrupt one or more proteins contributing to CRS. In various embodiments, the modified cells have reduced or absent expression and / or activity (e.g., knockout or silencing) of CRS-related cytokines.

[0079] In various embodiments, the modified cells have reduced or abolished expression of the IL-4 gene and / or reduced or abolished expression and / or activity of the IL-4 protein. In various embodiments, the modified cells have reduced or abolished expression of the IL-6 gene and / or reduced or abolished expression and / or activity of the IL-6 protein. In various embodiments, the IL-6 knockout prevents the unwanted encapsulation of IL-6 into BioNV and reduces the contribution of BioNV to localized (and concentrated by biomarker targeting) and / or potential systemic CRS events. In various embodiments, the modified cells have reduced or abolished expression of the IL-10 gene and / or reduced or abolished expression and / or activity of the IL-10 protein. In various embodiments, the modified cells have reduced or abolished expression of the IL-16 gene and / or reduced or abolished expression and / or activity of the IL-16 protein. In various embodiments, the reduction or abolition of the interleukin reduces the likelihood of CRS.

[0080] Serine protease inhibitor B9 (SerpinB9) is a member of the serine protease inhibitor superfamily. SerpinB9 has been reported to protect cells from the immune killing effect of granzyme B. In embodiments where BioNV is not designed to deliver granzyme, the modified cells from which BioNV is derived express SerpinB9 or have increased expression thereof. In embodiments where BioNV is designed to deliver granzyme, the modified cells from which BioNV is derived have SerpinB9 knocked out and / or silenced. In various embodiments, the modified cells have reduced or abolished expression and / or activity of the SerpinB9 gene and / or reduced or abolished expression and / or activity of the SerpinB9 protein.

[0081] In various embodiments, overexpression of SerpinB9 suppresses the function of granzyme B associated with immune activation responses, such as apoptosis of target cells and / or diseased cells. In various embodiments, granzyme B is inhibited in cell types such as activated lymphocytes, NK cells, macrophages, and follicular DCs. In various embodiments, for example, in the case of BioNV intended to deliver a non-granzyme payload, such as a gene editing payload, the modified cells can express SerpinB9 and / or have increased expression thereof.

[0082] In various embodiments, methods of treating, preventing, and / or ameliorating cancer use BioNVs derived from cells modified to be hypoimmunogenic by the expression or increased expression of one or more immunoprotective proteins. In various embodiments, the modified cells express or have increased expression of the CD34 gene and / or gene product. In various embodiments, the modified cells express or have increased expression of the CCL2 gene and / or gene product. In various embodiments, the modified cells express or have increased expression of the PD-L1 gene and / or gene product, and the modified cells are not activated, for example, the resulting BioNVs do not encapsulate perforin and / or granzyme. In various embodiments, the modified cells have reduced or absent expression of the PD-L1 gene and / or gene product, and the modified cells are activated, for example, the resulting BioNVs encapsulate perforin and / or granzyme. In various embodiments, the modified cells express or have increased expression of the H2-M3 gene and / or gene product.

[0083] In various embodiments, the modified cells express the CD47 gene and / or gene product, or have increased expression thereof. Exosomes and cell-derived vesicles (CDVs) are readily removed from the body by macrophages through phagocytosis. Phagocytosis has a great impact on the therapeutic benefit and efficacy of CDVs. Without wishing to be bound by theory, in order to prevent macrophage depletion by BioNV, in various embodiments, BioNV has a CD47 tag added to its surface. CD47tg (tag) provides a "do not eat me" signal, which in various embodiments increases the half-life and serum stability of BioNV in a subject. In various embodiments, the molecular CD47 isoform 2 (the isoform that interacts with the SIRPα receptor on macrophages) is incorporated into the modified cells (e.g., iPSC cell line). Without CD47tg, the half-life of BioNV is shortened by phagocytosis inhibition, resulting in the need for higher doses and / or more frequent administrations. In various embodiments, preventing the potential inhibitory phenotype of CD47 expression across the entire cell is done by interfering with the inhibitory mechanism of action of a series of microRNAs against the 3'UTR of the CD47 gene. This is done by deleting this region in a stable construct or by eliminating / inhibiting the expression of the microRNA. In various embodiments, this may solve the problem of inhibition caused by microRNAs across a subset of differentiated cells.

[0084] In various embodiments, the modified cells overexpress CD24. CD24 is a sialoglycoprotein expressed on mature granulocytes and B cells and is also an anti-phagocytic protein. CD24 prevents phagocytosis through interaction with Siglec-G / 10 on macrophages. In various embodiments, the modified cells express the CD24 gene and / or gene product or have increased expression thereof. In various embodiments, the modified cells express the chimeric CD24 / CD47 gene and / or gene product or have increased expression thereof. In various embodiments, the modified cells express a chimeric CD24 / CD47 having a tethered transmembrane domain. In various embodiments, the domains of CD47 isoform 2 and CD24 can be expressed separately or linked to form a two-leaf chimeric protein. In various embodiments, the modified cells are fibroblast-derived iPSCs rather than ABO cells.

[0085] In some embodiments, the modified cells express or have increased expression of a chimeric CD200 gene and / or gene product. In some embodiments, the CD200 tag minimizes phagocytosis by macrophages and prevents granulocyte activation. In some embodiments, when it is not desirable to suppress granulocytes, e.g., in the TME, the modified cells do not express CD200. This is to complement the mechanism of action of BioNV designed such that granulocyte activation releases granzymes and perforin. However, in some embodiments, when a CD47 or CD24 tag is used, or when a CD24 / CD47 chimeric bifunctional protein tag (each preventing phagocytosis) is used in combination with overexpressed H2-M3 (attenuating the NK response), clearance of the appropriate BioNV can be achieved without CD200 while achieving stability. In some embodiments, when granzymes and perforin are not selected as therapeutic biomolecules, CD200 can be expressed to prevent granulocyte activation while, on the other hand, a CD47 tag or a CD24 tag (but not both tags) can be removed. In some embodiments, the modified cells express or have increased expression of a chimeric CD24 / CD200 gene and / or gene product, or a chimeric CD47 / CD200 gene and / or gene product.

[0086] In some embodiments, the modified cells (or cells differentiated therefrom) do not express all three of CD47, CD24, and CD200. In some embodiments, the modified cells from which BioNV is derived are engineered to a degree such that BioNV is stabilized but not prevented from being eliminated from the body. A BioNV that is too stable may ultimately cause a humoral response, resulting in a limited number of administrations or treatments.

[0087] In some embodiments, the modified cells express a chimeric CTLA-4 gene and / or gene product, or have increased expression thereof. In some embodiments, the modified cells express a chimeric MFG-E8 gene and / or gene product, or have increased expression thereof. In some embodiments, the modified cells express an NCAM gene and / or gene product, or have increased expression thereof. In some embodiments, the modified cells express a chimeric α-phagocytic integrin gene and / or gene product, or have increased expression thereof.

[0088] In some embodiments, the modified cells express an antibody or antibody format molecule (anti-IL-6R) that targets the IL-6 surface receptor, or have increased expression and / or activity thereof. In some embodiments, the iPSC cell line for generating the anti-cancer BioNV has an anti-IL-6R antibody incorporated into the cell line. In some embodiments, the BioNV retains α-IL-6R, thereby blocking the activation of the signaling pathway on localized immune cells in the tumor environment.

[0089] In some embodiments, the modified cells express a FasL gene and / or gene product, or have increased expression thereof. In some embodiments, the modified cells do not overexpress the FasL gene and / or gene product. In some embodiments, in the membrane of the BioNV, for example, after processing by continuous extrusion, enrichment of native expression levels of FasL was observed, thus avoiding overexpression of FasL in the modified cells. If the concentration of FasL is too high, it can have the opposite effect and may prevent the recruitment of T cells to solid tumors and / or may cause premature death of T cells.

[0090] In various embodiments, the modified cell can express one or more fusion proteins of one or more portions of any of the immunoprotective proteins herein. For example, in an embodiment, the construct can be made when an appropriate portion of a selected ligand is linked to a transmembrane domain, such that the entire protein (e.g., an intracellular signaling domain) is not required. In various embodiments, the construct can be made when biologically relevant portions of two or more proteins are linked together and / or to a transmembrane domain.

[0091] In various embodiments, the modified cell has reduced or absent expression and / or activity of one or more immunogenic proteins, such as proteins that cause an immune response, donor-recipient mismatch, HLA alloimmunity, inflammation, CRS, etc. in a subject, such as MHC class I proteins, MHC class II proteins, HLA proteins, TCR proteins, CRS proteins, etc. In various embodiments, the modified cell has reduced or absent expression 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.

[0092] In various embodiments, the modified cells have the expression of one or more immune-protective proteins, such as proteins that result in preventing or reducing the immune response in a subject, preventing or reducing the early clearance of BioNV in a subject, preventing or reducing phagocytosis, conferring barrier-passing function, etc., such as CD47, CD24, CD200, CD34, CCL2, H2-M3, MFG-E8, PD-L1 (for non-activated cells), CTLA-4, etc., or have increased expression and / or activity thereof. In various embodiments, the modified cells have the expression of 3 or more immune-protective proteins, 4 or more immune-protective proteins, 5 or more immune-protective proteins, 6 or more immune-protective proteins, 7 or more immune-protective proteins, 8 or more immune-protective proteins, 9 or more immune-protective proteins, or 10 or more immune-protective proteins, or have increased expression thereof.

[0093] In various embodiments, as used herein, "increased expression" and "increased expression and / or activity" refer to an increase in expression and / or activity compared to natural or wild-type homologous cells in hypoimmunogenic cells and / or the resulting BioNV. For example, in various embodiments, the increased expression and / or activity of one or more biomolecules described herein can confer hypoimmunogenic properties to iPSCs compared to iPSCs with different protein expression patterns or levels. In various embodiments, "increased expression" is due to genetic modification such as knock-in.

[0094] In various embodiments, the modified cells are allogeneic. In various embodiments, the modified cells do not cause an immune response in the patient to whom the cells or the BioNV derived therefrom are administered.

[0095] In various embodiments, the modified cells are subjected to specific processing such as activation of the modified cells (e.g., via TCR / CD3, CD28, etc. to increase the expression of granzyme, perforin, etc.), differentiation of the modified cells (e.g., from iPSCs to T cells, NK cells, macrophages, etc.), and / or activation of the CAR before the manufacture of BioNV. In various embodiments, the modified cells are differentiated before BioNV formation. In various embodiments, the modified cells express the CAR by a controllable expression element. In various embodiments, the CAR is activated before BioNV formation. In various embodiments, the CAR is activated via its target and / or through another receptor and / or virus. In various embodiments, the modified cells are expanded after genetic manipulation. Any small-scale expansion culture method or large-scale feeder system expansion culture method known in the art can be used.

[0096] In various embodiments, the type of cancer to be targeted drives the differentiation into a given cell line. In various embodiments, BioNV can be derived from activated macrophages / monocytes when brain cancer is targeted. In various embodiments, BioNV inherits some phenotypic characteristics from the cells from which they are derived (their parent cells). For example, in various embodiments, macrophages / monocytes can easily cross the BBB, and this property (presumably receptor-driven) can be transmitted to BioNV. In various embodiments, BioNV can be generated from tumor-infiltrating lymphocytes (TILs) for high-density tumors.

[0097] In various embodiments, BioNV is formed from modified cells, or their differentiated cells (e.g., after activation) by sonication, adaptive focused acoustic technology, French press, extrusion, continuous extrusion, cell lysis with detergents, enzymatic cleavage of cells (trypsin treatment), and / or electroporation. In various embodiments, BioNV is formed from modified cells or their differentiated cells by continuous extrusion.

[0098] In various embodiments, BioNV is sized from about 10 nm to 1200 nm. In various embodiments, BioNV is sized 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 various embodiments, BioNV ranges in size from about 10 nm to 100 nm, from about 100 nm to 200 nm, from about 200 nm to 500 nm, or from about 500 nm to 1200 nm.

[0099] In various embodiments, in the methods of treating, preventing, and / or ameliorating cancer described herein, the size of BioNV is adapted to the cancer type, tumor type, disease progression (e.g., metastasis, spread, pathology, etc.), and tissue type. In various embodiments, methods of treating very high density / rubbery tumor types such as retinoblastoma utilize BioNV of a lower size (e.g., less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, or less than about 50 nm) for higher permeability. In various embodiments, methods of treating loose tumor types such as lung cancer utilize BioNV of a larger size (e.g., greater than about 500 nm, greater than about 600 nm, greater than about 700 nm, greater than about 800 nm, greater than about 900 nm, or greater than about 1000 nm).

[0100] In various embodiments, BioNV substantially lacks any one protein and / or activity of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, and HLA-E or HLA-G.

[0101] In various embodiments, BioNV substantially lacks any one protein and / or activity of HLA-A, HLA-B, HLA-C, HLA-F, CIITA, IL-6, TRAC, TRBC, SerpinB9, and HLA-E or HLA-G.

[0102] In various embodiments, BioNV substantially lacks any 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 proteins and / or activities of IL-4, IL-10, and IL-16.

[0103] In various embodiments, BioNV comprises membrane-embedded α-gulphagocytic integrin, CCL2, H2-M3, FasL, MFG-E8, an anti-IL-6R antibody or antibody format, and PD-L1 (in BioNV derived from non-activated cells) and / or CTLA-4, and any one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or any two of CD24, CD47, and CD200.

[0104] In various embodiments, BioNV comprises membrane-embedded α-gulphagocytic integrin, CCL2, H2-M3, FasL, MFG-E8, an anti-IL-6R antibody or antibody format, SerpinB9, and PD-L1 (in BioNV derived from non-activated cells) and / or CTLA-4, and any one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or any two of CD24, CD47, and CD200.

[0105] In some embodiments, BioNV has a membrane-embedded CD200 protein and is substantially lacking in either CD24 or CD47 protein. In some embodiments, BioNV is substantially lacking in the proteins and / or activities of SerpinB9 and CD200.

[0106] In some embodiments, the BioNV CAR comprises an antibody or antibody format selected from one or more of monoclonal antibodies, polyclonal antibodies, antibody fragments, VNARs, VHHs, Fabs, Fab’, Fab’-SH, F(ab’)2, Fvs, single-chain Fvs (scFvs), diabodies, nanobodies, linear antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, or fusion proteins containing the antigen-binding portion of an antibody. In some embodiments, the antibody format is an scFv. In some embodiments, the CAR comprises a viral ligand.

[0107] In some embodiments, the CAR comprises a transmembrane domain derived from CD28, CD3ζ, CD4, CD8α, ICOS, or fragments and / or combinations thereof. In some embodiments, the CAR further comprises an intracellular domain comprising the intracellular signaling domain of the CD3ζ chain and / or optionally one or more costimulatory molecules selected from CD28, 4-1BB, ICOS, CD27, and OX40. In some embodiments, the CAR lacks an intracellular domain and / or a costimulatory domain.

[0108] In various embodiments, the CAR targeting is directed against cancer-specific antigens, for example, but not limited to, CAR targeting AFP (alpha-fetoprotein) in a method for treating hepatocellular carcinoma (HCC); CAR targeting mesothelin in a method for treating mesothelioma; CAR targeting ER (estrogen receptor), PR (progesterone receptor), or HER-2 / neu in a method for treating breast cancer; CAR targeting EGFR (epidermal growth factor receptor) in a method for treating non-small cell lung cancer; CAR targeting KRAS in a method for treating non-small cell lung cancer and / or colorectal cancer; CAR targeting UGT1A1 in a method for treating colorectal cancer; CAR targeting c-KIT in a method for treating blood cancer, gastric cancer, and / or skin cancer; CAR targeting CD30 in a method for treating lymphoma; CAR targeting PDGFR (platelet-derived growth factor receptor) in a method for treating gastrointestinal cancer; CAR targeting TEM8 (tumor endothelial marker 8) in a method for treating lung cancer, breast cancer, and colorectal cancer; CAR targeting EIIIB in a method for treating various solid tumors; CAR targeting CA-125 in a method for treating ovarian cancer; CAR targeting CD9 in a method for treating renal cell carcinoma; CAR targeting CD11b and / or CD18 in a method for treating various tumors; CAR targeting CD19, CD20, and / or CD21 in a method for treating lymphoma; CAR targeting CD22 in a method for treating endometrial cancer, intestinal cancer, skin cancer, and / or leukemia; CAR targeting CD24 in a method for treating B-cell cancer; CAR targeting CD30 in a method for treating lymphoma; CAR targeting CD32a in a method for treating cancer driven by resting T cells carrying a virus; CAR targeting CD41 in a method for treating ovarian cancer; CAR targeting CD42b in a method for treating lymphoma; CAR targeting CD43 in a method for treating breast cancer, colon cancer, gastric cancer, and / or lung cancer; CAR targeting CD51 and / or CD54 in a method for treating colorectal cancer; CAR targeting CD61 in a method for treating breast cancer and / or ovarian cancer;CAR targeting CD63 in a method of treating lung cancer; CAR targeting CD66a / b / c / e in a method of treating cancer metastasis; CAR targeting CLEC-2 in a method of treating hepatocellular carcinoma and / or gastric cancer; CAR targeting CD147 in a method of treating bladder cancer; CAR targeting GPVI in a method of treating cancer-induced thrombosis. In various embodiments, the CAR is targeted to a cancer-specific antigen of one or more targets in Table 1 and / or Table 2.;

[0109] In various embodiments, BioNV encapsulates a payload, such as a "lumen-filled" (i.e., a payload that BioNV can fill into the lumen, the space within the biomimetic nanovesicle) payload. In various 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 various embodiments, the payload is a therapeutic payload for the cancer type targeted by the CAR. In various embodiments, the payload comprises one or more of an alkylating agent, an anthracycline, an antimetabolite, an antitumor antibiotic, an antitumor antibody or antibody format, a corticosteroid, a plant alkaloid, a topoisomerase inhibitor, and / or a checkpoint inhibitor.;

[0110] In various embodiments, the payloads are Abecma, Abemaciclib, Abiraterone Acetate, Abraxane, ABVD, ABVE, ABVE-PC, AC, Acalabrutinib, AC-T, Actemra, Adcetris, ADE, Ado-Trastuzumab Emtansine, Adriamycin, Afatinib Dimaleate, Afinitor, Akynzeo, Aldara, Aldesleukin, Alecensa, Alectinib, Alemtuzumab, Alimta, Aliqopa, Alkeran for Injection, Alkeran Tablets, Aloxi, Alpelisib, Alunbrig, Ameluz, Amifostine, Aminolevulinic Acid Hydrochloride, Amivantamab-vmjw, Anastrozole, Apalutamide, Aprepitant, Aranesp, Aredia, Arimidex, Aromasin, Arranon, Arsenic Trioxide, Arzerra, Asciminib Hydrochloride, Asparaginase Erwinia Chrysanthemi, Asparlas, Atezolizumab, Avapritinib, Avastin, Avelumab, Axicabtagene Ciloleucel, Axitinib, Ayvakit, Azacitidine, Azedra, Balversa, Bavencio, BEACOPP, Belantamab Mafodotin-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, BrexucabtageneAutoleucel, Breyanzi, Brigatinib, Brukinsa, BuMel, Busulfan, Busulfex, Cabazitaxel, Cablivi, Cabometyx, Cabozantinib-S-Malate, CAF, Calaspargase Pegol-mknl, Calquence, Campath, Camptosar, Capecitabine, Caplacizumab-yhdp, Capmatinib Hydrochloride, 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, 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, Darolutamide, Darzalex, Darzalex Faspro, Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposome, Daurismo, Decitabine, Decitabine and Cedazuridine, DefibrotideSodium, Defitelio, Degarelix, Denileukin Diftitox, Denosumab, Dexamethasone, Dexrazoxane Hydrochloride, Dinutuximab, Docetaxel, Dostarlimab-gxly, Doxil, Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome, 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, Erwinaze, Ethyol, Etopophos, Etoposide, Etoposide Phosphate, Everolimus, Evista, Evomela, Exemestane, Exkivity, 5-FU, 5-FU, Fam-Trastuzumab Deruxtecan-nxki, Fareston, Faslodex, FEC, Fedratinib Hydrochloride, Femara, Filgrastim, Firmagon, Fludarabine Phosphate, Fluoroplex, Fluorouracil Injection, Fluorouracil-Topical, Flutamide, FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRI-CETUXIMAB, FOLFIRINOX, FOLFOX, Folotyn, FostamatinibDisodium, Fotivda, Fulphila, FU-LV, Fulvestrant, Fyarro, Gamifant, Gardasil (Recombinant HPV Quadrivalent Vaccine), Gardasil 9 (Recombinant HPV Nonavalent Vaccine), Gavreto, Gazyva, Gefitinib, Gemcitabine Hydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINE-OXALIPLATIN, Gemtuzumab Ozogamicin, Gemzar, Gilotrif, Gilteritinib Fumarate, Glasdegib Maleate, Gleevec, Gliadel Wafer, Glucarpidase, Goserelin Acetate, Granisetron, Granisetron Hydrochloride, Granix, Halaven, Hemangeol, Herceptin Hylecta, Herceptin, HPV Bivalent Vaccine, Recombinant, HPV Nonavalent Vaccine, Recombinant, HPV Quadrivalent Vaccine, Recombinant, Hycamtin, Hydrea, Hydroxyurea, Hyper-CVAD, Ibrance, Ibritumomab Tiuxetan, Ibrutinib, ICE, Iclusig, Idamycin PFS, Idarubicin Hydrochloride, Idecabtagene Vicleucel, Idelalisib, Idhifa, Ifex, Ifosfamide, Interleukin-2 (recombinant), Imatinib Mesylate, Imbruvica, Imfinzi, Imiquimod, Imlygic, Infigratinib Phosphate, Infugem, Inlyta, Inotuzumab Ozogamicin, Inqovi, Inrebic, Interferon Alfa-2b, Intron A, Iobenguane I131, Ipilimumab, Iressa, IrinotecanHydrochloride、Isatuximab-irfc、Istodax、Ivosidenib、Ixabepilone、Ixazomib Citrate、Ixempra、Jakafi、JEB、Jelmyto、Jemperli、Jevtana、Kadcyla、Kepivance、Keytruda、Kimmtrak、Kisqali、Koselugo、Kymriah、Kyprolis、Lanreotide Acetate、Lapatinib Ditosylate、Larotrectinib Sulfate、Lenalidomide、Lenvatinib Mesylate、Lenvima、Letrozole、Leucovorin Calcium、Leukeran、Leuprolide Acetate、Levulan Kerastik、Libtayo、Lisocabtagene Maraleucel、Lomustine、Loncastuximab Tesirine-lpyl、Lonsurf、Lorbrena、Lorlatinib、Lumakras、Lumoxiti、Lupron Depot、Lurbinectedin、Luspatercept-aamt、Lutathera、Lutetium(Lu 177-Dotatate)、Lynparza、Margenza、Margetuximab-cmkb、Marqibo、Matulane、Mechlorethamine Hydrochloride、Megestrol Acetate、Mekinist、Mektovi、Melphalan、Melphalan Hydrochloride、Mercaptopurine、Mesna、Mesnex、Methotrexate Sodium、Methylnaltrexone Bromide、Midostaurin、Mitomycin、Mitoxantrone Hydrochloride、Mobocertinib Succinate、Mogamulizumab-kpkc、Monjuvi、MOPP、Moxetumomab Pasudotox-tdfk、Mozobil、MVAC、Mvasi、Myleran、Mylotarg、NanoparticlePaclitaxel, Naxitamab-gqgk, Necitumumab, Nelarabine, Neratinib Maleate, Nerlynx, Netupitant, Neulasta, Neupogen, Nexavar, Nilandron, Nilotinib, Nilutamide, Ninlaro, Niraparib Tosylate Monohydrate, Nivestym, Nivolumab, Nplate, Nubeqa, Nyvepria, Obinutuzumab, Odomzo, OEPA, Ofa tumumab, OFF, Olaparib, Omacetaxine Mepesuccinate, Oncaspar, Ondansetron Hydrochloride, Onivyde, Ontak, Onureg, Opdivo, OPPA, Orgovyx, Osimertinib Mesylate, Oxaliplatin, Paclitaxel, Paclitaxel Albumin-stabilized Nanoparticle Formulation, PAD, Padcev, Palbociclib, Palifermin, Palonosetron Hydrochloride, Pamidronate Disodium, Panitumumab, Paraplatin, Pazopanib Hydrochloride, PCV, PEB, Pegaspargase, Pegfilgrastim, Peginterferon Alfa-2b, PEG-Intron, Pemazyre, Pembrolizumab, Pemetrexed Disodium, Pemigatinib, Perjeta, Pertuzumab, Pexidartinib Hydrochloride, Phesgo, Piqray, Plerixafor, Polatuzumab Vedotin-piiq, Polivy, Pomalidomide, Pomalyst, Ponatinib Hydrochloride, Portrazza, Poteligeo, Pralatrexate, Pralsetinib, Prednisone, Procarbazine Hydrochloride, Procrit, Proleukin, Prolia, Promacta, Propranolol Hydrochloride, Provenge, Purinethol, Purixan, Qinlock, Radium 223 Dichloride, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, Ravulizumab-cwvz, Reblozyl, R-CHOP, R-CVP, Recombinant Human Papillomavirus(HPV) Bivalent Vaccine, Recombinant HumanPapillomavirus(HPV)Nonavalent Vaccine、Recombinant Human Papillomavirus(HPV)Quadrivalent Vaccine、Recombinant Interferon Alfa-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、Sacituzumab Govitecan-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 V、Sterile Talc Powder、Steritalc、Stivarga、Sunitinib Malate、Sustol、Sutent、Sylatron、Sylvant、Synribo、Tabloid、Tabrecta、TAC、Tafasitamab-cxix、Tafinlar、Tagraxofusp-erzs、Tagrisso、Talazoparib Tosylate、Talimogene Laherparepvec、Talzenna、Tamoxifen Citrate、Tarceva、Targretin、Tasigna、Tavalisse、Taxotere、TazemetostatHydrobromide, Tazverik, Tebentafusp-tebn, Tecartus, Tecentriq, Temodar, Temozolomide, Temsirolimus, Tepadina, Tepmetko, Tepotinib Hydrochloride, Thalidomide, Thalomid, Thioguanine, Thiotepa, Tibsovo, Tisagenlecleucel, Tisotumab Vedotin-tftv, Tivdak, Tivozanib Hydrochloride, Tocilizumab, Tolak, Topotecan Hydrochloride, Toremifene, Torisel, Totect, TPF, Trabectedin, Trametinib Dimethyl Sulfoxide, Trastuzumab, Trastuzumab and Hyaluronidase-oysk, Treanda, Trexall, Trifluridine and Tipiracil Hydrochloride, Trisenox, Trodelvy, Truseltiq, Truxima, Tucatinib, Tukysa, Turalio, Tykerb, Ukoniq, Ultomiris, Umbralisib Tosylate, Undencyca, Unituxin, Uridine Triacetate, VAC, Valrubicin, Valstar, Vandetanib, VAMP, Varubi, Vectibix, VeIP, Velcade, Vemurafenib, Venclexta, Venetoclax, Verzenio, Vidaza, Vinblastine Sulfate, Vincristine Sulfate, Vincristine Sulfate Liposome, VinorelbineIt is selected from one or more of 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 (Bevcizumab), Ziv-Aflibercept, Zofran, Zoladex, Zoledronic Acid, Zolinza, Zometa, Zyclara, Zydelig, Zykadia, Zynlonta, and Zytiga.

[0111] In some embodiments, the payload is one or more of pidilizumab, BMS-936559, tremelimumab, AGEN1884, and / or RG2077.

[0112] In some embodiments, the nucleic acid molecule encodes one or more of CRISPR / Cas components, guide RNA (gRNA), tracer RNA (tracrRNA), microRNA (miRNA), RNA interference (RNAi), small interfering RNA (siRNA), double-stranded RNA, Piwi-interacting RNA (piRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), antisense oligonucleotide (ASO), locked nucleic acid (LNA), splice-switching oligonucleotide (SSO), tRNA, ribosomal RNA (rRNA), short hairpin (shRNA) complementary messenger RNA, repeat-associated small interfering RNA (rasiRNA), and / or small non-coding RNA.

[0113] In various embodiments, 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, archaeal 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 editors. In various embodiments, the gene editor may also include a gRNA, which refers to a guide RNA as used herein. In various embodiments, the gRNA can be a sequence complementary to a coding or non-coding sequence and can be tailored to the specific sequence to be targeted. In various embodiments, the gRNA sequence can be a sense or antisense sequence. In various embodiments, when the gene editor composition is administered herein, preferably, but not limited to, it includes two or more gRNAs. However, a single gRNA can also be used.

[0114] In various embodiments, BioNV delivers a gene editing payload that includes a trans-activation response region (TAR) loop system. In various embodiments, BioNV expresses a gene editor and encapsulates a plasmid that contains a TAR loop sequence between the 5' end of the promoter and the gene editor / guide cassette, and this TAR loop sequence functions as a barrier to block transcription. In various embodiments, transcription is induced only in cells that are infected and contain the HIV Tat protein. In various embodiments, the Tat protein binds to the TAR loop, relaxes it, liberates the promoter for transcription, thereby expressing the editor and its guide.

[0115] In various embodiments, BioNV encapsulates one or more perforin molecules. In various embodiments, BioNV encapsulates one or more granzyme molecules. In various embodiments, the granzyme molecule is selected from granzyme A, B, H, K, and M. In various embodiments, BioNV encapsulates one or more perforin molecules and / or one or more granzyme molecules derived from the cells from which BioNV is derived. In various embodiments, BioNV encapsulates one or more perforin molecules and one or more granzyme molecules that are exogenously added to BioNV. During the cell processing step (e.g., continuous extrusion), in various embodiments, the chamber between the extrusion filters can be filled with a concentration gradient of extracellular, purified perforin, granzyme, etc. that can be encapsulated in BioNV during extrusion. In various embodiments, BioNV expressing PD-L1 is derived from non-activated cells, and BioNV is substantially lacking in perforin and / or granzyme.

[0116] In various embodiments, methods of treating, preventing, and / or ameliorating cancer include co-administering whole cell therapies (e.g., T cell, NK cell, TIL, macrophage therapy). In various embodiments, supplementing whole cell therapy with BioNV can reduce the effective amount of whole cell therapy required and reduce CRS, the likelihood of teratoma, off-target effects, etc.

[0117] In various embodiments, methods of treating or preventing cancer herein include administering an additional therapeutic agent. In various embodiments, the additional therapeutic agent can be any additional anti-cancer agent, analgesic, and / or non-steroidal anti-inflammatory agent (NSAID).

[0118] In some embodiments, BioNV can be frozen at -80°C or it is suitable for storage at about -80°C and / or lyophilization (e.g., for reconstitution in buffer). In some embodiments, BioNV is stable at approximately ambient temperature, at about -20°C, at about 4°C, at about 25°C, or at 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 1 week, or at least about 1 month or more.

[0119] In some embodiments, the PD-1 inhibitor is an antibody targeted against PD-1 or pembrolizumab, nivolumab, or cemiplimab. In some embodiments, the PD-L1 or PD-L2 inhibitor is an antibody targeted against PD-L1 or PD-L2, optionally atezolizumab, avelumab, or durvalumab, or an antigen-binding domain thereof, conjugated to the surface of BioNV. In some embodiments, the PD-1 inhibitor is an antibody targeted against PD-1 or pembrolizumab, nivolumab, or cemiplimab. In some embodiments, the PD-L1 or PD-L2 inhibitor is an antibody targeted against PD-L1 or PD-L2, optionally atezolizumab, avelumab, or durvalumab, or an antigen-binding domain thereof, membrane-anchored via transmembrane domain fusion. In some embodiments, the antibody can be membrane-anchored via any transmembrane domain suitable for a CAR. In some embodiments, "conjugated" can refer to the chemical association of the antibody to the lipid layer of BioNV, for example, by covalent conjugation via a linker, and linkers and antibody-drug conjugates for antibodies are known in the art.

[0120] In various embodiments, the method of treatment comprises a BioNV having an incorporated antibody or antibody format (e.g., pembrolizumab or nivolumab). In various embodiments, "incorporated" may refer to incorporating the antibody into the lipid layer of the BioNV. In various embodiments, the antibody may be expressed on the surface of the modified cell from which the BioNV is derived. In various embodiments, the antibody is engineered to have a transmembrane domain that allows the antibody or antibody format to be surface-exposed on the cell prior to BioNV formation.

[0121] In various embodiments, the cancer is one or more of carcinoma, sarcoma, myeloma, leukemia, lymphoma, mixed cancer, and / or metastatic cancer.

[0122] In various embodiments, the cancer is acute biphenotypic leukemia, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid dendritic cell leukemia, acute myeloid leukemia, lung adenocarcinoma, adrenal tumor, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, alveolar soft part sarcoma and cardiac sarcoma, amyloidosis, anal cancer, anaplastic large cell lymphoma, angioimmunoblastic T cell lymphoma, appendiceal cancer, astrocytoma, ataxia telangiectasia, attenuated familial adenomatosis, B-cell prolymphocytic leukemia, basal cell carcinoma, Beckwith-Wiedemann syndrome, bile duct cancer, Birt-Hogg-Dube syndrome, bladder cancer, bone cancer, central nervous system cancer, brain stem glioma, brain stem glioma, brain tumor, breast cancer, bronchial adenoma / carcinoid, Burkitt lymphoma, carcinoid tumor, carcinoid tumors, Carney complex, central nervous system tumor, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, childhood desmoplastic ganglioglioma, cholangiocarcinoma, chondrosarcoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloid leukemia, chronic T-cell lymphocytic leukemia, colon cancer, colorectal cancer, Cowden syndrome, craniopharyngioma, cutaneous T-cell lymphoma, dermatofibrosarcoma protuberans, desmoplastic small round cell tumor, diffuse gastric cancer, diffuse large B-cell lymphoma, endocrine cancer, endocrine tumor, endometrial cancer, eosinophilic leukemia, epithelioma, epitheloid hemangioendothelioma (EHE), esophageal cancer, Ewing sarcoma, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, eyelid cancer, fallopian tube cancer, familial adenomatosis, 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 cancer and gynecological cancer, germ cell tumor, 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, islet cell carcinoma (pancreatic endocrine),Pancreatic cell tumor, juvenile polyposis syndrome, Kaposi's sarcoma, keratoacanthoma, renal cancer, lacrimal gland tumor, large granular lymphocyte leukemia, laryngeal cancer and hypopharyngeal cancer, leiomyomatosis and renal cell carcinoma, leiomyosarcoma, Li-Fraumeni syndrome (LFS), liposarcoma, liver cancer, lung cancer, primary cutaneous lymphoma, lymphomatoid granulomatosis, lymphoplasmacytic lymphoma, Lynch syndrome, malignant fibrous histiocytoma of bone, mantle cell lymphoma, marginal zone B-cell lymphoma, mast cell leukemia, mastocytosis, mediastinal large cell type B-cell lymphoma, medullary carcinoma, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, mixed polyposis syndrome, mucosa-associated lymphoid tissue lymphoma, Muir-Torre syndrome (MTS), multiple endocrine neoplasia syndrome, multiple endocrine neoplasia type 1, multiple endocrine neoplasia type 2, multiple myeloma, multiple myeloma / plasma cell tumor, fungating polypoid tumor, myelodysplastic syndrome, MYH-related polyposis, myxosarcoma, nasal and paranasal cavity cancer, nephroblastoma, nasopharyngeal cancer, nasopharyngeal carcinoma, neuroblastoma, neuroendocrine tumor, 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 carcinoma, anaplastic glioma, optic glioma, oral cancer and / or oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, papillary renal cell carcinoma, paranasal cavity and nasal cancer, parathyroid cancer, pelvic cancer, penile cancer, Peutz-Jeghers 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, primary neuroectodermal tumor, prostate cancer, rectal cancer, renal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, primary cutaneous sarcoma, sebaceous adenocarcinoma, Sézary syndrome, skin appendage tumor, skin cancer, small bowel cancer, small cell lung cancer, small intestine cancer, splenic marginal zone lymphoma, squamous cell carcinoma of the lung, squamous cell carcinoma, squamous cell skin cancer, stomach cancer, superficial epithelial stromal tumor, T-cell prolymphocytic leukemia, testicular cancer, thoracic and respiratory cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell carcinoma, transitional cell carcinoma adenoid cystic carcinoma, tuberous sclerosis syndrome, tubular carcinoma, Turcot syndrome, carcinoma of unknown primary origin, unclassified carcinoma, ureteral cancer, urethral cancer, uterine cancer, uterine sarcoma, uveal melanomaIt is selected from vaginal cancer, optic chiasma hypothalamic glioma, von Hippel-Lindau (VHL) syndrome, vulvar cancer, Wilms tumor, and xeroderma pigmentosum.,

[0123] In various embodiments, BioNV can target cancer cells associated with adenoid cystic carcinoma, adrenal tumor, amyloidosis, anal cancer, appendiceal cancer, astrocytoma, ataxia telangiectasia, attenuated familial adenomatous polyposis, Beckwith-Wiedemann syndrome, bile duct cancer, Birt-Hogg-Dubé syndrome, bladder cancer, bone cancer, brainstem glioma, brain tumor, breast cancer, carcinoid tumor, Carney complex, central nervous system tumor, cervical cancer, colorectal cancer, Cowden syndrome, craniopharyngioma, desmoplastic infantile ganglioglioma, endocrine tumor, epithelioma, esophageal cancer, Ewing sarcoma, 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 tumor, germ cell tumor, 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 cancer, islet cell tumor, juvenile polyposis syndrome, kidney cancer, lacrimal gland tumor, laryngeal cancer and hypopharyngeal cancer, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, B-cell prolymphocytic 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-Torre syndrome, multiple endocrine neoplasia type 1, multiple endocrine neoplasia type 2, multiple myeloma, myelodysplastic syndrome, MYH-related polyposis, nasal and paranasal cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine tumor, neurofibromatosis type 1, neurofibromatosis type 2, nevoid basal cell carcinoma syndrome, oral and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, Peutz-Jeghers syndrome, pituitary tumor, pleuropulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, alveolar soft part sarcoma and cardiac sarcoma, Kaposi sarcoma, skin cancer, small intestine cancer, gastric cancer, testicular cancer, thymoma, thyroid cancer, tuberous sclerosis syndrome, Turcot syndrome, cancer of unknown primary origin, uterine cancer, vaginal cancer, von Hippel-Lindau syndrome, Wilms tumor, or xeroderma pigmentosum.

[0124] In various embodiments, treating, preventing, and / or ameliorating cancer comprises one or more of clinical remission, reduction of tumor volume, reduction of angiogenesis, reduction of the size and number of metastases, increase of tumor infiltration, reduction of tumor volume, or improvement of cancer-related clinical symptoms within about 2 weeks, about 4 weeks, about 6 weeks, about 12 weeks, about 18 weeks, about 24 weeks, about 6 months, about 1 year, or about 2 years from administration of the composition and methods using such composition.

[0125] Dosage and Administration The dosages and dosing schedules of BioNVs disclosed herein can depend on a variety of parameters and factors including, but not limited to, the particular BioNV, the cancer being treated, the severity of the condition, whether the condition is being treated or prevented, the age, weight, and overall health of the subject, and the discretion of the administering physician. Additionally, pharmacogenomics (the effect of genotype on the pharmacokinetics, pharmacodynamics, or efficacy profile of a therapeutic agent) information regarding a particular subject can affect the dosage used. Further, the exact individual dosage can be somewhat adjusted according to various factors such as the specific combination of agents being administered, the duration of administration, the route of administration, the nature of the formulation, the rate of excretion, the disease being treated, the severity of the disorder, and the anatomical location of the disorder. Some variation in dosage can be expected.

[0126] In various embodiments, delivery of BioNVs can be similar to delivery by vesicles, particularly liposomes (see Langer, 1990, Science 249:1527-1533; Treat et al., in Liposomes in Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989)).

[0127] Methods of treating and / or preventing cancer using BioNV as described herein include, in various embodiments, dosage ranges at concentrations of the number of BioNV per kilogram (kg) of the subject's body weight. In various embodiments, suitable dosage ranges for the methods described herein are from about 10 3 BioNV / kg to about 10 12 BioNV / kg. In various embodiments, BioNV is present in the composition at a concentration of from about 10 3 BioNV / mL to about 10 14 BioNV / mL. Alternatively, in various embodiments, the BioNV composition is present in the composition as a weight / volume in the range of from about 5 ng / mL to about 500 mg / mL. In various embodiments, the dosage of BioNV is based on the size of the BioNV used for treatment; for example, 1000 nm BioNV is provided in an amount that is about 5 to 10 times less than 100 nm BioNV to obtain an equivalent dosage.

[0128] In various embodiments, the BioNV disclosed herein is administered by controlled or sustained release means or by delivery of devices well known to those of skill in the art. Examples include, but are not limited to, those described in U.S. Patent 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 can be useful for providing controlled or sustained release of one or more active ingredients, using, for example, hydroxypropylmethylcellulose, other polymeric matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, microspheres, or combinations thereof, to provide the desired release profile at various rates. The controlled or sustained release of the 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, enzyme concentration or availability, water concentration or availability, or other physiological conditions or compounds.

[0129] In various embodiments, polymeric materials are 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).

[0130] In various embodiments, the controlled release system is placed near the target area to be treated and thus requires only a fraction of the systemic dose (see, e.g., Goodson, in 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.

[0131] In various embodiments, methods of using BioNV include applying BioNV to the surface of a device (e.g., a catheter) or enclosing it within a pump, patch, or other drug delivery device. Excipients or carriers can be selected based on the mode of administration and route of administration. Suitable pharmaceutical carriers and essential ingredients for pharmaceutical formulations are described in the well-known references Remington’s Pharmaceutical Sciences (E.W. Martin) and USP / NF (United States Pharmacopeia and the National Formulary) in the art.

[0132] Pembrolizumab is a humanized anti-PD-1 monoclonal antibody used in the treatment of cancers such as melanoma, lung cancer, head and neck cancer, Hodgkin lymphoma, gastric cancer, cervical cancer, and breast cancer. It binds to PD-1 on cancer cells and blocks the interaction with lymphocyte receptors. In various embodiments, this allows the immune system to target cancer cells that can no longer avoid a cytotoxic response while preventing the immune system from attacking healthy cells. In various embodiments, pembrolizumab can be administered at a dose of 200 mg to 400 mg, or 2 mg / kg.

[0133] Nivolumab is a humanized anti-PD-1 monoclonal antibody used in the treatment of cancers such as melanoma, lung cancer, malignant pleural mesothelioma, renal cell carcinoma, colon cancer, esophageal squamous cell carcinoma, liver cancer, gastric cancer, and esophageal or gastroesophageal junction cancer. In various embodiments, nivolumab acts in the same manner as pembrolizumab. In various embodiments, nivolumab can be administered at a dose of 240 mg to 480 mg, or 3 mg / kg.

[0134] In some embodiments, BioNV can be administered at a dose that matches the dose of pembrolizumab and / or nivolumab. In some embodiments, BioNV can be administered at a dose adapted to the dose of whole cells, for example, based on the CAR concentration. In some embodiments, the typical concentration range of CAR protein per microgram of T cells is 0.20 ng to 0.70 ng, while a single BioNV may have a total number of CARs that is 1 / 5 to 1 / 10,000 of the whole cells. As a result, when the mass of BioNV is converted to CAR concentration, the CAR concentration can be assumed to be equivalent to (such as in the case of exosomes) or increased (such as in the case of BioNV) to the cells from which it is derived (e.g., T cells). In some embodiments, the concentration and / or surface density of the targeting agent (e.g., CAR) increases on BioNV compared to the whole cells from which it is derived. In some embodiments, the concentration and / or surface density of the targeting agent (e.g., CAR) is concentrated by continuous extrusion processing of whole cells. In some embodiments, the concentration and / or surface density of cell surface molecules such as the targeting agent (e.g., CAR) on BioNV increases 2-fold to 100-fold compared to whole cells.

[0135] The dosing regimen using any BioNV disclosed herein can be selected according to various factors such as the type, species, age, weight, gender, and medical condition of the cancer in the subject; the severity of the condition being treated; the route of administration; the renal or hepatic function of the subject; the individual pharmacogenomic constitution; and the specific composition of the present disclosure being used. Any BioNV disclosed herein may be administered once a day or the total daily dose may be administered in divided doses of 2, 3, or 4 times a day. Further, any BioNV disclosed herein may be administered continuously rather than intermittently throughout the dosing regimen.

[0136] In some embodiments, BioNV is administered in continuous dosing at about every 1 hour, about every 2 hours, about every 6 hours, about every 12 hours, about every 24 hours, about every 2 days, about every 4 days, about every 7 days, about every 2 weeks, about every 4 weeks, or about every 1 month.

[0137] In various embodiments, a mixed remission or clinical remission of cancer, a reduction in tumor volume, a reduction in angiogenesis, a reduction in the size and number of metastases, an increase in tumor infiltration, a reduction in primary tumor volume, or an improvement in clinical symptoms is achieved within about 24 weeks, within about 18 weeks, within about 12 weeks, within about 8 weeks, within about 6 weeks, within about 4 weeks, within about 2 weeks, or within about 1 week from the administration of the composition and the method using such composition.

[0138] The composition of BioNV In various aspects, the present disclosure relates to a composition for the treatment and / or prevention of cancer, comprising an allogeneic BioNV comprising a membrane-embedded CAR targeted to a cell surface marker, and a conjugate and / or a membrane anchor targeting agent targeted to PD-L1 or PD-L2, wherein the agent targeted to PD-L1 or PD-L2 is atezolizumab, avelumab, or durvalumab, and the CAR is not targeted to PD-1, PD-L1, or PD-L2.

[0139] In various aspects, the present disclosure relates to a composition for the treatment and / or prevention of cancer, comprising a first membrane-embedded CAR targeted to a cell surface, and at least a second membrane-embedded CAR targeted to PD-L1 or PD-L2, wherein the first CAR is not targeted to PD-1, PD-L1, or PD-L2.

[0140] In various aspects, the present disclosure relates to a composition for the treatment and / or prevention of cancer, comprising a cell surface marker of cancer cells, and a bispecific membrane-embedded CAR targeted to PD-L1 or PD-L2, wherein the cell surface marker of the cancer cells is not PD-1, PD-L1, or PD-L2.

[0141] In some embodiments, the composition comprises BioNV. In some embodiments, the composition comprises BioNV and at least an anti-cancer therapeutic agent as described herein. In some embodiments, BioNV can adsorb a therapeutic molecule on the surface of NV and / or can encapsulate a therapeutic payload within the aqueous compartment of NV. In some embodiments, the composition comprises BioNV encapsulated within an aqueous core and at least one checkpoint inhibitor. In some embodiments, the composition comprises a therapeutically effective amount of BioNV. In some embodiments, BioNV and the checkpoint inhibitor(s) can be combined in solution or can be in separate solutions for co-administration. In some embodiments, the composition comprises BioNV loaded with a cytotoxic molecule such as perforin, granzyme, etc.

[0142] In some embodiments, the composition is derived from iPSCs (or other cell types) modified to reduce the expression of immunogenic molecules and / or increase the expression of immune-protective molecules.

[0143] In various embodiments, the composition is allogeneic and / or hypoimmunogenic. For example, in various embodiments, the composition does not elicit an inflammatory response and / or an immune response when administered. In various embodiments, BioNV is allogeneic and / or hypoimmunogenic. In various embodiments, when administered to a subject, the composition, optionally BioNV in the composition, elicits, for example, compared to a counterpart of an allogeneic whole cell therapy, at most 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 less than about 1% of an inflammatory or immune response as measured by cytokines, chemokines, or immunomodulatory enzyme concentrations such as, but not limited to, 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, or any combination thereof.

[0144] In various embodiments, BioNV is present in the composition at a concentration of about 10 3 BioNV / mL to about 10 14 BioNV / mL. Alternatively, in various embodiments, the BioNV composition is present in the composition as a weight / volume in the range of about 5 ng / mL to about 500 mg / mL.

[0145] In various embodiments, the composition is substantially free of one or more bacteria, viruses, fungi, spores, mycoplasma, pyrogens, and in more particular embodiments, substantially free of all of the foregoing. In various embodiments, the composition is substantially free of whole cells and intracellular cell components including cell organelles such as nuclei, mitochondria, Golgi, and / or substantially free of non-CAR expressing NVs, and / or substantially free of ruptured and damaged NVs. In various embodiments, the composition is substantially free of cell chromatin, nucleosomes, genomic DNA, especially cell genetic material and non-therapeutic nucleic acids.

[0146] Pharmaceutical Composition and BioNV Formulation In various aspects, the composition is a pharmaceutical composition. In various embodiments, the pharmaceutical composition of the present disclosure is formulated to provide a therapeutically effective amount of BioNV as an active ingredient. In various embodiments, the pharmaceutical composition of the present disclosure is formulated to provide, as a payload within BioNV as an active ingredient, a therapeutically effective amount of one or more anti-cancer therapeutic agents. Typically, the pharmaceutical composition also includes one or more pharmaceutically acceptable excipients, carriers, such as inert solid diluents and fillers, diluents, such as sterile aqueous solutions and various organic solvents, penetration enhancers, solubilizers, and adjuvants.

[0147] Pharmaceutical excipients can be liquids such as water and oils, including, for example, those derived from petroleum, animals, plants, or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Pharmaceutical excipients can be, for example, physiological saline, acacia gum, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. In addition, adjuvants, stabilizers, thickeners, lubricants, and coloring agents may be used. Pharmaceutically acceptable excipients are generally sterile when administered to a subject. Water is a useful excipient when any of the drugs disclosed herein are administered intravenously. Aqueous solutions of physiological saline as well as dextrose and glycerol can also be used as liquid excipients, particularly for injection solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. Any composition disclosed herein can also be formulated with a wetting agent or emulsifying agent, or a pH buffering agent, if desired. Other examples of suitable pharmaceutical excipients are described in Remington’s Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995), which is incorporated herein by reference.

[0148] In various embodiments, the composition includes an excipient or a carrier. In various embodiments, the diluent is a pharmaceutically acceptable excipient or carrier.

[0149] In various embodiments, the pharmaceutical composition includes a pharmaceutically acceptable diluent. Non-limiting examples of diluents include liquid diluents such as water, ethanol, propylene glycol, glycerin, and various combinations thereof, as well as inert solid diluents such as calcium carbonate, calcium phosphate, or kaolin. In various embodiments, the diluent includes one or more of physiological saline, phosphate buffered saline, Dulbecco's Modified Eagle Medium (DMEM), Alpha Modified Minimal Essential Medium (Alpha MEM), Roswell Park Memorial Institute Medium 1640 (RPMI Media1640), HBSS, human albumin, Ringer's solution, etc., or any combination thereof.

[0150] In various embodiments, the active ingredient is typically mixed with, diluted by, or encapsulated within such a carrier in the form of, for example, capsules, tablets, sachets, paper or other containers. When the excipient functions as a diluent, the excipient can be a solid, semi-solid, or liquid material (e.g., physiological saline) and acts as a vehicle, carrier, or medium for the active ingredient. In various embodiments, the composition can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), lotions, creams, ointments, gels, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders. As is known in the art, the type of diluent can vary depending on the intended route of administration. In various embodiments, the resulting composition can include additional agents such as preservatives, cryopreservatives (e.g., DMSO), and / or lyoprotectants (e.g., polyols, salts). In various embodiments, the carrier can be or can include lipid-based or polymer-based colloids. In various embodiments, the carrier material can be formulated as a colloid in the form of liposomes, hydrogels, microparticles, nanoparticles, or block copolymer micelles. In various embodiments, the carrier material can form a capsule, and the material can be a polymer-based colloid.

[0151] In various embodiments, the pharmaceutical composition comprising BioNV includes a solubilizing agent. In various embodiments, the pharmaceutical composition comprising BioNV includes a cryoprotectant such as DMSO or glycerol, or an agent that improves thermal stability. In various embodiments, the pharmaceutical composition can be delivered using a suitable vehicle or delivery device known in the art.

[0152] In various embodiments, the composition includes a scaffold. In various embodiments, the scaffold includes a biomaterial. In a non-limiting example, the three-dimensional biomaterial includes BioNV attached to, dispersed within, or captured within the scaffold or embedded in the extracellular matrix embedded within the scaffold. In various embodiments, the biomaterial is biodegradable and / or synthetic.

[0153] In various embodiments, the scaffold comprises a biodegradable biomaterial. Non-limiting examples of biodegradable biomaterials include fibrin, collagen, elastin, gelatin, vitronectin, fibronectin, laminin, reconstituted basement membrane matrix, starch, dextran, alginate, hyaluronic acid, chitin, chitosan, agarose, sugars, hyaluronic acid, poly(lactic acid), poly(glycolic acid), polyethylene glycol, decellularized tissue, self-assembling peptides, polypeptides, glycosaminoglycans, their derivatives and mixtures. Other useful biodegradable polymers or polymer species include polydioxanone, polycarbonate, polyoxalate, poly(α-ester), polyanhydride, polyacetate, polycaprolactone, poly(orthoester), polyamino acids, polyamides, and their mixtures and copolymers, L-lactic acid and D-lactic acid stereopolymers, copolymers of bis(p - carboxyphenoxy)propanoic acid and sebacic acid, sebacic acid copolymers, caprolactone copolymers, poly(lactic acid) / poly(glycolic acid) / polyethylene glycol copolymers, polyurethanes and poly(lactic acid) copolymers, polyurethanes and poly(lactic acid) copolymers, α - amino acid copolymers, α - amino acid and caproic acid copolymers, Α - benzylglutamic acid and polyethylene glycol copolymers, succinate and poly(glycol) copolymers, polyphosphazenes, polyhydroxyalkanoates and mixtures thereof, but are not limited thereto. Binary and ternary systems are also contemplated. In various embodiments, the scaffold comprises one or more of collagen, various proteoglycans, alginate-based substrates, and chitosan. In various embodiments, the scaffold comprises one or more of hydrogels, silk, Matrigel, acellular and / or decellularized scaffolds, poly - ε - caprolactone scaffolds, absorbable scaffolds, and nanofiber hydrogel composites.

[0154] In various embodiments, the scaffold comprises a synthetic biomaterial. Non-limiting examples of synthetic biomaterials include lactone-based polyesters or copolyesters, such as polylactic acid, polycaprolactone glycolide, 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, polyvinylpyrrolidone), fluorinated polymers, such as polytetrafluoroethylene and cellulose esters.

[0155] In various embodiments, the composition can be prepared by any method well known in the pharmaceutical art and can be administered by various routes (e.g., subcutaneous, intravenous, etc.) depending on whether local or systemic treatment is desired and the area being treated. In various embodiments, administration can be local (including ophthalmic, as well as delivery to mucous membranes including intranasal, intravaginal, and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols including those by nebulizer; intratracheal, intranasal, epithelial, and transdermal), ocular, oral, or parenteral. In various embodiments, the method can include intravitreal delivery, topical administration (eye drops), subconjunctival, periocular or intravitreal injection, or introduction by balloon catheter, or an ophthalmic insert surgically placed in the conjunctival sac. In various embodiments, parenteral administration includes intravenous, arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion, or intracranial, e.g., intrathecal or intraventricular administration. In various embodiments, parenteral administration can be in the form of a single bolus dose or, for example, by continuous infusion pump.

[0156] In various embodiments, pharmaceutical compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, solutions, powders, etc. In various embodiments, methods of treating and / or preventing cancer include the use of pharmaceutical carriers, aqueous, powder or oily bases, thickeners, and the like.

[0157] In various embodiments, the pharmaceutical composition contains, as an active ingredient, the nucleic acids and vectors described herein in combination with one or more pharmaceutically acceptable carriers. In various embodiments, the term "pharmaceutically acceptable" (or "pharmacologically acceptable") refers to molecular entities and compositions that do not cause a harmful, allergic, or other undesirable response when administered, as appropriate, to animals or humans. The methods and compositions disclosed herein can be applied to a wide range of species, such as, for example, humans, non-human primates (e.g., monkeys), horses, or other livestock, dogs, cats, ferrets, or other mammals kept as pets, rats, mice, or other laboratory animals. In various embodiments, the term "pharmaceutically acceptable carrier" includes any solvent, dispersion medium, coating agent, antibacterial agent, isotonic and absorption delaying agent, buffer, excipient, binder, lubricant, gel, surfactant, etc. that can be used as a vehicle for pharmaceutically acceptable substances.

[0158] In various embodiments, the composition can be applied to the surface of a device (e.g., a catheter) or can be contained within a pump, patch, or other drug delivery device. In various embodiments, the composition can be administered alone or as a mixture in the presence of a pharmaceutically acceptable excipient or carrier (e.g., physiological saline). The excipient or carrier is selected based on the mode of administration and the route of administration. Suitable pharmaceutical carriers and essential components for pharmaceutical formulations are described in the well-known references Remington’s Pharmaceutical Sciences (E.W. Martin) and USP / NF (United States Pharmacopeia and the National Formulary) in the art.

[0159] In various embodiments, the compositions disclosed herein, such as pharmaceutical compositions, are resuspended in a physiological saline buffer (including but not limited to TBS, PBS, etc.).

[0160] The present technology includes BioNV disclosed in the formulations of various pharmaceutical compositions. In various embodiments, the BioNV disclosed herein can take the form of a solution, suspension, emulsion, drops, tablets, pills, pellets, capsules, liquid-containing capsules, powders, sustained-release formulations, emulsions, aerosols, sprays, suspensions, or any other form suitable for use.

[0161] The pharmaceutical compositions containing BioNV described herein can be conveniently provided in unit dosage forms and can be prepared by any of the methods well-known in the pharmaceutical art. Such methods generally include the step of associating a therapeutic agent with a carrier constituting one or more accessory components. Typically, the pharmaceutical composition is prepared uniformly and intimately by associating the therapeutic agent with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the dosage form of the desired formulation (e.g., wet or dry granulation, powder blending, etc., and then tableting using conventional methods known in the art).

[0162] In various embodiments, any BioNV disclosed herein is formulated according to conventional methods as a pharmaceutical composition adapted to the mode of administration disclosed herein.

[0163] Additional therapeutic agents In various embodiments, the compositions or methods described herein further comprise a therapeutically effective amount of one or more additional therapeutic agents. In various embodiments, the therapeutically effective amount of one or more additional therapeutic agents may be in a solution containing BioNV, adsorbed on the surface of NV, or a payload encapsulated within BioNV. In various embodiments, the additional therapeutic agent is one or more of a checkpoint inhibitor, an analgesic, and / or an anti-infective agent.

[0164] In various embodiments, the composition or method contemplates other additional therapeutic agents, such as, for example, an analgesic agent to assist in the treatment of inflammation or pain at the administration site, or an anti-infective agent to prevent infection at the treatment site by the composition.Non-limiting examples of additional therapeutic agents include analgesics such as non-steroidal anti-inflammatory drugs, opioid agonists and salicylates; anti-infective agents such as anthelmintics, anti-anaerobic agents, antibiotics, aminoglycoside antibiotics, antifungal antibiotics, cephalosporin antibiotics, macrolide antibiotics, other beta-lactam antibiotics, penicillin antibiotics, quinolone antibiotics, sulfonamide antibiotics, tetracycline antibiotics, anti-mycobacterial drugs, anti-tuberculosis mycobacterium drugs, anti-protozoal drugs, anti-malaria protozoal drugs, antiviral agents, anti-retroviral agents, anti-scabies drugs, anti-inflammatory agents, corticosteroid anti-inflammatory agents, antipruritic / local anesthetics, topical anti-infective agents, antifungal topical anti-infective agents, antiviral topical anti-infective agents; electrolytes and renal agents such as acidifying agents, alkalinizing agents, diuretics, carbonic anhydrase inhibitor diuretics, loop diuretics, osmotic diuretics, potassium-sparing diuretics, thiazide diuretics, electrolyte replenishers, and uric acid excretants; enzymes such as pancreatic enzymes and thrombolytic enzymes; gastrointestinal drugs such as antidiarrheals, antiemetics, gastrointestinal anti-inflammatory agents, salicylic gastrointestinal anti-inflammatory agents, antacid anti-ulcer agents, gastric acid pump inhibitor anti-ulcer agents, gastric mucosal anti-ulcer agents, H2 blocker anti-ulcer agents, gallstone dissolving agents, digestive agents, emetics, laxatives and stool softeners, and gastrointestinal motility promoters; general anesthetics such as inhaled anesthetics, halogenated inhaled anesthetics, intravenous anesthetics, barbiturate intravenous anesthetics, benzodiazepine intravenous anesthetics, and opioid agonist intravenous anesthetics; hormone regulators such as abortifacients, adrenocortical agents, corticosteroid agents, androgens, anti-androgens; immunobiological agents such as immunoglobulins, immunosuppressants, toxoids, and vaccines; local anesthetics such as amide-type local anesthetics and ester-type local 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), salicylic 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.

[0165] Additional non-limiting examples of useful therapeutic agents of the above categories include: (1) analgesics in general such as lidocaine or its derivatives, and NSAID analgesics such as 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) anti-infective agents such as mupirocin; (6) anti-anaerobic anti-infective agents such as chloramphenicol and clindamycin; (7) antifungal antibiotic anti-infective agents such as amphotericin b, clotrimazole, fluconazole, and ketoconazole; (8) macrolide antibiotic anti-infective agents such as azithromycin and erythromycin; (9) other β-lactam antibiotic anti-infective agents such as aztreonam and imipenem; (10) penicillin antibiotic anti-infective agents such as nafcillin, oxacillin, penicillin G, and penicillin V; (11) quinolone antibiotic anti-infective agents such as ciprofloxacin and norfloxacin; (12) tetracycline antibiotic anti-infective agents such as doxycycline, minocycline, and tetracycline; (13) anti-tuberculosis mycobacterium anti-infective agents such as isoniazid (INH) and rifampin; (14) anti-protozoal anti-infective agents such as atovaquone and dapsone; (15) anti-malaria protozoal anti-infective agents such as chloroquine and pyrimethamine; (16) anti-retroviral anti-infective agents such as ritonavir and zidovudine; (17) anti-viral anti-infective agents such as acyclovir, ganciclovir, interferon alpha, remdesivir, and rimantadine; (18) antifungal topical anti-infective agents such as amphotericin B, clotrimazole, miconazole, and nystatin; (19) anti-viral topical anti-infective agents such as acyclovir; (20) electrolytes and renal agents such as lactulose; (21) loop diuretics such as furosemide; (22) potassium-sparing diuretics such as triamterene; (23) thiazide diuretics such as hydrochlorothiazide (HCTZ); (24) uric acid excretants such as probenecid; (25) enzymes such as RNase and DNase; (26) anti-emetics such as prochlorperazine;(27) Salicylic acid gastrointestinal anti-inflammatory agents such as sulfasalazine; (28) Gastric acid pump inhibitor anti-ulcer agents such as omeprazole; (29) H2 blocker anti-ulcer agents such as cimetidine, famotidine, nizatidine, and ranitidine; (30) Digestants such as pancreatin; (31) Gastrointestinal motility promoters such as erythromycin; (32) Ester-type local anesthetics such as benzocaine and procaine; (33) Skeletal system corticosteroid anti-inflammatory agents such as beclomethasone, betamethasone, cortisone, dexamethasone, hydrocortisone, and prednisone; (34) Skeletal system anti-inflammatory immunosuppressive agents such as azathioprine, cyclophosphamide, and methotrexate; (35) Skeletal system non-steroidal anti-inflammatory drugs (NSAIDs) such as diclofenac, ibuprofen, ketoprofen, ketorolac, 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 are included.;

[0166] Anti-cancer BioNV In various aspects, the present disclosure includes BioNV. BioNV has a size of about 20 to 1200 nm and contains a membrane-embedded CAR that can bind to a target molecule and faces outward. Without wishing to be bound by theory, the biomimetic quality depends on the nanovesicle composition derived from the plasma membrane of allogeneic low-immunogenic modified cells. In some embodiments, BioNV includes a lipid bilayer derived from the plasma membrane and completely encloses an aqueous core that can accommodate various cell-derived molecules including perforin, granzyme, cytokines, etc. In some embodiments, the aqueous core of NV can further encapsulate therapeutic agents such as exogenous biological agents, fluorescent proteins, tracer dyes, radionuclides, and small molecules.;

[0167] To ensure the proper orientation of the CAR and eliminate BioNV lacking the CAR, in various embodiments, an HPLC-based affinity chromatography technique can be used to select and concentrate only BioNV with a sufficient surface concentration of CAR exposed to the solvent. Using an HPLC-based affinity chromatography technique, the concentration of NV having contaminating cellular material and immunogenic cell surface markers can be reduced by either positive selection or negative selection.

[0168] The CAR construct can include various structural molecules. The structure-function of a prototype CAR includes an extracellular (or outward-facing) binding portion (e.g., scFv) connected by a hinge peptide (e.g., CH2 / CH3 domain from the IgG Fc region, Gly-Gly-Ser peptide bond, CD28 peptide, CD8α peptide, etc.) to a transmembrane domain (e.g., CD28, CD3ζ, CD4, CD8α, ICOS, etc.), followed by various intracellular signaling domains (e.g., 4-1BB, CD3ζ, CD28, 4-1BB, ICOS, CD27, OX40, etc.) in a fusion protein. In various embodiments, since BioNV lacks the intracellular machinery of whole cells, intracellular signaling molecules are not required in the CAR design (primary CAR construct). In various embodiments, the CAR construct includes an extracellular scFV binding portion fused to the transmembrane domain of CD28 with an IgG CH2 / CH3 linker and substantially lacks an intracellular domain or functionality. In various embodiments, the CAR construct can replace the intracellular domain of the prototype or otherwise be fused to an anchor protein, e.g., the PLA2 domain from AAV, a fusion protein, a radionuclide binding domain, a cytoskeletal element, a small molecule transport domain, etc., which can assist in fusion to target cells and / or encapsulation and release of a therapeutic payload.

[0169] In various embodiments, the CAR antigen-binding molecule comprises various binding moieties including antibody-based or antibody-format binding domains. In various embodiments, BioNV comprises an antibody or antibody-format binding moiety selected from one or more of monoclonal antibodies, polyclonal antibodies, antibody fragments, Fab, Fab’, Fab’-SH, F(ab’)2, Fv, single-chain Fv (scFv), diabodies, nanobodies, linear antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, and fusion proteins comprising antigen-binding portions of antibodies. In various embodiments, the CAR construct comprises a bispecific T cell engager (BiTE), a variable heavy chain IgG fragment VHH, a VNAR, or a binding moiety via an engineered T cell receptor (TCR). In various embodiments, the binding moiety can be a viral epitope recognition receptor (VERR) derived from one or more oncolytic virus receptors and / or viral ligands.

[0170] In various embodiments, BioNV is formed by disrupting the cell membrane of engineered iPSCs. In various embodiments, hypo-iPSCs are characterized by a B2M− / −, CIITA− / −, CD47+ / +, PD1− / − plasma membrane profile and can be used for the generation of the present BioNV. Hypo-BioNV can be generated from a parental iPSC cell line by methods such as sonication, adaptive focused acoustic technology, French press, extrusion, continuous extrusion, cell lysis with surfactants, and electroporation. In various embodiments, continuous extrusion is the method used to generate Hypo-BioNV. Continuous extrusion of iPSCs can produce BioNV that is tgCD47+ and HLA1 / HLA2 negative (low immunogenicity), showing elimination of PD1 resistance.

[0171] In various embodiments, BioNV can analyze size uniformity by methods of determining particle size such as dynamic light scattering (DLS), flow cytometry, mass photometry, etc. In various embodiments, BioNV can filter by particle size or particle size range to optimize renal clearance and other clinically relevant NV properties. In various embodiments, BioNV is sized from about 20 nm to 1200 nm. In various embodiments, BioNV is sized 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. The size of BioNV ranges from about 10 nm to 20 nm, about 20 nm to 30 nm, about 30 nm to 40 nm, about 40 nm to 50 nm, about 50 nm to 60 nm, about 60 nm to 70 nm, about 70 nm to 80 nm, about 80 nm to 90 nm, about 90 nm to 100 nm, about 10 nm to 100 nm, about 100 nm to 200 nm, about 200 nm to 400 nm, about 400 nm to 600 nm, about 600 nm to 800 nm, about 800 nm to 1000 nm, or about 1000 nm to 1200 nm.

[0172] In some embodiments, iPSC-derived BioNVs can include NVs having only the outer plasma membrane leaflet, only the inner plasma membrane leaflet, and / or both leaflets of an intact plasma membrane lipid bilayer. In some embodiments, iPSC-derived NVs are added with additional lipid additives (e.g., phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, ceramide, lecithin, etc.), nonionic surfactants (e.g., sorbitan 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, etc.) to improve physicochemical properties such as thermal stability and encapsulation / release of therapeutic payloads. The amount of cholesterol and the length and saturation of the hydrocarbon chains of phospholipids can affect the rigidity and stability of the bilayer, and thus can affect the ability of NVs to receive and release drugs, biomolecules, and other therapeutic payloads. In some embodiments, BioNVs also incorporate zwitterionic lipids and methods of using zwitterionic lipids as described, for example, in U.S. Patent Publication US20130216607, the content of which is incorporated herein by reference in its entirety. Correspondingly, by functionalizing the hydrophilic head of the lipid with a polymer or biomolecule, new features can be provided on the vesicle surface, and interactions with blood components, tissues, and the immune system can be formed in vivo.

[0173] In some embodiments, the CAR target includes various cell surface markers including markers associated with specific cancers, and the BioNVs are cancer-specific.

[0174] In various embodiments, BioNV is configured to encapsulate various therapeutic payloads. In various embodiments, the first-generation targeted BioNV can be used to deliver a small molecule therapeutic payload. In various embodiments, second-generation (or third-generation or fourth-generation) CAR-containing BioNVs derived from activated lymphocytes can contain cytokines and other cytotoxic peptides. In various embodiments, BioNV can be formatted to encapsulate and deliver plasmid DNA, for example, to express gene editing nucleases and gRNAs in target cells. Alternatively, or additionally, in various embodiments, BioNV can encapsulate nucleases and gRNAs. In various embodiments, the targeted second-generation (or third-generation or fourth-generation) BioNV can be designed to encapsulate and deliver an additional therapeutic protein or peptide of interest.

[0175] In various embodiments, BioNV delivers a gene editing payload that includes a trans-activation response region (TAR) loop system. In various embodiments, BioNV expresses a gene editor and encapsulates a plasmid that contains a TAR loop sequence between the 5' end of the promoter and the gene editor / guide cassette, and this TAR loop sequence functions as a barrier to block transcription. In various embodiments, transcription is induced only in cells that are infected and contain the HIV Tat protein. In various embodiments, the Tat protein binds to the TAR loop, relaxes it, liberates the promoter for transcription, thereby expressing the editor and its guide.

[0176] In various embodiments, BioNV is CD34+ or is derived from CD34+ cells such as human CD34+ umbilical cord blood. CD34+ umbilical cord blood-derived cell lines can function as base cell lines for the development, production, and manufacture of BioNV for the delivery of gene editing therapeutics. CD34+ umbilical cord blood-derived hypoimmunogenic cell lines have been experimentally confirmed for low expression of HLA1 / 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).

[0177] In various embodiments, BioNV and / or a composition comprising BioNV is administered in combination with one or more additional compounds. In various embodiments, BioNV is pre-treated with one or more additional compounds, for example, prior to administration to a subject.

[0178] In various embodiments, BioNV is modular and allogeneic (off-the-shelf) due to the lack of immunogenicity derived from engineered iPSCs. In various embodiments, due to the lack of whole cell signaling components, BioNV is easily adjustable for target specificity and resistance to immunosuppressive signals. In various embodiments, BioNV lacks gene elements that contribute to cytokine storm rampage, minimizing the risk of cytokine release syndrome (CRS) in patients. In various embodiments, the amounts of active cytokines, perforin, granzyme, interferon, interleukin, etc. encapsulated within BioNV are regulated during upstream (prior to BioNV induction) cell processes. In various embodiments, BioNV is derived from cells that can pass through biological barriers and / or virus receptors known to facilitate passage.

[0179] While not wishing to be bound by theory, BioNVs generated from iPSC-engineered allogeneic-based cell lines represent immune-invisible BioNVs with the potential for multiple administrations. Neutralization via BioNV antibodies is minimized and clearance via immune cells (T cells and macrophages) is avoided. In various embodiments, BioNVs do not contain viable genetic material from cells that would cause CRS or teratomas. In various embodiments, natural T cells can be mobilized by increasing the expression of certain cytokines encapsulated within BioNVs. In various embodiments, BioNVs can be derived from modified cell types with or without barrier-penetrating ligands to further control their activity after injection.

[0180] BioNV Formation Method In various embodiments, the modified cells are hypoimmunogenic cells derived from iPSCs engineered such that the expression and / or activity of immunogenic proteins is reduced or eliminated and / or the expression of immunoprotective proteins is expressed or increased. In various embodiments, the iPSCs are reverted from a somatic cell state using microRNA technology instead of small molecule transactivators. The use of microRNAs provides a more stringent differentiation system, which results in higher quality iPSCs. While not wishing to be bound by theory, such high-quality iPSCs are less likely to undergo expression suppression (of proteins such as CD47 after genetic engineering) and genetic drift, and have excellent quality / quantity of culture splitting (can be split more times than other methods before problems occur with cell integrity).

[0181] In various embodiments, BioNVs derived from iPSC-derived hypoimmunogenic cells retain the functions of the hypoimmunogenic cells, such as, but not limited to, the ability to cross the blood-brain barrier in the case of macrophages / monocytes or tissue-specific factors in the case of cardiomyocytes, hepatocytes, etc.

[0182] In various embodiments, in allogeneic iPSCs, important proteins involved in the expression of MHC class I and MHC class II complexes, such as B2M, which is a serum protein found associated with the MHC class I heavy chain on almost all nucleated cell surfaces and is involved in peptide antigen presentation to the immune system, are knocked out, and the MHC class I and MHC class II complexes are disrupted.

[0183] In various embodiments, when B2M knockout (KO), CIITA KO, IL-6 KO, and CD47tg knock-in (KI) are incorporated into iPSCs, the TRAC and TRBC genes can be knocked out. In various embodiments, only one gene of each, rather than both on separate alleles, is knocked out. In various embodiments, the TRAC and TRBC genes can be knocked out as described herein. The purpose of knocking out the TRAC and TRBC genes is to eliminate the T cell receptor. In various embodiments, the modified cells differentiate into a subset of T cells lacking the T cell receptor for inducing BioNV. By genetically modifying the cells to substantially lack the TCR, the possibility of generating competing ligands that can non-specifically target another tissue against the CAR construct can be reduced. Thus, in various embodiments, as a strategy for reducing the off-target effects of BioNV, the TCR gene is knocked out. In various embodiments, TRAC / TRBC knockout generally reduces the likelihood of CRS and the toxicity of BioNV.

[0184] In various embodiments, the modified cells are expanded in culture after genetic manipulation. Any small-scale expansion culture method or large-scale feeder system expansion culture method known in the art can be used.

[0185] In various embodiments, after constructing B2M KO, CIITA KO, IL-6 KO, CD47tgKI, and an IL-2 promoter-driven green fluorescent protein (GFP) (IL-2p GFP) reporter, the CAR construct can be incorporated / engineered into cells. In various embodiments, the CAR construct can be knocked into the TRAC / TRBC gene and simultaneously knock out the remaining TRAC / TRBC gene to obtain cells that are CAR+ and TRAC / TRBC− / −. In various embodiments, the CAR construct can be knocked into the TRAC / TRBC locus simultaneously at both loci to obtain cells that are CAR+ / + and TRAC / TRBC− / −.

[0186] In various embodiments, when B2M KO, CIITA KO, IL-6 KO, CD47tg KI, IL-2p GFP KI, and CAR-modified cells (e.g., iPSCs) are engineered, the immune synapse (IS) quality between the CAR recognition domain and the biomarker is measured. In various embodiments, the quality of the BioNV's IS may be directly related to the efficacy in whole cell therapy.

[0187] In various embodiments, BioNV, or its derived low immunogenicity, contains nucleic acids encoding GFP (among other fluorescent proteins). In various embodiments, when B2M KO, CIITA KO, CD47tgKI, IL-6 KO, TRAC / TRBC single KO are incorporated into iPSCs, GFP molecules are incorporated into the modified cell lines. In various embodiments, this functions as a control cell line. In various embodiments, non-control cell lines (therapeutic cell lines) do not have GFP. In various embodiments, the nucleic acid encoding GFP is operably linked to a promoter derived from one or more of IL-2, perforin, granzyme, alarmin, TNF, INF, combinations thereof, and / or any other cell-specific gene or reporter gene. When lymphocytes are widely / globally activated from various stimuli, the IL-2 promoter is constitutively activated. In various embodiments, more focused activation / suppression (regulation) is used. In various embodiments, the IL-2p GFP reporter gene functions as an indicator of the degree of widespread / global activation of cells (as part of the BioNV induction process). In various embodiments, by combining GFP signal and immunoblot analysis of cytokine levels (such as perforin, granzyme, alarmin, TNF, and INF), the degree of widespread / global activation of lymphocytes upon exposure to activating antigens can be efficiently regulated. In various embodiments, GFP is used to compare the degree of activation between manufacturing lots and ensure consistency in therapeutic drug development.

[0188] In various embodiments, the hypoimmunogenic cells are CD34+ or are derived from CD34+ cells such as human CD34+ cord blood. In various embodiments, the CD34+ cord blood-derived cell line can function as a base cell line for the development, production, and manufacture of BioNV for the delivery of gene editing therapeutics. In various embodiments, the CD34+ cord blood-derived hypoimmunogenic cell line has been experimentally confirmed for low expression of HLA1 / 2 and overexpression of CD47 (Deuse.et al. “Hypoimmunogenic derivatives of induced pluripotent stem cells evade immune rejection in fully immunocompetent allogeneic recipients.” Nat.Biotechnol.Vol.37, No.3, 2019:pp.252-258.doi:10.1038 / s41587-019-0016-3).

[0189] In various embodiments, the hypoimmunogenic cells can be engineered using a plurality of hypoimmunogenic engineering techniques described, for example, by Deuse et al., Han et al., Xu et al., Harding et al., as well as Deuse's published US patent applications Nos. 20190376045, 20190376045, 20210308183, and 20210292715, Nagy's US20210161971, Strominger's US20180141992, and Poirot's published European patent application No. 3693384 (each of which is incorporated herein by reference in its entirety), (Han X, et al. “Generation of hypoimmunogenic human pluripotent stem 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).

[0190] In various embodiments, BioNV is derived from cells in which the HLA genes encoding MHC membrane glycoproteins that result in an immune response associated with the GVHD rejection response have been eliminated. The HLA gene cluster can be classified into three categories: 1) the MHC class I pathway, 2) the MHC class II pathway, and 3) the MHC class III pathway. Only the MHC class I and II pathways in GVHD express protein complexes that cause an immune response, and the MHC class III complex is not involved in immune activity.

[0191] When the MHC class protein complex is removed, NK cells and macrophages may be induced into an active clearance mode, after which the cells are destroyed. To avoid this killing mechanism, in various embodiments, by incorporating an addition of a transmembrane molecular protein tag of CD47 isoform 2 into the cell membrane of the modified cells, NK and macrophage-mediated killing responses can be avoided. For example, it is 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: pp. 6662-7. doi: 10.1073 / pnas.1121623109).In various embodiments, cells can be engineered to prevent these responses using additional mechanisms as described below: 1) a CD24 transmembrane molecular 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. “A membrane-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 to prevent T cell responses. In various embodiments, BioNVs derived from “activated” cells encapsulate and / or release perforin and / or granzyme, resulting in targeted cell death. In various embodiments, activated cells produce perforin and / or granzyme encapsulated in BioNVs. In various embodiments, hypoimmunogenic cells to be activated do not express PD-L1 to avoid the resulting BioNVs targeting PD-1 on T cells. In various embodiments, this reduces the likelihood that perforin and / or granzyme are released and unwanted T cell death occurs. In various embodiments, PD-L1 is overexpressed in BioNVs derived from non-activated cells not filled with apoptotic cytokines. In various embodiments, hypoimmunogenic cells to be activated have PD-L1 downregulated, knocked out, or silenced in other ways.In various embodiments, for non-activated low immunogenic cells, i.e., BioNVs used for gene editor delivery, PD-L1 is upregulated. In various embodiments, CD47 can be utilized for immune tolerance to innate immune cells in genetically engineered iPSCs, as, for example, in Chhabra et al., Han et al., and Jaiswal et al. (Chhabra A, et al. Sci Transl Med. “Hematopoietic stem cell transplantation in immunocompetent hosts without radiation or chemotherapy.” 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 various embodiments, the cells can be modified as described in Jaiswal et al.'s U.S. Patent No. 8,562,997, which is incorporated herein by reference in its entirety.

[0192] In various embodiments, for example, as done in Xu et al. and Han et al., instead of completely knocking out all HLA genes, only HLA genes highly relevant to the immune response are knocked out, and HLA genes that attenuate macrophage or NK responses (e.g., HLA-E, HLA-F, and HLA-G) are left intact. This approach can also be used in various embodiments. This approach does not require the addition of a CD47 tag, and the modified cells can be engineered to generate BioNVs with or without CD47.

[0193] In various embodiments, the method improves the hypoimmunogenic approach of Table 3.

Table 3

[0194] In various embodiments, the development of allogeneic modified cells involves the removal of MHC class I and MHC class II protein complexes by disruption of certain HLA genes or B2M knockout followed by CIITA gene knockout. In various embodiments, due to the rapid mechanism of action, CRISPR gene editing approaches can be used to perform the knockout. In various embodiments, the knockout is performed using zinc finger nucleases (ZFNs) and / or TALENs. In various embodiments, modified cells are generated using the Cre / Lox recombinase system. In various embodiments, modified cells are generated using RNA silencing (RNAi, shRNA, microRNA, CRISPR Cas13a-d, etc.).

[0195] In various embodiments, the method of developing allogeneic hypoimmunogenic modified cells is different from the method of creating alloreactivity by Harding et al. Instead of deleting MHC class I / II genes and risking preventing long-term acceptance by the recipient, the method of Harding et al. involves alternative approaches based on immune evasion mechanisms that occur in nature. The method relies on the biomimicry of Harding et al. based on the horizontally transmissible cancer DFTD2 type that is common in the Tasmanian devil. In various embodiments, the development of allogeneic modified cells can include the expression or increased expression of the immunomodulatory proteins CCL21, PD-L1, FasL, SerpinB9, H2-M3, CD47, CD200, and / or MFG-E8 to protect cell derivatives from long-term immune rejection responses in mice (and humans) without deleting MHC class I / II proteins. In various embodiments, the modified cells express one or more of the proteins shown in Table 4 (including any splice variants and / or isoforms of any of the proteins shown, e.g., CD200 splice variants). In various embodiments, this system can be used to interfere with the activity of APCs, macrophages, NK cells, and T lymphocytes. In various embodiments, the modified cell line can also include the safe cell system developed by Liang et al., in which a cell division gene is linked to a suicide gene to prevent the runaway of teratomas leading to 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).

[0196] In various embodiments, the method improves the hypoimmunogenic approach of Table 4.

Table 4

[0197] In various embodiments, the low-immunogenicity cells derived from BioNV are engineered to have knockout of one or more of 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 of one or more of CCL2, PD-L1 (in BioNV derived from non-activated cell sources), CTLA-4, H2-M3, CD24, CD47 (removing the 3’UTR region or having an alternative 3’UTR region without binding sites for inhibitory microRNAs), MFG-E8, CD200, and / or any combination thereof.

[0198] In various embodiments, BioNV is generated from low-immunogenicity cells having one or more of the modifications in Table 3.

Table 5

[0199] In various embodiments, the inactivation / activation of genes is controlled by inducible promoters throughout the differentiation, activation, and manufacturing processes of BioNV. In various embodiments, disruption of MHC, TCR, and CRS genes generates allogeneic iPSCs that are - / -CRS and - / -TCR and have a plasma membrane that exhibits low immunogenicity when injected into a subject. CRS genes involved in the etiology of CRS include cytokines such as IL-6, IL-10, IFN-γ, monocyte chemoattractant protein 1 (MCP-1), granulocyte macrophage colony-stimulating factor (GM-CSF), and also tumor necrosis factor (TNF), IL-1, IL-2, IL-2 receptor-α, and IL-8. In various embodiments, one or more of these genes are inactivated, for example, in cells from which BioNV is derived.

[0200] In some embodiments, BioNVs are formed by disrupting the cell membranes of engineered iPSCs. In some embodiments, hypo-iPSCs are characterized by a B2M− / −, CIITA− / −, CD47+ / +, PD1− / − plasma membrane profile and can be used for the generation of BioNVs. Low immunogenic BioNVs can be generated from parental iPSC cell lines by methods such as sonication, adaptive focused acoustics technology, French press, extrusion, continuous extrusion, cell lysis with surfactants, and electroporation. In some embodiments, continuous extrusion is the method used to generate low immunogenic BioNVs. In some embodiments, continuous extrusion of iPSCs can produce BioNVs that are tgCD47+ and HLA1 / HLA2 negative (low immunogenic) and show elimination of PD1 resistance.

[0201] In some embodiments, genetic engineering of iPSCs includes gene editing techniques such as CRISPR-based gene editing systems, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and meganucleases for generating allogeneic / hypoimmunogenic iPSCs and / or for CAR cassette integration, among other gene editing methods. In some embodiments, genetic engineering of iPSCs can refer to a decrease or loss of transcription of any genetic element. Similarly, genetic engineering of iPSCs can refer to an increase or knock-in of the expression of any genetic element, including both endogenous and exogenous genetic elements.

[0202] For example, in various embodiments, stable cell integration (safe harbor gene locus) in iPSCs can be controlled by implementing a Tet-regulated CRISPRa and a targeted 3x transcription factor-targeted gRNA system. The CRISPR activation system for three upstream transcription factors can trigger signal cascade events that promote the production of a CAR in which the ORF of an endogenous antibody is replaced at a specified locus (or loci). This system can be made "tunable" by including a Tet control promoter and can vary the concentration of the CAR on the surface of the cell. Next, stable cell replacement with the CAR cassette of the CDR as well as the heavy and light chain antibody regions can be achieved by Cpf-1-induced homologous recombination repair (HDR). Finally, the stably integrated CAR cassette can include adjacent gRNA binding sites, which allows for the repeated exchange or alteration of scFv (such as other antibody formats) or VERR / viral ligands, enabling the rapid and consistent insertion of desired sequences.

[0203] In various embodiments, the allogeneic and hypoimmunogenic properties of iPSCs can be further improved by inducing the overexpression of immunoprotective molecules. For example, without limitation, the overexpression of CD47, among other cell surface integrins, can reduce the response rate of macrophage depletion of BioNV products from the blood. In various embodiments, further, the allogeneic and hypoimmunogenic properties of iPSCs can be improved by the expression of alpha and beta phagocytic integrins. In various embodiments, the overexpression of similar immunogenic protective cell surface markers that signal to white blood cells can be implemented as a strategy to increase the half-life of BioNV after injection.

[0204] In various embodiments, the iPSCs are genetically engineered for CAR cassette integration. The CAR cassette integration can include both integrated transgenes and non-integrated transgenes. Non-limiting examples of non-integrated transgene insertion include mRNA, non-integrated lentivirus, and endonuclease-targeted methods. Integrated CAR cassette insertion methods include stable retroviral vector insertion systems and transposase-based integration systems. Stable CAR cassette transduction can be achieved, for example, using a retroviral vector that can enable the iPSCs to maintain the genetic elements encoding the CAR throughout differentiation, proliferation, and activation. In various embodiments, clinically graded stable transduction of the CAR cassette into T cells is achieved with GRV vectors in brexucabtagene autoleucel (Tecartus®, Kite Pharma Inc.) and axicabtagene ciloleucel (Yescarta®, Kite Pharma Inc.), and tisagenlecleucel (Kymriah®, Novartis International AG) was formally transduced using a lentiviral vector (Labbe, R.P., et al. “Lentiviral Vectors for T Cell Engineering: Clinical Applications, Bioprocessing and Future Perspectives.” Viruses 13(2021);1528.doi:10.3390 / v13081528).

[0205] In various embodiments, the concentration of CAR on the surface of iPSC-based cell lines or any downstream differentiated cells (and the resulting BioNV) can be regulated using various transcriptional control elements such as a tetracycline on / off promoter (or a similar drug-regulated promoter) to drive the expression of the CRISPR activation / gRNA (CRISPRa) system. The CRISPRa system can then activate antibody-regulated transcription factors, such as Drm2, Fr5, and Bxp2, which regulate the expression of the engineered CAR cassette integrated at the site of the antibody locus (where the antibody gene has been replaced). Additionally, similar transcriptional control elements can be provided at defined manufacturing stages for purposes such as controlling the overexpression of genes (e.g., CD47), driving genes that control differentiation, and the like.

[0206] Subject and / or animal In various embodiments, the subject and / or animal is a mammal, such as a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or non-human primate. In various embodiments, the subject and / or animal is a non-mammal, such as a zebrafish, for example. In various embodiments, the subject and / or animal is a transgenic animal containing fluorescent cells, such as RPE cells and / or immune cells having GFP, for example. In various embodiments, the subject and / or animal is a human. In various embodiments, the BioNV is derived from fluorescent-tagged cells and / or encapsulated with a fluorescent-tagged protein or tag (e.g., with GFP). In various embodiments, the human is a pediatric human, adult, elderly, infant, or child. In other various embodiments, the human is referred to as a patient.

[0207] In various embodiments, the treatment method includes administering to a human of an age in the range of 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.

[0208] In various embodiments, the subject is a non - human animal, and thus, the present disclosure is for veterinary use. In various embodiments, the non - human animal is a household pet. In various embodiments, the non - human animal is a livestock animal.

[0209] In various embodiments, the serum and / or immune cells and / or tumor cells are evaluated and / or affected. In various embodiments, the immune cells include cells of the innate immune system of the subject and / or the animal. In various 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 present disclosure provides for detecting the presence, absence, or measuring the amount of tumor volume, tumor cells, metastases, cDNA, or RNA in a sample derived from a subject.

[0210] Kit In various embodiments, the present disclosure provides kits that can simplify the administration of any of the agents described herein. Exemplary kits of the present disclosure include any of the agents described herein in unit dosage forms. In various embodiments, the unit dosage form is a container such as a prefilled syringe that can be sterile and contains any of the agents described herein, as well as a pharmaceutically acceptable carrier, diluent, excipient, or vehicle. In various embodiments, the kit further includes a label or printed instructions that direct the use of any of the agents described herein. In various embodiments, the kit also includes an eyelid retractor, a local anesthetic, and a cleanser for the injection site. In various embodiments, the kit further includes one or more additional agents described herein.

[0211] In various aspects, the present disclosure includes a syringe containing one or more of the compositions of the present disclosure. In various embodiments, the syringe is prefilled with a certain amount of the composition. In various embodiments, the syringe is prefilled with a composition having a volume of from about 1 mL to about 10 mL. In various embodiments, the syringe is prefilled with a composition having 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.

[0212] In various embodiments, the syringe contains a composition having a storage stability in the range of about 1 hour to about 1 week. In various embodiments, the syringe contains a composition having a storage stability 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 various embodiments, the syringe contains a composition having a storage stability 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.

[0213] In various embodiments, the storage temperature is about -80°C. In various embodiments, the storage temperature is about -20°C. In various embodiments, the storage temperature is about 4°C. In various embodiments, the storage temperature is about 21°C. In various embodiments, the kit includes lyophilized BioNV.

[0214] In one embodiment, the kit includes a container containing a composition comprising the BioNV of the present disclosure, a therapeutically effective amount of an additional therapeutic agent as described herein, and instructions for use.

Examples

[0215] Example 1: Generation of BioNV by Continuous Extrusion Biomimetic nanovesicles (BioNV) can be produced from low immunogenic cell lines as shown in the scheme illustrated in Figure 17. The following protocol outlines experiments with mini-CAR BioNV. Mini-CAR BioNV can be derived from any cell type. The following outlines BioNV derived from either activated or inactivated natural killer lymphocytes (NK cells). BioNV can be designed to target any antigen receptor as described herein.

[0216] The level of CAR expression can be measured in low immunogenic cell lines using a combination of flow cytometry and iodixanol density gradient (e.g., step 1 in Figure 3).

[0217] Differentiation of iPSC-expressing surface CAR into lymphocytes can be analyzed by identification of lymphocyte markers such as cell surface markers such as CD4 / CD8 (T cells) or CD56 / CD16 (natural killer cells) (e.g., step 2 in Figure 3). The expression profile can be determined by flow cytometry, RT-PCR, and / or CRISPR-based analysis.

[0218] Next, lymphocyte activation can be achieved in two steps over a period of two weeks at a predetermined low concentration using biomarker antigen-coated beads (e.g., step 3 of FIG. 3). This process can also measure i) quantification of F-actin accumulation at the synapse formation site, ii) distribution of pZeta at the synapse, iii) clustering of antigens through the IS position, and / or iv) polarization of soluble granules (LG) containing perforin and granzyme, using a well-established protocol to analyze the quality of the immune synapse (IS) between the CAR and the antigen-coated beads.

[0219] After lymphocyte activation, the cells are grown using an established protocol (e.g., step 4 of FIG. 3). After growth, the levels of perforin and granzyme (or other luminal payload if applicable) are analyzed for each cell population to ensure consistent concentration levels for each batch. This is achieved using a series of qPCR, immunoblotting, flow cytometry, and / or mass spectrometry. If a cell population of sufficient size can be achieved in step 3, the growth step may not be necessary.

[0220] Once the cells are activated and produce the desired therapeutic protein(s), the cells are grown, harvested, washed several times, and then placed in a buffer extrusion medium. The cells are then fully processed by continuous extrusion through each step of a polycarbonate filter system with decreasing pore sizes (e.g., step 5 of FIG. 3). In the first extrusion step of the continuous extrusion process, nuclei (including nuclear components such as nuclear pores, genomic material, and transcription factors) and mitochondria are removed. The sample is then treated with an endonuclease, e.g., BENZONASE. BENZONASE is a non-specific recombinant endonuclease that cleaves any type of DNA and RNA variant into non-functional fragments less than 8 soluble base pairs. This leads to maximal reduction of nucleic acid load per sample and on a scalable basis, without interfering with the chemical properties of the BioNV membrane. This cleavage process also removes the viscosity of the nucleic acids, allowing subsequent filling of the material and passage through the next extrusion filter set.

[0221] The continuous extrusion process avoids the removal of other organelles such as the Golgi apparatus or the ER. The membrane systems of these organelles are highly evolved such that vesicles transport (release and uptake) between the folded membranes. For example, the cis and trans faces of the Golgi apparatus have unique lipid compositions that facilitate the absorption and release of low energy barriers in vesicle transport. These components are relatively less abundant in the plasma membrane. Therefore, isolating the plasma membrane to obtain BioNV is not very advantageous. BioNV is disrupted based on pore size and spontaneously formed when passing through a polycarbonate filter in the continuous extrusion process. This process yields BioNV containing membranes with a homogeneous mixture of the lipid content and protein components of the cytoplasm, Golgi, and ER, which can significantly increase the affinity for delivery and uptake into cells and tissues compared to BioNV that has been processed to remove these organelles. These characteristics may result in better and more consistent uptake of BioNV into target cells at much lower doses than systems that do not incorporate these properties.

[0222] After the extrusion step, BioNV passes through an α-CD3 HPLC (FPLC at scale-up) column to remove a low percentage (about 0.05%) of inverted BioNV that forms spontaneously during the continuous extrusion process (e.g., step 6 in Figure 3). This is done to ensure that the resulting BioNV has a uniform orientation with respect to the membrane. Since this step is a flow-through process that captures impurities, the yield loss during this step is low. When BioNV is collected after the HPLC / FPLC step, it is examined through a standardization process.

[0223] The standardization process includes one or more of the following assays.

[0224] Homogeneity of BioNV: By using nanoparticle flow cytometry (NanoFCM), the BioNV concentration, size uniformity, density of BioNV, and / or homogeneity of the lumen components of BioNV can be confirmed.

[0225] Concentration of lumen payload: Using NanoFM technology, the type and concentration of nucleic acid / protein encapsulated in the lumen of BioNV can be determined. These data can be confirmed in parallel with one or more methods including immunoblotting, mass spectrometry, and BCA analysis to determine the nucleic acid and protein content of BioNV.

[0226] Stability of BioNV: A combination of nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), and electron microscopy (EM), combined with immunoblotting and mass spectrometry, can be used to determine the physical and biochemical characteristics of BioNV over 8 - 10 months. Data from these assays may include protein expression profiles, the degree of intact BioNV membrane / enclosure, and / or the degree of aggregation.

[0227] Integrity of the membrane: The integrity of the BioNV membrane is evaluated using a calcein release assay combined with NanoFCM to assess membrane permeability. These results may provide insights into the leakage characteristics of BioNV relative to a standardized BioNV panel.

[0228] Quality of the lumen payload: The quality of the payload encapsulated in the lumen can be determined using multiple analytical assays depending on the nature of the payload. If the delivery is nucleic acid, qPCR and / or sequencing over 8 - 10 months can be used to confirm the integrity and amount of the nucleic acid payload. In the case of protein, the protein payload can be analyzed using an analysis of BioNV components using one or more of NanoFCM, mass spectrometry, and immunoblot analysis.

[0229] Quality and surface density of CAR: The CAR surface density can be determined using NanoFCM, mass spectrometry, and / or immunoblot analysis. In BioNV, the CAR surface density is expected to be at least about 5-fold to at least about 10-fold higher compared to the total cell surface density. This may significantly enhance the targeting to antigens compared to whole cells. The CAR quality can be determined at the cell stage as described above (e.g., as in step 3). Mathematical models can be used to extrapolate cell quality data and correlate it with the data results of the efficacy test for application to BioNV.

[0230] Functionality of BioNV: BioNV can be tested for basic functionality using multiple defined standardized assays, such as in vitro cell uptake into target cells when the antigen is expressed and not expressed, and the ability to pass through high-density tissues such as the human retina model. Following these basic functionality assays that can be performed immediately after the continuous extrusion process, preclinical trials address the remaining quality and functionality characteristics of BioNV.

[0231] Following the extrusion process, mini-CAR BioNV (as well as VERR and / or viral ligand BioNV) is segmented into the following groups based on activated immune synapse (IS) binding: 1) Low-quality IS pool of whole cells (activated) (where "low-quality" refers to the degree of IS binding), 2) Medium-quality IS pool of whole cells (activated) (where "medium-quality" refers to the degree of IS binding relative to "low" and "high" quality), 3) High-quality IS pool of whole cells (activated) (where "high-quality" refers to the degree of IS binding), 4) High-quality IS pool of whole cells (non-activated). BioNV can be cryopreserved at this stage.

[0232] Use statistical analysis to evaluate and address batch - to - batch consistency. Control upper and lower limits are based on data using a control (e.g., mean value, 95% confidence interval, range, and / or percentile distribution).

[0233] Example 2: In Vitro Evaluation of BioNV The experiments are performed in triplicate using each of low - quality, medium - quality, and high - quality activated mini - CAR NK BioNV and high - quality mini - CAR NK non - activated BioNV. The experiments are performed in combination with whole - cell CAR NK.

[0234] Cytotoxicity: mini - CAR NK BioNV (and whole - cell mini - CAR NK cells) are evaluated for cell cytotoxicity using a chromium - 51 ( 51 Cr) release assay with a plurality of cell lines derived from various cancers along with a control cell line. The target cells are 51 cultured with 51 Cr, which is incorporated into the cells. After treating the cancer cell lines (and / or whole - cells) with BioNV, 51 Cr is then released from the target cells during lysis. The supernatant can be collected and centrifuged to clarify the supernatant, and the amount of 51 Cr can be measured as a surrogate for cell lysis.

[0235] Using nanoparticle flow cytometry (NanoFCM), ELISA, and / or immunoblot analysis, monitor the concentration and presence of therapeutic payloads filled in the lumen, such as perforin, granzyme, p53, alarmin, TNF, and / or INF. The concentration and / or presence of these proteins can also be evaluated during the cell killing assay. Using PCR, monitor the activation status of apoptosis-related genes such as BAD, BAX, and caspase-3 during the treatment process. Measure the uptake of each subset of mini-CAR NK BioNV into cells by rhodamine staining. Use control treatment with "high-quality" inactivated mini-CAR NK BioNV for comparison.

[0236] Perform the evaluation in one or more of the following cell lines.

[0237] Lung cancer cell lines: A549: A widely used non-small cell lung cancer (NSCLC) cell line derived from lung cancer.

[0238] H1975: An NSCLC cell line derived from a patient with acquired resistance to gefitinib, an epidermal growth factor receptor (EGFR) inhibitor.

[0239] H1650: An NSCLC cell line with an EGFR exon 19 deletion mutation related to sensitivity to EGFR tyrosine kinase inhibitor (TKI).

[0240] HCC827: An NSCLC cell line carrying an EGFR exon 19 deletion mutation and highly sensitive to EGFR TKI.

[0241] H1299: A p53-null NSCLC cell line frequently used in lung cancer research.

[0242] Calu-3: A lung adenocarcinoma cell line derived from a metastatic site.

[0243] PC-9: An NSCLC cell line derived from a patient with lung adenocarcinoma having an EGFR exon 19 deletion. PC-9 cells are used to investigate EGFR targeted therapy.

[0244] Hepatocellular carcinoma cell lines: HepG2: A liver cancer cell line.

[0245] Huh7: A hepatocellular carcinoma cell line.

[0246] PLC / PRF / 5: A liver cancer cell line for investigating virus-related HCC.

[0247] Hep3B: A hepatocellular carcinoma cell line lacking functional p53.

[0248] SK-Hep-1: A hepatocellular carcinoma cell line derived from the lymph node metastasis site of a liver cancer patient.

[0249] Breast cancer cell lines: MCF-7: Hormone receptor positive (estrogen receptor positive and progesterone receptor positive).

[0250] T47D: A hormone receptor positive breast cancer cell line derived from the metastasis site of an invasive ductal carcinoma patient.

[0251] MDA-MB-231: A triple-negative breast cancer cell line lacking the expression of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2).

[0252] BT-474: A breast cancer cell line overexpressing the HER2 / neu receptor.

[0253] SK-BR-3: A HER2 positive breast cancer cell line derived from the pleural effusion of a metastatic breast adenocarcinoma patient.

[0254] ZR-75-1: An estrogen receptor positive breast cancer cell line derived from the metastasis site of an invasive ductal carcinoma patient.

[0255] Brain cancer cell lines: U87MG: A glioblastoma cell line.

[0256] U251: A glioma cell line derived from human glioblastoma tumors.

[0257] LN-18: A glioma cell line derived from human glioblastoma used in the study of glioma invasion.

[0258] T98G, SF268, D54, A172: Glioblastoma cell lines derived from human glioblastoma tumors.

[0259] Renal cell carcinoma cell lines: 786-O: A renal cell carcinoma cell line derived from the primary renal tumor of a clear cell carcinoma patient.

[0260] ACHN: A renal cell carcinoma cell line derived from human renal adenocarcinoma.

[0261] Caki-1, 769-P: Renal cell carcinoma cell lines derived from human clear cell carcinoma of the kidney.

[0262] A498: A renal cell carcinoma cell line derived from human renal cell carcinoma with a clear cell histotype.

[0263] RCC-MF: (Renal cell carcinoma metastasis to the calf) A renal cell carcinoma cell line derived from metastatic lesions in the calf of a renal cell carcinoma patient.

[0264] Lymphoma cell lines: Raji, Daudi: Burkitt lymphoma cell lines that serve as models for malignant B-cell lymphoma.

[0265] Jurkat: A T-cell lymphoma cell line derived from a patient with acute T-cell leukemia.

[0266] SU-DHL-4: A diffuse large B-cell lymphoma (DLBCL) cell line.

[0267] HUT78: A cutaneous T-cell lymphoma (CTCL) cell line derived from a patient with Sézary syndrome.

[0268] OCI-LY10: A DLBCL cell line derived from DLBCL patients.

[0269] Toledo: A mantle cell lymphoma cell line derived from mantle cell lymphoma patients.

[0270] Leukemia cell lines: K-562: A CML cell line derived from a patient with chronic myelogenous leukemia (CML) in the acute transformation phase.

[0271] HL-60: A promyelocytic leukemia cell line derived from patients with acute promyelocytic leukemia.

[0272] THP-1: A monocytic leukemia cell line derived from patients with acute monocytic leukemia.

[0273] Jurkat: A T cell leukemia cell line derived from patients with acute T cell leukemia.

[0274] MV4-11: A myelomonocytic leukemia cell line derived from AML patients.

[0275] NALM-6: A B cell precursor leukemia cell line derived from patients with B cell acute lymphoblastic leukemia (ALL).

[0276] REH: A B cell precursor leukemia cell line derived from patients with B cell ALL.

[0277] Melanoma cell lines: A375, SK-MEL-28, SK-MEL-2, WM-266-4, UACC-62: Melanoma cell lines derived from metastatic sites of melanoma patients.

[0278] M14, MeWo: Melanoma cell lines derived from primary tumors of melanoma patients.

[0279] Example 3: In vivo evaluation of mini-CAR NK cells and BioNV efficacy The experiment is conducted with mini-CAR NK whole cells and BioNV against GPC3. GPC3 CAR NK cells are used to establish a standard curve for immune synapse (IS) quality.

[0280] Mouse models: One or more of the following mouse models are used to test BioNV and / or whole cells: hGPC3TG-NSG: hepatocellular carcinoma (HCC) that expresses 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: patient-derived xenograft mice with transplanted hepatocellular carcinoma (HCC); and severe combined immunodeficiency (SCID) mice. The mice may have a C57BL / 6J background.

[0281] Tumor transplantation and visualization: When establishing in vivo tumors, tumor cells are fluorescently labeled before administration to enable visualization and measurement using an in vivo animal imaging device, such as a dual positron emission tomography (PET) scan and computed tomography (CT) scan (PET / CT), e.g., IVIS known dose, high-resolution micro-CT (Perkin Elmer). The tumor cells are transduced with a retrovirus encoding one or more luciferase genes, such as the RLuc luciferase gene (Renilla reniformis), FLuc, and teLuc, under puromycin selection. To evaluate the efficacy and persistence of GPC3 CAR NK in vivo, several subsets, including GPC3 CAR NK with "low", "medium", and "high" IS quality (e.g., as described above), are administered, and the cells are transduced with plasmid eGFP (high-sensitivity green fluorescent protein). 5×10 6 fluorescently labeled tumor cells suspended in a cancer cell suspension matrix, e.g., MATRIGEL (Becton Dickinson), are injected intraperitoneally (IP) into 8- to 10-week-old mice (e.g., hGPC3TG-NSG, HCC NSG, and / or HCC PDX mice). Approximately 5 to 10 days later, when a palpable tumor becomes detectable (about 50 mm2 ) Administer 5 × 10 6 individual GPC3 CAR NK and control GCP3-CAR-NK cells via IP injection. For in vivo imaging of GPC3 CAR NK expressing eGFP and tumor cells expressing the luciferase gene, IP inject mice with D-luciferin (about 150 mg / kg, substrate for eGFP-FLuc) and coelenterazine (about 1 mg / kg, substrate for RLuc), respectively. Since eGFP and RLuc utilize different substrates, these can be combined as dual reporters to monitor both tumors and GPC3 CAR NK.

[0282] Visualize and measure tumors in hGPC3TG-NSG, HCC NSG, and / or HCC PDX mouse models using PET / CT, e.g., 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).

[0283] Tumor permeability: Evaluate the tumor penetration characteristics of each IS quality of mini-CAR NK GPC3 BioNV at various stages of tumor growth and compare with total cell infiltration of the tumor. The permeability of BioNV tumors can also be compared with other systems such as oncolytic viruses, AAV, and / or exosomes. Mini-CAR NK BioNV (and cells) derived from the three sets described, as well as from activated and non-activated total cells, are tested by tumor cross-section and / or immunostaining over various stages of tumor growth and over time.

[0284] In vivo antitumor activity: Evaluate the homing, persistence, and antitumor activity of each quality of mini-CAR NK BioNV (alone and in combination with whole-cell CAR NK) in chip-based tumor models and animal models. Use severe combined immunodeficiency (SCID) and patient-derived xenograft (PDX) mouse models together with an in vivo imaging system (e.g., PET / CT and / or IVIS, Perkin Elmer). Measure efficacy in a dose-dependent response manner over time using one or more of tumor volume, tumor weight, and tumor inhibition. Monitor each tumor for the release of mini-CAR therapeutics cargos such as perforin, granzyme, and p53. Evaluate apoptosis gene activity such as BAD, BAX, and caspase-3 during the course of treatment using PCR, ELISA, and / or immunoblotting.

[0285] Example 4: In vitro and in vivo evaluation of CRS-related toxicity Cytokine activity: Evaluate the degree of in vitro cytokine activity in one or more of the cell lines outlined herein with each quality of mini-CAR NK GCP3 BioNV and CAR NK GCP3 and one or more control CAR cell lines (e.g., CAR-T-T1E, CAR-T cells having a CAR targeting pan-ErbB). Co-culture cancer cell lines with CD8 T cells. Add mini-CAR NK GCP3 BioNV and CAR NK GCP3 cells at increasing concentrations and assay the degree of cytokine activity over time. Measure the levels of the molecules IL-6, IL-2, and / or INF-γ via PCR, immunoblot, ELISA, and / or flow cytometry. Furthermore, evaluate the cytotoxicity of the cells as described above, 51 by a Cr release assay.

[0286] Decrease in CRS: Using in vivo models, measure the degree of cytokine release syndrome (CRS) caused by each quality of mini-CAR NK GCP3 BioNV, CAR NK GCP3 cells, and their combination therapies compared to the standardized CRS cause, CAR-T-T1E cells, and CAR-EXO-T1E BioNV. The degree of CRS (in vivo) is assayed by measuring the serum levels of various cytokines (molecular IL-6, IL-2, and / or INF-γ) and comparing them to the degree of cytokine release by whole cell controls. Increase the concentrations of mini-CAR NK GCP3 BioNV, CAR NK GCP3 cells, and the control in a dose-dependent manner until CRS induction is observed.

[0287] Lymphocyte mobilization and PD-L1 blockade: Mini-CAR BioNV targeting checkpoint inhibitors (CPI) such as PDL1 or CTLA4 (e.g., via single-specific or bispecific CAR constructs or transmembrane antibodies) are assayed for BioNV uptake and cell / tumor killing as described above. BioNV with anti-CPI CARs are also for natural lymphocyte mobilization and / or lymphocyte activation. Evaluate lymphocyte mobilization and / or activity in the tumor microenvironment (TME) via flow cytometry and / or measurement of cytokine concentrations related to lymphocyte migration via PCR, ELIA, immunoblot, and / or NanoFCM.

[0288] Animal models: Use one or more of the following animal models: SCID (CB17) or SCID (Beige) models: Use SCID mice with a CB17 or Beige background. These mice have a non-functional immune system.

[0289] NOD-SCID models: NOD-SCID (non-obese diabetic SCID) mice have combined immunodeficiency and are commonly used in xenotransplantation tests. They allow for the engraftment of human lung cancer cells or tumor tissues.

[0290] NSG (NOD-SCID IL-2Rγ null) model: NSG mice are highly immunodeficient and lack the IL-2Rγ receptor, making them permissive for human cell engraftment.

[0291] Rag2- / -γc- / - model: Rag2- / -γc- / - mice are double knockout mice lacking the Rag2 gene and the common gamma chain (γc) gene.

[0292] NOG (NOD / Shi-scid / IL2Rγ null) model: NOG mice are highly immunodeficient and lack both T cells and B cells.

[0293] The following xenograft models (or tumor models adapted to chips - "tumors on chips") can be used to test the effect of mini-CAR BioNV in vivo (alone or as co-therapy with whole cell CAR therapy).

[0294] Lung cancer tumors: A549 xenograft model: A human lung adenocarcinoma cell line used to establish a xenograft model in mice.

[0295] H460 xenograft model: A human lung cancer cell line used to establish a xenograft model in mice.

[0296] HCC827 xenograft model: A human lung adenocarcinoma cell line carrying a mutation in the epidermal growth factor receptor (EGFR).

[0297] NCI-H1975 xenograft model: A human lung adenocarcinoma cell line carrying mutations in both EGFR and T790M related to resistance to EGFR targeted therapy.

[0298] Patient-derived xenograft (PDX) model: The PDX model involves directly transplanting patient-derived lung cancer tissue into mice. This model retains the characteristics and heterogeneity of the original patient's tumor.

[0299] Hepatocellular carcinoma tumors: HepG2 and Huh7 xenograft models: Human hepatocellular carcinoma cell lines used to establish xenograft models in mice.

[0300] PLC / PRF / 5 xenograft model: A human hepatocellular carcinoma cell line derived from a liver cancer patient.

[0301] SK-HEP-1 xenograft model: A human hepatocellular carcinoma cell line commonly used in biological research and investigation of treatment approaches for HCC.

[0302] Patient-derived xenograft (PDX) model: The PDX model involves directly transplanting patient-derived HCC tissue into mice. This model retains the characteristics and heterogeneity of the original patient's tumor.

[0303] FRG (Fah- / -Rag2- / -Il2rg- / -) model: FRG mice are triple knockout mice lacking the fumarate acetohydrolase (Fah) gene, Rag2 gene, and Il2rg gene. These mice have liver impairment and are highly permissive for the engraftment of human HCC cells.

[0304] Breast cancer tumors: MCF-7, MDA-MB-231, and 4T1 xenograft models: Human breast cancer cell lines used in xenograft models. Cells can be transplanted into the mammary fat pads of nude mice, where they proliferate and form tumors.

[0305] Brain cancer tumors: U87MG U251 xenograft model: Human glioblastoma multiforme, a malignant form of brain cancer.

[0306] GL261 xenograft model: Mouse glioblastoma used as a syngeneic mouse xenograft model.

[0307] D283 Med xenograft model: A pediatric brain tumor called medulloblastoma.

[0308] BT-474 Xenograft Model: Originally derived from breast cancer patients, the BT-474 cell line is also used as a xenograft model for brain metastasis to colonize the brain.

[0309] D283 Med: Pediatric medulloblastoma.

[0310] Patient-Derived Xenograft (PDX) Model: The PDX model involves directly transplanting patient-derived lung cancer tissue into mice. This model retains the characteristics and heterogeneity of the original patient's tumor.

[0311] Renal cell carcinoma: Renca Xenograft Model: A mouse renal adenocarcinoma cell (RCC) line derived from BALB / c mice.

[0312] ACHN, Caki-1, 786-O Xenograft Models: Human RCC cell lines.

[0313] OS-RC-2 Xenograft Model: A human RCC cell line derived from a clear cell RCC patient.

[0314] Lymphoma tumors: Raji and Ramos Xenograft Models: Derived from Burkitt lymphoma patients used in the study of B cell lymphoma.

[0315] SU-DHL-4 Xenograft Model: A diffuse large B cell lymphoma (DLBCL) cell line.

[0316] Granta-519 Xenograft Model: An MCL cell line for the growth, invasion, and response to treatment of mantle cell lymphoma (MCL) tumors.

[0317] Pfeiffer Xenograft Model: Derived from primary effusion lymphoma (PEL) patients with unique characteristics such as being associated with human herpesvirus 8 (HHV-8).

[0318] Leukemia tumors: Subcutaneous xenograft model: Human leukemia cells are subcutaneously injected into immunodeficient mice, typically SCID, NOD-SCID, NSG, or NOG mice.

[0319] Orthotopic xenograft model: The orthotopic model involves injecting leukemia cells into anatomically relevant sites such as the bone marrow or spleen of immunodeficient mice.

[0320] Intravenous xenograft model: Human leukemia cells are directly injected into the bloodstream of immunodeficient mice.

[0321] Patient-derived xenograft (PDX) model: The PDX model involves transplanting patient-derived leukemia cells into immunodeficient mice. These models recapitulate the heterogeneity and characteristics of human leukemia in mice.

[0322] Genetically engineered model: In addition to directly transplanting human leukemia cells, genetically engineered mouse models (GEMMs) can be used to develop leukemia.

[0323] Melanoma tumors: Subcutaneous xenograft model: Human melanoma cells are subcutaneously injected into immunodeficient mice, typically SCID, NOD-SCID, NSG, or NOG mice.

[0324] Orthotopic xenograft model: Injection of melanoma cells into anatomically relevant sites such as the skin or dermis of immunodeficient mice. This model mimics the microenvironment of melanoma and shows tumor infiltration, angiogenesis, and interaction with surrounding tissues.

[0325] Metastatic xenograft model: Melanoma cells are injected directly into the bloodstream or via organ-specific injections such as intravenous or splenic injection to promote metastatic spread.

[0326] Patient-derived xenograft (PDX) model: The PDX model involves transplanting patient-derived melanoma cells or tumor cell fragments into immunodeficient mice.

[0327] Genetic manipulation model: Using a genetically engineered mouse model (GEMM), specific genetic changes, such as mutations in genes such as BRAF and / or NRAS, can be activated to develop melanoma and initiate melanoma formation.

[0328] Cell lines can be used in combination or as controls. For example, in mini-CAR EGFR BioNV therapy experiments targeting lung cancer, EGFR+ve models and EGFR-ve models are used to test specificity.

[0329] Any cell line and / or animal model described herein can be used for the evaluation of BioNV and cell lines used to generate BioNV, as well as the evaluation of their controls.

[0330] Definitions The following definitions are used in connection with the present disclosure. Unless otherwise defined, 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 pertains.

[0331] "Effective amount" or "therapeutically effective amount" is an amount effective to treat, prevent, or ameliorate cancer, such as those described herein, or an amount intended to reduce the number of malignant cells, reduce the primary tumor volume, decrease angiogenesis, reduce the size and / or number of metastases, or improve cancer-related symptoms in a subject.

[0332] As used herein, "a", "an", or "the" may mean one or more than one.

[0333] As used herein, the term "comprising" and variations thereof are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other similar items that may be useful in the materials, compositions, devices, and methods of this technology. Similarly, the terms "can" and "may" and variations thereof are intended to be non-limiting, such that recitation that certain elements or features in some embodiments can or may be included is not to the exclusion of other embodiments of this technology that do not include those elements or features.

[0334] As used herein, for purposes of description and claims of this disclosure, the open-ended term "comprising" is used as a synonym for terms such as "including," "containing," or "having," although this disclosure or embodiments thereof may alternatively be described using alternative terms, such as "consisting of" or "consisting essentially of."

[0335] In various embodiments, "BioNV" refers to allogeneic, low immunogenic, and biomimetic nanovesicles (NVs) that include at least one surface-oriented membrane-embedded CAR. In various embodiments, the "nanovesicles (NVs)" referred to herein are lipid-bound vesicles sized from about 10 nm to about 1200 nm that encapsulate an aqueous core. In various embodiments, the lipid-bound NVs can be formed using a lipid monolayer, lipid bilayer, or can maintain a multi-layered morphology. In various embodiments, BioNV refers to biologically-derived nanosized vesicles that can have engineered biological functionality. In various embodiments, BioNV is "biomimetic" in that it is derived from endogenous cellular material and more specifically substantially reproduces the plasma membrane material found within cells. In various embodiments, the cells from which BioNV is derived can include any type of stem cell, including cell types differentiated from such stem cells. In various embodiments, BioNV substantially excludes encapsulated cellular debris that includes cellular genomic nucleic acids, organelles, or organelle parts. In various embodiments, BioNV is characterized by having one or more, two or more, three or more, four or more, five or more, or six or more of the following. a. Being sized from about 10 nm to about 1200 nm; b. Having a total volume of about 500 nm 3 to about 5 μm 3 assuming a spherical shape; c. Having a content of at least one phospholipid and cholesterol; d. The surface membrane has 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-phagocyte integrin, and / or an anti-6R antibody or antibody format, or a chimera of any one or more of them; the surface membrane is substantially lacking in T cell receptor components (TRAC and / or TRBC), MHC class I components, and / or MHC class II components, lacking one or more proteins 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 one or more proteins of IL-4, IL-6, IL-10, and / or IL-16 inside the vesicles; e. May contain an antibody or antibody format selected from one or more of monoclonal antibodies, polyclonal antibodies, antibody fragments, 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, fusion protein containing the antigen-binding portion of an antibody, bispecific T cell engager (BiTE), VERR / viral ligand, or a membrane-embedded CAR containing a target-binding portion via a T cell receptor (TCR), wherein the CAR can target a single biomarker, multiple biomarkers, or multiple portions of a single biomarker; H H or V NAR or a membrane-embedded CAR containing a target-binding portion via a T cell receptor (TCR), wherein the CAR can target a single biomarker, multiple biomarkers, or multiple portions of a single biomarker; f. Can adsorb and / or encapsulate one or more therapeutic payloads such as perforin, granzyme, cytokine, cytotoxic protein, checkpoint inhibitor, gene editing payload, antibody or antibody fragment, fusion protein, small molecule, biological agent, radionuclide, tracer, dye, fluorescent protein, and / or any combination thereof; and g. being able to not cause a harmful immune response in a subject.

[0336] In various embodiments, "induced pluripotent stem cell" or "iPSC" refers to a stem cell that can be generated directly from an adult cell. An iPSC can arise from a differentiated cell that is reprogrammed back to an embryonic-like pluripotent state. An iPSC can generally proliferate indefinitely and become any cell type of the organism from which it is derived.

[0337] In various embodiments, as used herein, "allogeneic" refers to biological materials, tissues, or cells that are not genetically similar and are originally immunologically incompatible, even though they are derived from the same species. For example, an allogeneic BioNV is a material derived from a first subject (iPSC donor) and can be provided to any number of different subjects that are not genetically identical.

[0338] In various embodiments, as used herein with respect to a modified cell and / or BioNV, "low immunogenicity" or "low immune" refers to a reduction in the ability to generate an immune response. In various embodiments, iPSCs and BioNVs can be hypoimmunogenic because they have reduced or absent expression and / or activity of one or more immunogenic cell surface proteins and / or secreted proteins, such as TCR proteins, CRS proteins, MHC class I or MHC class II proteins, etc. In various embodiments, iPSCs and BioNVs can be hypoimmunogenic by virtue of the expression of one or more immunoprotective cell surface proteins such as CD47, CD34, CD24, CD200, alpha - macrophage integrin. In various embodiments, BioNV can be hypoimmunogenic by not inducing CRS in a subject and / or not inducing HLA incompatibility.

[0339] In various embodiments, "knockout," "silencing," "inactivation," "disruption," or "blockade," and equivalents thereof, with respect to transcription, gene expression, or protein expression, refer to a decrease in the amount of transcription, gene, or protein expression, or a lessening relative to the normal state, in a particular subset of cells. This decrease can be significant such that gene expression does not occur at all or occurs at only a very low level.

[0340] In various embodiments, as used herein, "overexpression" refers to an increase in the amount of transcription, or gene or protein expression, or a greater amount relative to the normal state, in a particular subset of cells.

[0341] Equivalents One of ordinary skill in the art will recognize or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments specifically described herein. Such equivalents are intended to be encompassed by the following claims.

[0342] As used herein, all headings are for organizational purposes only and are not intended to limit the disclosure in any way. The content of any individual item may be equally applicable to all items.

[0343] Incorporation by reference All patents and publications referred to in this specification are hereby incorporated by reference in their entirety, including the published PCT application WO2020 / 227369 entitled "Tailored Hypoimmune Nanovesicle Delivery Systems for Cancer Tumors", filed on May 6, 2020, and the published US non-provisional application 20220040106A1 entitled "Tailored Hypoimmune Nanovesicular Delivery Systems for Cancer Tumors, Hereditary and Infectious Diseases.", filed on August 3, 2021.

Claims

1. A method for treating or preventing cancer, the method comprising administering to a subject in need of treatment or prevention of cancer (i) a therapeutically effective amount of biomimetic nanovesicles (BioNVs) comprising (a) a membrane-embedded chimeric antigen receptor (CAR) targeted to a cell surface marker, and (b) a PD-1, PD-L1, and / or PD-L2 inhibitor, or comprising administering said BioNVs, or (ii) administering a therapeutically effective amount of BioNVs comprising a membrane-embedded CAR targeted to a cell surface marker, wherein said 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 of treatment or prevention of cancer (i) a therapeutically effective amount of biomimetic nanovesicles (BioNVs) comprising (a) a membrane-embedded chimeric antigen receptor (CAR) targeted to a cell surface marker, and (b) a conjugate and / or membrane-anchored PD-L1 and / or PD-L2 inhibitor, or said BioNVs, or (ii) a therapeutically effective amount of BioNVs comprising (a) a membrane-embedded bispecific CAR targeted to a first cell surface marker and a second cell surface marker, and (b) a conjugate and / or membrane-anchored PD-L1 and / or PD-L2 inhibitor, said BioNVs, comprising administering.

3. A method for treating or preventing cancer, the method comprising administering to a subject in need of treatment or prevention of cancer (i) a therapeutically effective amount of biomimetic nanovesicles (BioNVs) comprising a bispecific chimeric antigen receptor (CAR) targeted to a first cell surface marker and either PD-L1 or PD-L2, wherein said first cell surface marker is not PD-1, PD-L1, or PD-L2, or (ii) a therapeutically effective amount of BioNVs comprising (a) a bispecific CAR targeted to said first cell surface marker and either PD-L1 or PD-L2, wherein said first cell surface marker is not PD-1, PD-L1, or PD-L2, said bispecific CAR, and (b) a PD-1, PD-L1, and / or PD-L2 inhibitor or comprising administering said BioNVs. (iii) administering a therapeutically effective amount of BioNV comprising either PD-L1 or PD-L2, and a bispecific CAR targeted to a first cell surface marker, wherein the subject has been treated with a PD-1, PD-L1, and / or PD-L2 inhibitor, and the first cell surface marker is not PD-1, PD-L1, or PD-L2, the method. **Claim 4** A method of treating or preventing cancer, the method comprising administering to a subject in need of treatment or prevention of cancer a therapeutically effective amount of a biomimetic nanovesicle (BioNV), the BioNV comprising (a) a bispecific chimeric antigen receptor (CAR) targeted to a first cell surface marker and a second cell surface marker, and (b) a PD-1, PD-L1, and / or PD-L2 inhibitor the method. **Claim 5** A method of treating or preventing cancer, the method comprising administering to a subject in need of treatment or prevention of cancer a therapeutically effective amount of a biomimetic nanovesicle (BioNV) comprising a bispecific chimeric antigen receptor (CAR) targeted to a first cell surface marker and a second cell surface marker, wherein the subject has been treated with a PD-1, PD-L1, and / or PD-L2 inhibitor, the method. **Claim 6** The method according to 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. **Claim 7** The method according to claim 3, wherein the first cell surface marker is selected from Table 1 and / or Table 2. **Claim 8** The method according to 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. **Claim 9** The method according to claim 8, wherein the first cell is a cancer cell and the second cell is an immune cell. **Claim 10** The method according to any one of the preceding claims, wherein the PD-1 inhibitor is an antibody targeted to PD-1, optionally pembrolizumab, nivolumab, or semaprimab. **Claim 11** The method according to any one of claims 1 to 9, wherein the PD-L1 and / or the PD-L2 inhibitor is an antibody targeted to PD-L1 and / or PD-L2, optionally atezolizumab, avelumab, or durvalumab. **Claim 12** The method according to any one of the preceding claims, wherein the BioNV is derived from a modified cell. **Claim 13** The method according to claim 12, wherein the modified cell is a differentiated cell derived from a stem cell, an induced pluripotent stem cell (iPSC), a reprogrammed pluripotent or multipotent cell, an embryonic stem cell, a mesenchymal stem cell, or any modified cell thereof. **Claim 14** The method according to claim 13, wherein the modified cell is an iPSC. **Claim 15** The method according to claim 12, wherein the modified cell is a T cell, a helper T cell, a T memory cell, or an NK cell. **Claim 16** The method according to claim 12, wherein the modified cell is a macrophage. **Claim 17** The method according to claim 12, wherein the modified cell is a monocyte. **Claim 18** The method according to any one of claims 12-17, wherein the modified cell substantially lacks one or more MHC class I proteins, MHC class II proteins, T cell receptor (TCR) proteins, and / or cytokine release syndrome (CRS) proteins. **Claim 19** The method according to any one of claims 12-17, wherein the modified cell has reduced or absent expression of the β2-microglobulin (B2M) gene and / or reduced or absent expression and / or activity of MHC class I proteins. **Claim 20** The method according to any one of claims 12-19, wherein the modified cell has reduced or absent expression of the CIITA gene and / or reduced or absent expression and / or activity of MHC class II proteins. **Claim 21** The method according to any one of claims 12-20, wherein the modified cell has reduced or absent expression of the HLA-A gene and / or reduced or absent expression and / or activity of HLA-A proteins. **Claim 22** The method according to any one of claims 12-21, wherein the modified cell has reduced or absent expression of the HLA-B gene and / or reduced or absent expression and / or activity of HLA-B proteins. **Claim 23** The method according to any one of claims 12-22, wherein the modified cell has reduced or absent expression of the HLA-C gene and / or reduced or absent expression and / or activity of HLA-C proteins. **Claim 24** The method according to any one of claims 12 to 23, wherein the modified cell has reduced or abolished expression of the HLA-E or HLA-G gene and / or reduced or abolished expression and / or activity of the HLA-E or HLA-G protein.

25. The method according to any one of claims 12 to 24, wherein the modified cell has reduced or abolished expression of the HLA-F gene and / or reduced or abolished expression and / or activity of the HLA-F protein.

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

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

28. The method according to any one of claims 12 to 27, wherein the modified cell has reduced or abolished expression of the PD-1 gene and / or reduced or abolished expression and / or activity of the PD-1 protein.

29. The method according to any one of claims 12 to 28, wherein the modified cell has reduced or abolished expression of the IL-4 gene and / or reduced or abolished expression and / or activity of the IL-4 protein.

30. The method according to any one of claims 12 to 29, wherein the modified cell has reduced or abolished expression of the IL-6 gene and / or reduced or abolished expression and / or activity of the IL-6 protein.

31. The method according to any one of claims 12 to 30, wherein the modified cell has reduced or abolished expression of the IL-10 gene and / or reduced or abolished expression and / or activity of the IL-10 protein.

32. The method according to any one of claims 12 to 31, wherein the modified cell has reduced or abolished expression of the IL-16 gene and / or reduced or abolished expression and / or activity of the IL-16 protein.

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

34. The method according to any one of claims 12 to 33, wherein the modified cell expresses the CD34 gene and / or gene product or has increased expression thereof.

35. The method according to any one of claims 12 to 34, wherein the modified cell expresses the CCL2 gene and / or gene product or has increased expression thereof.

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

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

38. The method according to any one of claims 12 to 37, wherein the modified cell expresses the CD47 gene and / or gene product or has increased expression thereof.

39. The method according to any one of claims 12 to 38, wherein the modified cell expresses the CD24 gene and / or gene product or has increased expression thereof.

40. The method according to any one of claims 12 to 39, wherein the modified cell expresses the CD24 / CD47 gene and / or gene product or has increased expression thereof.

41. The method according to any one of claims 12 to 40, wherein the modified cell expresses the CD200 gene and / or gene product or has increased expression thereof.

42. The method according to any one of claims 12 to 41, wherein the modified cell expresses the chimeric CD24 / CD200 gene and / or gene product or the chimeric CD47 / CD200 gene and / or gene product or has increased expression thereof.

43. The method according to any one of claims 12 to 42, wherein the modified cell expresses the CTLA-4 gene and / or gene product, or has increased expression thereof.

44. The method according to any one of claims 12 to 43, wherein the modified cell expresses the MFG-E8 gene and / or gene product, or has increased expression thereof.

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

46. The method according to any one of claims 12 to 45, wherein the modified cell expresses the chimeric α-phagocyte integrin gene and / or gene product, or has increased expression thereof.

47. The method according to any one of claims 12 to 46, wherein the modified cell expresses an antibody or antibody format molecule (anti-IL-6R) targeting the IL-6 surface receptor, or has increased expression and / or activity thereof.

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

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

50. The method according to any one of claims 12 to 49, wherein the modified cell has reduced or abolished 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.

51. The method according to any one of claims 12 to 50, wherein the modified cell expresses 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 has increased expression thereof.

52. The method according to any one of claims 12 to 51, wherein the modified cell is allogeneic.

53. The method according to any one of claims 12 to 52, wherein the modified cell does not cause an immune response in a patient to whom the cell or BioNV derived therefrom has been administered.

54. The method according to any one of claims 12 to 53, wherein the modified cell is differentiated before BioNV formation.

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

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

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

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

59. The method according to claim 58, wherein the BioNV is formed from the modified cell by continuous extrusion.

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

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

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

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

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

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

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

67. The method according to any one of the preceding claims, wherein the BioNV substantially lacks the expression and / or activity of any one of HLA-A, HLA-B, HLA-C, HLA-F, CII TA, 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 BioNV is a membrane-embedded α-phagocytic integrin, CCL2, H2-M3, FasL, MFG-E8, an anti-IL-6R antibody or antibody format, and PD-L1 and / or CTLA-4; and The method according to any one of the preceding claims, comprising any one of CD24, CD47, CD200, chimeric CD24 / CD47, chimeric CD24 / CD200, and chimeric CD47 / CD200, or any two of CD24, CD47, and CD200.

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

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

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

72. The CAR comprises one or more antibodies or antibody formats selected from monoclonal antibodies, polyclonal antibodies, antibody fragments, 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 containing the antigen-binding portion of an antibody, the method according to any one of the preceding claims.

73. The method according to claim 71, wherein the antibody format is the scFv.

74. The method according to any one of the preceding claims, wherein the CAR comprises a transmembrane domain derived from CD28, CD3ζ, CD4, CD8α, ICOS, or fragments and / or combinations thereof. **Claim 75** In some embodiments, the CAR further comprises an intracellular domain comprising the intracellular signaling domain of the CD3ζ chain and / or optionally one or more co-stimulatory molecules selected from CD28, 4-1BB, ICOS, CD27, and OX40, The method according to any one of the preceding claims. **Claim 76** The method according to any one of the preceding claims, wherein the CAR is targeted to a cancer-specific antigen. **Claim 77** The method according to any one of the preceding claims, wherein the BioNV, or the modified cell derived therefrom, comprises a nucleic acid encoding green fluorescent protein (GFP) and / or a GFP protein. **Claim 78** The method according to claim 77, wherein the nucleic acid encoding GFP is operably linked to a promoter derived from one or more of IL-2, perforin, granzyme, alarmin, TNF, INF, and / or combinations thereof. **Claim 79** The method according to any one of the preceding claims, wherein the BioNV encapsulates a payload. **Claim 80** The method according to claim 79, wherein 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 tracking dye, a radionuclide, and / or a small molecule. **Claim 81** The method according to claim 79, wherein the payload is a therapeutic payload for the cancer type targeted by the CAR. **Claim 82** The method according to claim 79, wherein the payload comprises an alkylating agent. **Claim 83** The method according to claim 79, wherein the payload comprises an anthracycline. **Claim 84** The method according to claim 79, wherein the payload comprises an antimetabolite. **Claim 85** The method according to claim 79, wherein the payload comprises an antitumor antibiotic. **Claim 86** The method according to claim 79, wherein the payload comprises an antitumor antibiotic or an antibody format. **Claim 87** The method according to claim 79, wherein the payload comprises an adrenocortical steroid. **Claim 88** The method according to claim 79, wherein the payload comprises a plant alkaloid. **Claim 89** The method according to claim 79, wherein the payload comprises a topoisomerase inhibitor.

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

91. The payload is Abecma, Abemaciclib, Abiraterone Acetate, Abraxane, ABVD, ABVE, ABVE-PC, AC, Acalabrutinib, AC-T, Actemra, Adcetris, ADE, Ado-Trastuzumab Emtansine, Adriamycin, Afatinib Dimaleate, Afinitor, Akynzeo, Aldara, Aldesleukin, Alecensa, Alectinib, Alemtuzumab, Alimta, Aliqopa, Alkeran for Injection, Alkeran Tablets, Aloxi, Alpelisib, Alunbrig, Ameluz, Amifostine, Aminolevulinic Acid Hydrochloride, Amivantamab-vmjw, Anastrozole, Apalutamide, Aprepitant, Aranesp, Redia, Arimidex, Aromasin, Arranon, Arsenic Trioxide, Arzerra, Asciminib Hydrochloride, Asparaginase Erwinia Chrysanthemi, Asparlas, Atezolizumab, Avapritinib, Avastin, Ave lumab, Axicabtagene Ciloleucel, Axitinib, Ayvakit, Azacitidine, Azedra, Balversa, Bavencio, BEACOPP, Belantamab Mafodotin-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, BrexucabtageneAutoleucel, Breyanzi, Brigatinib, Brukinsa, BuMel, Busulfan, Busulfex, Cabazitaxel, Cabivi, Cabometyx, Cabozantinib-S-Malate, CAF, Calaspargase Pegol-mknl, Calquence, Campath, Camptosar, Capecitabine, Caplacizumab-yhdp, Capmatinib Hydrochloride, CAPOX, Carac, Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, Carmustine, Carmustine Implant, Casodex, CEM, Cemiplimab-rwlc, Ceritinib, Cerubidine, Cervarix (Recombinant HPV Bivalent Vaccine), Cetuximab, CEV, Chlorambucil, CHLORAMBUCIL-PREDNISON, CHOP, Cisplatin, Cladribine, Clofarabine, Clolar, CMF, Cobimetinib Fumarate, Cometriq, Copanlisib Hydrochloride, COPDAC, Copiktra, COPP, COPP-ABV, Cosmege, Cotellic, Crizotinib, CVP, Cyclophosphamide, Cyramza, Cytarabine, Dabrafenib Mesylate, Dacarbazine, Dacogen, Dacomitinib, Dactinomycin, Danazol, Daratumumab, Daratumumab and Hyaluronidase-fihj, Darbepoetin Alfa, Darolutamide, Darzalex, Darzalex Faspro, Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposome, Daurismo, Decitabine, Decitabine and Cedazuridine, DefibrotideSodium, Defitelio, Degarelix, Denileukin Diftitox, Denosumab, Dexamethasone, Dexrazoxane Hydrochloride, Dinutuximab, Docetaxel, Dostarlimab-gxly, Doxil, Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome, Durvalumab, Duvelisib, Efudex, Eligard, Elitek, Ellence, Elotuzumab, Oxaliplatin, Eltrombopag Olamine, Elzonris, Emapalumab-lzsg, Emend, Empliciti, Enasidenib Mesylate, Encorafenib, Enfortumab Vedotin-ejfv, Enhertu, Entrectinib, Enzalutamide, Epirubicin Hydrochloride, EPOCH, Epogen, Erbitux, Erdafitinib, Eribulin Mesylate, Erivedge, Erleada, Erlotinib Hydrochloride, Erwinaze, Ethyol, Etophos, Etoposide, Etoposide Phosphate, Everolimus, Evista, Evomela, Exemestane, Exkivity, 5-FU, 5-FU, Fam-Trastuzumab Deruxtecan-nxki, Fareston, Faslodex, FEC, Fedratinib Hydrochloride, Femara, Filgrastim, Firmagon, Fludarabine Phosphate, Fluoroplex, Fluorouracil Injection, Fluorouracil-Topical, Flutamide, FOLFIRI, FOLFIRI-Bevacizumab, FOLFIRI-Cetuximab, FOLFIRINOX, FOLFOX, Folotyn, FostamatinibDisodium, Fotivda, Fulphila, FU-LV, Fulvestrant, Fyarro, Gamifant, Gardasil (Recombinant HPV Quadrivalent Vaccine), Gardasil 9 (Recombinant HPV Nonavalent Vaccine), Gavreto, Gazyva, Gefitinib, Gemcitabine Hydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINE-OXALIPLATIN, Gemtuzumab Ozogamicin, Gemzar, Gilotrif, Gilteritinib Fumarate, Glasdegib Maleate, Gleevec, Gliadel Wafer, Glucarpidase, Goserelin Acetate, Granisetron, Granisetron Hydrochloride, Granix, Halaven, Hemangeol, Herceptin Hylecta, Herceptin, HPV Bivalent Vaccine, Recombinant, HPV Nonavalent Vaccine, Recombinant, HPV Quadrivalent Vaccine, Recombinant, Hycamtin, Hydrea, Hydroxyurea, Hyper-CVAD, Ibrance, Ibritumomab Tiuxetan, Ibrutinib, ICE, Iclusig, Idamycin PFS, Idarubicin Hydrochloride, Idecabtagene Vicleucel, Idelalisib, Idhifa, Ifex, Ifosfamide, Interleukin-2 (recombinant), Imatinib Mesylate, Imbruvica, Imfinzi, Imiquimod, Imlygic, Infgratiniib Phosphate, Infugem, Inlyta, Inotuzumab Ozogamicin, Inqovi, Inrebic, Interferon Alfa-2b, Intron A, Iobenguane I131, Ipilimumab, Iressa, IrinotecanHydrochloride, Isatuximab-irfc, Istodax, Ivosidenib, Ixabepilone, Ixazomib Citrate, Ixempra, Jakafi, JEB, Jelmyto, Jemperli, Jevtana, Kadcyla, Kepivance, Keytruda, Kimtrak, Kisqali, Koselugo, Kymriah, Kyprolis, Lanreotide Acetate, Lapatinib Ditosylate, Larotrectinib Sulfate, Lenalidomide, Lenvatinib Mesylate, Lenvima, Letrozole, Leucovorin Calcium, Leukeran, Leuprolide Acetate, Levulan Kerastik, Libtayo, Lisocabtagene Maraleucel, Lomustine, Loncastuximab Tesirine-lpyl, Lonsurf, Lorbrena, Lorlatinib, Lumakras, Lumoxiti, Lupron Depot, Lurbinectedin, Luspaterecept-aamt, Lutathera, Lutetium (Lu 177-Dotatate), Lynparza, Margenza, Margatuximab-cmkb, Marqibo, Matulane, Mechlorethamine Hydrochloride, Megestrol Acetate, Mekinist, Mektov, Melphalan, Melphalan Hydrochloride, Mercaptopurine, Mesna, Mesnex, Methotrexate Sodium, Methylnaltrexone Bromide, Midostaurin, Mitomycin, Mitoxantrone Hydrochloride, Mobocertinib Succinate, Mogamulizumab-kpk, Monjuvi, MOPP, Moxetumomab Pasudotox-tdfk, Mozobil, MVAC, Mvas, Myleran, Mylotarg, NanoparticlePaclitaxel, Naxitamab-gqgk, Necitumumab, Nelarabine, Neratinib Maleate, Nerlynx, Netupitant, Neulasta, Neupogen, Nexavar, Nilandron, Nilotinib, Nilutamide, Ninlaro, Niraparib Tosylate Monohydrate, Nivestym, Nivolumab, Nplate, Nubeqa, Nyvepria, Obinutuzumab, Odomzo, OEPA, Ofatumumab b, OFF, Olaparib, Omacetaxine Mepesuccinate, Oncaspar, Ondansetron Hydrochloride, Onivyde, Ontak, Onureg, Opdivo, OPPA, Orgovyx, Osimertinib Mesylate, Oxaliplatin, Paclitaxel, Paclitaxel Albumin-stabilized Nanoparticle Formulation, PAD, Padcev, Palbociclib, Palifermin, Palonosetron Hydrochloride, Pamidronate Disodium, Panitumumab, Paraplatine, Pazopanib Hydrochloride, PCV, PEB, Pegaspargase, Pegfilgrastim, Peginterferon Alfa-2b, PEG-Intron, Pemazyre, Pembrolizumab, Pemetrexed Disodium, Pemigatinib, Perjeta, Pertuzumab, Pexidartinib Hydrochloride, Phesgo, Piqray, Plerixafor, Polatuzumab Vedotin-piiq, Polivy, Pomalidomide, Pomalyst, Ponatinib Hydrochloride, Portrazza, Poteligeo, Pralatrexate, Pralsetinib, Prednisone, Procarbazine Hydrochloride, Procrit, Proleukin, Prolia, Promacta, Propranolol Hydrochloride, Provenge, Purinethol, Purixan, Qinlock, Radium 223 Dichloride, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, Ravulizumab-cwvz, Reblozyl, R-CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant HumanPapillomavirus (HPV) Nonavalent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Interferon Alfa-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, Ryadapt, Rylaze, Sacituzumab Govitecan-hziy, Sancuso, Sarclisa, Sclerosol Intrapleural Aerosol, Selinexor, Selpercatinib, Selumetinib Sulfate, Scenblik, Siltuximab, Sipuleucel-T, Sirolimus Protein-Bound Particles, Soltamox, Somatuline Depot, Sonidegib, Sorafenib Tosylate, Sotorasib, Sprycel, STANFORD Sterile Talc Powder, Steritalc, Stivarga, Sunitinib Malate, Sustol, Sutent, Sylatron, Sylvanant, Synribo, Tabloid, Tabrecta, TAC, Tafasitamab-cxix, Tafinlar, Tagraxofusp-perzs, Tagrisso, Talazoparib Tosylate, Talimogene Laherparepvec, Talzenna, Tamoxifen Citrate, Tarceva, Targretin, Tasigna, Tavalisse, Taxotere, TazemetostatHydrobromide, Tazverik, Tebentafusp-tebn, Tecartus, Tecentriq, Temodar, Temozolomide, Temsirolimus, Tepadina, Tepmetko, Tepotinib Hydrochloride, Thalidomide, Thalomid, Thioguanine, Thiotepa, Tibsovo, Tisagenlecleucel, Isotumab Vedotin-tftv, Tivdak, Tivozanib Hydrochloride, Tocilizumab, Tolak, Topotecan Hydrochloride, Toremifene, Torisel, Totec, TPF, Trabectedin, Trametinib Dimethyl Sulfoxide, Trastuzumab, Trastuzumab and Hyaluronidase-oysk, Treanda, Trexall, Trifluridine and Tipiracil Hydrochloride, Trisenox, Trodelvy, Truseltiq, Truxima, Tucatinib, Tucysa, Turalio, Tykerb, Ukonix, Ultomiris, Umbralisib Tosylate, Undencyca, Unituxin, Uridine Triacetate, VAC, Valrubicin, Valsar, Vandetanib, VAMP, Varubi, Vectibix, VeIP, Velcade, Vemurafenib, Venclexta, Venetoclax, Verzenio, Vidaza, Vinblastine Sulfate, Vincristine Sulfate, Vincristine Sulfate Liposome, VinorelbineThe method according to claim 79, selected from Tartrate, VIP, Vismodegib, Vistogard, Vitrakvi, Vizimpro, Voraxaze, Vorinostat, Votrient, Vyxeos, Welireg, Xalkori, Xatmeb, Xeloda, XELIRI, XELOX, Xgeva, Xofigo, Xospata, Xpovio, Xtandi, Yervoy, Yescarta, Yondelis, Yonsa, Zaltrap, Zanubrutinib, Zarxio, Zejula, Zelboraf, Zepzelca, Zevalin, Ziextenzo, Zinecard, Zirabev (Bevcizumab), Ziv-Aflibercept, Zofran, Zoladex, Zoledronic Acid, Zolinza, Zometa, Zyclara, Zydelig, Zykadia, Zynlonta, and Zytiga.

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

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

94. The method according to claim 80, wherein the gene editing payload is one or more of TALEN, ZFN, RNase P RNA, C2c1, C2c2, C2c3, Cas9, Cpf1, TevCas9, Archaeal 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 according to any one of the preceding claims, wherein the BioNV encapsulates one or more perforin molecules.

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

97. The method according to claim 96, wherein the granzyme molecule is selected from granzyme A, B, H, K, and M.

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

99. The method according to 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 according to any one of the preceding claims, further comprising co-administering a whole cell therapy.

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

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

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

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

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

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

107. The method according to claim 2 or any one of claims 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 via a transmembrane domain fusion.

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

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

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

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

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

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

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

115. wherein the cancer is acute biphenotypic leukemia, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid dendritic cell leukemia, acute myeloid leukemia, lung adenocarcinoma, adrenal tumor, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, alveolar soft part sarcoma and cardiac sarcoma, amyloidosis, anal cancer, anaplastic large cell lymphoma, angioimmunoblastic T cell lymphoma, appendiceal cancer, astrocytoma, ataxia telangiectasia, attenuated familial adenomatosis coli, B-cell prolymphocytic leukemia, basal cell carcinoma, Beckwith-Wiedemann syndrome, bile duct cancer, Birt-Hogg-Dubé syndrome, bladder cancer, bone cancer, cancer of the nervous system, brain stem glioma, brain stem glioma, brain tumor, breast cancer, bronchial adenoma / carcinoid, Burkitt lymphoma, carcinoid tumor, carcinoid tumors, Carney complex, central nervous system tumor, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, childhood desmoplastic ganglioglioma, bile duct cancer, chondrosarcoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myelogenous leukemia, chronic T-cell lymphocytic leukemia, colon cancer, colorectal cancer, Cowden syndrome, craniopharyngioma, cutaneous T cell lymphoma, dermatofibrosarcoma protuberans, desmoplastic small round cell tumor, diffuse gastric cancer, diffuse large B-cell lymphoma, endocrine cancer, endocrine tumor, endometrial cancer, eosinophilic leukemia, epithelioma, epithelioid hemangioendothelioma (EHE), esophageal cancer, Ewing sarcoma, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, eyelid cancer, fallopian tube cancer, familial adenomatosis coli, 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 cancer and gynecological cancer, germ cell tumor, 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, islet cell carcinoma (pancreatic endocrine), islet cell tumor,Hereditary polyposis syndrome, Kaposi's sarcoma, keratoacanthoma, renal cancer, lacrimal gland tumor, large granular lymphocyte leukemia, laryngeal cancer and hypopharyngeal cancer, leiomyoma and renal cell carcinoma, leiomyosarcoma, Li-Fraumeni syndrome (LFS), liposarcoma, liver cancer, lung cancer, primary cutaneous lymphoma, lymphomatoid granulomatosis, 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 (MTS), multiple endocrine neoplasia syndrome, multiple endocrine neoplasia type 1, multiple endocrine neoplasia type 2, multiple myeloma, multiple myeloma / plasma cell tumor, fungating polyp, myelodysplastic syndrome, MYH-related polyposis, myxosarcoma, nasal and paranasal cavity cancer, nephroblastoma, nasopharyngeal cancer, nasopharyngeal carcinoma, neuroblastoma, neuroendocrine tumor, 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 carcinoma, anaplastic glioma, optic nerve glioma, oral cancer and / or oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, papillary renal cell carcinoma, paranasal cavity and nasal cancer, parathyroid cancer, pelvic cancer, penile cancer, Peutz-Jeghers 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, primary neuroectodermal tumor, prostate cancer, rectal cancer, renal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, primary cutaneous sarcoma, sebaceous adenocarcinoma, Sézary syndrome, skin appendage tumor, skin cancer, small bowel cancer, small cell lung cancer, small intestine cancer, splenic marginal zone lymphoma, squamous cell carcinoma of lung, squamous cell carcinoma, squamous cell skin cancer, stomach cancer, surface epithelial stromal tumor, T-cell prolymphocytic leukemia, testicular cancer, thoracic and respiratory cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell carcinoma, transitional cell carcinoma adenoid cystic carcinoma, tuberous sclerosis syndrome, tubular carcinoma, Turcot syndrome, carcinoma of unknown primary, unclassified carcinoma, ureteral cancer, urethral cancer, uterine cancer, uterine sarcoma, choroidal melanoma, vaginal cancerThe method according to any one of claims 1 to 107, selected from optic pathway 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) targeted to a cell surface marker; and (b) a conjugate and / or a membrane anchor targeting agent targeted to PD-L1 or PD-L2, wherein the agent targeted to PD-L1 or PD-L2 is atezolizumab, avelumab, or durvalumab, and wherein the CAR is not targeted to PD-L1 or PD-L2.

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

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

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