Method and composition for stimulating gamma delta T cells

Abstract

Compositions and methods for stimulating the proliferation and cytotoxicity of γδ T cells are described. Therapeutic compositions and methods using the expanded and stimulated γδ T cells are described.

Description

[Background technology]

[0001] Cell therapies utilizing innate effector populations such as γδT cells offer a promising therapeutic platform for various diseases. Because these immune cells are present in the blood in limited quantities, the application of cell therapy requires efficient methods of cell proliferation that enable the generation of sufficient cell volumes to produce therapeutic doses. Challenges to fully realizing the clinical potential of γδT cell therapy include obtaining a large number of robust and healthy γδT cells exhibiting high cytotoxicity, the ability to target γδT cells to disease targets, and ensuring sufficient in vivo persistence of γδT cells to achieve therapeutic effects once introduced into a patient. What is needed are compositions and methods for proliferation of γδT cells, as well as their use for the treatment of diseases. [Overview of the Initiative]

[0002] The present invention, as conceived herein, encompasses compositions for proliferating γδT cells and their use, comprising the Fc domain of an antibody suitable for agonizing an Fc receptor (e.g., CD16) conjugated on feeder cells, engineered particles, exosomes, or several other solid supports. The feeder cells, engineered particles, exosomes, and other solid supports having a conjugated Fc domain may also comprise one or more additional γδT cell effector agents, such as membrane-bound IL-21, 4-1BBL, other cytokines, adhesion molecules, and / or other stimulant (or possibly inhibitory) receptors and corresponding signaling pathways, such as γδT cell activators. Engagement of the Fc receptor (e.g., CD16) by the aforementioned agents results in the proliferation of an initial population of γδT cells, the cells produced through proliferation having higher cytotoxicity than the initial population of cells. In addition, the method may result in co-proliferation of γδT cells and NK cells if NK cells are not removed before proliferation. The combination of these two populations may result in broader antitumor function and therefore better efficacy. Such γδT cells or γδT / NK cell mixtures may be used as therapeutic agents for the treatment of diseases.

[0003] In some embodiments, disclosed herein are methods for inducing, activating, and / or proliferating γδT cells, comprising contacting at least one γδT cell with an engineered feeder cell, engineered plasma membrane particle, exosome, or solid support containing an Fc domain bound to its outer surface via a transmembrane domain. The transmembrane domain may be a neuraminidase transmembrane domain, a parainfluenza virus hemagglutinin-neuraminidase-derived signal anchor sequence, a transferrin receptor-derived signal anchor sequence, an MHC class II invariant chain-derived signal anchor sequence, a P-glycoprotein-derived signal anchor sequence, an asialoglycoprotein receptor-derived signal anchor sequence, and a neutral endopeptidase-derived signal anchor sequence. In some embodiments, the transmembrane domain includes a parainfluenza virus hemagglutinin-neuraminidase (NA) peptide sequence containing at least 81% identical to Sequence ID No. 1. The transmembrane domain and the Fc domain may be linked via a peptide linker.

[0004] In some embodiments, the Fc domain comprises an immunoglobulin Fc domain selected from IgG1, IgG2, IgG3, IgG4, IgA, and IgE. In some embodiments, the Fc domain binds to CD16.

[0005] Feeder cells may be peripheral blood mononuclear cells (PBMCs), fibroblasts, epithelial cells, endothelial cells, antigen-presenting cells, or microbial cells, or cell lines, and the cell lines may be RPMI8866, HFWT, 721.221, K562, or EBV-LCL.

[0006] In some embodiments, the method of any of the aforementioned embodiments further comprises contacting at least one γδT cell with at least one γδT cell effector agent, the at least one γδT cell effector agent being expressed on or bound to engineered feeder cells (i.e., membrane binding (mb)), engineered plasma membrane particles, exosomes, or the outer surface of a solid support. The at least one γδT cell effector agent may be a cytokine, adhesion molecule, or γδT cell activator. In some embodiments, at least one γδ T cell effector agent includes an agonist (e.g., an agnostic antibody) or ligand of 4-1BBL;CD80;CD86;MICA;UBLP;2B4;LFA-1;NKG2D, NKp46, NKp44, NKp30, or DNAM-1; an agonist (e.g., an agnostic antibody) or ligand of Notch, BCM / SLAMF2, or TLR; IL-2;IL-12;IL-18;IL-15; or IL-21; or any combination thereof. In some embodiments, at least one γδ T cell effector agent comprises 4-1BBL, IL-18, IL-15, or IL-21, or any combination thereof (e.g., 4-1BBL and IL-21; 4-1BBL and IL-15; 4-1BBL and IL-18; 4-1BBL, IL-15, and IL-21; 4-1BBL, IL-18, and IL-21; 4-1BBL, IL-15, and IL-18; or 4-1BBL, IL-15, IL-18, and IL-21, etc.), which include membrane-bound 4-1BBL, IL-18, IL-21, IL-18, IL-21, etc. This includes, but is not limited to, L-15, IL-21, or combinations thereof (e.g., mb4-1BBL and mbIL-21; mb4-1BBL and mbIL-15; mb4-1BBL and mbIL-18; mb4-1BBL, mbIL-15, and mbIL-21; mb4-1BBL, mbIL-18, and mbIL-21; mb4-1BBL, mbIL-15, and mbIL-18; or mb4-1BBL, mbIL-15, mbIL-18, and mbIL-21, etc.), as well as combinations of membrane-bound and unbound effector agents.

[0007] In some embodiments, any of the methods described above include contacting at least one γδT cell with a feeder cell, manipulated particle, exosome, or solid support in vitro, in vivo, or ex vivo. In some embodiments, the proliferated γδT cells include the Vδ2 subtype and / or the Vδ1 subtype. The γδT cells may be autologous, haplotype-identical, or allogeneic γδT cells. In some embodiments, the γδT cells are proliferated for at least 14 days, and at least about 5%, 10%, 20%, 30%, 40%, 50%, or 60% of the proliferated cells are Vδ2 subtype γδT cells.

[0008] In some embodiments, γδT cells proliferate at a faster rate over 14 days than the control γδT cell population.

[0009] γδT cells grown according to any of the aforementioned methods may be an isolated cell population or in a mixed cell population. The mixed cell population may be depleted of NK cells before, during, or after the proliferation of γδT cells.

[0010] In some embodiments, disclosed herein are methods for treating, reducing, inhibiting, mitigating, improving, and / or preventing cancer, metastasis, or infectious disease in a subject, comprising administering to the subject a therapeutically effective amount of γδT cells that have been proliferated, activated, or induced in accordance with any of the methods described herein.

[0011] In some embodiments, disclosed herein are methods for treating, reducing, inhibiting, mitigating, improving, and / or preventing cancer, metastasis, or infectious disease in a subject, a. Obtain at least one γδT cell, b. Contacting at least one γδT cell with an engineered feeder cell, engineered plasma membrane particle, exosome, or solid support containing an Fc domain bound to its outer surface. c. A method comprising administering a therapeutically effective amount of contacted γδT cells to a subject.

[0012] In some embodiments, step b further comprises inducing, activating, and / or proliferating at least one γδT cell after contact with an engineered feeder cell, engineered plasma membrane particle, exosome, or solid support containing an externally bound Fc domain, wherein the γδT cell is induced, activated, and / or proliferated for at least 14 days.

[0013] In some embodiments, the engineered feeder cells, engineered plasma membrane particles, exosomes, or solid support may further comprise at least one γδT cell effector agent, the at least one γδT cell effector agent comprising: agonists (e.g., agnostic antibodies) or ligands of 4-1BBL;CD80;CD86;MICA;UBLP;2B4;LFA-1;NKG2D, NKp46, NKp44, NKp30, or DNAM-1; agonists (e.g., agnostic antibodies) or ligands of Notch, BCM / SLAMF2, or TLR; IL-2;IL-12;IL-18;IL-15; or IL-21; or any combination thereof. In some embodiments, at least one γδ T cell effector agent comprises 4-1BBL, IL-18, IL-15, or IL-21, or any combination thereof (e.g., 4-1BBL and IL-21; 4-1BBL and IL-15; 4-1BBL and IL-18; 4-1BBL, IL-15, and IL-21; 4-1BBL, IL-18, and IL-21; 4-1BBL, IL-15, and IL-18; or 4-1BBL, IL-15, IL-18, and IL-21, etc.), which include membrane-bound 4-1BBL, IL-18, IL-21, IL-18, IL-21, etc. This includes, but is not limited to, L-15, IL-21, or combinations thereof (e.g., mb4-1BBL and mbIL-21; mb4-1BBL and mbIL-15; mb4-1BBL and mbIL-18; mb4-1BBL, mbIL-15, and mbIL-21; mb4-1BBL, mbIL-18, and mbIL-21; mb4-1BBL, mbIL-15, and mbIL-18; or mb4-1BBL, mbIL-15, mbIL-18, and mbIL-21, etc.), as well as combinations of membrane-bound and unbound effector agents.

[0014] In some embodiments, disclosed herein are methods for treating, reducing, inhibiting, mitigating, improving, and / or preventing cancer, metastasis, or infectious disease in a subject by promoting, inducing, and / or activating endogenous γδT cells in the subject, the method comprising administering to the subject engineered plasma membrane particles, exosomes, or solid supports comprising an externally bound Fc domain, wherein the engineered feeder cells, engineered plasma membrane particles, exosomes, or solid supports contain at least one γδT cell effector agent Furthermore, the formulation may include at least one γδ T cell effector agent comprising: an agonist (e.g., agnostic antibody) or ligand of 4-1BBL;CD80;CD86;MICA;UBLP;2B4;LFA-1;NKG2D, NKp46, NKp44, NKp30, or DNAM-1; an agonist (e.g., agnostic antibody) or ligand of Notch, BCM / SLAMF2, or TLR; IL-2;IL-12;IL-18;IL-15; or IL-21; or any combination thereof. In some embodiments, at least one γδ T cell effector agent comprises 4-1BBL, IL-18, IL-15, or IL-21, or any combination thereof (e.g., 4-1BBL and IL-21; 4-1BBL and IL-15; 4-1BBL and IL-18; 4-1BBL, IL-15, and IL-21; 4-1BBL, IL-18, and IL-21; 4-1BBL, IL-15, and IL-18; or 4-1BBL, IL-15, IL-18, and IL-21, etc.), which include membrane-bound 4-1BBL, IL-18, IL-21, IL-18, IL-21, etc. This includes, but is not limited to, L-15, IL-21, or combinations thereof (e.g., mb4-1BBL and mbIL-21; mb4-1BBL and mbIL-15; mb4-1BBL and mbIL-18; mb4-1BBL, mbIL-15, and mbIL-21; mb4-1BBL, mbIL-18, and mbIL-21; mb4-1BBL, mbIL-15, and mbIL-18; or mb4-1BBL, mbIL-15, mbIL-18, and mbIL-21, etc.), as well as combinations of membrane-bound and unbound effector agents.

[0015] In some embodiments, any of the methods described above further comprises administering to a subject an ex vivo composition comprising a fusion protein having a transmembrane domain ligated to the amino terminus of an Fc domain and bound to engineered feeder cells, engineered plasma membrane particles, exosomes, or a solid support, by contacting the ex vivo composition with an isolated mixed cell population comprising at least one γδT cell containing CD16 or a functional fragment thereof. In some embodiments, the ex vivo composition further comprises at least one γδ T cell effector agent, the at least one γδ T cell effector agent comprising an agonist (e.g., agnostic antibody) or ligand of 4-1BBL;CD80;CD86;MICA;UBLP;2B4;LFA-1;NKG2D, NKp46, NKp44, NKp30, or DNAM-1; an agonist (e.g., agnostic antibody) or ligand of Notch, BCM / SLAMF2, or TLR; IL-2;IL-12;IL-18;IL-15; or IL-21; or any combination thereof. In some embodiments, at least one γδ T cell effector agent comprises 4-1BBL, IL-18, IL-15, or IL-21, or any combination thereof (e.g., 4-1BBL and IL-21; 4-1BBL and IL-15; 4-1BBL and IL-18; 4-1BBL, IL-15, and IL-21; 4-1BBL, IL-18, and IL-21; 4-1BBL, IL-15, and IL-18; or 4-1BBL, IL-15, IL-18, and IL-21, etc.), which include membrane-bound 4-1BBL, IL-18, IL-21, IL-18, IL-21, etc. This includes, but is not limited to, L-15, IL-21, or combinations thereof (e.g., mb4-1BBL and mbIL-21; mb4-1BBL and mbIL-15; mb4-1BBL and mbIL-18; mb4-1BBL, mbIL-15, and mbIL-21; mb4-1BBL, mbIL-18, and mbIL-21; mb4-1BBL, mbIL-15, and mbIL-18; or mb4-1BBL, mbIL-15, mbIL-18, and mbIL-21, etc.), as well as combinations of membrane-bound and unbound effector agents.The manipulated plasma film particles may comprise a plasma film and a plurality of microparticles or support surfaces, wherein the plasma film coats the plurality of microparticles or support surfaces. In some embodiments, the plurality of microparticles or surfaces include at least one of magnetic microparticles, silica beads, polystyrene beads, latex beads, microstructures, contrast agents, and cancer treatment agents.

[0016] The methods disclosed herein are for the treatment of cancer, which is selected from the group consisting of hematological cancers, lymphomas, colorectal cancers, colon cancers, lung cancers, head and neck cancers, ovarian cancers, prostate cancers, testicular cancers, kidney cancers, skin cancers, cervical cancers, pancreatic cancers, and breast cancers. In one embodiment, cancer includes solid tumors. In another embodiment, cancer is selected from acute myeloid leukemia, myelodysplastic syndromes, chronic myeloid leukemia, acute lymphoblastic leukemia, myelofibrosis, and multiple myeloma. In yet another embodiment, cancer is selected from leukemias, lymphomas, sarcomas, and carcinomas, and may originate from the bone marrow, brain, lungs, breasts, pancreas, liver, head and neck, skin, reproductive tract, prostate, colon, kidney, intraperitoneal, bone, joints, and eyes.

[0017] In some embodiments, any of the methods described above further comprises administering to a subject a combination of at least one cancer treatment agent, the at least one cancer treatment agent being abemaciclib, abiraterone acetate, abitrexate (methotrexate), Abraxane (paclitaxel albumin-stabilized nanoparticle formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (brentuximab vedotin), ADE, Ado-trastuzumab emtansine, Adriamycin (doxorubicin hydrochloride), afatinib dimareate, Afinitor (everolimus), Akynzeo (netupitant and palonosetron hydrochloride), Aldara (imiquimod), Aldesleukin, Alecensa (alecinib) Alectinib, Alemtuzumab, Alimta (pemetrexed disodium), Aliqopa (copanlisib hydrochloride), Alkeran for injection (melphalan hydrochloride), Alkeran tablets (melphalan), Aloxi (palonosetron hydrochloride), Alunbrig (brigatinib), Ambochlorin (chlorambucil), Ambochlorin (chlorambucil), Amifostine, aminolevulinic acid, Anastrozole, Aprepitant, Aredia (pamidronate disodium), Arimidex (anastrozole), Aromasin (exemestane), Alanone (nelarabine), Arsenic trioxide, Arzerra (ofatumumab), Asparaginase Erwinia Chrysanthemi, atezolizumab, Avastin (bevacizumab), avelumab, axitinib, azacitidine, Bavencio (avelumab), BEACOPP, Becenum (carmustine), Beleodaq (belinostat), bendamustine hydrochloride, BEP, Besponsa (inotuzumab ozogamicin), bevacizumab, bexarotene, Bexxar (tositumomab and iodine I131 tositumomab), bicalutamide, BiCNU (carmustine), bleomycin, blinatumomab,Blincyto (blinatumomab), bortezomib, Bosulif (bosutinib), bosutinib, brentuximab vedotin, brigatinib, BuMel, busulfan, busulfex (busulfan), cabazitaxel, Cabometyx (cabozantinib-S-malate), cabozantinib-S-malate, CAF, Camppath (aremtuzumab), Camptosar (irinotecan hydrochloride), capecitabine, CAPOX, Carac (fluorouracil-topical), carbopra Chloramine, carboplatin-taxol, carfilzomib, Carmubris (carmustine), carmustine, carmustine implant, Casodex (bicalutamide), CEM, ceritinib, cerubidine (daunorubicin hydrochloride), Cervarix (recombinant HPV bivalent vaccine), cetuximab, CEV, chlorambucil, chlorambucil-prednisone, CHOP, cisplatin, cladribine, Clafen (cyclophosphamide), clofarabine, Clofarex (clofarabine) Clofarabine, Chloral (clofarabine), CMF, Cobimetinib, Cometriq (cabozantinib-S-malate), Copanlisib hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (dactinomycin), Cotellic (cobimetinib), Crizotinib, CVP, Cyclophosphamide, Cyfos (ifosfamide), Cyramza (ramucirumab), Cytarabine, Cytarabine liposome, Cytosar-U (cytarabine), Cytoxan (cyclophosphamide) Sphamide), dabrafenib, dacarbazine, Dacogen (decitabine), dactinomycin, daratumumab, Darzalex (daratumumab), dasatinib, daunorubicin hydrochloride, daunorubicin hydrochloride and cytarabine liposomes, decitabine, defibrotide sodium, Defitelio (defibrotide sodium), degarelix, denileukin diffitox, denosumab, DepoCyt (cytarabine liposomes), dexamethasone, dexrazoxane hydrochloride, dinutuximab, docetaxel,Doxil (doxorubicin hydrochloride liposome), doxorubicin hydrochloride, doxorubicin hydrochloride liposome, Dox-SL (doxorubicin hydrochloride liposome), DTIC-Dome (dacarbazine), durvalumab, Efudex (fluorouracil - topical), Elitek (rasburicase), Ellence (epirubicin hydrochloride), elotuzumab, Eloxatin (oxaliplatin), Eltrombopagolamine, Emend (aprepitant), Empliciti (elotuzumab), Enasidenib mesylate, Enzalutamide, Epirubicin hydrochloride, EPOCH, Erbitux (cetuximab), Eribulin mesylate, Erivedge (vismodegib), Erlotinib hydrochloride, Erwinase (asparaginase Erwinia) Chrysanthemi), Ethyol (amifostine), Etopophos (etoposide phosphat), Etoposide, Etoposide phosphat, Evacet (doxorubicin hydrochloride liposome), Everolimus, Evista (raloxifene hydrochloride), Evomela (melphalan hydrochloride), Exemestane, 5-FU (fluorouracil injection), 5-FU (fluorouracil - topical), Far Eston (toremifene), Farydak (panobinostat), Faslodex (fulvestrant), FEC, Femara (letrozole), filgrastim, Fludara (fludarabine phosphate), fludarabine phosphate, Fluoroplex (fluorouracil - topical), fluorouracil injection, fluorouracil - topical, flutamide, Folex (methotrexate), Folex PFS (methotrexate), FOLFIRI, FOLFIRI-bevacizumab, FOLFIRI-cetuximab, FOLFIRINOX, FOLFOX, Folotyn (pralatrexate), FU-LV, fulvestrant, Gardasil (recombinant HPV tetravalent vaccine), Gardasil 9 (recombinant HPV nonavalent vaccine), Gazyva (obinutuzumab), gefitinib, gemcitabine hydrochloride, gemcitabine-cisplatin, gemcitabine-oxaliplatin, gemtuzumab ozogamicin,Gemzar (gemcitabine hydrochloride), Zirotrif (afatinib dimareate), Gleevec (imatinib mesylate), Gliadel (carmustine implant), Gliadel wafer (carmustine implant), glucarpidase, goserelin acetate, Halaven (eribulin mesylate), Hemangeol (propranolol hydrochloride), Herceptin (trastuzumab), HPV bivalent vaccine, recombinant, HPV nonavalent vaccine, recombinant, HPV tetravalent vaccine, recombinant, Hycamtin (topotecan hydrochloride), Hydrea (hydroxyl Siurea), Hydroxyurea, Hyper-CVAD, Ibrance (palbociclib), Ibritumomab tiuxetan, Ibrutinib, ICE, Iclusig (ponatinib hydrochloride), Idamycin (idarubicin hydrochloride), Idarubicin hydrochloride, Ideralisib, Idhifa (enasidenib mesylate), Ifex (ifosfamide), ifosfamide, Ifosfamidum (ifosfamide), IL-2 (aldesleukin), Imatinib mesylate, Im Bruvica (ibrutinib), Imfinzi (durvalumab), imiquimod, Imlygic (tarimodine laherpalepbec), Inlyta (axitinib), inotuzumab ozogamicin, interferon alfa-2b, recombinant, interleukin-2 (aldesleukin), intron A (recombinant interferon alfa-2b), iodine I131 tositumomab and tositumomab, ipilimumab, Iressa (gefitinib), irinotecan hydrochloride, irinotecan hydrochloride liposome, Istodax (romine Depsin), Ixabepirone, Ixazomib citrate, Ixempra (Ixabepirone), Jakafi (Ruxolitinib phosphat), JEB, Jevtana (Cabazitaxel), Kadcyla (Ado-Trastuzumab Emtansine), Keoxifene (Raloxifene Hydrochloride), Kepivance (Palifermin), Keytruda (Pembrolizumab), Kisqali (Ribociclib), Kymriah (Tisagenlecleucel), Kyprolis (Carfilzomib),Lanreotide acetate, lapatinib ditosylate, Lartruvo (Olaratumab), lenalidomide, lenvatinib mesylate, Lenvima (lenvatinib mesylate), letrozole, leucovorin calcium, Leukeran (chlorambucil), leuprolide acetate, leustatin (cladribine), Levulan (aminolevulinic acid), linfollizin (chlorambucil), LipoDox (doxorubicin hydrochloride liposome), lomustine, Lonsurf (trifluri (Zin and tipiracil hydrochloride), Lupron (leuprolide acetate), Lupron Depot (leuprolide acetate), Lupron Depot Ped (leuprolide acetate), Lynparza (olaparib), Marquivo (vincristine sulfate liposome), Matulane (procarbazine hydrochloride), mechloretamine hydrochloride, megestrol acetate, Mekinist (trametinib), melphalan, melphalan hydrochloride, mercaptopurine, Mesna, Mesnex (mesna), metazolastone (methazolastone) Mozolomide, methotrexate, methotrexate LPF (methotrexate), methylnaltrexone bromide, Mexate (methotrexate), Mexate-AQ (methotrexate), midostaurine, mitomycin C, mitoxantrone hydrochloride, mitozytrex (mitomycin C), MOPP, mozovir (plelixafor), mustargen (mechloretamine hydrochloride), mutamycin (mitoma Icin C), Myleran (busulfan), Mylosar (azacitidine), Mylotarg (gemtuzumab ozogamicin), nanoparticle paclitaxel (paclitaxel albumin-stabilized nanoparticle formulation), Navelbine (vinorelbine turtrate), necitumumab, nelarabine, Neosar (cyclophosphamide), neratinib maleate, Nerlinx (neratinib maleate), netupitant and palonosetron hydrochloride, Neulasta (pegfilgrastim), Neupogen (filgrastim),Nexavar (sorafenib tosylate), Nilandron (niltamide), nilotinib, niltamide, Ninlaro (ixazomib citrate), niraparib tosylate monohydrate, nivolumab, Nolvadex (tamoxifen citrate), Nplate (romiplostim), obinutuzumab, Odomz, o (sonidegib), OEPA, ofatumumab, OFF, olaparib, olalatumab, omasetaxin mepesuccinate, Oncasper (peguaspargase), ondansetron hydrochloride, Onivyde (irinotecan hydrochloride liposome), Ontak (denileukin difutitox), Opdivo (nivolumab), OPPA, osimertinib, oxaliplatin, paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation, PAD, palbociclib, parifermin, palonosetron hydrochloride, palonosetron hydrochloride and Netupitant, Pamidronate disodium, Panitumumab, Panobinostat, Paraplat (Carboplatin), Paraplatin (Carboplatin), Pazopanib hydrochloride, PCV, PEB, Pegaspargase, Pegfilgrastim, Peginterferon alfa-2b, PEG-Intron (Peginterferon alfa-2b), Pembrolizumab, Pemetrexed disodium, Perjeta (Pertuzumab), Pertuzumab, Platinol (Cisplatin), Platinol-AQ (Cisplatin), Preliquid Plerixafor, Pomalidomide, Pomalist (Pomalidomide), Ponatinib hydrochloride, Portraza (Necitumumab), Pralatrexate, Prednisone, Procarbazine hydrochloride, Proleukin (Aldesleukin), Prolia (Denosumab), Promacta (Eltrombopagolamine), Propranolol hydrochloride, Provenge (Cyplucel-T), Purinetol Mercaptopurine, Purixan (mercaptopurine), radium-223 dichloride, raloxifene hydrochloride, ramucirumab, rasburicase, R-CHOP, R-CVP, recombinant human papillomavirus (HPV) bivalent vaccine, recombinant human papillomavirus (HPV) nonavalent vaccine, recombinant human papillomavirus (HPV) tetravalent vaccine, recombinant interferon alpha-2b, regorafenib, Relistor (methyltrexone bromide), R-EPOCH, Revlimid (lenalidomide), Rheumatrex (methotrexate),Ribociclib, R-ICE, Rituxan (Rituximab) Hycela (rituximab and hyaluronidase), rituximab, rituximab and hyaluronidase, lorapitant hydrochloride, romidepsin, romiplostim, rubidomycin (daunorubicin hydrochloride), Rubraca (rucaparib cansylate), rucaparib cansylate, ruxolitinib phosphate, Rydapt (midostaurine), Sclerosol intrapleural aerosol (talc), siltuximab, ciplucel-T, somatuline depot (lanreotide acetate), sonidegib, sorafenib tosylate, Sprycel (dasatinib), STANFORD V, sterile talc powder (talc), Steritalc (talc), Stivarga (regorafenib), sunitinib malate, Sutent (sunitinib malate), Sylatron (pegylated interferon alfa-2b), Sylvant (siltuximab), Synribo (omacetaxin mepesuccinate), Tabloid (thioguanine), TAC, Tafinlar (dabrafenib), Tagrisso (osimertinib), talc, tarimodine laherpalepvec, tamoxifen citrate, tarabine PFS (cytarabine), tarceva (erlotinib hydrochloride), targretin (bexarotene), tasigna (nilotinib), Taxol (pacli) Taxel, Taxotere (docetaxel), Tecentriq (atezolizumab), Temodal (temozolomide), temozolomide, temsirolimus, thalidomide, thalomid (thalidomide), thioguanine, thiotepa, tisagenlecleucel, Tolak (fluorouracil - topical), topotecan hydrochloride, toremifene, Toli Cell (temsirolimus), tositumomab and iodine-I131 tositumomab, Totect (dexrazoxane hydrochloride), TPF, trabectedin, trametinib, trastuzumab, Treanda (bendamustine hydrochloride), trifluridine and tipiracil hydrochloride, Trisenox (arsenic trioxide), Tykerb (lapatinib ditosylate),Unituxin (dinutuximab), uridine triacetate, VAC, vandetanib, VAMP, Barbit (Varubi) (rolapitant hydrochloride), Vectibix (panitumumab), VeIP, Velban (vinblastine sulfate), Velcade (bortezomib), Velsar (vinblastine sulfate), vemurafenib, Benclexta (venetoclax), venetoclax, Verzenio (abemaciclib), Viadur (leuprolide acetate), Vidaza (azacitidine), vinblastine sulfate, Vincasar PFS (vincristine sulfate), vincristine sulfate, vincristine sulfate liposome, vinorelvin tartrate, VIP, bismodegib, Vistagoard (uridine triacetate), Voraxaze (glucarpidase), vorinostat, Votrient (pazopanib hydrochloride), Vyxeos (daunorubicin hydrochloride and cytarabine liposome), Wellcovorin (leucovorin calcium), Xalkori (crizotinib), Xeloda (capecitabine), XELIRI, XELOX, Xgeva (denosumab), Xofigo (radium-223 dichloride), Xtandi (enzalutaminum The following are selected from the group consisting of (D), Yervoy (ipilimumab), Yondelis (trabectedin), Zaltrap (Ziv-aflibercept), Zarxio (filgrastim), Zejura (niraparib tosylate monohydrate), Zelboraf (vemurafenib), Zevalin (ibritumomab tiuxetan), Zinecard (dexrazoxane hydrochloride), Ziv-aflibercept, Zofran (ondansetron hydrochloride), Zoladex (goserelin acetate), zoledronic acid, Zolinza (vorinostat), Zometa (zoledronic acid), Zydelig (idelalisib), Zykadia (ceritinib), and / or Zytiga (abiraterone acetate). In some embodiments, at least one cancer treatment agent is selected from chemotherapeutic agents (e.g., CHOP, FLAG, 7+3), drug-based preparation regimens, or combinations thereof (Cy-Flu, Bu-Flu, Flu-Mel).

[0018] In some embodiments, the manipulated particles further comprise one or more γδT effector agents. In some embodiments, the manipulated particles further comprise at least one γδT cell effector agent, the γδT cell effector agent being IL-21. In another embodiment, the manipulated particles further comprise at least two γδT cell effector agents, one of which is IL-2.

[0019] In some embodiments, the methods disclosed herein are for treating infectious diseases caused by viral infections, including herpes simplex virus-1, herpes simplex virus-2, varicella-zoster virus, Epstein-Barr virus, cytomegalovirus, human herpesvirus-6, smallpox virus, varicella-stomatitis virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, rhinovirus, coronavirus, influenza A virus, influenza B virus, measles virus, polyomavirus, human papillomavirus, respiratory syncytial virus, and adenowyi virus. This includes infection with Russ virus, coxsackievirus, dengue virus, mumps virus, poliovirus, rabies virus, Rouss sarcoma virus, reovirus, yellow fever virus, Zika virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern equine encephalitis virus, Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, rotavirus A, rotavirus B, rotavirus C, Sindbis virus, simian immunodeficiency virus, human T-cell leukemia virus type 1, hantavirus, rubella virus, simian immunodeficiency virus, human immunodeficiency virus type 1, or human immunodeficiency virus type 2.

[0020] In some embodiments, the methods disclosed herein are for treating infectious diseases caused by bacterial infections, including Mycobaterium tuberculosis, Mycobaterium bovis, Mycobaterium bovis strain BCG, BCG substrain, Mycobaterium avium, Mycobaterium intracellular, Mycobaterium africanum, Mycobaterium kansasii, Mycobaterium marinum, Mycobaterium ulcerans, Mycobaterium avium subspecies paratuberculosis, Nocardia asteroides, other Nocardia species, Legionella pneumophila, other Legionella species, Acetinobacter baumanii, Salmonella typhi, Salmonella enterica, other Salmonella species, Shigella boydii, Shigella dysenteriae, Shigella sonnei, Shigella flexneri, other Shigella species, and Yersinia. pestis, Pasteurella haemolytica, Pasteurella multocida, other Pasteurella species, Actinobacillus pleuropneumoniae, Listeria monocytogenes, Listeria ivanovii, Brucella abortus, other Brucella species, Cowdria ruminantium, Borrelia burgdorferi, Bordetella avium, Bordetella pertussis, Bordetella bronchiseptica, Bordetella trematum, Bordetella hinzii, Bordetella pteri, Bordetella parapertussis, Bordetella ansorpii, other Bordetella species, Burkholderia mallei, BurkholderiaInfections including those caused by Pseudomonas mallei, Burkholderia cepacian, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Coxiella burnetii, Rickettsial species, Ehrlichia species, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Escherichia coli, Vibrio cholerae, Campylobacter species, Neiserria meningitidis, Neiserria gonorrhea, Pseudomonas aeruginosa, other Pseudomonas species, Haemophilus influenzae, Haemophilus ducreyi, other Hemophilus species, Clostridium tetani, Clostridium difficile, other Clostridium species, Yersinia enterolitica, and other Yersinia species, as well as Mycoplasma species.

[0021] In some embodiments, the methods disclosed herein are for the treatment of infectious diseases caused by fungal infections, which include infections by Candida albicans, Cryptococcus neoformans, Histoplama capsulatum, Aspergillus fumigatus, Coccidiodes immitis, Paracoccidiodes brasiliensis, Blastomyces dermitidis, Pneumocystis carinii, Penicillium marneffi, or Alternaria alternate.

[0022] In some embodiments, the methods disclosed herein are for the treatment of infectious diseases caused by parasitic infections, including those caused by Toxoplasma gondii, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, other Plasmodium species, Entamoeba histolytica, Naegleria fowleri, Rhinosporidium seeberi, Giardia lamblia, Enterobius vermicularis, Enterobius gregorii, Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus, and Cryptosporidium spp.Infection with Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, other Leishmania species, Diphyllobothrium latum, Hymenolepis nana, Hymenolepis diminuta, Echinococcus granulosus, Echinococcus multilocularis, Echinococcus vogeli, Echinococcus oligarthrus, Diphyllobothrium latum, Clonorchis sinensis; Clonorchis viverrini, Fasciola hepatica, Fasciola gigantica, Dicrocoelium dendriticum, Fasciolopsis buski, Metagonimus yokogawai, Opisthorchis viverrini, Opisthorchis felineus, Clonorchis sinensis, Trichomonas vaginalis, Acanthamoeba species, Schistosoma intercalatum, Schistosoma haematobium, Schistosoma japonicum, Schistosoma mansoni, other Schistosoma species, Trichobilharzia regenti, Trichinella spiralis, Trichinella britovi, Trichinella nelsoni, Trichinella nativa, or Entamoeba histolytica is included.

[0023] In some embodiments, the γδ T cells administered by the method of any of the foregoing embodiments are formulated in a pharmaceutically acceptable carrier and a pharmaceutically acceptable excipient.

[0024] In some embodiments, the method of any of the foregoing embodiments includes administering the γδ T cells parenterally, intravenously, intraperitoneally, or subcutaneously, or via arterial infusion, intravenous infusion, or catheter-mediated infusion.

[0025] It should be further understood that any of the treatment methods described herein is also considered a medical use of any of the compositions disclosed herein for the treatment of any of the cancers or infectious diseases disclosed herein. [Brief explanation of the drawing]

[0026] [Figure 1] Figure 1 shows the effect of Fc on T cell proliferation using CSTX-002 feeder cells. Inclusion of Fc on the CSTX-002 (K562-mb21-41BBL) cell line resulted in increased T cell proliferation over 14 days. Fc was immobilized to the cell membrane using neuraminidase (NA) stalks of different lengths, with NA2 being the shortest and NA4 the longest. An increase in T cell content was observed in all cultures stimulated with Fc-containing CSTX-002 cells, with the longest NA fragment resulting in the highest final T cell content. [Figure 2] Figure 2 shows that stimulation with CSTX-002-Fc induces proliferation of γδ T cells. Follow-up experiments using PBMCs derived from different donors confirmed that including NA-Fc on CSTX-002 cells resulted in T cell proliferation. T cell phenotyping showed that the majority of these cells were γδ T cells, and that Vδ2 cells preferentially proliferated upon stimulation with NA-Fc-expressing CSTX-002 feeder cells. The construct with the longest NA stock yielded the highest final content of Vδ2 cells. [Figure 3] Figure 3 shows the effect of NA stalk length and starting material on γδ T cell proliferation. Longer NA stalks from NA-Fc constructs result in greater proliferation of Vδ2 subtype γδ T cells. NA4 is longer than NA2. [Figure 4]Figure 4 shows that Fc selectively induces the proliferation of Vδ2 subtype γδT cells. Proliferation is independent of the presence of NK cells. PBMCs obtained from four different donors were stimulated with CSTX-002 cells that were either CD56-depleted (to eliminate NK cells) or not, and that expressed or did not express the Fc domain on their cell surface. Vδ2T cell content was regularly monitored over a 14-day culture period. Figure 4 shows the cumulative theoretical proliferation of Vδ2 cells for all four donors. Inclusion of Fc on CSTX-002 cells resulted in T cell proliferation in all donors tested. T cell phenotyping showed that the majority of these cells were γδT cells, and that Vδ2 cells preferentially proliferated upon stimulation with NA-Fc-expressing CSTX-002 feeder cells. NK cell depletion did not negatively affect Vδ2 cell proliferation. [Figure 5] Figure 5 shows that Fc selectively induces the proliferation of Vδ2 subtype γδ T cells. Proliferation is independent of the presence of NK cells. PBMCs obtained from two different donors were stimulated with CSTX-002 cells that were either CD56-depleted (to eliminate NK cells) or not, and that expressed or did not express the Fc domain on their cell surface. Vδ2 T cell content was regularly monitored over a 14-day culture period. The figure above shows the cumulative theoretical proliferation of Vδ2 cells on day 14. Inclusion of Fc on CSTX-002 cells resulted in significant proliferation of Vδ2 T cells in all donors tested (p=001). NK cell depletion did not negatively affect Vδ proliferation. [Figure 6]Figure 6 shows that Fc selectively induces the proliferation of Vδ2 subtype γδ T cells. Proliferation is independent of the presence of NK cells. PBMCs obtained from four different donors were stimulated with CSTX-002 cells that were either CD56-depleted (to eliminate NK cells) or not, and that expressed or did not express the Fc domain on their cell surface. The figure shows an example of the final cell content of the culture at day 14 for one of the donors. Inclusion of Fc in CSTX-002 cells significantly increased the Vδ2 T cell content. NK cell depletion did not negatively affect Vδ2 cell proliferation. [Figure 7] Figures 7A and 7B show the construction of membrane-bound immune cell target ligands containing an uncleaved signal anchor. Figure 7A shows the structures of type I and type II essential membrane proteins with different orientations with respect to the N-terminus and C-terminus. Figure 7B shows the structure of an NA-Fc chimeric protein used as a membrane-bound immune cell target ligand, consisting of a neuraminidase transmembrane domain, a stalk region, and a human IgG'Fc region that function as a membrane anchor. [Figure 8] Figure 8 shows alternative constructs of membrane-bound immune cell target ligands containing a neuraminidase (NA) signaling anchor and an Fc domain that increases NA stalk length. [Figure 9] Figure 9 shows an example of a membrane-bound immune cell target ligand having an NA signaling anchor fused to the IgG Fc domain by an RS linker. [Figure 10] Figures 10A and 10B show the amino acid sequences (Figure 10A) and nucleic acid sequences (Figure 10B) of the original, clone 1, and consensus sequence (SEQ ID NO: 36) of NA1-Fc. [Figure 11] Figures 11A and 11B show the amino acid sequence (Figure 11A) and nucleic acid sequence (Figure 11B) of NA2-Fc. [Figure 12] Figures 12A and 12B show the amino acid sequence (Figure 12A) and nucleic acid sequence (Figure 12B) of NA3-Fc. [Figure 13]Figures 13A and 13B show the amino acid sequence (Figure 13A) and nucleic acid sequence (Figure 13B) of NA4-Fc. [Modes for carrying out the invention]

[0027] Before the compounds, compositions, articles, devices, and / or methods of the present invention are disclosed and described, it should be understood that, unless otherwise specified, they are not limited to specific synthesis methods or specific recombinant biotechnology methods, nor are they limited to specific reagents, and are therefore, of course, subject to change. It should also be understood that the terms used herein are intended solely to describe specific embodiments and are not intended to limit them.

[0028] definition Throughout this application, various publications are referenced. The disclosures of these publications, in their entirety, are incorporated herein by reference to provide a more complete description of the state of the art relating to this application. The disclosed references are also discussed in the texts relating thereto, and the materials contained therein are incorporated herein by reference individually and specifically.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the meanings generally understood by those skilled in the art to which this invention pertains. The following references provide general definitions of many terms used in this invention to those skilled in the art: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd Ed. 1994), The Cambridge Dictionary of Science and Technology (Walker ed., 1988), The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991), and Hale & Marham, The Harper Collins Dictionary of Biology (1991). Where used herein, the following terms have the meanings associated therewith unless otherwise specified.

[0030] When describing elements of this disclosure or preferred embodiments thereof, the articles “a,” “an,” “the,” and “said” are intended to mean that one or more of the elements exist. The terms “comprising,” “including,” and “having” are intended to be comprehensive and mean that additional elements other than those listed may exist.

[0031] In this specification, a range may be expressed as “about” one particular value and / or “about” another particular value. Where such a range is expressed, another embodiment includes one particular value and / or another particular value. Similarly, where the use of the antecedent “about” expresses a value as an approximation, it will be understood that a particular value forms another embodiment. It will be further understood that each endpoint of a range is important both in relation to and independently of the other endpoints. There are several values ​​disclosed herein, and each value is also disclosed herein as “about” that particular value, in addition to the value itself. For example, where the value “10” is disclosed, then “about 10” is also disclosed. As will be well understood by those skilled in the art, where a value is disclosed, it will also be understood that “less than or equal to that value,” “greater than or equal to that value,” and possible ranges between that value are also disclosed. For example, where the value “10” is disclosed, “less than or equal to 10” and “greater than or equal to 10” are also disclosed. Furthermore, throughout this application, the data is provided in several different formats, and it is understood that this data also represents the range of endpoints and starting points, as well as any combination of data points. For example, if a specific data point "10" and a specific data point 15 are disclosed, it is understood that not only the range between 10 and 15 is disclosed, but also the ranges greater than 10, 10 or greater, less than 10, 10 or less, and equal to 10, greater than 15, 15 or greater, less than 15, 15 or less, and equal to 15. It is also understood that each unit between two specific units is disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0032] As used herein, the terms “optional” or “optional” mean that the events or circumstances described thereafter may or may not occur, and this description includes both cases in which such events or circumstances occur and cases in which they do not occur.

[0033] The term "linker" refers to at least a divalent portion having a binding site for one polypeptide and a binding site for another polypeptide. For example, a polypeptide can be bound to a linker via one of its functional groups, either at its N-terminus, its C-terminus, or its side chain. The linker is sufficient to separate two polypeptides by at least one atom, and in some embodiments by one or more atoms.

[0034] As used herein, “N-terminal side” or “amino-terminal” may refer to the orientation of a peptide, polypeptide, or protein, and not necessarily the N-terminus. In some embodiments, when a chimeric or fusion peptide, polypeptide, or protein is considered, the N-terminal side may refer only to a component of the chimeric or fusion peptide, polypeptide, or protein, and not to the entire structure. For example, when an Fc domain is considered and described as fused to its amino-terminus or an N-terminal side facing the cell, what is intended herein is a chimeric or fusion peptide, polypeptide, or protein in which a single anchor is at the N-terminus of the chimeric or fusion construct and actually spans the cell membrane. Thus, in such a chimera, the transmembrane anchor is bound to the amino-terminal side of the Fc domain, and the orientation of the Fc domain has the N-terminal side facing the cell, which is inverted relative to the Fc domain on a typical B cell, which typically has a carboxyl terminus extending into the cell membrane and an amino terminus extending into the extracellular matrix.

[0035] The terms "peptide," "polypeptide," and "protein" are used interchangeably to refer to polymers of amino acid residues.

[0036] As used herein, the term “sequence identity” refers to a quantitative measure of the degree of identity between two sequences of substantially equal length. Whether nucleic acid or amino acid sequences, the percentage of identity between two sequences is calculated by multiplying the number of exact matches between the two aligned sequences by the length of the shorter sequence by 100. Approximate alignment of nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be applied to amino acid sequences by using a scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, MOD Dayhoff ed., 5 suppl.3:353-358, National Biomedical Research Foundation, Washington, DC, USA, and normalized by Gribskov, Nucle. Acids Res. 14(6):6745-6763 (1986). An exemplary implementation of this algorithm for determining the percentage of sequence identity is provided by the Genetics Computer Group (Madison, Wis.) in the “Best Fit” utility application. Other programs suitable for calculating the percentage of identity or similarity between sequences are generally known in the art, for example, another alignment program, BLAST, is used with default parameters. For example, BLASTN and BLASTP can be used with the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; prediction=10; matrix=BLOSUM62; description=50 sequences; sorting=high score; database=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBankCDS translation+Swiss protein+Spupdate+PIR. Details of these programs can be found on the GenBank website.Generally, substitutions are conservative amino acid substitutions and are limited to exchanges within the following groups: Group 1: glycine, alanine, valine, leucine, and isoleucine; Group 2: serine, cysteine, threonine, and methionine; Group 3: proline; Group 4: phenylalanine, tyrosine, and tryptophan; and Group 5: aspartic acid, glutamic acid, asparagine, and glutamine.

[0037] Techniques for determining nucleic acid and amino acid sequence identity are known in the art. Typically, such techniques involve determining the nucleotide sequence of a gene's mRNA and / or the amino acid sequence encoded therein, and comparing these sequences with a second nucleotide or amino acid sequence. Genomic sequences can also be determined and compared in this manner. Generally, identity refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. Two or more sequences (polynucleotides or amino acids) can be compared by determining their identity percentage.

[0038] Since various modifications can be made to the cells and methods described above without departing from the scope of the present invention, all matters contained in the above description and the examples given below are intended to be interpreted as illustrative and not as limiting.

[0039] "Increase" can refer to any change that results in a greater quantity of symptoms, disease, composition, condition, or activity. An increase can be a statistically significant increase in any individual, median, or mean of a condition, symptom, activity, or composition. Thus, an increase can be an increase of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%, insofar as the increase is statistically significant.

[0040] "Reduction" can refer to any change that results in a smaller amount of symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene if the genetic output of the gene product containing that substance is less than the genetic output of the gene product without that substance. For example, a reduction could also be a change in the symptoms of a disorder, such that the symptoms are less severe than previously observed. A reduction can be a statistically significant reduction in any individual, median, or mean reduction of a condition, symptom, activity, or composition. Thus, a reduction can be a reduction of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%, insofar as the reduction is statistically significant.

[0041] "Inhibit," "to inhibit," and "inhibition" mean reducing activity, response, condition, disease, or other biological parameter. This may include, but is not limited to, the complete elimination of activity, response, condition, or disease. This may include, for example, a 10% reduction of activity, response, condition, or disease compared to natural or control levels. Thus, the reduction may be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount in between, compared to natural or control levels.

[0042] "To reduce" or other forms of the word, such as "to reduce" or "reduction," means a decrease in an event or characteristic (e.g., tumor growth). This is typically relative to some standard or expected value, in other words, it is relative, but it is understood that the standard or relative value is not necessarily required to be referenced. For example, "to reduce tumor growth" means to reduce the rate of tumor growth compared to a standard or control.

[0043] "Prevent" or other forms of the word, such as "preventing" or "prevention," means to stop a particular event or characteristic, to stabilize or slow the development or progression of a particular event or characteristic, or to minimize the likelihood of a particular event or characteristic occurring. Prevention is typically more absolute than reduction, for example, and therefore does not require comparison with a contrast. As used herein, something may be reduced but not prevented, while something that is reduced may be prevented. Similarly, something may be prevented but not reduced, while something that is prevented may be reduced. When reduction or prevention is used, it should be understood that the use of other words is also expressly disclosed unless specifically designated otherwise.

[0044] The term "subject" refers to any individual that is the target of administration or treatment. A subject may be a vertebrate, e.g., a mammal. In one embodiment, a subject may be a human, a non-human primate, a cattle, a horse, a pig, a dog, or a cat. A subject may also be a guinea pig, a rat, a hamster, a rabbit, a mouse, or a mole. Thus, a subject may be a human or a veterinary patient. The term "patient" refers to a subject under the treatment of a clinician, e.g., a physician.

[0045] The term "therapeutically effective" means that the amount of the composition used is sufficient to alleviate one or more causes or symptoms of a disease or disorder. Such alleviation may only require reduction or modification, and does not necessarily have to be elimination.

[0046] The term “treatment” refers to the medical management of a patient with the intention of curing, alleviating, stabilizing, or preventing a disease, pathological condition, or disorder. This term includes active treatment, i.e., treatment specifically directed toward improvement of a disease, pathological condition, or disorder, and also includes causal treatment, i.e., treatment directed toward the removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, i.e., treatment designed to alleviate symptoms rather than cure a disease, pathological condition, or disorder; preventive treatment, i.e., treatment directed toward minimizing, or partially or completely inhibiting, the onset of the associated disease, pathological condition, or disorder; and adjunct treatment, i.e., treatment used to supplement another specific therapy directed toward improvement of the associated disease, pathological condition, or disorder.

[0047] "Administration" to a subject includes any route for introducing or delivering the drug to the subject. Administration can be carried out by any preferred route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-articular, parenteral, intra-arterial, intradermal, intraventricular, intracranial, intraperitoneal, intrafocal, intranasal, rectal, vaginal, inhalation, via implanted reservoir, and parenteral (e.g., subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intrafocal, and intracranial injection or infusion techniques). As used herein, "concurrent administration," "administration in combination," "simultaneous administration," or "administered simultaneously" means that the compounds are administered at the same time or essentially immediately after each other. In the latter case, the two compounds are administered at times close enough that the observed results are indistinguishable from the results obtained if they were administered at the same time. "Systemic administration" refers to the introduction or delivery of a drug to a subject via a route that introduces or delivers the drug to a large area of ​​the subject's body (e.g., more than 50% of the body), for example, through an entry point into the circulatory or lymphatic system. In contrast, "local administration" refers to the introduction or delivery of a drug to a subject via a route that introduces or delivers the drug to the area of ​​the administration site or an area directly adjacent thereto, and does not introduce the drug systemically in a therapeutically significant amount. For example, a locally administered drug is readily detectable in the local vicinity of the administration site, but is undetectable or detectable in negligible amounts in distal parts of the subject's body. Administration includes self-administration and administration by another person. In some embodiments, the compositions disclosed herein are administered parenterally, intravenously, intraperitoneally, or subcutaneously, or via arterial infusion, intravenous infusion, or artificial catheter-mediated infusion.

[0048] When used herein, “treat,” “treating,” “treatment,” and their grammatical variations include the administration of a composition intended or aimed at partially or completely preventing, delaying, curing, resolving, alleviating, relieving, altering, remedying, ameliorating, improving, stabilizing, mitigating, and / or reducing the intensity or frequency of one or more diseases or conditions, the symptoms of a disease or condition, or the underlying causes of a disease or condition. Treatments according to the present invention may be applied preventively, prophylactically, palliatively, or therapeutically. Prophylactic treatment is administered to a subject before the onset of symptoms (e.g., before obvious signs of cancer), during early onset (e.g., at the time of the initial signs and symptoms of cancer), or after the development of established cancer. Prophylactic administration may be carried out from one day (several days) to several years before the onset of symptoms of a disease or infection.

[0049] Compositions and methods The present invention, as conceived herein, is a composition for proliferating γδT cells and its use, comprising the Fc domain of an antibody suitable for agonizing an Fc receptor (e.g., CD16) conjugated on feeder cells, engineered particles, exosomes, or several other solid supports. Feeder cells, engineered particles, exosomes, and other solid supports having a conjugated Fc domain may also be included with other stimulants such as membrane-bound IL-21, 4-1BBL, other cytokines, or other stimulant (or inhibitory) receptors and other chemical moieties that engage simultaneously with the corresponding signaling pathways. As described above, challenges remain in utilizing γδT cells for clinical applications. The compositions and methods disclosed herein demonstrate remarkable efficacy in the induction, activation, and / or proliferation of γδT cells in vivo and / or in vitro. Proliferated γδT cells are effective in treating diseases such as cancer or infectious diseases.

[0050] I. Peptides Accordingly, disclosed herein are γδT cell proliferation compositions, the compositions comprising engineered feeder cells, engineered plasma membrane particles, exosomes, and engineered lymphocytes (e.g., lymphocytes engineered to express an Fc domain to stimulate γδT cells, such as T cells), and solid supports (as specified herein, Fc-bound feeder cells, Fc-bound engineered plasma membrane particles, and Fc-bound exosomes and Fc-bound lymphocytes, respectively), the Fc fusion peptide comprising a transmembrane peptide main linked to the amino or carboxyl terminus of the Fc domain. In one embodiment, the transmembrane domain of the Fc fusion peptide may contain cleaved or uncleaved signal anchor sequences, such as the transmembrane domain of neuraminidase, a signal anchor derived from paranfluenza virus hemagglutinin-neuraminidase, a signal anchor derived from a transferrin receptor, a signal anchor derived from an MHC class II invariant chain, a signal anchor derived from P-glycoprotein, a signal anchor derived from an asialoglycoprotein receptor, or a signal anchor derived from a neutral endopeptidase. In one example, the transmembrane domain contains a paranfluenza virus hemagglutinin-neuraminidase (NA) peptide sequence. The transmembrane neuraminidase (NA) peptide domain may couple or bind the Fc domain to the outer surface of a feeder cell. In another embodiment, the transmembrane neuraminidase (NA) peptide domain is used to couple or bind the Fc domain to the outer surface of engineered feeder cells, engineered plasma membrane nanoparticles, exosomes, or a solid support. In some embodiments, the NA peptide domain consists of an N-terminal cytoplasmic tail, an uncleaved signal anchor functioning as a transmembrane domain, and a stalk region extending from the plasma membrane. The length of the stalk region can be varied, and it will be understood that the length of the stalk region affects the effectiveness of the surface-bound Fc domain-NA peptide in stimulating γδT cell proliferation.

[0051] In some embodiments, the transmembrane domain contains a paranfluenza virus hemagglutinin-neuraminidase (NA) peptide sequence. In some embodiments, the NA peptide domain contains a sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. The transmembrane domain and the Fc domain may be linked via a peptide linker.

[0052] The Fc domain is a ligand to which the γδT cell surface receptor CD16 (FcγRIII) binds. CD16 is one of the primary receptors on γδT cells that bind to the Fc portion of the Fc domain of antibodies (e.g., IgG1, IgG2, IgG3, and / or IgG4). In another embodiment, the Fc domain (IgG1, IgG2, IgG3, and / or IgG4) can also bind to CD16 receptors on other immune cells such as mast cells, macrophages, or antigen-presenting cells. In another embodiment, other types of cells may be engineered to bind to Fc. This disclosure provides Fc-binding engineered feeder cells, Fc-binding engineered plasma membrane particles, Fc-binding exosomes, or Fc-binding solid supports.

[0053] In some embodiments, the Fc domain comprises an immunoglobulin Fc domain selected from IgG1, IgG2, IgG3, IgG4, IgA, and IgE. In some embodiments, the Fc domain binds to CD16.

[0054] In another embodiment, other Fc immunoglobulin isotypes other than IgG (IgA, IgE, IgM) may be used to stimulate different Fc receptors corresponding to the stimulation of other immune cell types. For example, domain FcαRI(CD89) specifically binds to IgA on macrophages, neutrophils, and eosinophils; FcγRI(CD64) specifically binds to IgG on monocytes and macrophages; and FcεRII(CD23) specifically binds to IgE on B cells. Fc binds to CD64 on monocytes or macrophages and thus stimulates them. Thus, fusion peptides, Fc-binding feeder cells (FCs), Fc-binding lymphocytes, Fc-binding engineered plasma membrane (PM) particles, Fc-binding engineered exosomes, and compositions containing them may also be used to substantially proliferate mast cells and / or macrophages according to the methods herein for proliferating γδT cells.

[0055] In one embodiment, disclosed herein is a fusion peptide comprising a transmembrane domain (e.g., an immunoglobulin Fc domain fused to an NA peptide domain, e.g., IgG1, IgG2, IgG3, IgG4, IgA, and / or IgE Fc domains) as described above. The Fc domain may be presented as a monomer, dimer, or multimer construct. In one embodiment, the Fc domain may be further modified to optimize or enhance the proliferation and / or activation of γδ T cells. For example, the Fc domain may be modified to increase its affinity for CD16. Thus, for example, the Fc domain may include one or more mutations such as T256A, K290A, S298A, E333A, K334A, L235V, F243L, R292P, Y300L, and / or P396L. Similarly, the Fc domain may be further modified to increase the selectivity of binding to activating (IIIa) versus inhibitory Fc(IIb) receptors. Therefore, for example, the Fc domain may contain one, two, three, four, five, six, seven, eight or more mutations, or alternative forms such as S239D, I332E, A330L, F243L, R292P, V305I, and / or P396L. For example, in one embodiment, the Fc domain may be modified to include R292L, Y300L, V305I, and P396L. In another example, the Fc domain may be modified to include S239D, I332E, and A330L.

[0056] Transmembrane domains, such as NA peptide domains, can be linked to Fc domains directly or indirectly via linkers. Direct chemical bonds are, for example, covalent bonds (e.g., peptide bonds, ester bonds, etc.) or, alternatively, non-covalent bonds (e.g., ionic, electrostatic, hydrogen, hydrophobic, van der interaction, or π effect). Indirect linkage can be achieved using linkers, i.e., chemical groups that connect one or more other chemical groups via at least one covalent bond. Suitable linkers include amino acids, peptides, nucleotides, nucleic acids, dimeric hinge Fc, organic linker molecules (e.g., maleimide derivatives, N-ethoxybenzylimidazole, biphenyl-3,4',5-tricarboxylic acid, p-aminobenzyloxycarbonyl, etc.), disulfide linkers, and polymer linkers (e.g., PEG). The linker may contain one or more spacing groups, including but not limited to alkylene, alkenylene, alkynylene, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, aralkyl, aralkenyl, and aralquinyl. The linker may be neutral or have a positive or negative charge. In addition, the linker may be cleavable so that the covalent bond of the linker connecting to another chemical group can be broken or cleaved under specific conditions, including pH, temperature, salt concentration, light, catalyst, or enzyme. In one embodiment, the NA peptide domain may be NA4-fc siadel (S239D / I332E / A330L).

[0057] In one embodiment, the linker may be a peptide linker. Examples of suitable peptide linkers are well known in the art, and programs for designing linkers are readily available (e.g., Crasto et al., Protein Eng., 2000, 13(5):309-312). Peptide linkers may be, for example, restriction site linkers such as short sequence RS, or flexible amino acid linkers (e.g., including small, nonpolar or polar amino acids). Non-limiting examples of flexible linkers include LEGGGS (SEQ ID NO: 5), TGSG (SEQ ID NO: 6), GGSGGGSG (SEQ ID NO: 7), (GGGGS). 1-4(Sequence 8), (GGGS) 1-4 (Sequence 9), (GSGGGG) 1-4 (Sequence ID 10), and (Gly) 6-8 (Sequence ID 11) is an example. Alternatively, the peptide linker may be a rigid amino acid linker. Such a linker may be (EAAAK) 1-4 (Sequence ID 12), A(EAAAK) 2-5 A (sequence number 13), PAPAP (sequence number 14), and (AP) 6-8 (Sequence ID 15) is an example. The Fc domain can be ligated to the N-terminus, C-terminus, and / or internal position of the NA peptide. In one embodiment, the peptide linker may be a short amino acid sequence of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In another embodiment, the peptide linker may be an amino acid sequence of any length of 2-10, 2-8, or 2-6 amino acids.

[0058] In some embodiments, the Fc fusion peptide is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of MNPNQKITTIGSICLVVGLISLILQIGNIISIWISHSIQTGSQNHTGICNRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV It has an amino acid sequence containing DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 16).

[0059] In some embodiments, the Fc fusion peptide is MNPNQKITTIGSICLVVGLISLILQIGNIISIWISHSIQTGSQNHTGICNQNIITYKNSTWVKDTTSVILTGNSSLCPIRRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA It has an amino acid sequence that has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 17).

[0060] In some embodiments, the Fc fusion peptide is MNPNQKITTIGSICLVVGLISLILQIGNIISIWISHSIQTGSQNHTGICNQNIITYKNSTWVKDTTSVILTGNSSLCPIRGWAIYSKDNSIRIGSKGDVFRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP It has an amino acid sequence that has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 18).

[0061] In some embodiments, the Fc fusion peptide is MNPNQKITTIGSICLVVGLISLILQIGNIISIWISHSIQTGSQNHTGICNQNIITYKNSTWVKDTTSVILTGNSSLCPIRGWAIYSKDNSIRIGSKGDVFVIREPFISCSHLECRTFFLTRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY It has an amino acid sequence that has at least approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 19).

[0062] To target the placement of the Fc domain on the plasma membrane, a well-characterized membrane targeting domain derived from influenza virus neuraminidase protein (NA) can be used, which consists of an N-terminal cytoplasmic tail, an uncleaved signal anchor functioning as a transmembrane domain, and a stalk region extending from the plasma membrane. Figures 7A and 7B are schematic diagrams showing the construction of membrane-bound immune cell targeting ligands containing the uncleaved signal anchor sequence. Figure 7A shows the structures of type I and type II essential membrane proteins, as well as their respective signal anchors. Figure 7B shows the structure of the uncleaved signal anchor derived from the type II essential membrane protein used in the membrane-bound immune cell targeting ligand. As shown in Figure 7B, exemplary but non-limiting constructs according to this disclosure consist of NA-Fc chimeras in which an Fc domain (IgG1) is linked to the uncleaved NA stalk region via a short linker. In particular, NA-Fc chimeras can be inserted into recombinant P / V / F viruses to generate novel oncolytic viruses that are specific to tumor versus normal cells (due to P / V mutations) and can enhance ADCC by NK cells. Figure 8 shows alternative structures of NA-Fc chimeras that increase NA stalk length.

[0063] The NA-Fc construct consists of an NA peptide domain (SEQ ID NO: 1), a linker (e.g., an RS linker), a hinge region DKTHTCPPCPAPELL (SEQ ID NO: 20) or TCPPCPAPELL (SEQ ID NO: 21), a CH2 domain GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 23), and a CH3 domain GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS The FC region containing DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 24) may include GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 22). It is understood and intended herein that NA-Fc chimeras can contain membrane targeting domains of any length derived from well-characterized influenza virus neuraminidase protein (NA), including MNPNQKITTIGSICLVVGLISLILQIGNIISIWISHSIQTGSQNHTGICNQNIITYKNSTWVKDTTSVILTGNSSLCPIR (SEQ ID NO: 2), MNPNQKITTIGSICLVVGLISLILQIGNIISIWISHSIQTGSQNHTGICNQNIITYKNSTWVKDTTSVILTGNSSLCPIRGWAIYSKDNSIRIGSKGDVF (SEQ ID NO: 3), and MNPNQKITTIGSICLVVGLISLILQIGNIISIWISHSIQTGSQNHTGICNQNIITYKNSTWVKDTTSVILTGNSSLCPIRGWAIYSKDNSIRIGSKGDVFVIREPFISCSHLECRTFFLT (SEQ ID NO: 4).

[0064] As described above, the Fc region may contain one or more mutations such as L234Y, L235V, L235Q, G236W, S239D, S239M, F243L, T256A, K290A, R292P, N297Q, S298A, Y300L, V305I, A330L, I332E, E333A, K334A, and / or P396L. Therefore, what is specifically disclosed herein is leucine (L) or tyrosine (Y) at residue 234, leucine (L), glutamine or valine (V) at residue 235, glutamine (G) or tryptophan (W) at residue 236, serine (S), methionine (M) or asparagine (D) at residue 239, and phenylalanine (F) or leucine (L) at residue 243, threonine (T) or alanine (A) at residue 256, histidine (H) or asparagine (D) at residue 268, asparagine (D) or glutamic acid (E) at residue 270, lysine (K) or alanine (A) at residue 290, arginine (R) or at residue 292 The Fc region includes proline (P), serine (S) or alanine (A) at residue 298, asparagine or glutamine at residue 297, tyrosine (Y) or leucine (L) at residue 300, valine (V) or isoleucine (I) at residue 305, lysine (K) or aspartin (D) at residue 326, alanine (A), methionine (M), or leucine (L) at residue 330, and isoleucine (I) or glutamic acid (E) at residue 332, glutamic acid (E) or alanine (A) at residue 333, lysine (K), glutamic acid (E), or alanine (A) at residue 334, and / or proline (P) or leucine (L) at residue 396. It is particularly understood that the Fc region may be free of substituents, or any one of the substitutions referred to herein, or a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17.Accordingly, one embodiment disclosed herein is a fusion protein comprising substitutions of the Fc region at F243L, R292P, Y300L, V305I, and P396L, wherein the sequence of NA4-Fc is MNPNQKITTIGSICLVVGLISLILQIGNIISIWISHSIQTGSQNHTGICNQNIITYKNSTWVKDTTSVILTGNSSLCPIRGWAIYSKDNSIRIGSKGDVFVIREPFISCSHLECRTFFLTDKTHTCPPCPAPELLG Includes GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 25).In one embodiment, the NA4-Fc fusion is the sequence GGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAK (SEQ ID NO: 27) and MNPNQKITTIGSICLVVGLISLILQIGNIISIWISHSIQTGSQNHTGICNQNIITYKNSTWVKDTTSVILTGNSSLCPIRGWAIYSKDNSIRIGSKGDVFVIREPFISCSHLECRTFFLTDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYT The substitutions include S293D, I332E, and A330L, which have an Fc domain containing the sequence GGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 26), which has a CH2 domain containing the complete sequence of LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 28).

[0065] In another example, the Fc region of the NA-Fc fusion is, 23 CH2 domain having F243, R292, Y300, and V305 as described in GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK, and Sequence ID24 GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID) 22 The NA-Fc fusion has an Fc domain having the sequence MNPNQKITTIGSICLVVGLISLILQIGNIISIWISHSIQTGSQNHTGICNQNIITYKNSTWVKDTTSVILTGNSSLCPIRGWAIYSKDNSIRIGSKGDVFVIREPFISCSHLECRTFFLTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID) 29 ) may include. In another example, the Fc region of the NA-Fc fusion is, 23 CH2 domains having F243L, R292P, Y300L, and V305I substitutions as described in GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK, and Sequence ID 24P396L in the CH3 domain described in GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, and sequence GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID) 22 A full-length Fc domain having the sequence MNPNQKITTIGSICLVVGLISLILQIGNIISIWISHSIQTGSQNHTGICNQNIITYKNSTWVKDTTSVILTGNSSLCPIRGWAIYSKDNSIRIGSKGDVFVIREPFISCSHLECRTFFLTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID) 30 It includes an NA-Fc fusion having ).

[0066] In one embodiment, the NA4-Fc fusion protein may contain two Fc domains linked via a hinge region. For example, the NA-Fc fusion may have a sequence (SEQ ID NO:). 31 ) may include.

[0067] In another embodiment, the Fc domains may be asymmetric variants, for example, one heavy chain Fc domain may contain L234Y / L235Q / G236W / S239M / H268D / D270E / S298A, and the other Fc domain may contain D270E / K326D / A330M / K334E.

[0068] Generally, any amino acid substitution is conservative and limited to substitutions within the following groups: Group 1: glycine, alanine, valine, leucine, and isoleucine; Group 2: serine, cysteine, threonine, and methionine; Group 3: proline; Group 4: phenylalanine, tyrosine, and tryptophan; and Group 5: aspartic acid, glutamic acid, asparagine, and glutamine.

[0069] In some embodiments, the NA-Fc fusion is GAATTCCAGGGGGTTTAAAATGAATCCAAATCAGAAAATAACAACCATTGGATCAATCTGTCTGGTAGTCGGACTAATTAGCCTAATATTGCAAATAGGGAATATAATCTCAATATGGATTAGCCATTCAATTCAAACTGGAAGTCAAAACCATACTGGAATATGCAACAGATCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCTAGCTGG (SEQ ID NO 32)、GAATTCCAGGGGGTTTAAAATGAATCCAAATCAGAAAATAACAACCATTGGATCAATCTGTCTGGTAGTCGGACTAATTAGCCTAATATTGCAAATAGGGAATATAATCTCAATATGGATTAGCCATTCAATTCAAACTGGAAGTCAAAACCATACTGGAATATGCAACCAAAACATCATTACCTATAAAAATAGCACCTGGGTAAAGGACACAACTTCAGTGATATTAACCGGCAATTCATCTCTTTGTCCCATCCGTAGATCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCTAGCTGG(SEQ ID NO 333435 Coded by ).

[0070] This disclosure also intends to provide nucleic acids encoding any fusion protein disclosed herein, vectors comprising such nucleic acids as described in the claims, and cells comprising such vectors. Vectors and cells comprising such vectors may be prepared using methods known in the art.

[0071] II. Manipulated feeder cells, manipulated plasma membrane particles, and manipulated exosomes containing membrane-bound Fc The compositions of this disclosure include compositions comprising Fc-binding feeder cells (FCs), compositions comprising Fc-binding engineered plasma membrane (PM) particles, and compositions comprising Fc-binding engineered exosomes. Fc-binding engineered PM particles include PM nanoparticles derived from Fc-binding feeder cells. Fc-binding engineered exosomes include exosomes or other extracellular vesicles derived from Fc-binding feeder cells, as described in more detail below. Alternatively, exosomes may be derived from other sources such as platelets and megakaryocytes.

[0072] As used herein, the term “Fc binding” should be understood to refer to the coupling of a reverse-bound (i.e., inward-facing amino-terminus) Fc domain to the outer surface of a feeder cell or engineered particle via a transmembrane peptide. This can be achieved using the Fc fusion peptides disclosed herein. Thus, one aspect of the present disclosure provides a feeder cell composition comprising at least one Fc-bound feeder cell, i.e., a feeder cell comprising an Fc domain bound to the outer surface of the feeder cell, as described in further detail below. For example, the feeder cell may be genetically modified to express an Fc domain bound to the outer surface of the feeder cell, i.e., to express an Fc fusion peptide, as described in further detail below. Another aspect of the present disclosure provides an NK cell proliferation composition that does not comprise a feeder cell, comprising at least one Fc-bound engineered particle, i.e., an engineered particle comprising an Fc domain bound in reverse to the outer surface of a feeder cell. In some aspects, the feeder cell may be engineered to express an agonist (e.g., an agnostic antibody) or ligand (e.g., CD20) that can be tagged with a humanized antibody. In some embodiments, feeder cells are engineered to express CD20 and then opsonized with rituxan.

[0073] In a feeder cell composition, at least one Fc-bound feeder cell optionally contains at least one γδT cell effector agent, the at least one γδT cell effector agent containing a cytokine, adhesion molecule, or γδT cell activator. For example, an Fc-bound feeder cell contains an agonist (e.g., agnostic antibody) or ligand of 4-1BBL;CD80;CD86;MICA;UBLP;2B4;LFA-1;NKG2D, NKp46, NKp44, NKp30, or DNAM-1; an agonist (e.g., agnostic antibody) or ligand of Notch, BCM / SLAMF2, or TLR; IL-2;IL-12;IL-18;IL-15; or IL-21; or any combination thereof, at least one γδT cell effector selected from the group. In one example, at least one γδ T cell effector agent includes 4-1BBL, IL-18, IL-15, or IL-21, or any combination thereof. In one example, the Fc-binding feeder cells include one γδ T cell effector which is IL-15 or IL-21. In one example, the Fc-binding feeder cells may include at least two or more different γδ T cell effector agents. For example, the Fc-bound feeder cells include at least one γδ T cell effector selected from membrane-bound 4-1BBL, IL-2, or IL-21 or a combination thereof (e.g., mb4-1BBL and mbIL-21; mb4-1BBL and mbIL-2; mbIL-2 and mbIL-21; or mb4-1BBL, mbIL-2, and mbIL-21, etc.), as well as combinations of membrane-bound and unbound effector agents, including but not limited to IL-2, IL-21, or 4-1BBL, or a combination thereof (e.g., 4-1BBL and IL-21; 4-1BBL and IL-2; IL-21 and IL-2; or 4-1BBL, IL-2, and IL-21, etc.).

[0074] In a γδT cell proliferation composition that does not contain feeder cells, the Fc-binding plasma membrane particles optionally contain at least one γδT cell effector agent, the at least one γδT cell effector agent containing a cytokine, adhesion molecule, or γδT cell activator. For example, the Fc-binding particles contain at least one γδT cell effector selected from the group consisting of 4-1BBL;CD80;CD86;MICA;UBLP;2B4;LFA-1;NKG2D, NKp46, NKp44, NKp30, or DNAM-1 agonists (e.g., agnostic antibodies) or ligands; Notch, BCM / SLAMF2, or TLR agonists (e.g., agnostic antibodies) or ligands; IL-2;IL-12;IL-18;IL-15; or IL-21; or any combination thereof. In one example, at least one γδ T cell effector agent includes 4-1BBL, IL-18, IL-15, or IL-21, or any combination thereof. In another example, an Fc-binding engineered particle contains one γδ T cell effector which is either IL-15 or IL-21. An Fc-binding engineered PM particle may contain at least two or more different γδ T cell effector agents. In one example, the Fc-binding manipulated particles contain at least one γδ T cell effector selected from membrane-bound 4-1BBL, IL-2, or IL-21 or a combination thereof (e.g., mb4-1BBL and mbIL-21; mb4-1BBL and mbIL-2; mbIL-2 and mbIL-21; or mb4-1BBL, mbIL-2, and mbIL-21, etc.), as well as combinations of membrane-bound and unbound effector agents, including but not limited to IL-2, IL-21, or 4-1BBL, or a combination thereof (e.g., 4-1BBL and IL-21; 4-1BBL and IL-2; IL-21 and IL-2; or 4-1BBL, IL-2, and IL-21, etc.).

[0075] (a) Fc-bound feeder cells This disclosure provides feeder cells containing the above-described Fc fusion peptide. γδ T cell feeder cells for use in the methods disclosed herein and in the production of PM particles and exosomes disclosed herein may be irradiated autologous or allogeneic peripheral blood mononuclear cells (PBMCs), fibroblasts, epithelial cells, endothelial cells, antigen-presenting cells (e.g., dendritic cells, B cells, mast cells, macrophages, monocytes), T cells, NK cells, microbial cells, or any of the non-irradiated autologous or allogeneic PBMCs, RPMI8866, HFWT, 721.221, or K562 cells, and EBV-LCL, other non-HLA or low-HLA expressing cell lines, or patient-derived primary tumors that can be used as tumor vaccines. Microbial cells may be bacterial cells. Microcaries may be non-disease-causing cells (e.g., Bacillus Calmette-Guerin). In some embodiments, the microbial cells are cells of probiotics (e.g., Lactobacillus, Bifidobacterium, Streptococcus, Bacillus, Lactococcus, Enterococcus, Pediococcus, Propionibacterium, Peptostreptococcus, or Saccharomyces). Fc-bound feeder cells can be prepared by transfecting or transfecting feeder cells with any of the Fc fusion peptides described herein using standard transduction or transfection techniques well known in the art. For example, a cDNA vector of an Fc fusion peptide disclosed herein can be ligated into an expression plasmid that enables expression in bacterial (E. coli), insect, or mammalian cells. The cDNA vector may be FLAG-tagged or HIS-tagged.Suitable transfection methods include nuclear fusion (or electroporation), calcium phosphate-mediated transfection, cationic polymer transfection (e.g., DEAE-dextran or polyethyleneimine), viral transfection, virosomal transfection, virion transfection, liposome transfection, cationic liposome transfection, immunoliposome transfection, non-liposomal lipid transfection, dendrimer transfection, heat shock transfection, magnetofection, lipofection, gene gun delivery, impalefection, sonoporation, optical transfection, and proprietary drugs with enhanced nucleic acid uptake. Transfection methods are well known in the art (see, for example, “Current Protocols in Molecular Biology” Ausubel et al., John Wiley & Sons, New York, 2003 or “Molecular Cloning: A Laboratory Manual” Sambrook & Russell, Cold Spring Harbor Press, Cold Spring Harbor, NY, 3rd edition, 2001). Alternatively, molecules can be introduced into cells by microinjection. For example, molecules can be injected into the cytoplasm or nucleus of the target cell. The amount of each molecule introduced into the cell may vary, but those skilled in the art are familiar with means for determining appropriate amounts.

[0076] For example, the feeder cells used in the present invention may be peripheral blood mononuclear cells (PBMCs), fibroblasts, epithelial cells, endothelial cells, antigen-presenting cells, or microbial cells, or cell lines, the cell lines may be RPMI8866, HFWT, 721.221, K562, or EBV-LCL.

[0077] It will be understood that various molecules can be introduced into cells simultaneously or sequentially. For example, an Fc fusion peptide and one or more membrane-bound γδ T cell effectors can be introduced into feeder cells simultaneously. Alternatively, one may be introduced first, and then other molecules may be introduced into the cells later. For example, feeder cells that have been transfected or transduced once with an Fc fusion peptide may be further transfected with membrane-bound γδ T cell effectors such as IL-2, IL-15 and / or IL-21 and / or 41BBL, and / or infected with EBV-LCL and / or other γδ T cell effectors. Alternatively, feeder cells may be transfected or transduced simultaneously with the Fc fusion peptide and membrane-bound γδ T cell effectors such as IL-2, IL-15 and / or IL-21 and / or 41BBL, and / or EBV-LCL and / or other γδ T cell effectors. Alternatively, feeder cells that have been previously transfected or transduced to express membrane-bound γδ T cell effectors such as IL-2, IL-15 and / or IL-21 and / or 41BBL, and / or infected with EBV-LCL and / or other γδ T cell effectors, can be transfected or transduced with the Fc fusion peptide. It will also be understood that membrane-bound Fc can be achieved using other means, such as chemical conjugation methods known in the art.

[0078] In general, apart from contact with the compositions disclosed herein, cells are maintained under conditions suitable for cell proliferation and / or maintenance. Suitable cell culture conditions are well known in the art and are described, for example, in Santiago et al., Proc. Natl. Acad. Sci. USA, 2008, 105:5809-5814, Moehle et al., Proc. Natl. Acad. Sci. USA, 2007, 104:3055-3060, Urnov et al., Nature, 2005, 435:646-651, and Lombardo et al., Nat. Biotechnol., 2007, 25:1298-1306. Those skilled in the art will understand that methods for culturing cells are known in the art and may and will vary depending on the cell type. In all cases, optimization of routine practices can be used to determine the best technique for a particular cell type.

[0079] Fc-bound feeder cells can be used in cell culture to directly stimulate γδT cells, or to prepare plasma membrane particles or exosomes derived from feeder cells.

[0080] (b) Fc-bonded plasma particles Fc-binding manipulated PM (plasma membrane) particles include Fc-binding PM particles that can be prepared from Fc-binding γδ T cell feeder cells using known methods. PM particles are vesicles prepared from the cell's plasma membrane or artificially prepared vesicles (i.e., liposomes). PM particles may contain a lipid bilayer or simply a single lipid layer. PM particles may be prepared in monolayer, multilayer, or inverted forms. PM particles may be prepared from Fc-binding feeder cells as described herein using known plasma membrane preparation protocols or protocols for preparing liposomes, such as those described in U.S. Patent No. 9,623,082 (the full disclosure thereof is incorporated herein by reference). In certain embodiments, the PM particles disclosed herein have an average diameter in the range of about 170 to about 300 nm.

[0081] (c) Fc-bonded plasma particles The Fc-bound exosomes disclosed herein may be prepared from exosome-secreting cells that can be prepared from Fc-bound feeder cells using known methods, and the exosomes are extracellular products of exosome-secreting cells, as described in U.S. Patent Application Publication No. 20170333479 (the full disclosure thereof is incorporated herein by reference). Exosomes comprise lipids and proteins, and the identity of the proteins found in a particular exosome depends on the cell that produces them. The exosomes disclosed herein comprise the Fc-fusion peptide disclosed herein (i.e., Fc-bound) and, optionally, one or more stimulating peptides (γδT cell effectors) present in the exosome membrane. Exosomes may be produced, for example, from cell lines engineered for improved exosome formation or release. Such cell lines include, but are not limited to, the Fc-bound cell lines described above in Section II(a). Non-limiting cell lines include Fc-bound K562-mb15-41BBL and Fc-bound K562-mb21-41BBL. In certain embodiments, the exosomes disclosed herein have an average diameter in the range of about 30 to about 100 nm or about 160 nm. In one embodiment, the exosomes have an average diameter of about 60 to 80 nm. The ability to have exosomes that achieve a particle size smaller than that readily achieved with PM particles means that exosomes can be readily adapted to uses where smaller size is preferred. For example, exosomes may be preferred in applications requiring diffusion across physiological barriers, enhanced in vivo distribution across tissue compartments, or intravenous injection.

[0082] III. Composition This disclosure provides various γδT cell proliferation compositions comprising the Fc-binding feeder cells disclosed above, and, in other embodiments, γδT cell proliferation compositions that do not comprise feeder cells, comprising one or more manipulated Fc-binding particles such as PM particles or exosomes disclosed above. Either the Fc-binding feeder cells or Fc-binding manipulated PM particles used in the composition optionally comprises at least one, two or more different γδT cell effector agents. In one embodiment, one γδT cell effector agent is IL-21, and in several embodiments, one γδT cell effector agent is IL-21 and a second T cell effector agent is 4-1BBL. The Fc-binding feeder cells or Fc-binding manipulated PM particles optionally comprise one or more of the above-mentioned additional γδT cell effector agents.

[0083] A γδ T cell proliferation composition containing PM particles with a plasma membrane may further contain multiple microparticles / nanoparticles, the plasma membrane coating the multiple microparticles. The microparticles / nanoparticles may include magnetic microparticles, silica beads, polystyrene beads, latex beads, microparticle contrast agents, microparticle cancer treatment agents, or any combination thereof.

[0084] This disclosure also envisions a γδT cell proliferation infusion formulation comprising any of the γδT cell proliferation compositions disclosed herein, combined with a pharmaceutically acceptable carrier.

[0085] Therapeutic pharmaceutical compositions may be prepared by combining Fc-bound feeder cells or engineered PM particles disclosed herein with pharmaceutically acceptable carriers known in the art, such as those described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. ARGennaro, Mack Publishing Company, Easton, Pa. 1995. Examples of pharmaceutically acceptable carriers include, but are not limited to, sterile water, saline, Ringer's solution, dextrose solution, and buffer solutions at physiological pH. For example, the pH of the solution is preferably about 5 to about 8, more preferably about 7 to about 7.5.

[0086] It will be apparent to those skilled in the art that certain carriers may be more preferable, for example, depending on the route of administration and the concentration of the composition being administered. Pharmaceutical compositions can be suitably prepared for administration to mammals, particularly humans, via any of several known routes of administration, depending on whether topical or systemic treatment is desired and the area being treated. Administration may be topical (including ocular, vaginal, rectal, and nasal), oral, inhaled, or parenteral, for example, by intravenous infusion or injection, or by subcutaneous, intraperitoneal, or intramuscular injection.

[0087] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including physiological saline and buffer media. Parenteral vehicles include sodium chloride solution, ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixative oils. Intravenous vehicles include fluids and nutritional supplements, electrolyte supplements (such as those based on ringer's dextrose), etc. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may also be present.

[0088] Topical formulations may include ointments, lotions, creams, gels, intravenous infusions, suppositories, sprays, liquids, and powders. Conventional drug carriers, aqueous, powder, or oily bases, and thickeners may be necessary or desirable.

[0089] Some of these compositions may potentially be administered as pharmaceutically acceptable acids or base addition salts formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with inorganic bases such as sodium hydroxide, ammonium hydroxide, and potassium hydroxide, and organic bases such as mono, di, trialkyl, and arylamines, and substituted ethanolamines.

[0090] Therefore, γδT cell proliferation infusion formulations can be formulated for parenteral infusion, arterial infusion, intravenous infusion, artificial catheter-mediated infusion, intravenous, intraperitoneal, subcutaneous injection, oral, or local delivery. In some embodiments, any of the methods described above include administering γδT cells parenterally, intravenously, intraperitoneally, or subcutaneously, or via arterial infusion, intravenous infusion, or artificial catheter-mediated infusion.

[0091] In one embodiment, the present disclosure envisions any γδT cell proliferation composition to be prepared in vitro or ex vivo as disclosed herein and administered to or injected into a subject requiring γδT cell proliferation. It is understood and envisioned herein that injection may be performed in vitro using a commercially available source of γδT cells or ex vivo from a donor source (e.g., an allogeneic or autologous donor source, i.e., a recipient subject that has received the proliferated γδT cells).

[0092] In another embodiment, the Disclosure envisions an in vitro γδT cell population in contact with the Fc-binding feeder cell composition disclosed herein, or a γδT cell composition comprising an Fc-binding NK cell proliferation composition that does not include the feeder cells disclosed herein.

[0093] In another embodiment, the disclosure envisions a population of proliferated γδT cells exposed in vitro to a γδT cell proliferation composition, the composition not containing feeder cells and comprising at least one Fc-bound engineered particle disclosed herein, comprising at least two γδT effector agents, the at least two γδT cell effector agents being selected from IL-2, IL-21, IL-15, or 4-1BBL, or any combination thereof. In another embodiment, the disclosure envisions a population of proliferated γδT cells exposed in vitro to a γδT cell proliferation composition, the composition not containing feeder cells and comprising at least one Fc-bound engineered particle disclosed herein, comprising at least two γδT cell effector agents, one of which is IL-21 or 4-1BBL. In one example, the γδT effector agent is IL-2. In one example, the γδT effector agent is IL-21. In one example, the γδT effector is IL-15. In another example, the γδT effector is 4-1BBL. The proliferated population of γδT cells may show increased cytotoxicity compared to non-proliferated γδT cells.

[0094] In different embodiments, a population of proliferated γδT cells may exhibit at least approximately 2, 5, or 10 times greater cytotoxicity than a non-proliferated γδT cell population, which may be determined by an increased percentage of the γδT cell population producing cytotoxic effectors (e.g., IFNγ, TNFα, perforin, granzyme), or by an increased production / expression level of cytotoxic effectors (e.g., IFNγ, TNFα, perforin, granzyme), or by an increased expression level of molecules for killing (e.g., FasL, TRAIL).

[0095] In some embodiments, any of the methods described above include contacting at least one γδT cell with a feeder cell, manipulated particle, exosome, or solid support in vitro, in vivo, or ex vivo. In some embodiments, the proliferated γδT cells include the Vδ2 subtype and / or the Vδ1 subtype. The γδT cells may be autologous, haplotype-identical, or allogeneic γδT cells. In some embodiments, the γδT cells are proliferated for at least 14 days, and at least about 5%, 10%, 20%, 30%, 40%, 50%, or 60% of the proliferated cells are Vδ2 subtype γδT cells.

[0096] In some embodiments, γδT cells proliferate at a faster rate over 14 days than the control γδT cell population. It should be understood that, as used herein, the term “control γδT cell population” refers to γδT cells before contact with the Fc-binding feeder cells, exosomes, engineered plasma membrane particles, or solid support disclosed herein, or to γδT cells in contact with the Fc-binding feeder cells, exosomes, engineered plasma membrane particles, or solid support disclosed herein.

[0097] γδT cells proliferated according to any of the aforementioned embodiments may be an isolated cell population or a mixed cell population. The mixed cell population may deplete NK cells before, during, or after γδT cell proliferation. Therefore, the method may result in co-proliferation of γδT cells and NK cells if NK cells are not removed before proliferation. The combination of these two populations may result in broader antitumor function and therefore better efficacy. Such γδT cells or γδT / NK cell mixtures may be used as therapeutic agents for the treatment of diseases.

[0098] In another aspect, the present disclosure provides, optionally in combination with a pharmaceutically acceptable carrier, a composition comprising a therapeutic dose of γδT cells, comprising a population of expanded γδT cells disclosed herein. The population of expanded γδT cells may exhibit other advantageous properties such as higher CD16 and higher cytotoxicity. The amount of γδT cells providing a therapeutic dose will vary depending on several factors as understood by those skilled in the art, and it is discussed, for example, in U.S. Patent No. 9,907,820, the entire disclosure of which is incorporated herein by reference. Factors include the age, sex, and diagnosis of the subject, as well as the route of administration, which can be, but is not limited to, oral, buccal, mucosal, and intravenous routes. For example, a dose suitable for a therapeutic effect is, for example, preferably at least 10 4 or about 10 4 to about 10 10 cells per dose, about 10 4 to about 10<, 8 cells per dose, or about 10 5 to about 10 7 cells per dose. An exemplary dosing regimen consists of four one-week escalating dose administration cycles starting at at least about 10 10 cells on day 0 and increasing stepwise to a target dose of about 10 5 cells within a few weeks from the start of the patient-internal dose escalation scheme. Suitable administration forms include intravenous, subcutaneous, intra-cavity (e.g., by a reservoir access device), intraperitoneal, and direct injection into the tumor mass. It will be understood that the equivalents of the therapeutic doses represented above can alternatively be expressed as an amount per total body surface area.

[0099] The range of the glycoprotein glycoprotein is 4-1BBL(HGNC:11939 Entrez Gene:8744). Ensemble:ENSG00000125657 OMIM:606182 UniProtKB:P41273)、CD80(HGNC:1700 Enter Gene:941 Ensembl:ENSG00000121594 OMIM:112203 UniProtKB:P33681)、CD86(HGNC:1705 Entrance Gene:942 Set:ENSG00000114013 OMIM:601020 UniProtKB:P42081);MICA(HGNC:7090 Entrance Gene:100507436 Ensembl:ENSG00000204520 OMIM:600169 UniProtKB:Q29983)、UBLP、2B4(HGNC:18171 Enter Gene:51744 Ensemble:ENSG00000122223 OMIM:605554 UniProtKB:Q9BZW8) and LFA-1(CD11a / CD18) and CD11a (HGNC:6148 Entrez Gene:3683 Set:ENSG00000005844 OMIM:153370 UniProtKB:P20701) and CD18 (HGNC:6155 Entrez Gene:3689 Ensemble:ENSG00000160255 OMIM:600065 UniProtKB:P05107)、ICAM-1(HGNC:5344 Entrez Gene:3383 Ensemble:ENSG00000090339 OMIM:147840 UniProtKB:P05362) of NKG2D (HGNC:18788 Entrez Gene:22914 Ensemble:ENSG00000213809 OMIM:611817 UniProtKB:P26718) NKp46(HGNC:6731 Entrez Gene:9437 Set:ENSG00000189430 OMIM:604530 UniProtKB:O76036)、NKp44(HGNC:6732 Entrez Gene:9436 Set:ENSG00000096264OMIM:604531 UniProtKB:O95944) and is NKp30(HGNC:19077 Entrez Gene:259197 Set:ENSG00000204475 OMIM:611550 UniProtKB:O14931) is a DNAM-1 fragment (HGNC:16961 Entrez Gene:10666 Ensemble:ENSG00000150637 OMIM:605397 UniProtKB:Q15762)、IL-2(HGNC:6001 Enter Gene:3558 Ensemble:ENSG00000109471 OMIM:147680 UniProtKB:P60568)、IL-12(HGNC:5969 Entrance Gene:3592 Set:ENSG00000168811 OMIM:161560 UniProtKB:P29459); Ensemble:ENSG00000150782 OMIM:600953 UniProtKB:Q14116) 、IL-15(HGNC:5977 Entrez Gene:3600 Ensemble:ENSG00000164136 OMIM:600554 UniProtKB:P40933、IL-21(HGNC:6005 Entrance Gene:59067 Ensemble:ENSG00000138684 OMIM:605384 UniProtKB:Q9HBE4)、CD69(HGNC:1694 Entrance Gene:969 Ensemble:ENSG00000110848 OMIM:107273 UniProtKB:Q07108)、CD25(HGNC:6008 Entrez Gene:3559 Ensemble:ENSG00000134460 OMIM:147730 UniProtKB:P01589).

[0100] IV.Experience in the snow (a) Methods and uses for increasing the cytotoxicity of γδT cells In one embodiment, the Disclosure provides a method for increasing the cytotoxicity of γδT cells by growing a population of early γδT cells using a γδT cell proliferation composition or formulation disclosed herein. Alternatively, the Disclosure provides the use of a γδT cell proliferation composition or formulation disclosed herein for increasing the cytotoxicity of γδT cells by growing a population of early γδT cells. The methods and uses of the Disclosure provide a simple proliferation platform that avoids complex alternative processes for proliferation, such as coating a solid support with a monoclonal antibody and using soluble cytokines in solution. Instead, in the methods and uses disclosed herein, a population of early γδT cells is obtained from a donor and exposed to a γδT cell proliferation composition disclosed herein. In the therapeutic method, exposure may be in vitro or in vivo. In the use, exposure may be in vitro or ex vivo. In either of the methods and uses, γδT cells are brought into contact with one or more Fc-binding feeder cells, Fc-binding PM particles, or Fc-binding exosomes, or any combination thereof. The exposed Fc domains bind to CD16 on the surface of γδT cells, stimulating them to proliferate more rapidly and / or more efficiently, producing γδT cells with higher antitumor toxicity and a more favorable overall phenotype.

[0101] In either method or use, the composition in contact with γδT cells may include any of the Fc-binding feeder cells, Fc-binding engineered PM particles, or Fc-binding engineered exosomes disclosed herein. The engineered PM particles may be Fc-binding PM particles. In one embodiment, the optionally present γδT cell effector is IL-21 or IL-15. The optionally present second γδT cell effector may be selected from 4-1BBL, IL-2, IL-12, IL-15, IL-18, IL-21, MICA, UBLP, 2B4, LFA-1, Notch ligand, NKp46, or agonists (e.g., agnostic antibodies) or ligands of BCM1 / SLAMF2, or agonists (e.g., agnostic antibodies) or ligands of TLR and NKG2D. In one embodiment, the second NK cell effector is 4-1BBL. The crude product may further comprise at least one additional (i.e., third, fourth, fifth, etc.) γδ T cell effector agent selected from agonists (e.g., agnostic antibodies) or ligands of IL-2, IL-12, IL-15, IL-18, IL-21, MICA, UBLP, 2B4, LFA-1, Notch ligand, NKp46, or BCM1 / SLAMF2, and agonists (e.g., agnostic antibodies) or ligands of TLR and NKG2D. In some embodiments, the γδ T cell effector agent comprises 4-1BBL, IL-18, IL-15, or IL-21, or any combination thereof, and γδ T cell proliferation carried out in this manner can be achieved much more significantly (about 3-4 times) in 10 days. Rather, the proliferation of γδ T cells by this method can achieve an increase in the number of γδ T cells of at least approximately 100 times, approximately 200 times, approximately 300 times, approximately 400 times, approximately 500 times, approximately 600 times, approximately 700 times, approximately 800 times, approximately 900 times, approximately 1100 times, approximately 1200 times, approximately 1300 times, approximately 1400 times, approximately 1500 times, approximately 1600 times, approximately 1700 times, approximately 1800 times, approximately 1900 times, and approximately 2000 times over a period of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 1300 times, approximately 1400 times, approximately 1500 times, approximately 1600 times, approximately 1700 times, approximately 1800 times, approximately 1900 times, and approximately 2000 times.Therefore, the disclosed methods and uses are useful for the scale-up production of γδT cells. Sources of γδT cells may include peripheral blood, spleen γδT cells, lymphocyte preparations such as buffy coat, iPSC-derived γδT cells, and genetically modified / manipulated γδT cells, or any genetically modified γδT cells, including but not limited to those derived from polymorphisms of the Fc receptor such as Phe or Val at position 158, as known in the art and described, for example, in Blood (1997) 90:1109-14 and J Clin Invest. (1997) 100:1059-70. Such genetically modified γδT cell sources may be manipulated using methods known in the art. Alternatively, γδT cells may be derived from cell donors having the desired polymorphism, as well as donor cells used as a population of initial γδT cells to be proliferated by using the methods and compositions described herein. Therefore, in this context, "genetically modified" includes naturally occurring γδT cells that have polymorphisms. This method may be applied to γδT cells of human origin or other animal origin.

[0102] In some embodiments, any of the methods or uses described above involve contacting at least one γδT cell with a feeder cell, manipulated particle, exosome, or solid support in vitro, in vivo, or ex vivo. In some embodiments, the proliferated γδT cells include the Vδ2 subtype and / or the Vδ1 subtype. The γδT cells may be autologous, haplotype-identical, or allogeneic γδT cells. In some embodiments, the γδT cells are proliferated for at least 14 days, and at least about 5%, 10%, 20%, 30%, 40%, 50%, or 60% of the proliferated cells are Vδ2 subtype γδT cells.

[0103] In some embodiments, γδT cells proliferate at a faster rate over 14 days than the control γδT cell population. It should be understood that, as used herein, the term “control γδT cell population” refers to γδT cells before contact with the Fc-binding feeder cells, exosomes, engineered plasma membrane particles, or solid support disclosed herein, or to γδT cells in contact with the Fc-binding feeder cells, exosomes, engineered plasma membrane particles, or solid support disclosed herein.

[0104] γδT cells proliferated according to any of the aforementioned methods or uses may be isolated cell populations or mixed cell populations. Mixed cell populations may deplete NK cells before, during, or after γδT cell proliferation. Therefore, the method may result in co-proliferation of γδT cells and NK cells if NK cells are not removed before proliferation. The combination of these two populations may result in broader antitumor function and therefore better efficacy. Such γδT cells or γδT / NK cell mixtures may be used as therapeutic agents for the treatment of diseases.

[0105] Furthermore, the disclosed methods and uses have the additional benefit of providing cells with higher cytotoxicity. Populations of early γδT cells proliferated according to the methods of this disclosure produce proliferated γδT cell populations exhibiting at least approximately twice the cytotoxicity of the early γδT cell population, at least approximately four times the cytotoxicity of the early γδT cell population, at least approximately five times the cytotoxicity of the early γδT cell population, at least approximately eight times the cytotoxicity of the early γδT cell population, or at least approximately ten times the cytotoxicity of the early γδT cell population. As a non-limiting example, the relative cytotoxicity of proliferated γδT cells can be assessed compared to non-proliferated γδT cells or γδT cells proliferated under other conditions by using higher expression of ADCC-related proteins such as CD16, or as a non-limiting example, other γδT cell ligands such as NKG2D, NKp46, CD62L, ICAM-1. Markers such as CD69, CD25, and RANKL are indicators of activated γδT cells. In combination, markers can provide signals of increased cytotoxicity even when cytotoxicity cannot be directly assessed. For example, a population of proliferated γδT cells disclosed herein may show increased killing of tumor targets, or secrete greater amounts of antitumor or antipathogen cytokines (e.g., IFNγ, TNFα, perforin, granzyme), or express increased levels of killing molecules (e.g., FasL, TRAIL) compared with non-proliferated γδT cells. In another embodiment, a population of proliferated γδT cells disclosed herein may show increased expression of CD69, CD25, NKG2D, NKp46, and / or CD16 compared with non-proliferated γδT cells. Various means for detecting the amount of specific proteins to assess the activation state of γδT cells are known and can be used in the art, including spectroscopic methods such as flow cytometry, or immunodetection methods such as Western blotting, enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation, immunoelectrophoresis, or immunostaining.

[0106] Furthermore, the proliferated γδT cell populations disclosed herein may withstand cryopreservation, retain their viability and cytotoxicity, and exhibit improved capabilities following freezing and thawing.

[0107] (b) Treatment methods and use The compositions and methods disclosed herein may be used in a variety of therapeutic, diagnostic, industrial, and research applications. In some embodiments, cancer may be treated using this disclosure. Thus, in one embodiment, disclosed herein is a method for treating, inhibiting, reducing, and / or preventing cancer, cancer recurrence, or metastasis, or infectious diseases such as viral or bacterial infections in a subject, comprising administering an effective amount of the composition described herein or a population of proliferated γδ T cells to a subject in need thereof.

[0108] Therefore, in some embodiments, what is disclosed herein is a method for treating, reducing, inhibiting, mitigating, improving, and / or preventing cancer, metastasis, or infectious disease in a subject, a. Obtain at least one γδT cell, b. Contacting at least one γδT cell with an engineered feeder cell, engineered plasma membrane particle, exosome, or solid support containing an Fc domain bound to its outer surface. c. A method comprising administering a therapeutically effective amount of contacted γδT cells to a subject.

[0109] In some embodiments, step b further comprises inducing, activating, and / or proliferating at least one γδT cell after contact with an engineered feeder cell, engineered plasma membrane particle, exosome, or solid support containing an externally bound Fc domain, wherein the γδT cell is induced, activated, and / or proliferated for at least 14 days.

[0110] In some embodiments, the engineered feeder cells, engineered plasma membrane particles, exosomes, or solid support may further comprise at least one γδT cell effector agent, the at least one γδT cell effector agent comprising: 4-1BBL;CD80;CD86;MICA;UBLP;2B4;LFA-1;NKG2D, NKp46, NKp44, NKp30, or DNAM-1 agonists (e.g., agnostic antibodies) or ligands; Notch, BCM / SLAMF2, or TLR agonists (e.g., agnostic antibodies) or ligands; IL-2;IL-12;IL-18;IL-15, or IL-21; or any combination thereof. In some embodiments, the at least one γδT cell effector agent comprises 4-1BBL, IL-18, IL-15, or IL-21, or any combination thereof.

[0111] In some embodiments, disclosed herein are methods for treating, reducing, inhibiting, mitigating, improving, and / or preventing cancer, metastasis, or infectious disease in a subject by promoting, inducing, and / or activating endogenous γδT cells in the subject, the method comprising administering to the subject engineered plasma membrane particles, exosomes, or solid supports comprising an externally bound Fc domain, wherein the engineered feeder cells, engineered plasma membrane particles, exosomes, or solid supports contain at least one γδT cell effector agent The following may further be included, and at least one γδ T cell effector agent may include an agonist (e.g., agnostic antibody) or ligand of 4-1BBL;CD80;CD86;MICA;UBLP;2B4;LFA-1;NKG2D, NKp46, NKp44, NKp30, or DNAM-1; an agonist (e.g., agnostic antibody) or ligand of Notch, BCM / SLAMF2, or TLR; IL-2;IL-12;IL-18;IL-15; or IL-21; or any combination thereof. In some embodiments, at least one γδ T cell effector agent may include 4-1BBL, IL-18, IL-15, or IL-21, or any combination thereof.

[0112] In some embodiments, any of the methods described above further comprises administering to a subject an ex vivo composition comprising a fusion protein having a transmembrane domain ligated to the amino terminus of an Fc domain and bound to engineered feeder cells, engineered plasma membrane particles, exosomes, or a solid support, by contacting the ex vivo composition with an isolated mixed cell population comprising at least one γδT cell containing CD16 or a functional fragment thereof. In some embodiments, the ex vivo composition further comprises at least one γδ T cell effector agent, the at least one γδ T cell effector agent comprising an agonist (e.g., agnostic antibody) or ligand of 4-1BBL;CD80;CD86;MICA;UBLP;2B4;LFA-1;NKG2D, NKp46, NKp44, NKp30, or DNAM-1; an agonist (e.g., agnostic antibody) or ligand of Notch, BCM / SLAMF2, or TLR; IL-2;IL-12;IL-18;IL-15; or IL-21; or any combination thereof. In some embodiments, the at least one γδ T cell effector agent comprises 4-1BBL, IL-18, IL-15, or IL-21, or any combination thereof. The manipulated plasma film particles may comprise a plasma film and a plurality of microparticles or support surfaces, wherein the plasma film coats the plurality of microparticles or support surfaces. In some embodiments, the plurality of microparticles or surfaces include at least one of magnetic microparticles, silica beads, polystyrene beads, latex beads, microstructures, contrast agents, and cancer treatment agents.

[0113] Cancer may be selected from, but is not limited to, hematological cancers, lymphomas, colorectal cancers, colon cancers, lung cancers, head and neck cancers, ovarian cancers, prostate cancers, testicular cancers, kidney cancers, skin cancers, cervical cancers, pancreatic cancers, and breast cancers. In one embodiment, cancer includes solid tumors. In another embodiment, cancer is selected from acute myeloid leukemia, myelodysplastic syndromes, chronic myeloid leukemia, acute lymphoblastic leukemia, myelofibrosis, and multiple myeloma. In yet another embodiment, cancer is selected from leukemias, lymphomas, sarcomas, and carcinomas, and may originate from the bone marrow, brain, lungs, breasts, pancreas, liver, head and neck, skin, reproductive tract, prostate, colon, kidney, intraperitoneal, bone, joints, and eyes.

[0114] Any of the therapeutic methods disclosed herein may further include administering an additional therapeutic agent or regimen (concurrently, simultaneously, or as a single formulation) to a subject in combination with an effective amount of the composition herein or a population of proliferated γδ T cells. The additional therapeutic agent may be a drug-based preparation regimen such as Cy-Flu, Bu-Flu, Flu-Mel, or similar with adjustments to the dosage or administration. Alternatively, the additional therapeutic agent or regimen may be selected from chemotherapeutic agents and regimens known by the acronyms CHOP, FLAG (FLAG-Ida or FLAG-IDA or IDA-FLAG or Ida-FLAG, and FLAG-Mito or FLAG-MITO or Mito-FLAG or MITO-FLAG or FLANG), IA or IAC, or 7+3. Alternatively, an effective amount of any of the disclosed compositions and / or proliferated γδ T cell populations described herein may be used, at the discretion of the user, concurrently, simultaneously, or as a single formulation in combination with the use of additional therapeutic agents or regimens in the treatment of any disease described herein. In such use, the additional therapeutic agent may be a drug-based formulation regimen such as Cy-Flu, Bu-Flu, Flu-Mel, or similar with adjustments to the dosage or administration. Alternatively, the additional therapeutic agent or regimen may be selected from chemotherapeutic agents and regimens known by the acronyms CHOP, FLAG (FLAG-Ida or FLAG-IDA or IDA-FLAG or Ida-FLAG, and FLAG-Mito or FLAG-MITO or Mito-FLAG or MITO-FLAG or FLANG), IA or IAC, or 7+3.

[0115] For example, disclosed methods for inhibiting, reducing, and / or preventing cancer metastasis and / or recurrence include abemaciclib, abiraterone acetate, abitrexate (methotrexate), Abraxane (paclitaxel albumin-stabilized nanoparticle formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (brentuximab vedotin), ADE, Ado-trastuzumab emtansine, Adriamycin (doxorubicin hydrochloride), afatinib dimareate, Afinitor (everolimus), Akynzeo (netupitant and palonosetron hydrochloride), Aldara (imiquimod), Aldesleukin, Alecensa (alecinib), alectinib, alemtuzumab, and Alim Ta (pemetrexed disodium), Aliqopa (copanlisib hydrochloride), Alkeran for injection (melphalan hydrochloride), Alkeran tablets (melphalan), Aloxi (palonosetron hydrochloride), Alunbrig (brigatinib), Ambochlorin (chlorambucil), Ambochlorin (chlorambucil), Amifostine, Aminolevulinic acid, Anastrozole, Aprepitant, Aredia (pamidronate disodium), Arimidex (anastrozole), Aromasin (exemestane), Alanone (nelarabine), Arsenic trioxide, Arzerra (ofatumumab), Asparaginase Erwinia Chrysanthemi, atezolizumab, Avastin (bevacizumab), avelumab, axitinib, azacitidine, Bavencio (avelumab), BEACOPP, Becenum (carmustine), Belenostat (belinostat), bendamustine hydrochloride, BEP, Besponsa (inotuzumab ozogamicin), bevacizumab, bexarotene, Bexxar (tositumomab and iodine I131 tositumomab), bicalutamide, BiCNU (carmustine), bleomycin, blinatumomab, Blincyto (blinatumomab), bortezomib, Bosulif (bosutinib), bosutinib,Brentuximab vedotin, Brigatinib, BuMel, Busulfan, Busulfex (Busulfan), Cabazitaxel, Cabometyx (Cabozantinib-S-Malate), Cabozantinib-S-Malate, CAF, Camppath (Alemtuzumab), Camptosar (Irinotecan Hydrochloride), Capecitabine, CAPOX, Carac (Fluorouracil - Topical), Carboplatin, Carboplatin-Taxol, Carfilzomib, Carmubris (Carmustine), Car Mustine, Carmustine Implant, Casodex (bicalutamide), CEM, Ceritinib, Cerubidine (daunorubicin hydrochloride), Cervarix (recombinant HPV bivalent vaccine), Cetuximab, CEV, Chlorambucil, Chlorambucil-prednisone, CHOP, Cisplatin, Cladribine, Clafen (cyclophosphamide), Clofarabine, Clofarex (clofarabine), Chloral (clofarabine), CMF, Cobimetinib, Cometriq (cabozantinib) (B-S-malate), Copanlisib hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (Dactinomycin), Cotellic (Cobimetinib), Crizotinib, CVP, Cyclophosphamide, Cyfos (Ifosfamide), Cyramza (Ramucirumab), Cytarabine, Cytarabine liposome, Cytosar-U (Cytarabine), Cytoxan (Cyclophosphamide), Dabrafenib, Dacarbazine, Dacogen (Decitabine), Dactinomycin (Da ctinomycin), daratumumab, Darzalex (daratumumab), dasatinib, daunorubicin hydrochloride, daunorubicin hydrochloride and cytarabine liposomes, decitabine, defibrotide sodium, Defitelio (defibrotide sodium), degarelix, denileukin diffitox, denosumab, DepoCyt (cytarabine liposomes), dexamethasone, dexrazoxane hydrochloride, dinutuximab, docetaxel, doxil (doxorubicin hydrochloride liposomes), doxorubicin hydrochloride, doxorubicin hydrochloride liposomes,Dox-SL (doxorubicin hydrochloride liposome), DTIC-Dome (dacarbazine), durvalumab, Efudex (fluorouracil - topical), Elitek (rasburicase), Ellence (epirubicin hydrochloride), elotuzumab, Eloxatin (oxaliplatin), eltrombopagolamine, Emend (aprepitant), Empliciti (elotuzumab), enasidenib mesylate, enzalutamide, epirubicin hydrochloride, EPOCH, Erbitux (cetuximab), eribulin mesylate, Erivedge (vismodegib), erlotinib hydrochloride, Erwina (asparaginase) Chrysanthemi), Ethyol (amifostine), Etopophos (etoposide phosphat), Etoposide, Etoposide phosphat, Evacet (doxorubicin hydrochloride liposome), Everolimus, Evista (raloxifene hydrochloride), Evomela (melphalan hydrochloride), Exemestane, 5-FU (fluorouracil injection), 5-FU (fluorouracil - topical), Far Eston (toremifene), Farydak (panobinostat), Faslodex (fulvestrant), FEC, Femara (letrozole), filgrastim, Fludara (fludarabine phosphate), fludarabine phosphate, Fluoroplex (fluorouracil - topical), fluorouracil injection, fluorouracil - topical, flutamide, Folex (methotrexate), Folex PFS (methotrexate), FOLFIRI, FOLFIRI-bevacizumab, FOLFIRI-cetuximab, FOLFIRINOX, FOLFOX, Folotyn (pralatrexate), FU-LV, fulvestrant, Gardasil (recombinant HPV tetravalent vaccine), Gardasil 9 (recombinant HPV nonavalent vaccine), Gazyva (obinutuzumab), gefitinib, gemcitabine hydrochloride, gemcitabine-cisplatin, gemcitabine-oxaliplatin, gemtuzumab ozogamicin, gemzar (gemcitabine hydrochloride), zirotrif (afatinib dimareate), gleevec (imatinib mesylate),Gliadel (carmustine implant), Gliadel wafer (carmustine implant), glucarpidase, goserelin acetate, Halaven (eribulin mesylate), Hemangeol (propranolol hydrochloride), Herceptin (trastuzumab), HPV bivalent vaccine, recombinant, HPV nonavalent vaccine, recombinant, HPV tetravalent vaccine, recombinant, Hycamtin (topotecan hydrochloride), Hydrea (hydroxyurea), hydroxyurea, Hyper-CVAD, Ibrance (palbociclib), ibritumomab tiuxetan, Ibrutinib, ICE, Iclusig (ponatinib hydrochloride), Idamycin (idarubicin hydrochloride), idarubicin hydrochloride, idelalisib, Idhifa (enasidenib mesylate), Ifex (ifosfamide), ifosfamide, ifosfamidum (ifosfamide), IL-2 (aldesleukin), imatinib mesylate, Imbruvica (ibrutinib), Imfinzi (durvalumab), imiquimod, Imlygic (tarimodine laherpalebube ), Inlyta (axitinib), Inotuzumab ozogamicin, Interferon alfa-2b, Recombinant, Interleukin-2 (Aldesleukin), Intron A (Recombinant Interferon alfa-2b), Iodine I131 Tositumomab and Tositumomab, Ipilimumab, Iressa (gefitinib), Irinotecan hydrochloride, Irinotecan hydrochloride liposome, Istodax (romidepsin), Ixabepirone, Ixazomib citrate, Ixempra (Ixabepirone), Jakafi (Ruxolitinib Phosphate) Fart), JEB, Jevtana (cabazitaxel), Kadcyla (Ado-trastuzumab emtansine), Keoxifene (raloxifene hydrochloride), Kepivance (palifermin), Keytruda (pembrolizumab), Kisqali (ribociclib), Kymriah (tisagenlecleucel), Kyprolis (carfilzomib), lanreotide acetate, lapatinib ditosylate, Lartruvo (olalatumab),Lenalidomide, lenvatinib mesylate, Lenvima (lenvatinib mesylate), letrozole, leucovorin calcium, Leukeran (chlorambucil), leuprolide acetate, leustatin (cladribine), Levulan (aminolevulinic acid), lympholizin (chlorambucil), LipoDox (doxorubicin hydrochloride liposome), lomustine, Lonsurf (trifluridine and tipiracil hydrochloride), Lupron (leuprolide acetate), Lupron Depot (leuprolide acetate), Lupron Depot Ped (leuprolide acetate), Lynparza (olaparib), Marquivo (vincristine sulfa liposome), Matulane (procarbazine hydrochloride), mechloretamine hydrochloride, megestrol acetate, Mekinist (trametinib), melphalan, melphalan hydrochloride, mercaptopurine, Mesna, Mesnex (mesna), Methazolastone (temozolomide), methotrexate, methotrexate LPF (methotrexate), methylnaltrexone bromide, Mexate ( Mexate (methotrexate), Mexate-AQ (methotrexate), midostaurine, mitomycin C, mitoxantrone hydrochloride, mitozytrex (mitomycin C), MOPP, mozovir (plelixafor), mustargen (mechloretamine hydrochloride), mutamycin (mitomycin C), myleran (busulfan), mylosar (azacitidine), mylotarg (gemtuzma Buozogamicin), nanoparticle paclitaxel (paclitaxel albumin-stabilized nanoparticle formulation), navelbine (vinorelbine tartrate), necitumumab, nelarabine, Neosar (cyclophosphamide), neratinib maleate, Nerlinx (neratinib maleate), netupitant and palonosetron hydrochloride, Neulasta (pegfilgrastim), Neupogen (filgrastim), Nexavar (sorafenib tosylate), Nilandron (nilutamide), nilotinib, nilutamide,Ninlaro (ixazomib citrate), niraparib tosylate monohydrate, nivolumab, nolvadex (tamoxifen citrate), Nplate (romiplostim), obinutuzumab, Odomzo (sonidegib), OEPA, ofatumumab, OFF, olaparib, olalatumab, (Olaratumab), Omasetaxin mepesuccinate, Oncasper (peguaspar gauze), Ondansetron hydrochloride, Onivyde (irinotecan hydrochloride liposome), Ontak (denileukin difutitox), Opdivo (nivolumab), OPPA, Osimertinib, Oxaliplatin, Paclitaxel, Paclitaxel albumin-stabilized nanoparticle formulation, PAD, Palbociclib, Palifermin, Palonosetron hydrochloride, Palonosetron hydrochloride and Netupitant, Pamidronate disodium, Panitumumab, Panobinostat, Para Paraplatin (carboplatin), Paraplatin (carboplatin), pazopanib hydrochloride, PCV, PEB, Peguaspargase, pegfilgrastim, pegylated interferon alfa-2b, PEG-intron (pegylated interferon alfa-2b), pembrolizumab, pemetrexed disodium, Perjeta (pertuzumab), pertuzumab, Platinol (cisplatin), Platinol-AQ (cisplatin), Plerixafor, Pomalidomide, Pomalist (Pomalidomide), Ponatinib hydrochloride, Portraza (Necitumumab), Pralatrexate, Prednisone, Procarbazine hydrochloride, Proleukin (Aldesleukin), Prolia (Denosumab), Promacta (Eltrombopagolamine), Propranolol hydrochloride, Provenge (Cyplucel-T), Prinethol (Mercaptopurine), Purixan (Mercaptopurine), Radium-223 dichloride D, raloxifene hydrochloride, ramucirumab, rasburicase, R-CHOP, R-CVP, recombinant human papillomavirus (HPV) bivalent vaccine, recombinant human papillomavirus (HPV) nonavalent vaccine, recombinant human papillomavirus (HPV) tetravalent vaccine, recombinant interferon alpha-2b, regorafenib, Relistor (methyltrexone bromide), R-EPOCH, Revlimid (lenalidomide), Rheumatrex (methotrexate), ribociclib, R-ICE, Rituxan (rituximab),Rituxan Hycela (rituximab and hyaluronidase), rituximab, rituximab and hyaluronidase, lorapitant hydrochloride, romidepsin, romiplostim, rubidomycin (daunorubicin hydrochloride), Rubraca (rucaparib cansylate), rucaparib cansylate, ruxolitinib phosphate, Rydapt (midostaurine), Sclerosol intrapleural aerosol (talc), siltuximab, ciplucel-T, somatuline depot (lanreotide acetate), sonidegib, sorafenib tosylate, sprycel (dasatinib), STANFORD V, sterile talc powder (talc), Steritalc (talc), Stivarga (regorafenib), sunitinib malate, Sutent (sunitinib malate), Sylatron (pegylated interferon alpha-2b), Sylvant (siltuximab), Synribo (omacetaxin mepesuccinate), Tabloid (Ta bloid (thioguanine), TAC, Tafinlar (dabrafenib), Tagrisso (osimertinib), talc, tarimodine laherpalepbec, tamoxifen citrate, tarabine PFS (cytarabine), tarceva (erlotinib hydrochloride), targretin (bexarotene), tasigna (nilotinib), taxol (paclitaxel), taxotere (docetaxel), te Centriq (atezolizumab), Temodar (temozolomide), temozolomide, temsirolimus, thalidomide, thalomid (thalidomide), thioguanine, thiotepa, tisagenlecleucel, Tolak (fluorouracil - topical), topotecan hydrochloride, toremifene, Torisel (temsirolimus), tositumomab and iodine I131 tositumomab Totect (dexrazoxane hydrochloride), TPF, trabectedin, trametinib, trastuzumab, Treanda (bendamustine hydrochloride), trifluridine and tipiracil hydrochloride, Trisenox (arsenic trioxide), Tykerb (lapatinib ditosylate), Unituxin (dinutuximab), uridine triacetate, VAC, vandetanib, VAMP,Barbit (Varubi) (Rolapitant hydrochloride), Vectibix (panitumumab), VeIP, Velban (vinblastine sulfate), Velcade (bortezomib), Velsar (vinblastine sulfate), vemurafenib, Benclexta (venetoclax), venetoclax, Verzenio (abemaciclib), Viadur (leuprolide acetate), Vidaza (azacitidine), vinblastine sulfate, Vincasar PFS (vincristine sulfate), vincristine sulfate, vincristine sulfate liposome, vinorelvin tartrate, VIP, bismodegib, Vistagoard (uridine triacetate), Voraxaze (glucarpidase), vorinostat, Votrient (pazopanib hydrochloride), Vyxeos (daunorubicin hydrochloride and cytarabine liposome), Wellcovorin (leucovorin calcium), Xalkori (crizotinib), Xeloda (capecitabine), XELIRI, XELOX, Xgeva (denosumab), Xofigo (radium-223 dichloride), Xtandi (enzalutamide), Yervoy (ipilimumab), Yondelis This specification intends to include, but is not limited to, the administration of any anticancer agent known in the art, including (trabectedin), Zaltrap (Ziv-aflibercept), Zarxio (filgrastim), Zejura (niraparib tosylate monohydrate), Zelboraf (vemurafenib), Zevalin (ibritumomab tiuxetan), Zinecard (dexrazoxane hydrochloride), Ziv-aflibercept, Zofran (ondansetron hydrochloride), Zoladex (goserelin acetate), zoledronic acid, Zolinza (vorinostat), Zometa (zoledronic acid), Zydelig (idelalisib), Zykadia (ceritinib), and / or Zytiga (abiraterone acetate). This specification also refers to PD1 / PDL1 blocking inhibitors (e.g., lambrolizumab, nivolumab, pembrolizumab, pidilizumab, BMS-936559, atezolizumab, durvalumab,Chemotherapy agents such as avelumab are also intended. In this specification, the disclosed use of the disclosed compositions and / or proliferated γδ T cell populations for inhibiting, reducing, and / or preventing cancer metastasis and / or recurrence is also intended to include use in combination with any anticancer agents known in the art, including but not limited to those agents described above.

[0116] In some embodiments, all use of the therapeutic methods and compositions disclosed herein is for the treatment of infectious diseases caused by viral infections, including herpes simplex virus-1, herpes simplex virus-2, varicella-zoster virus, Epstein-Barr virus, cytomegalovirus, human herpesvirus-6, smallpox virus, varicella-stomatitis virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, rhinovirus, coronavirus, influenza A virus, influenza B virus, measles virus, polyomavirus, human papillomavirus, and respiratory syncytial virus. This includes infection with adenovirus, coxsackievirus, dengue virus, mumps virus, poliovirus, rabies virus, Roussarcoma virus, reovirus, yellow fever virus, Zika virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern equine encephalitis virus, Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, rotavirus A, rotavirus B, rotavirus C, Sindbisvirus, simian immunodeficiency virus, human T-cell leukemia virus type 1, hantavirus, rubella virus, simian immunodeficiency virus, human immunodeficiency virus type 1, or human immunodeficiency virus type 2.

[0117] Alternatively, in either a method of treatment or use for treatment, additional therapeutic agents may be antiviral agents selected from, but not limited to, 5-substituted 2-deoxyuridine analogs, nucleoside analogs, (non-nucleoside) pyrophosphate analogs, nucleoside reverse transcriptase (RT) inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), and integrase inhibitors, entry inhibitors, and acyclic guanosine analogs, acyclic nucleoside phosphonate (ANP) analogs, hepatitis C virus (HCV) NS5A and NS5B inhibitors, influenza virus inhibitors, immunostimulants, interferons, oligonucleotides, and anti-mitotic inhibitors. Non-exclusive examples of antiviral agents include acyclovir, famciclovir, valacyclovir, penciclovir, ganciclovir, ritonavir, lopinavir, saquinavir, cimetidine, ranitidine, captopril, metformin, bupropion, fexofenadine, oxcarbazepine, rebeteracetam, tramadol, or any of their isomers, tautomers, analogs, polymorphs, solvates, derivatives, or pharmaceutically acceptable salts.

[0118] In some embodiments, all use of the methods and compositions disclosed herein is for the treatment of infectious diseases caused by bacterial infections, including Mycobaterium tuberculosis, Mycobaterium bovis, Mycobaterium bovis strain BCG, BCG substrain, Mycobaterium avium, Mycobaterium intracellular, Mycobaterium africanum, Mycobaterium kansasii, Mycobaterium marinum, Mycobaterium ulcerans, Mycobaterium avium subspecies paratuberculosis, Nocardia asteroides, other Nocardia species, Legionella pneumophila, other Legionella species, Acetinobacter baumanii, Salmonella typhi, Salmonella enterica, other Salmonella species, Shigella boydii, Shigella dysenteriae, Shigella sonnei, Shigella flexneri, other Shigella species, and Yersinia. pestis, Pasteurella haemolytica, Pasteurella multocida, other Pasteurella species, Actinobacillus pleuropneumoniae, Listeria monocytogenes, Listeria ivanovii, Brucella abortus, other Brucella species, Cowdria ruminantium, Borrelia burgdorferi, Bordetella avium, Bordetella pertussis, Bordetella bronchiseptica, Bordetella trematum, Bordetella hinzii, Bordetella pteri, Bordetella parapertussis, Bordetella ansorpii, other Bordetella species, Burkholderia mallei, Burkholderiapsuedomallei, Burkholderia cepacian, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Coxiella burnetii, Rickettsial species, Ehrlichia species, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Escherichia coli, Vibrio cholerae, Campylobacter species, Neiserria meningitidis, Neiserria gonorrhea, Pseudomonas aeruginosa, other Pseudomonas species, Haemophilus influenzae, Haemophilus ducreyi, other Hemophilus species, Clostridium tetani, Clostridium difficile, other Clostridium species, Yersinia This includes infections of enterolitica, other Yersinia species, and Mycoplasma species.

[0119] In some embodiments, all use of the methods and compositions disclosed herein is for the treatment of infectious diseases caused by fungal infections, including infections of Candida albicans, Cryptococcus neoformans, Histoplama capsulatum, Aspergillus fumigatus, Coccidiodes immitis, Paracoccidiodes brasiliensis, Blastomyces dermitidis, Pneumocystis carinii, Penicillium marneffi, or Alternaria alternate.

[0120] In some embodiments, all use of the methods and compositions disclosed herein is for the treatment of infectious diseases caused by parasitic infections, including Toxoplasma gondii, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, other Plasmodium species, Entamoeba histolytica, Naegleria fowleri, Rhinosporidium seeberi, Giardia lamblia, Enterobius vermicularis, Enterobius gregorii, Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus, and Cryptosporidium spp., Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, other Leishmania species, Diphyllobothrium latum, Hymenolepis nana, Hymenolepis diminuta, Echinococcus granulosus, Echinococcus multilocularis, Echinococcus vogeli, Echinococcus oligarthrus, Diphyllobothrium latum, Clonorchis sinensis; Clonorchis viverrini, Fasciola hepatica, Fasciola gigantica, Dicrocoelium dendriticum, Fasciolopsis buski, Metagonimus yokogawai, Opisthorchis viverrini, Opisthorchis felineus, Clonorchis sinensis, Trichomonas vaginalis, Acanthamoeba species, Schistosoma intercalatum, Schistosoma haematobium, Schistosoma japonicum, Schistosoma mansoni, other Schistosoma species, Trichobilharzia regenti, Trichinella spiralis, Trichinella britovi, Trichinella nelsoni, Trichinella nativa, or Entamoeba histolytica infections.

[0121] Alternatively, in either the method or use, the additional therapeutic agent may be an antibiotic selected from, but not limited to, penicillin, tetracycline, cephalosporin, lincomycin, macrolide, sulfonamide, glycopeptide, aminoglycoside, and carbapenem. Non-limited examples of antiviral agents include amoxicillin, doxycycline, cephalexin, ciprofloxacin, clindamycin, metronidazole, azithromycin, sulfamethoxazole and trimethoprim, clavulanat, and levofloxacin.

[0122] In some embodiments, the γδT cells administered or used in any of the aforementioned methods or uses are formulated in a pharmaceutically acceptable carrier and a pharmaceutically acceptable excipient.

[0123] Because the timing of cancer, metastatic conditions, or infections is often unpredictable, it should be understood that any use of the disclosed methods for treating, preventing, reducing, and / or inhibiting cancer, metastatic conditions, or infections, or any of the disclosed compositions or combinations for such treatment, prevention, reduction, and / or inhibition of cancer, metastatic conditions, or infections, may be carried out before or after the onset of cancer, metastatic conditions, or infections in order to treat, prevent, inhibit, and / or reduce muscle diseases. In one embodiment, the disclosed method or use is to simultaneously with cancer, metastatic condition, or infection, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 days prior, 60, 48, 36, 30, 24, 18, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2 hours prior, 60, 45, 30, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 minute prior, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 , 30, 35, 40, 45, 50, 55, 60, 75, 90, 105, 120 minutes later, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 24, 30, 36, 48, 60 hours later, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 It can be used 22, 23, 24, 25, 26, 27, 28, 29, 30, 45, 60, 90 days later, 4, 5, 6, 7, 9, 10, 11, 12 months later, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 years later or later.

[0124] V. Kit Further aspects of this disclosure provide a kit comprising at least one of the fusion peptides detailed above, and / or at least one of the Fc-bound feeder cells, and / or at least one of the Fc-bound engineered particles (PM particles and / or exosomes) detailed above. The fusion peptide may be supplied in a suitable container together with other kit components such as cell reagents, cell growth media, selective media, protein purification reagents, and buffers. The kits provided herein generally include instructions for carrying out the methods detailed below. Instructions included in the kit may be affixed to the packaging or included as accompanying documentation. Instructions are typically, but not limited to, written or printed materials. Any medium capable of storing such instructions and communicating them to end users is contemplated by this disclosure. Such mediums include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD-ROMs), etc. As used herein, the term “instructions” may include the address of an internet site providing the instructions. [Examples]

[0125] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the disclosed invention pertains. Publications cited herein and materials from which they are cited are incorporated specifically by reference.

[0126] Those skilled in the art will recognize, or can verify by mere routine experimentation, many equivalents to the specific embodiments of the present invention described herein. While the present invention is described with reference to specific embodiments and implementations, it will be understood that various modifications and additional variations may be made, and equivalents may be substituted for their elements, without departing from the scope of the invention or its inventive concept. Furthermore, many modifications may be made to adapt the teachings of the present invention to specific situations or devices, without departing from the essential scope of the invention. Such equivalents are intended to be encompassed by the following claims. The present invention is not limited to the specific implementations disclosed herein, and is intended to include all implementations within the scope of the appended claims.

[0127] 1) Materials and methods Proliferating peripheral blood mononuclear cells (PBMCs) of γδ T cells using CSTX-2-Fc feeder cells were characterized for their NK and T cell content and seeded at 200,000 NK cells per mL in RPMI1640 medium supplemented with 10% fetal bovine serum, 1% antibiotic-antifungal agent, and 100 U / mL of hIL-2 (Peprotec). On day 0, 1 × 10⁶ cells per mL were observed. 6 Cells were co-cultured with irradiated or mitomycin C-treated (50 μg / mL for 30 minutes) CSTX-002 or CSTX-002-Fc feeder cells, which were added to the culture at a rate of 500,000 feeder cells per mL on day 7. Cells were counted every other day and maintained at a concentration of 250,000 NK cells per mL. In experiments testing the effect of NK cells on γδT cells, PBMCs or CD56-depleted PBMCs were given 1 × 10⁶ cells per mL on day 0. 6The culture was seeded with 100 total cells, restimulated with feeder cells on day 7, and maintained with 250,000 total cells per mL every other day. For experiments using PBMCs with depleted TCRα / β as the starting material, the culture was seeded with 70,000 TCRγ / δ+ T cells per mL on day 0, restimulated with feeder cells on day 7, and maintained with 250,000 NK cells per mL every two days.

[0128] Flow cytometry. To determine the cell surface phenotype, 50,000–100,000 cells were stained with a fluorescently labeled antibody in 50 μL of flow cytometry buffer containing 0.5% bovine serum albumin (BSA) + 2 mM EDTA in Dubbelco's phosphate-buffered saline (DPBS) for 25 minutes at 4°C. After washing the samples with flow buffer, they were analyzed using a CytoFlex (Beckman Coulter) flow cytometer. The following pre-conjugated antibodies were used for detection. CD3-PacBlue (clone UCHT1), CD8a-PE-Cy7 (clone RPA-T8), CD56-PE (clone 5.1H11), TCRVδ2-APC-Fire750 (clone B6), and TCRαβ-APC (clone IP26) purchased from Biolegend, as well as TCRVδ1-FITC (clone REAL277) from Miltenyi Biotech.

[0129] 2) Results Gamma-delta (γδ) T cells have surface expression of CD16 (FcγRIIIa), a low-affinity receptor for IgG, and can respond to stimulation of IgG1 at the Fc domain. To mimic antibody-opsonized target cells, K562 cell lines containing other T and NK stimulants (membrane-bound IL-21, 41BBL), and cells called CSTX-002, were transduced to express the surface-anchored Fc domain of IgG1 via a neuraminidase (NA) domain (Figure 7). These CSTX-002-Fc cells were used in co-culture with healthy donor PBMCs to stimulate γδ T cells via CD16 engagement. Since antibody / ligand interactions result in large molecular complexes that can play a role in steric effects in CD16 / Fc interactions, we first tested the effect of NA stalk length on γδ T cell proliferation. PBMCs were stimulated with CSTX-002 cells or CSTX-002 cells expressing Fc fused to NAs of varying lengths, with NA2 being the shortest and NA4 the longest (Figure 8). Including Fc on the surface of CSTX-002 feeder cells resulted in an increase in T cell content of co-cultured PBMC cells on day 14 (1.5% for CSTX-002 vs. 16.3% for CSTX2-002 NA2-4-Fc, left panel of Figure 1), with the highest T cell content observed in cultures stimulated with Fc anchored to cells surfaced by the longest NA-stalk (NA4) (right panel of Figure 1). Characterization of T cell content revealed a decrease in αβ T cells and an increase in γδ T cells, particularly the Vγ9Vδ2 subtype, in cultures stimulated with Fc-expressing CSTX-002 cells compared to CSTX-002 controls (Figure 2). The Vγ9Vδ2 T cell content was highest in cultures stimulated with Fc cells fused to the longest NA stalks (NA4-Fc). To test whether the removal of other T cell subpopulations competing with nutrients and other factors could further improve γδ T cell proliferation, αβ T cells were depleted before co-culture with feeder cells, and the effect of neuraminidase stalk length (NA2 vs. NA4) on proliferation was compared again.Depletion of αβ T cells resulted in a higher proliferation rate of Vδ2 T cells (2990x PBMC vs. 6460x αβ depletion in NA4-Fc, and 810x PBMC vs. 1200x αβ depletion in NA2-Fc) (Figure 3). Regardless of the presence of αβ T cells, increased NA length resulted in a higher proliferation rate of Vδ2 T cells. To test whether Vδ2 T cell proliferation was dependent on another CD16-expressing cell type, NK cells, CD56, were cultured. +Cells were depleted before co-culture with feeder cells, and γδ T cell proliferation was monitored and compared to cultures using uncontacted PBMCs. In both co-cultures of all PBMCs and CD56-depleted cells, exposure to CSTX-002-Fc cells resulted in higher levels of Vδ2 T cell proliferation compared to CSTX-002 controls (Figure 4). Compared to less than 300-fold proliferation when co-cultured with CSTX2, Vδ2 T cell proliferation exceeded 2,500-fold in both uncontacted PBMC cultures and CD56-depleted PBMC cultures when co-cultured with CSTX-002-Fc, demonstrating that proliferation was independent of the presence of NK cells in the starting material (Figure 5). Furthermore, depletion of the CD56-expressing cell population from the starting material resulted in increased preferential proliferation of Vδ2 T cells and higher purity of the final product when stimulated with Fc-CSTX-2 cells. On day 14, the Vδ2 T cell content in Fc-stimulated cultures increased from 40% to 65% when CD56-depleted PBMCs were used as the starting material instead of uncontacted PBMCs (Figure 6). Stimulation with Fc resulted in preferential proliferation of Vδ2 T cells, with Vδ2 T cells accounting for 64% of the cells in CD56-depleted PBMC cultures exposed to CSTX-002-Fc after 14 days, compared to 17% when co-cultured with CSTX-002 control cells lacking Fc (Figure 6). The increase in Vδ2 T cells was consistent with the decrease in the αβ T cell population, which decreased from 44% in CSTX-002 control cultures to 17% in CSTX-002-Fc cultures after 14 days when CD56-depleted PBMCs were used as the starting material. These results clearly support the use of CSTX2-Fc feeder cells for ex vivo proliferation of γδT cells, demonstrating that longer NA-anchores and the use of γδT cell-enriched starting materials can indeed have a positive impact on the overall level of proliferation.

Claims

1. A method for inducing, activating, and / or proliferating γδ T cells, The method comprises contacting at least one γδT cell with an engineered feeder cell, engineered plasma membrane particle, engineered exosome, or solid support containing an antibody Fc domain, ex vivo or in vitro. The antibody Fc domain is capable of agonizing the Fc receptor and is bound via a transmembrane domain to the manipulated feeder cells, the manipulated plasma membrane particles, the manipulated exosomes, or the outer surface of the solid support, thereby inducing, activating, and / or proliferating the at least one γδT cell. The γδ T cells are proliferated for at least 14 days, and at least about 5%, 10%, 20%, 30%, 40%, 50%, or 60% of the proliferated cells are Vδ2 subtype γδ T cells. A method characterized by the following:

2. The method according to claim 1, characterized in that the transmembrane domain comprises a signal anchor sequence selected from a transmembrane domain of neuraminidase, a signal anchor sequence derived from parainfluenza virus hemagglutinin-neuraminidase, a signal anchor sequence derived from a transferrin receptor, a signal anchor sequence derived from an MHC class II invariant chain, a signal anchor sequence derived from P-glycoprotein, a signal anchor sequence derived from an asialoglycoprotein receptor, and a signal anchor sequence derived from a neutral endopeptidase.

3. The method according to claim 2, characterized in that the transmembrane domain comprises a parainfluenza virus hemagglutinin-neuraminidase (NA) peptide sequence.

4. The method according to claim 3, characterized in that the NA peptide sequence contains at least 95% of the same sequence as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO:

4.

5. The method according to claim 2 or 3, characterized in that the transmembrane domain and the amino-terminal side of the Fc domain are linked via a peptide linker, and the amino-terminal side of the Fc domain faces the manipulated feeder cell, the manipulated plasma membrane particle, the manipulated exosome, or the solid support.

6. A method according to claim 1 or 2, characterized in that the Fc domain comprises an immunoglobulin Fc domain selected from IgG1, IgG2, IgG3, IgG4, IgA, and IgE.

7. The method according to claim 1, characterized in that the Fc domain binds to CD16.

8. The method according to claim 1, characterized in that the feeder cells include peripheral blood mononuclear cells (PBMCs), fibroblasts, epithelial cells, endothelial cells, antigen-presenting cells, K562 cells, or microbial cells.

9. The method according to claim 8, characterized in that the feeder cells include RPMI8866, HFWT, 721.221, or EBV-LCL.

10. The method according to claim 1, further comprising contacting the at least one γδT cell with at least one γδT cell effector agent, (a) The at least one γδ T cell effector agent is expressed on or bound to the engineered feeder cells, the engineered plasma membrane particles, the engineered exosomes, or the outer surface of the solid support, and (b) A method characterized in that the at least one γδ T cell effector agent comprises a cytokine, an adhesion molecule, or a γδ T cell activator.

11. The method according to claim 10, characterized in that the at least one γδ T cell effector agent comprises a ligand for 4-1BBL, CD80, CD86, MICA, UBLP, 2B4, LFA-1, NKG2D, a ligand for DNAM-1, IL-2, IL-12, IL-18, IL-15, or IL-21, or any combination thereof.

12. The method according to claim 11, characterized in that the at least one γδ T cell effector agent comprises 4-1BBL, IL-18, IL-15, or IL-21, or any combination thereof.

13. In the method according to claim 1, (a) The proliferated γδ T cells further include the Vδ1 subtype; (b) The γδ T cells are autologous, haplotype-identical, or allogeneic γδ T cells; and / or (c) The γδ T cells are in a mixed cell population that includes NK cells. A method characterized by the following:

14. The method according to claim 1, characterized in that the γδ T cells proliferate at a faster rate over 14 days than the control γδ T cell population.

15. The method according to claim 1, characterized in that the γδ T cells are in a mixed cell population including NK cells, and the mixed cell population depletes the NK cells before, during, or after the proliferation of the γδ T cells.

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