Multiple receptor natural killer cells

Abstract

Embodiments of this disclosure include methods and compositions for human-mediated modification of NK cells to express heterologous a) polypeptides containing a CD16 Fc-binding domain, b) T cell receptors (TCRs, e.g., invariant T cell receptors (iTCRs)), and c) CD3 coreceptors not naturally expressed on NK cells. Such modified NK cells effectively act with monospecific, bispecific, and / or multispecific antibodies having modified or unmodified Fc domains.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 500,423, filed on 5 May 2023, and U.S. Provisional Patent Application No. 63 / 601,150, filed on 20 November 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence List This application includes a sequence listing filed in ST26 format, which is incorporated into this application in its entirety by reference. The ST26 copy, created on 2 May 2023, is named MDAC_1358_Sequence_Listing.xml and has a size of 256,797 bytes.

[0003] This disclosure relates at least to the fields of immunology, cell biology, molecular biology, and medicine (including at least cancer medicine). [Background technology]

[0004] Natural killer (NK) cells have been studied as potential antitumor effectors, but many barriers, primarily related to a lack of antigen specificity, limit their therapeutic use. One approach to overcome this is to transduce NK cells with chimeric antigen receptors (CARs) or engineered T cell receptors (TCRs) to target desired antigens. In T cells, bispecific or multispecific antibodies can be utilized, such as bispecific T cell engagers (BiTEs) that bind to CD3 on the T cell surface and also to antigens on the surface of target cells (e.g., cancer cells). CD3 is composed of four distinct chains; in mammals, the complex contains CD3γ chain, CD3δ chain, and two CD3ε chains. These chains bind to the T cell receptor (TCR) and ζ (zeta) chain, generating activation signals in T lymphocytes. However, since NK cells do not naturally express the CD3 receptor complex or TCR, they cannot be effectively utilized even when used in combination with BiTEs. NK cells naturally express FcγR proteins such as FcγRIII(CD16), but polymorphisms in the extracellular Fc-recognition domain can result in a lack (or poor) binding of non-Fc-domain modification (e.g., glycosylation) antibodies to the NK cell surface. [Overview of the project] [Problems that the invention aims to solve]

[0005] This disclosure addresses a long-standing need in the art to improve the efficacy, safety profile, and targeting capabilities of immunotherapies, including NK cell-based immunotherapy. [Means for solving the problem]

[0006] Autologous anti-CD19 chimeric antigen receptor (CAR) T cells have been shown to induce remission in 57-71% of patients with chronic lymphocytic leukemia (CLL), 52-82% of patients with diffuse large B-cell lymphoma (DLBCL), and 78-92% of patients with low-grade non-Hodgkin lymphoma (LG-NHL). In fact, multiple autologous anti-CD19 CAR T cell products are currently FDA-approved and available for clinical use. However, CAR T cells are recognized to have limitations such as treatment costs and the time required to harvest T cells and manufacture the product. Furthermore, some patients treated with CAR T cells develop toxicities such as cytokine release syndrome (CRS), neurotoxicity, or hemophagocytic lymphohistiocytosis (HLH), each with significant morbidity. These limitations require administration by a specialized team and further restrict access to life-saving treatment. Therefore, there is great interest in the development of cost-effective, safe, and potent off-the-shelf cell therapies. Natural killer (NK) cells play an important role in cancer immune surveillance by downregulating HLA class I molecules or targeting cancer cells that non-antigen-specifically express stress markers. These cells can be engineered to express various transgenes and can be safely administered without the need for HLA matching, eliminating the need to manufacture immunotherapy products on an individual patient basis. This property makes NK cells particularly attractive as an off-the-shelf therapy and thus allows for reduction of manufacturing costs and expansion of access to these potentially life-saving treatments for more patients.

[0007] Embodiments of the present disclosure include, but are not limited to, methods and compositions for treating individuals having disorders such as autoimmune disorders, cancer, and / or infectious diseases using adoptive cell therapy. In a specific embodiment, an individual is provided with a therapeutically effective amount of a bipartite therapy comprising a modified immune cell (e.g., a modified NK cell) and an antibody capable of binding to the NK cell to initiate signaling, activation, and / or killing of target cells. The present disclosure relates to NK cells modified to express a plurality of proteins that do not naturally occur in NK cells, but that function in concert, including heterologous proteins on the NK cell surface that do not naturally occur in NK cells. The present disclosure also relates to NK cells modified to overexpress proteins that naturally occur in NK cells.

[0008] As used herein, polynucleotides are provided that can include sequences that are at least about 80%, 85%, 90%, 95%, 98%, or 100% identical to the transcription reading frames including SEQ ID NOs: 171-175. The polynucleotide encoding the sequence of interest can be housed in a vector. The vector can include at least about 80%, 85%, 90%, 95%, 98%, or 100% sequence identity to SEQ ID NOs: 177-181.

[0009] This specification provides polynucleotides comprising sequences encoding T cell receptor (TCR) alpha and TCR beta polypeptides, and / or TCR gamma and TCR delta polypeptides, as well as polypeptides containing a CD16-derived Fc-binding domain. The TCR polypeptide may be an immutable TCR (iTCR) polypeptide. The iTCRα and / or iTCRβ polypeptide and / or the polynucleotide encoding it may comprise sequences that are at least about 80%, 85%, 90%, 95%, 98%, or 100% identical to one or more of SEQ ID NOs. 51-149. The iTCRβ polypeptide may comprise polynucleotides encoding a Vβ-DJ region that is at least about 80%, 85%, 90%, 95%, 98%, or 100% identical to one or more of SEQ ID NOs. 75-149. The iTCRβ polypeptide and / or the polynucleotide encoding it may contain sequences that are at least approximately 80%, 85%, 90%, 95%, 98%, or 100% identical to one or more of sequence numbers 59-74. The encoded iTCRβ polypeptide may be at least approximately 80%, 85%, 90%, 95%, 98%, or 100% identical to sequence number 60. The polynucleotide encoding the iTCRβ polypeptide may be at least approximately 80%, 85%, 90%, 95%, 98%, or 100% identical to sequence number 59. The iTCRα polypeptide and / or the polynucleotide encoding it may contain sequences that are at least approximately 80%, 85%, 90%, 95%, 98%, or 100% identical to sequence numbers 51-52.

[0010] A polynucleotide containing a sequence encoding a CD16-derived Fc-binding domain may contain the encoding sequence of the human CD16A Fc-binding domain. A polynucleotide encoding a polypeptide containing a CD16-derived Fc-binding domain may contain an Fc-binding domain fused in an N-terminus-to-C-terminus order to any hinge domain, a transmembrane domain (TMD), and one or more any intracellular signaling domains (ICD). The TMD may be derived from CD16 or CD3ζ. The TMD may contain or consist of a sequence having at least about 90% identity with SEQ ID NO: 163 or 167. The hinge domain may be derived from CD32. In certain particular cases, the hinge domain may be a hinge domain as previously described in the art. The hinge domain may contain or consist of a sequence having at least about 90% identity with SEQ ID NO: 161. The ICD may be derived from CD16 and / or CD3ζ. Polynucleotides may contain a CD16-derived Fc-binding domain that does not contain mutations that make the CD16-derived Fc-binding domain resistant to cleavage. The coding sequence of a polypeptide containing a CD16-derived Fc domain may be at least about 80%, 85%, 90%, 95%, 98%, or 100% identical to one or more of sequence numbers 150-154. The polynucleotides provided herein may contain sequences that encode one or more cytokine sequences. Cytokines may include IL-15 and / or IL-21.

[0011] Furthermore, this specification provides engineered NK cells containing polynucleotides disclosed herein. In addition, this specification provides a method for treating a disease in an individual, the method comprising administering engineered NK cells containing one or more polynucleotides disclosed herein to an individual in need thereof.

[0012] This specification provides engineered immune cells comprising one or more transgenic polynucleotides, each encoding a CD3 protein complex comprising a) some or all of a single-strand or any combination of CD3δ, CD3ε, CD3γ, or CD3ζ; b) optionally at least one cytokine; c) at least one TCRα and TCRβ chain, and / or TCRγ and TCRδ chain; and d) a polypeptide comprising a CD16 Fc-binding domain. One or more transgenic polynucleotides may include a multi-cistronic transcription open reading frame. NK cells can be modified to express some or all of CD3δ, and two of CD3ε, CD3γ, and / or CD3ζ. One or more of CD3δ, CD3ε, CD3γ, and / or CD3ζ can be ligated to one or more heterologous intracellular signaling domains. The heterologous intracellular signaling domain may be selected from the group consisting of CD16, NKG2D, DAP10, DAP12, 2B4, 4-1BB, CD2, CD28, and combinations thereof. The heterologous intracellular signaling domain may include the DAP10 intracellular signaling domain. The heterologous intracellular signaling domain may include an amino acid sequence that is at least approximately 85% identical to SEQ ID NO: 42. The heterologous intracellular signaling domain may include the CD28 intracellular signaling domain. The heterologous intracellular signaling domain may include an amino acid sequence that is at least approximately 85% identical to SEQ ID NO: 43. The heterologous intracellular signaling domain may include both the DAP10 and CD28 intracellular signaling domains. The heterologous intracellular signaling domain may include an amino acid sequence that is at least approximately 85% identical to SEQ ID NO: 44.

[0013] The engineered immune cells provided herein may include coding sequences for a CD3 protein complex and at least one cytokine, which may be constituted in a first multi-cistronic construct, and coding sequences for polypeptides including at least one TCRα and TCRβ chain, and / or TCRγ and TCRδ chains, and a CD16 Fc-binding domain may also be encoded by a second multi-cistronic construct. The engineered immune cells may include coding sequences for cytokines, which include IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, and / or GMCSF. The engineered immune cells may be modified to express polynucleotide sequences that are at least 85% identical to UT-NK15-DAP10 (SEQ ID NO: 45), UT-NK15-28 (SEQ ID NO: 47), or UTNK15-28-DAP10 (SEQ ID NO: 49). The cytokines may include IL-15 and / or IL-21. The cytokine may contain IL-15 and may contain a polypeptide sequence and / or a polynucleotide sequence encoding it that is at least 85% identical to one or more of sequence numbers 182-183. The cytokine may contain IL-21 and may contain a polypeptide sequence and / or a polynucleotide sequence encoding it that is at least 85% identical to one or more of sequence numbers 184-187.

[0014] The manipulated immune cells provided herein may contain TCRs, and the TCR polypeptides are invariant TCR (iTCR) polypeptides. The iTCRα and iTCRβ polypeptides and / or the polynucleotides encoding them may contain sequences that are at least about 80%, 85%, 90%, 95%, 98%, or 100% identical to one or more of SEQ ID NOs. 51-149. The iTCRβ polypeptide may contain polynucleotides encoding a Vβ-DJ region that is at least about 80%, 85%, 90%, 95%, 98%, or 100% identical to one or more of SEQ ID NOs. 75-149. The iTCRβ polypeptide and / or the polynucleotides encoding it may contain sequences that are at least about 80%, 85%, 90%, 95%, 98%, or 100% identical to one or more of SEQ ID NOs. 59-74. The encoded iTCRβ polypeptide may be at least approximately 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 60. The polynucleotide encoding the iTCRβ polypeptide may be at least approximately 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 59. The iTCRα polypeptide and / or the polynucleotide encoding it may contain sequences that are at least approximately 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NOs: 51-52.

[0015] The manipulated immune cells provided herein may comprise polypeptides containing a CD16 Fc-binding domain (e.g., a CD16 extracellular domain), the CD16 Fc-binding domain may include a human CD16 Fc-binding domain. Polypeptides containing a CD16-derived Fc-binding domain may comprise a human CD16A Fc-binding domain. Polypeptides containing a CD16 Fc-binding domain may be fused (from N-terminus to C-terminus) to any hinge domain, a transmembrane domain (TMD), and one or more any intracellular signaling domains (ICD). The TMD may be derived from CD16 or CD3ζ. The TMD may contain or consist of a sequence having at least about 90% identity with SEQ ID NO: 163 or 167. The hinge domain may be derived from CD32. The hinge domain may contain or consist of a sequence having at least about 90% identity with SEQ ID NO: 161. The ICD may be derived from CD16 and / or CD3ζ. The manipulated immune cells provided herein may contain coding sequences that are at least approximately 80%, 85%, 90%, 95%, 98%, or 100% identical to one or more of sequence numbers 171-175.

[0016] The manipulated immune cells provided herein may be natural killer (NK) cells. NK cells may originate from umbilical cord blood (CB), peripheral blood (PB), bone marrow, stem cells, or a combination thereof. NK cells may be primary NK cells and may not originate from stem cells and / or induced pluripotent stem cells (iPSCs).

[0017] Furthermore, this specification provides compositions comprising engineered NK cells loaded (e.g., complexed) with one or more antibodies. The one or more antibodies may be monospecific, bispecific, or multispecific antibodies. At least one antibody may contain a glycosylated Fc domain having high affinity for wild-type CD16. At least one antibody may contain an unglycosylated Fc domain having low affinity for wild-type CD16 Fc-binding domains. The unglycosylated Fc domains may be loaded (complexed) onto a transgenic polypeptide containing a CD16 Fc-binding domain. The one or more antibodies may contain IgG1 and / or IgG4 Fc domains.

[0018] One or more antibody target antigens may be CD3, CD16, CD28, EGFR, c-MET, CD30, PSMA, MUC17, CD33, FLT3, STEAP1, BCMA, CLDN18.2, CD123, CD19, CD20, EpCAM, CEA, GPC3, CD38, CD33, CD22, HER2, GPA33, GD2, MUC16, GPRC5D, DLL-3, CLEC12A, and / or SSTR. One or more antibody targets may be CD3, CD16, CD28, CD19, CD20, CD30, HER2, GPRC5D, EGFR, c-MET, FcRH5, and / or BCMA. One or more antibodies include [fam]-trastuzumab deruxtecan, absiximab, adalimumab, ad-trastuzumab emtansine, aducanumab, alemtuzumab, alirocumab, amivantamab, aniflorumab, ansuvimab, atezolizumab, atorutibimab and maftibimab and odesibimab-ebgn (also known as immazeb), avelumab, basiliximab, verantamab mahodotin, belimumab, benralizumab, bevacizumab, bezlotoxumab, bimekizumab, blinatumomab, and brentuximab. Vedotin, Brodalumab, Brolucizumab, Brosumab, Canakinumab, Caplacizumab, Casiribimab + Imdevimab, Catumaxomab, Semiprimab, Certolizumab Pegol, Cetuximab, Cebostamab, Chryzanlizumab, Daclizumab, Daratumumab, Denosumab, Dinutuximab, Donanemab, Dostarimab, Dupilumab, Durvalumab, Eculizumab, Edrecolomab, Efalizumab, Elotuzumab, Emapalmab, Emicizumab, Enfortzumab Vedotin, eptinezumab, erenumab, evinacumab, evolocumab, falisimab, fremanezumab, galcanezumab, gemtuzumab, gemtuzumab-ozogamicin, golimumab, guselkumab, ibalizumab, ibritumomab, tiucetan, idarucizumab, imugatuzumab, inebilizumab, infliximab, inorimomab, inotuzumab, inotuzumab-ozogamicin, IPH61, ipilimumab, isatuximab, ixekizumab, lanadelmab, lecanemab, ronkastuximab, tesirin, margetuximab, mepolizumab, milbetuximabSorabtansine, mogamulizumab, mosnetuzumab, moxetumomab Pasdotox, muromonab-CD3, nalsoprimab, natalizumab, naxitamab, nevacumab, necitumumab, nirsevimab, nivolumab, obiltoxaximab, obinutuzumab, ocrelizumab, ofatumumab, oraratumab, omalizumab, omblutamab, oporutuzumab Monatox, palivizumab, panitumumab, pembrolizumab, pemprimab, pertuzumab, polatuzumab Vedotin, Ramucirumab, Ranibizumab, Rabrizumab, Laxibakumab, Regdanvimab, Relatrimab, Reslizumab, Letifanlimab, Risankizumab, Rituximab, Romosozumab, Sacituzumab Govitecan, Sarilumab, Satralizumab, Secukinumab, Siltuximab, Syntilimab, Sotrovimab, Spesolimab, Stimulimab, Tafacitamab, Teventafusp, Tecristamab, Teprizumab, Teprotumumab, Tezeperumab, Childraquizumab, Tislerizumab, Tisotumab Vedotin, tixagevimab, silgavimab, tocilizumab, tripalimab, tositumomab-I131, tralokinumab, trastuzumab, tremelimumab, ubrituximab, ustekinumab, vedolizumab, AMG 160 / akapatamab, AMG 199 / TNB 585, AMG 330, AMG 427 / emirodatamab, AMG 509, AMG 701, AMG 910, APVO414 / ES414 / MOR209, APVO436, Katsumakisomab / Removab, CC-1, CC-93269 / EM801, Sibisatamab / RG7802 / RO6958688, CLN-049, Elranatamab / PF-06863135, EMB-06, Epcolitamab / GEN3013, ERY974, Flotetuzumab / MGD006, Grofitamab / RG6026 / RO7082859, ISB1342 / GBR1342, JNJ-63709178, JNJ-63898081, JNJ-67571244, JNJ-75348780, Limbocertamab / REGNThis may include 5458, M701, M802, MGD007, mosnetuzumab / RG7828, nivatorotamab / Hu3F8-BsAb, odronextamab / REGN1979, REGN4018, REGN5459, REGN7075, REGN5678, talketamab / JNJ-64407564, tarlatamab / AMG 757, tepozitamab / MCLA-117, TNB-383B, TNB-486, TNB-585, XmAb13676 / pramotamab, XmAb14045 / vibecotamab, XmAb18087 / tidutamab, and / or AFM13. One or more antibodies may include erranatamab, imugatuzumab, margetuximab, amivantamab, blinatumomab, obinutuzumab, IPH61 (also known as IPH6101 or SAR443579), tecristamab, cetuximab, talketamab, pertuzumab, trastuzumab, tafacitamab, brentuximab, and / or rituximab. One or more antibodies may include or consist of erranatamab. One or more antibodies may include or consist of imugatuzumab. One or more antibodies may include or consist of margetuximab. One or more antibodies may include or consist of amivantamab. One or more antibodies may include or consist of blinatumomab. One or more antibodies may include or consist of obinutuzumab. One or more antibodies may include or consist of IPH61. One or more antibodies may include or consist of teclistamagb. One or more antibodies may include or consist of cetuximab. One or more antibodies may include or consist of rituximab. NK cells may be manipulated to express one or more antibodies. One or more antibodies may include or consist of talketamab. One or more antibodies may include or consist of pertuzumab. One or more antibodies may include or consist of trastuzumab. One or more antibodies may include or consist of tafacitamab. One or more antibodies may include or consist of brentuximab.

[0019] Engineered NK cells may be modified to express one or more additional heterologous proteins selected from the group consisting of antigen receptors, cytokines, homing receptors, chemokine receptors, and combinations thereof. Engineered NK cells may be pre-activated with one or more cytokines. Pre-activated cytokines may be IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, or any combination thereof. Engineered NK cells may contain one or more engineered mutations in endogenous genes. Engineered mutations in endogenous genes may be mutations in GR, TGFBR2, CISH, and / or CD38. Engineered NK cells provided herein may be contained in a composition, which may optionally contain pharmaceutically acceptable excipients and / or be contained in a delivery device.

[0020] Furthermore, this specification provides methods for treating diseases in individuals. Methods for treating individuals may include administering to an individual a therapeutically effective amount of one or more of the cells or compositions described herein. Diseases may be autoimmune diseases, infections, and / or cancers. A disease may be cancer. Cancer may express tumor-associated antigens (TAAs). Cancer may express CD19, CD20, CD30, HER2, GPRC5D, EGFR, c-MET, and / or BCMA. Cancers may include pancreatic cancer, colorectal cancer, ovarian cancer, kidney cancer, glioblastoma, breast cancer, renal cancer, myeloma, and / or leukemia. Methods for treating individuals may further include administering to an individual one or more monospecific antibodies, bispecific antibodies, and / or multispecific antibodies simultaneously or at different times. One or more antibodies may include erranatamab, imugatuzumab, margetuximab, amibantamab, blinatumomab, obinutuzumab, IPH61 (also known as IPH6101 or SAR443579), tecristamag, cetuximab, and / or rituximab. One or more antibodies may be administered concurrently with engineered NK cells, and / or one or more antibodies may be conjugated with engineered NK cells before administration to an individual. One or more antibodies may be administered before, after, and / or concurrently with engineered NK cells. One or more antibodies may be administered more than once before, after, and / or concurrently with engineered NK cells.

[0021] Certain embodiments of the present invention are characterized by the following aspects.

[0022] Embodiment 1 is a polynucleotide comprising a sequence that is at least about 80%, 85%, 90%, 95%, 98%, or 100% identical to a transcription reading frame comprising sequence numbers 171-175.

[0023] Embodiment 2 is the polynucleotide according to Embodiment 1, wherein the polynucleotide is contained in a vector having at least about 80%, 85%, 90%, 95%, 98%, or 100% sequence identity with SEQ ID NOs. 177-181.

[0024] Embodiment 3 is a polynucleotide comprising a sequence encoding T cell receptor (TCR) alpha and TCR beta polypeptides, and / or TCR gamma and TCR delta polypeptides, as well as a polypeptide containing a CD16-derived Fc-binding domain.

[0025] Embodiment 4 is the polynucleotide according to Embodiment 3, wherein the TCR polypeptide is an invariant TCR (iTCR) polypeptide.

[0026] Embodiment 5 is the polynucleotide according to Embodiment 4, wherein the iTCRα and iTCRβ polypeptides and / or the polynucleotide encoding them include a sequence that is at least about 80%, 85%, 90%, 95%, 98%, or 100% identical to one or more of the sequence numbers 51 to 149.

[0027] Embodiment 6 is a polynucleotide according to any one of Embodiments 3 to 5, wherein the iTCRβ polypeptide comprises a polynucleotide encoding a Vβ-DJ region which is at least approximately 80%, 85%, 90%, 95%, 98%, or 100% identical to one or more of the sequence numbers 75 to 149.

[0028] Embodiment 7 is a polynucleotide according to any one of Embodiments 3 to 6, wherein the iTCRβ polypeptide and / or the polynucleotide encoding it includes a sequence that is at least about 80%, 85%, 90%, 95%, 98%, or 100% identical to one or more of the sequence numbers 59 to 74.

[0029] Embodiment 8 is a polynucleotide according to any one of Embodiments 3 to 7, wherein the encoded iTCRβ polypeptide is at least about 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 60.

[0030] Embodiment 9 is the polynucleotide according to Embodiment 8, wherein the polynucleotide encoding the iTCRβ polypeptide is at least about 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 59.

[0031] Embodiment 10 is a polynucleotide according to any one of Embodiments 3 to 9, wherein the iTCRα polypeptide and / or the polynucleotide encoding it comprises a sequence that is at least about 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NOs. 51-52.

[0032] Embodiment 11 is a polynucleotide according to any one of Embodiments 3 to 10, wherein the polypeptide containing a CD16-derived Fc-binding domain contains a human CD16A Fc-binding domain.

[0033] Embodiment 12 is a polynucleotide according to any one of embodiments 3 to 11, further comprising an Fc-binding domain fused in an N-terminus-to-C-terminus order to any hinge domain, a transmembrane domain (TMD), and one or more any intracellular signaling domains (ICD).

[0034] Embodiment 13 is the polynucleotide according to Embodiment 12, wherein the TMD is derived from CD16 or CD3ζ.

[0035] Embodiment 14 is a polynucleotide according to Embodiment 12 or 13, wherein the TMD comprises or consists of a sequence having at least about 90% identity with SEQ ID NO: 163 or 167.

[0036] Embodiment 15 is a polynucleotide according to any one of Embodiments 12 to 14, wherein the hinge domain is derived from CD32.

[0037] Embodiment 16 is a polynucleotide according to any one of embodiments 12 to 15, comprising a hinge domain having at least about 90% identity with sequence number 161.

[0038] Embodiment 17 is a polynucleotide according to any one of Embodiments 12 to 16, comprising an ICD derived from CD16 and / or CD3ζ.

[0039] Embodiment 18 is a polynucleotide according to any one of Embodiments 3 to 17, wherein the polypeptide containing the CD16-derived Fc-binding domain does not contain a mutation that makes the CD16-derived Fc-binding domain resistant to cleavage.

[0040] Embodiment 19 is a polynucleotide according to any one of Embodiments 3 to 18, comprising a coding sequence that is at least approximately 80%, 85%, 90%, 95%, 98%, or 100% identical to one or more of the sequence numbers 150 to 154.

[0041] Embodiment 20 is a polynucleotide according to any one of embodiments 3 to 19, wherein the polynucleotide further encodes one or more cytokine sequences.

[0042] Embodiment 21 is the polynucleotide according to Embodiment 20, wherein the cytokine comprises IL-15 and / or IL-21.

[0043] Embodiment 22 is an engineered NK cell containing the polynucleotide described in any one of Embodiments 1 to 22.

[0044] Embodiment 23 is a method for treating a disease in an individual, comprising administering the manipulated NK cells described in Embodiment 22 to an individual in need of treatment.

[0045] Embodiment 24 is an engineered immune cell comprising one or more transgenic polynucleotides encoding a) a CD3 protein complex comprising a single-strand or any combination of CD3δ, CD3ε, CD3γ, or CD3ζ, part or all of it; b) optionally at least one cytokine; c) at least one TCRα and TCRβ chain, and / or TCRγ and TCRδ chain, and d) a polypeptide comprising a CD16 Fc-binding domain.

[0046] Embodiment 25 is an engineered immune cell according to Embodiment 24, wherein one or more transgenic polynucleotides include a multi-cistronic transcription open reading frame.

[0047] Embodiment 26 is an engineered immune cell according to Embodiment 24 or 25, wherein the cell is modified to express two CD3δ, two CD3ε, one CD3γ, and / or one or all of the CD3ζ.

[0048] Embodiment 27 is an engineered immune cell according to any one of embodiments 24 to 26, wherein one or more of CD3δ, CD3ε, CD3γ, and / or CD3ζ are linked to one or more heterologous intracellular signaling domains.

[0049] Embodiment 28 is an engineered immune cell according to Embodiment 27, wherein the heterologous intracellular signaling domain is selected from the group consisting of CD16, NKG2D, DAP10, DAP12, 2B4, 4-1BB, CD2, CD28, and combinations thereof.

[0050] Embodiment 29 is an engineered immune cell according to any one of Embodiments 27 to 28, wherein the heterologous intracellular signaling domain includes a DAP10 intracellular signaling domain.

[0051] Embodiment 30 is the manipulated immune cell according to Embodiment 29, wherein the heterologous intracellular signaling domain contains an amino acid sequence that is at least about 85% identical to SEQ ID NO: 42.

[0052] Embodiment 31 is an engineered immune cell according to any one of Embodiments 27 to 30, wherein the heterologous intracellular signaling domain includes a CD28 intracellular signaling domain.

[0053] Embodiment 32 is the manipulated immune cell according to Embodiment 31, wherein the heterologous intracellular signaling domain contains an amino acid sequence that is at least about 85% identical to SEQ ID NO: 43.

[0054] Embodiment 33 is an engineered immune cell according to any one of Embodiments 27 to 32, wherein the heterologous intracellular signaling domain includes the DAP10 and CD28 intracellular signaling domains.

[0055] Embodiment 34 is the manipulated immune cell according to Embodiment 33, wherein the heterologous intracellular signaling domain contains an amino acid sequence that is at least about 85% identical to SEQ ID NO: 44.

[0056] Embodiment 35 is an engineered immune cell according to any one of embodiments 24 to 34, wherein the coding sequence of a CD3 protein complex and at least one cytokine is contained in a first multi-cistronic construct, and the coding sequence of a polypeptide comprising at least one TCRα and TCRβ chain, and / or TCRγ and TCRδ chain, and a CD16 Fc-binding domain is encoded by a second multi-cistronic construct.

[0057] Embodiment 36 is an engineered immune cell according to any one of embodiments 24 to 35, comprising a cytokine coding sequence, wherein the cytokine comprises IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, and / or GMCSF.

[0058] Embodiment 37 is the engineered immune cell according to Embodiment 36, wherein the cell is modified to express a polynucleotide sequence that is at least 85% identical to UT-NK15-DAP10 (SEQ ID NO: 45), UT-NK15-28 (SEQ ID NO: 47), or UTNK15-28-DAP10 (SEQ ID NO: 49).

[0059] Embodiment 38 is the engineered immune cell according to Embodiment 37, wherein the cytokines include IL-15 and / or IL-21.

[0060] Embodiment 39 is an engineered immune cell according to Embodiment 38, wherein the cytokine comprises IL-15 and a polypeptide sequence and / or a polynucleotide sequence encoding it that is at least 85% identical to one or more of SEQ ID NOs. 182-183.

[0061] Embodiment 40 is an engineered immune cell according to Embodiment 38, wherein the cytokine comprises IL-21 and a polypeptide sequence and / or a polynucleotide sequence encoding it that is at least 85% identical to one or more of SEQ ID NOs. 184 to 187.

[0062] Embodiment 41 is an engineered immune cell according to any one of Embodiments 24 to 40, wherein the TCR polypeptide is an invariant TCR (iTCR) polypeptide.

[0063] Embodiment 42 is an engineered immune cell according to Embodiment 41, wherein the iTCRα and iTCRβ polypeptides and / or the polynucleotides encoding them include sequences that are at least about 80%, 85%, 90%, 95%, 98%, or 100% identical to one or more of the sequence numbers 51-149.

[0064] Embodiment 43 is an engineered immune cell according to Embodiment 41 or 42, wherein the iTCRβ polypeptide comprises a polynucleotide encoding a Vβ-DJ region which is at least about 80%, 85%, 90%, 95%, 98%, or 100% identical to one or more of SEQ ID NOs. 75 to 149.

[0065] Embodiment 44 is an engineered immune cell according to any one of Embodiments 41 to 43, wherein the iTCRβ polypeptide and / or the polynucleotide encoding it comprises a sequence that is at least about 80%, 85%, 90%, 95%, 98%, or 100% identical to one or more of SEQ ID NOs. 59 to 74.

[0066] Embodiment 45 is an engineered immune cell according to any one of Embodiments 41 to 44, wherein the encoded iTCRβ polypeptide is at least about 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 60.

[0067] Embodiment 46 is an engineered immune cell according to Embodiment 45, wherein the polynucleotide encoding the iTCRβ polypeptide is at least about 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 59.

[0068] Embodiment 47 is an engineered immune cell according to any one of Embodiments 41 to 46, wherein the iTCRα polypeptide and / or the polynucleotide encoding it comprises a sequence that is at least about 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NOs. 51 to 52.

[0069] Embodiment 48 is an engineered immune cell according to any one of Embodiments 24 to 47, wherein the polypeptide containing the CD16 Fc-binding domain contains a human CD16-derived Fc-binding domain.

[0070] Embodiment 49 is an engineered immune cell according to any one of Embodiments 24 to 48, wherein the polypeptide containing the CD16-derived Fc-binding domain contains a human CD16A Fc-binding domain.

[0071] Embodiment 50 is an engineered immune cell according to any one of Embodiments 24 to 49, wherein the Fc-binding domain is fused to any hinge domain, transmembrane domain (TMD), and one or more any intracellular signaling domains (ICD) in an N-terminal to C-terminal order.

[0072] Embodiment 51 is the engineered immune cell according to Embodiment 50, wherein the TMD is derived from CD16 or CD3ζ.

[0073] Embodiment 52 is an engineered immune cell according to Embodiment 50 or 51, wherein the TMD contains or consists of a sequence having at least about 90% identity with SEQ ID NO: 163 or 167.

[0074] Embodiment 53 is an engineered immune cell according to any one of Embodiments 50 to 52, wherein the hinge domain is derived from CD32.

[0075] Embodiment 54 is an engineered immune cell according to any one of embodiments 50 to 53, wherein the hinge domain contains or consists of a sequence having at least about 90% identity with SEQ ID NO: 161.

[0076] Embodiment 55 is an engineered immune cell according to any one of embodiments 50 to 54, comprising an ICD derived from CD16 and / or CD3ζ.

[0077] Embodiment 56 is an engineered immune cell according to any one of Embodiments 24 to 55, comprising a coding sequence that is at least approximately 80%, 85%, 90%, 95%, 98%, or 100% identical to one or more of the sequence numbers 171 to 175.

[0078] Embodiment 57 is an engineered immune cell according to any one of Embodiments 24 to 56, wherein the cell is a natural killer (NK) cell.

[0079] Embodiment 58 is the engineered NK cells described in Embodiment 57, wherein the NK cells are derived from umbilical cord blood (CB), peripheral blood (PB), bone marrow, stem cells, or a combination thereof.

[0080] Embodiment 59 is an engineered NK cell according to any one of Embodiments 57 to 58, wherein the NK cell is a primary NK cell and is not derived from a stem cell and / or an induced pluripotent stem cell (iPSC).

[0081] Embodiment 60 is an engineered NK cell according to any one of embodiments 57 to 59, wherein the NK cell is loaded with (and complexed with) one or more antibodies.

[0082] Embodiment 61 is the manipulated NK cells according to Embodiment 60, wherein one or more antibodies are one or more monospecific antibodies, bispecific antibodies, or multispecific antibodies.

[0083] Embodiment 62 is an engineered NK cell according to Embodiment 60 or 61, wherein at least one antibody comprises a glycosylated Fc domain having high affinity for wild-type CD16.

[0084] Embodiment 63 is an engineered NK cell according to Embodiment 60 or 61, wherein at least one antibody comprises an unglycosylated Fc domain having low affinity for the wild-type CD16 Fc binding domain.

[0085] Embodiment 64 is the modified NK cell according to Embodiment 63, wherein the non-glycosylated Fc domain is loaded (complexed) with a transgenic polypeptide containing a CD16 Fc-binding domain.

[0086] Embodiment 65 is an engineered NK cell according to any one of embodiments 60 to 64, wherein one or more antibodies contain IgG1 and / or IgG4 Fc domains.

[0087] Embodiment 66 is an engineered NK cell according to any one of embodiments 60 to 65, wherein one or more antibodies target the antigens CD3, CD16, CD28, EGFR, c-MET, CD30, PSMA, MUC17, CD33, FLT3, STEAP1, BCMA, CLDN18.2, CD123, CD19, CD20, EpCAM, CEA, GPC3, CD38, CD33, CD22, HER2, GPA33, GD2, MUC16, GPRC5D, DLL-3, CLEC12A, FcRH5, and / or SSTR.

[0088] Embodiment 67 is an engineered NK cell according to any one of embodiments 60 to 66, wherein one or more antibodies target CD3, CD16, CD28, CD19, CD20, CD30, HER2, GPRC5D, EGFR, c-MET, and / or BCMA.

[0089] Embodiment 68 is a manipulated NK cell in which one or more antibodies are [fam]-trastuzumab deruxtecan, absiximab, adalimumab, ad-trastuzumab emtansine, aducanumab, alemtuzumab, alirocumab, amivantamab, aniflorumab, ansuvimab, atezolizumab, atorutibimab and maftibimab and odesibimab-ebgn (also known as immazeb), avelumab, basiliximab, verantamab mahodotin, belimumab, benralizumab, bevacizumab, bezlotoxumab, bimekizumab, blinatumomab, brentuximab Vedotin, Brodalumab, Brolucizumab, Brosumab, Canakinumab, Caplacizumab, Casiribimab + Imdevimab, Catumaxomab, Semiprimab, Certolizumab Pegol, Cetuximab, Cebostamab, Chryzanlizumab, Daclizumab, Daratumumab, Denosumab, Dinutuximab, Donanemab, Dostarimab, Dupilumab, Durvalumab, Eculizumab, Edrecolomab, Efalizumab, Elotuzumab, Emapalmab, Emicizumab, Enfortzumab Vedotin, eptinezumab, erenumab, evinacumab, evolocumab, falisimab, fremanezumab, galcanezumab, gemtuzumab, gemtuzumab-ozogamicin, golimumab, guselkumab, ibalizumab, ibritumomab, tiucetan, idarucizumab, imugatuzumab, inebilizumab, infliximab, inorimomab, inotuzumab, inotuzumab-ozogamicin, IPH61, ipilimumab, isatuximab, ixekizumab, lanadelmab, lecanemab, ronkastuximab, tesirin, margetuximab, mepolizumab, milbetuximab, sorabtansine, mogamulizumab, mosnetuzumab, moxetumomab Pasdotox, Muromonab-CD3, Narsoprimab, Natalizumab, Naxitamab, Nevacumab, Necitumumab, Nilsevimab, Nivolumab, Oviltoxaximab, Obinutuzumab, Ocrelizumab, Ofatumumab, Oraratumab, Omalizumab, Omblutamab, Oportuzumab Monatox, Palivizumab, Panitumumab, Pembrolizumab, Penprimab, Pertuzumab, PolatuzumabVedotin, Ramucirumab, Ranibizumab, Rabrizumab, Laxibakumab, Regdanvimab, Relatrimab, Reslizumab, Letifanlimab, Risankizumab, Rituximab, Romosozumab, Sacituzumab Govitecan, Sarilumab, Satralizumab, Secukinumab, Siltuximab, Syntilimab, Sotrovimab, Spesolimab, Stimulimab, Tafacitamab, Teventafusp, Tecristamab, Teprizumab, Teprotumumab, Tezeperumab, Childraquizumab, Tislerizumab, Tisotumab Vedotin, tixagevimab, silgavimab, tocilizumab, tripalimab, tositumomab-I131, tralokinumab, trastuzumab, tremelimumab, ubrituximab, ustekinumab, vedolizumab, AMG 160 / akapatamab, AMG 199 / TNB 585, AMG 330, AMG 427 / emirodatamab, AMG 509, AMG 701, AMG 910, APVO414 / ES414 / MOR209, APVO436, Katsumakisomab / Removab, CC-1, CC-93269 / EM801, Sibisatamab / RG7802 / RO6958688, CLN-049, Elranatamab / PF-06863135, EMB-06, Epcolitamab / GEN3013, ERY974, Flotetuzumab / MGD006, Grofitamab / RG6026 / RO7082859, ISB 1342 / GBR 1342, JNJ-63709178, JNJ-63898081, JNJ-67571244, JNJ-75348780, Limbocertamab / REGN The engineered NK cells described in any one of embodiments 60 to 67 include 5458, M701, M802, MGD007, mosnetuzumab / RG7828, nivatorotamab / Hu3F8-BsAb, odronectamab / REGN1979, REGN4018, REGN5459, REGN7075, REGN5678, talketamab / JNJ-64407564, tarulatamab / AMG 757, tepositamab / MCLA-117, TNB-383B, TNB-486, TNB-585, XmAb13676 / pramotamab, XmAb14045 / vibecotamab, XmAb18087 / tidutamab, and / or AFM13.

[0090] Embodiment 69 is an engineered NK cell according to any one of Embodiments 60 to 68, wherein one or more antibodies comprise erranatamab, imugatuzumab, margetuximab, amibantamab, blinatumomab, obinutuzumab, IPH61 (also known as IPH6101 or SAR443579), tecristamag, cetuximab, talketamab, pertuzumab, trastuzumab, tafacitamab, brentuximab, and / or rituximab.

[0091] Embodiment 70 is the engineered NK cells according to Embodiment 69, wherein one or more antibodies contain or consist of erranatamab.

[0092] Embodiment 71 is the engineered NK cells according to Embodiment 69, wherein one or more antibodies contain or consist of imugatuzumab.

[0093] Embodiment 72 is the engineered NK cells according to Embodiment 69, wherein one or more antibodies contain or consist of margetuximab.

[0094] Embodiment 73 is the engineered NK cells according to Embodiment 69, wherein one or more antibodies contain or consist of amibantamab.

[0095] Embodiment 74 is the engineered NK cells according to Embodiment 69, wherein one or more antibodies contain or consist of blinatumomab.

[0096] Embodiment 75 is the engineered NK cells according to Embodiment 69, wherein one or more antibodies contain or consist of obinutuzumab.

[0097] Embodiment 76 is the manipulated NK cells according to Embodiment 69, wherein one or more antibodies contain or consist of IPH61.

[0098] Embodiment 77 is the engineered NK cells according to Embodiment 69, wherein one or more antibodies contain or consist of teclistamaib.

[0099] Embodiment 78 is the engineered NK cells according to Embodiment 69, wherein one or more antibodies contain or consist of cetuximab.

[0100] Embodiment 79 is the engineered NK cells according to Embodiment 69, wherein one or more antibodies contain or consist of rituximab.

[0101] Embodiment 80 is the engineered NK cells according to Embodiment 69, wherein one or more antibodies contain or consist of talketamab.

[0102] Embodiment 81 is the engineered NK cells according to Embodiment 69, wherein one or more antibodies contain or consist of pertuzumab.

[0103] Embodiment 82 is the engineered NK cells according to Embodiment 69, wherein one or more antibodies contain or consist of trastuzumab.

[0104] Embodiment 83 is the engineered NK cells according to Embodiment 69, wherein one or more antibodies contain or consist of tafacitamab.

[0105] Embodiment 84 is the engineered NK cells according to Embodiment 69, wherein one or more antibodies contain or consist of brentuximab.

[0106] Embodiment 85 is an engineered NK cell according to any one of Embodiments 60 to 84, wherein the NK cell expresses one or more antibodies.

[0107] Embodiment 86 is an engineered NK cell according to any one of embodiments 57 to 85, wherein the NK cell is further modified to express one or more additional heterologous proteins selected from the group consisting of antigen receptors, cytokines, homing receptors, chemokine receptors, and combinations thereof.

[0108] Embodiment 87 is an engineered NK cell according to any one of embodiments 57 to 86, wherein the NK cell has been pre-activated with one or more cytokines.

[0109] Embodiment 88 is the engineered NK cell according to Embodiment 87, wherein the cytokine is IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, or a combination thereof.

[0110] Embodiment 89 is an engineered NK cell according to any one of embodiments 57 to 88, wherein the NK cell further comprises one or more engineered mutations in an endogenous gene.

[0111] Embodiment 90 is the engineered NK cell according to Embodiment 89, wherein the endogenous genes are GR, TGFBR2, CISH, and / or CD38.

[0112] Embodiment 91 is a composition comprising manipulated NK cells as described in any one of Embodiments 57 to 90.

[0113] Embodiment 92 is the composition according to Embodiment 91, further comprising a pharmaceutically acceptable excipient.

[0114] Embodiment 93 is the composition according to Embodiment 91 or 92, wherein the composition is contained in a delivery device.

[0115] Embodiment 94 is a method for treating a disease in an individual, comprising the step of administering to the individual an effective therapeutic amount of any one of the cells or compositions described in Embodiments 24 to 91.

[0116] Embodiment 95 is the method according to Embodiment 94, wherein the disease is an autoimmune disease, an infection, and / or cancer.

[0117] Embodiment 96 is the method described in Embodiment 95, wherein the disease is an autoimmune disease.

[0118] Embodiment 97 is the method according to Embodiment 95.1, wherein the autoimmune disease includes B cell-associated autoimmunity, systemic lupus erythematosus (SLE), systemic sclerosis (SSc), multiple sclerosis (MS), Graves' disease, rheumatoid arthritis (RA), myositis, dermatomyositis, myasthenia gravis, Sjögren's syndrome, diffuse sclerosis, inflammatory myopathy, ANCA-associated systemic vasculitis, antiphospholipid syndrome, immune nephritis, ITP, refractory POEMS syndrome, amyloidosis, autoimmune hemolytic anemia, and / or vasculitis.

[0119] Embodiment 98 is the method described in Embodiment 95, wherein the disease is cancer.

[0120] Embodiment 99 is the method according to Embodiment 98, wherein the cancer expresses CD19, CD20, CD30, HER2, GPRC5D, EGFR, c-MET, and / or BCMA.

[0121] Embodiment 100 is the method according to Embodiment 98 or 99, wherein the cancer is pancreatic cancer, colorectal cancer, ovarian cancer, kidney cancer, glioblastoma, breast cancer, renal cancer, myeloma, and / or leukemia.

[0122] Embodiment 101 is a method according to any one of embodiments 95 to 100, further comprising administering one or more monospecific antibodies, bispecific antibodies, and / or multispecific antibodies to an individual simultaneously or at different times.

[0123] Embodiment 102 is the method according to Embodiment 101, wherein one or more antibodies comprise erranatamab, imugatuzumab, margetuximab, amibantamab, blinatumomab, obinutuzumab, IPH61 (also known as IPH6101 or SAR443579), tecristamag, cetuximab, talketamab, pertuzumab, trastuzumab, tafacitamab, brentuximab, and / or rituximab.

[0124] Embodiment 103 is the method according to Embodiment 101 or 102, wherein one or more antibodies are administered simultaneously, and / or NK cells manipulated with one or more antibodies are complexed before administration to an individual.

[0125] Embodiment 104 is the method according to any one of Embodiments 101 to 103, wherein one or more antibodies are administered more than once, including at least once at the time following the administration of the manipulated NK cells.

[0126] Embodiment 105 is the method according to any one of Embodiments 101 to 104, wherein one or more antibodies are administered more than once, including at least once at the time prior to the administration of the manipulated NK cells.

[0127] Embodiment 106 is the method according to any one of Embodiments 101 to 105, wherein one or more antibodies are administered more than once, including at least once at the time before administration of the manipulated NK cells and at least once at the time after administration of the manipulated NK cells.

[0128] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that while the detailed description and specific examples illustrate specific embodiments of the present invention, various modifications and alterations within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description and are therefore given for illustrative purposes only.

[0129] The following drawings form part of this specification and are included to further illustrate certain aspects of the invention. The invention may be better understood by referring to one or more of these drawings in combination with the detailed description of the specific embodiments presented herein. Unless otherwise noted, two-way analysis of variance (ANOVA) was used for statistical analysis, with a p-value of less than 0.05 considered statistically significant. [Brief explanation of the drawing]

[0130] [Figure 1-1]Figures 1A–1D show the in vitro expression and cytotoxic function of transduced NK cells with invariant natural killer T cell receptors (iTCRs). Additional experimental details are provided in Example 6 below. Figure 1A depicts an exemplary flow cytometry plot on NK cells showing the expression of iTCRα (SEQ ID NO: 52), and three different iTCR pairs, each containing either iTCRβ chain 1 (SEQ ID NO: 54), iTCRβ chain 2 (SEQ ID NO: 56), or iTCRβ chain 3 (SEQ ID NO: 58), and CD3, using antibodies specific to the iTCR target Vα24 and Vβ11 regions (y and x axes, respectively). Figure 1B shows an exemplary flow cytometry plot showing the binding of blinatumomab (y axis) to NK cells expressing the three different CD3 / iTCR complexes described in (A), e.g., iTCR1, iTCR2, and iTCR3, respectively. NK cells were derived from three donors: cell donor 4 (CD4), cell donor 8 (CD8), or cell donor N (CDN). Blinatumomab binding to CD3 was confirmed by flow cytometry using the Miltenyi Biotech® CAR19 detection kit. Figure 1C shows the results of the IncuCyte® live-cell imaging assay used to measure the cytotoxicity of NK cells pre-loaded with blinatumomab and co-transfected with iTCR and UT-NK15 against GFP-expressing Raji tumor cells in a 3:1 effector-to-target ratio. T cells and iNKT cells were used as positive and negative controls, respectively. Decreased GFP expression indicates cell death. Three NK donor strains, CB152, CB153, and CB154, were used. NK cells were transduced with three different CD3 / iTCR complexes, e.g., iTCR1, iTCR2, or iTCR3, as shown in Figure 1A, or remained untransduced (NT) as a negative control. Figure 1D is a bar graph showing the residual tumor percentage (Y-axis) of the data shown in Figure 1C. Unpaired t-tests were used to individually compare iNKT cells with different iTCRs. CD3 / iTCR NK cells were significantly more cytotoxic (P=<0.01) than iNKT cells (N=3 replications). [Figure 1-2] Same as above.

[0131] [Figure 2-1]Figures 2A-2D show the cloning of iTCR sequences from NK T cells isolated from human umbilical cord blood, and the transgenic expression and efficacy of a subset of iTCR clones in transduced umbilical cord blood-derived NK cells. Additional experimental details are described in Example 6 below. Figure 2A describes the isolation of iTCR clones from purified iNKT cells from five umbilical cord blood donors and exemplary flow cytometry results showing iTCR expression on the iNKT cells of the said donors. iNKT cells were isolated from five umbilical cord blood donors using the iNKT isolation kit from Miltenyi Biotech®. The isolated cells were stimulated and expanded in irradiated umbilical cord blood PBMCs (40 Gry) in the presence of 100 ng / ml alpha-galactosylceramide and 200 U / ml IL-2. On day 7 after iNKT expansion, the purity of the iNKT cultures was confirmed by iNKT-specific antibodies against Vα24 and Vβ11. Total mRNA was extracted from iNKT cells, and the cDNA of the Vβ-DJ region was cloned and sequenced (100 individual clones). Figure 2B describes the sequence of the iTCRβ clone Vβ-DJ sequence. Figure 2C shows the transgenic expression of Vα24 and CD3 in a population of transgenic CD3 / iTCR-expressing NK cells from three umbilical cord blood donors. NK cells were co-transduced on day 5 with iTCR (eight randomly selected iTCRβ clones with coding sequences represented by SEQ ID NOs. 59, 61, 63, 65, 67, 69, 71, or 73, representing clones 3, 18, 24, 51, 56, 76, 93, and 96, respectively, and the iTCRα coding sequence represented by SEQ ID NO. 51) and UT-NK15. iTCR expression was confirmed by flow cytometry using an iTCR-specific antibody against Vα24 and an antibody against CD3. Figure 2D shows a graph illustrating the cytotoxicity of NK cells co-transduced with iTCR (described in Figure 2B) and UT-NK15, and pre-loaded with blinatumomab (37°C for 1 hour), measured using IncuCyte® live-cell imaging against Raji cells expressing GFP in a 3:1 target:effector ratio. T cells were used as a positive control, and untransduced (NT) NK cells were used as a negative control.A decrease in GFP expression indicates tumor cell death. [Figure 2-2] Same as above. [Figure 2-3] Same as above. [Figure 2-4] Same as above. [Figure 2-5] Same as above.

[0132] [Figure 3-1] Figures 3A–3C show polynucleotide construct layouts and schematic diagrams of exemplary uTNK15 and T cell receptor (TCR) / Fc receptor (FcR) (TCR / FcR) vectors (e.g., vectors containing coding sequences of one or more polypeptides comprising one or more TCR chains and Fc-binding domains derived from the Fc receptor). Figure 3A shows the vector map of the multicistronic construct uTNK15-28 (SEQ ID NO: 47) containing the open reading frame (ORF) coding regions of the CD3 complex protein and IL-15. Figure 3B shows the vector map of the multicistronic TCR / FcR constructs described herein, including the open reading frames of polypeptides containing alpha (α; SEQ ID NO: 51) and beta (β, clone 3; SEQ ID NO: 59) invariant T cell receptor (iTCR) chains and CD16 Fc-binding domains. Figure 3C shows schematic diagrams of constructs containing uTNK15-28 and iTCR / CD16 Fc-binding domain polypeptides: TCR / FcR#1 (in Open Reading Frame (ORF) SEQ ID NO: 171, Vector SEQ ID NO: 177), TCR / FcR#2 (in ORF SEQ ID NO: 172, Vector SEQ ID NO: 178), TCR / FcR#3 (in ORF SEQ ID NO: 173, Vector SEQ ID NO: 179), TCR / FcR#4 (in ORF SEQ ID NO: 174, Vector SEQ ID NO: 180), and TCR / FcR#5 (in ORF SEQ ID NO: 175, Vector SEQ ID NO: 181). [Figure 3-2] Same as above.

[0133] [Figure 4]Figure 4 shows CB-NK and T cells transduced with various TCR / FcR constructs. The top row shows T cells expanded with anti-CD3 / 28 beads in the presence of IL-2 (50 Iu / ML) and describes T cells transduced with different TCR / FcR constructs on day 3. Transduction efficiency was determined by measuring the surface expression of iTCRvb11 and CD16 after 48 hours. The middle row shows irradiated (100 Gy) UAPC feeder cells (feeder cell:NK ratio 2:1) and umbilical cord blood-derived NK cells expanded with recombinant human IL-2 (200 U / ML) in complete NK cell proliferation medium. Seven days after expansion, NK cells were transduced with one of the described iTCR3 or TCR / FcR constructs #1–#5, each containing a CD16 Fc-binding domain polypeptide variant, SEQ ID NOs. 155–159 (encoded by SEQ ID NOs. 150–154, respectively). Transduction efficiency was determined at 48 hours by measuring the surface expression of the heterologous CD16 Fc-binding domain containing the polypeptide, iTCRvb11, CD3 complex, and polypeptide, as described below (measured as an anti-flag antibody against flag-tagged CD16 to avoid endogenous CD16 background signaling).

[0134] [Figure 5-1]Figures 5A–5F show blinatumomab (Blina)-loaded TCR / FcR construct-transduced NK cells, demonstrating enhanced death of CD19+ (Raji cell line) tumor cells. Umbilical cord blood-derived NK cells were transduced with either uTNK15, iTCR3 (Figure 5A), or TCR / FcR constructs #1–#5 (Figures 5B–5F). Non-transduced (NT) umbilical cord blood-derived NK cells (NT NK cells) were used as a negative control, and non-transduced T cells were used as a positive control. Cells were loaded with or unloaded blinatumomab (final concentration 100 μg / ml) in PBS at room temperature for 1 hour, and then washed before co-culturing with Raji cells. Raji cells were labeled with chromium 51 and co-culturised with immune cells at various effector-versus-target (E:T) ratios (X axis). Cells were co-cultured for 4 hours, and chromium release (corresponding to the cytotoxicity of NK cells against cancer cells) was measured (Y axis). Compared to unloaded TCR / FcR-NK cells or loaded non-transduced (NT) NK cells, blinatumomab-loaded TCR / FcR-transduced NK cells showed increased cytotoxicity against CD19+ Raji cells. [Figure 5-2] Same as above. [Figure 5-3] Same as above.

[0135] [Figure 6]Figure 6 shows TCR / FcR-transduced NK cells loaded with blinatumomab and / or obinutuzumab, demonstrating enhanced death of CD19+++ / CD20+(Nalm6) tumor cells. Umbilical cord blood-derived NK cells were transduced with uTNK15 and either iTCR3 or TCR / FcR constructs #1-#5. Untransduced (NT) umbilical cord blood-derived NK cells (NT NK cells) were used as a negative control. NK cells were either left unloaded (PBS), loaded with blinatumomab (final concentration 100 μg / ml), loaded with obinutuzumab (final concentration 500 μg / ml), or loaded with blinatumomab (final concentration 100 μg / ml) and obinutuzumab (final concentration 500 μg / ml), left in PBS at room temperature for 1 hour, and then washed before co-culture with tumor cells. Immune cells and Nalm6 cells were co-cultured in a 1:1 E:T ratio, and the real-time cytotoxicity of effector cells against Nalm6 cells was measured every 2 hours for 40 hours. Compared to unloaded TCR / FcR-NK cells or loaded NT NK cells, blinatumomab and / or obinutuzumab-loaded TCR / FcR transduced NK cells showed increased cytotoxicity against CD19+ NALM6 cells.

[0136] [Figure 7-1]Figure 7 shows that the T cell engager tecristamag can bind to transduced NK cells and T cells with TCR / FcR, but not to untransduced NK cells. NK cells were isolated from umbilical cord blood and expanded in complete medium in the presence of irradiated (100 Gy) uAPC feeder cells (2:1 feeder cell:NK ratio) and recombinant human IL-2 (200 U / ml). Seven days after expansion, NK cells were either transduced with uTNK15 and either iTCR3 or TCR / FcR constructs #1-#5, or left untransduced (NT). T cells were used as a positive control and loaded with tecristamag (final concentration 200 μg / ml) in PBS at room temperature for 1 hour, then washed before verification of tecristamag binding. Flow cytometry analysis of anti-human IgG stained cells showed that tecristamag bound to transduced NK cells and T cells with uTNK15, iTCR3, or TCR / FcR#1-#5, but not to NT NK cells. [Figure 7-2] Same as above.

[0137] [Figure 8-1]Figures 8A–8F show teclistamagb (Tecli)-loaded TCR / FcR-transduced NK cells, demonstrating enhanced BCMA+ (MM.1S, myeloma) tumor cell death. Umbilical cord blood-derived NK cells were transduced with uTNK15 and either iTCR3 (Figure 8A) or TCR / FcR constructs #1–#5 (Figures 8B–8F). Non-transduced (NT) umbilical cord blood-derived NK cells (NT NK cells) were used as a negative control, and non-transduced T cells were used as a positive control. Cells were loaded with or unloaded teclistamagb (final concentration 200 μg / ml) in PBS at room temperature for 1 hour, and then washed before co-culturing with MM.1S cells. MM.1S cells were labeled with chromium 51 (cr51) and co-cultured with immune cells at various effector-versus-target (E:T) ratios (X axis). Cells were co-cultured for 4 hours, and chromium release (corresponding to the cytotoxicity of NK cells against cancer cells) was measured (Y axis). Compared to unloaded TCR / FcR-NK cells or loaded non-transduced (NT) NK cells, tecristamag-loaded TCR / FcR-transduced NK cells showed increased cytotoxicity against BCMA+ MM.1S cells. [Figure 8-2] Same as above. [Figure 8-3] Same as above.

[0138] [Figure 9-1]Figures 9A–9F show teclistamagb (Tecli)-loaded TCR / FcR-transduced NK cells, demonstrating enhanced BCMA+ (H929, myeloma) tumor cell death. Umbilical cord blood-derived NK cells were transduced with either uTNK15, iTCR3 (Figure 9A), or TCR / FcR constructs #1–#5 (Figures 9B–9F). Non-transduced (NT) umbilical cord blood-derived NK cells (NT NK cells) were used as a negative control, and non-transduced T cells were used as a positive control. Cells were loaded with or unloaded teclistamagb (final concentration 200 μg / ml) in PBS at room temperature for 1 hour, and then washed before co-culturing with MM.1S cells. H929 cells were labeled with chromium 51 and co-culturised with immune cells at various effector-versus-target (E:T) ratios (X axis). Cells were co-cultured for 4 hours, and chromium release (corresponding to the cytotoxicity of NK cells against cancer cells) was measured (Y axis). Compared to unloaded TCR / FcR-NK cells or loaded non-transduced (NT) NK cells, tecristamag-loaded TCR / FcR-transduced NK cells showed increased cytotoxicity against BCMA+ H929 cells. [Figure 9-2] Same as above. [Figure 9-3] Same as above.

[0139] [Figure 10]Figure 10 shows the binding of a low Fcγ receptor affinity antibody (e.g., cetuximab) to TCR / FcR transduced NK cells. NK cells were derived from umbilical cord blood and expanded in complete NK cell proliferation medium (Click's / RPMI) using irradiated (100 Gy) UAPC feeder cells (2:1 feeder cell:NK ratio) and recombinant human IL-2 (200 U / ml). NK cells were loaded with a low Fcγ receptor affinity antibody (e.g., cetuximab at a final concentration of 100 μg / ml) in Click's / RPMI medium at 37°C for 1 hour, followed by washing, and the binding of the low Fcγ receptor affinity antibody was verified by flow cytometry. NK cells were stained with Alexa-Fluor647 affinity purified F(ab')2 fragment goat anti-human IgG(H+L) antibody and analyzed by flow cytometry. The results showed that antibodies with an Fc domain that has low affinity for the Fcγ receptor (such as cetuximab) bound to high-affinity CD16 (CD16ha, (F158V)) TCR / FcR transduced NK cells at a high level compared to ITCR3 transduced NK cells, NT NK cells, or T cells.

[0140] [Figure 11]Figure 11 shows that cetuximab-loaded TCR / FcR transduced NK cells exhibit enhanced WiDR (colorectal cancer cell; CRC) death compared to unloaded TCR / FcR NK cells or loaded / unloaded non-transduced (NT) NK cells. NK cells were transduced with either uTNK15, iTCR3, or TCR / FcR constructs #1-#5. WiDR cells alone, non-transduced (NT) umbilical cord blood-derived NK cells (NT NK cells), and T cells were used as controls. Cells were loaded with cetuximab (final concentration 100 μg / ml) in Click's / RPMI medium at 37°C for 1 hour or unloaded, and then washed before co-culturing with tumor cells in a 1:1 E:T ratio. Compared to cetuximab alone, loaded / unloaded NT NK cells, and unloaded TCR / FcR NK cells, cetuximab-loaded TCR / FcR NK cells showed the highest cytotoxic activity against EGFR+CRC (WiDR) cell lines. A lower normalized cell index (Y-axis) indicates a higher degree of cytotoxicity. SDS was used as a positive control for cytotoxicity. NK cells transduced from TCR / FcR#4 or TCR / FcR#2 and loaded with cetuximab showed the highest level of cytotoxicity against WiDR cells.

[0141] [Figure 12-1]Figures 12A–12B show that cetuximab-loaded TCR / FcR transduced NK cells exhibit enhanced death of PATC148 cells (pancreatic ductal adenocarcinoma, PDAC) compared to unloaded TCR / FcR NK cells or loaded / unloaded non-transduced (NT) NK cells. NK cells were transduced with either uTNK15, iTCR3, or one of the TCR / FcR constructs #1–#5. PATC148 cells alone, non-transduced (NT) umbilical cord blood-derived NK cells (NT NK cells), and T cells were used as controls. Cells were loaded with cetuximab (final concentration 100 μg / ml) in Click's / RPMI medium at 37°C for 1 hour or unloaded, and then washed before co-culture with tumor cells at a 4:1 E:T ratio (Figure 12A) or a 2:1 ET ratio (Figure 12B). Compared to cetuximab alone, or non-transduced (NT) NK cells (both processed and unprocessed), or unprocessed TCR / FcR NK cells, cetuximab-processed TCR / FcR transduced NK cells showed the greatest cytotoxic activity against EGFR+PATC148(PDAC) cell lines. A lower normalized cell index (Y-axis) indicates a higher degree of cytotoxicity. SDS was used as a positive control for cytotoxicity. NK cells transduced to TCR / FcR#4 or TCR / FcR#2 and processed with cetuximab showed the highest levels of cytotoxicity against PATC148(PDAC) cells. [Figure 12-2] Same as above.

[0142] [Figure 13-1]Figures 13A-13B show that imugatuzumab-loaded TCR / FcR transduced NK cells exhibit enhanced PDAC or CRC cell death compared to unloaded TCR / FcR NK cells or loaded / unloaded non-transduced (NT) NK cells. NT NK cells or NK cells transduced with uTNK15 and TCR / FcR construct #2 were loaded with imugatuzumab (final concentration 10 μg / ml) in PBS at room temperature for 1 hour, and then washed before co-culturing with EGFR+ PDAC (PATC-148, Figure 13A) or EGFR+ colorectal cancer (WiDR, Figure 13B) tumor cells in a 1:1 E:T ratio. Compared to imugatuzumab alone, loaded / unloaded NT-NK cells, or unloaded TCR / FcR NK cells, imugatuzumab-loaded TCR / FcR transduced NK cells showed increased cytotoxic activity against EGFR+PDAC (PATC148) and colorectal cancer (WiDR) cell lines. [Figure 13-2] Same as above.

[0143] [Figure 14-1] Figures 14A-14C show that imugatuzumab-loaded TCR / FcR transduced NK cells enhance the death of 3D PDAC (PATC-148) tumor spheroids. Figure 14A is a representative image of PATC148 spheroids (GFP-transduced PDAC tumor cell line) that were left alone, treated with NT NK cells, or treated with NK cells transduced with uTNK15 and TCR / FcR construct #2. The NK cells were left unloaded or loaded with imugatuzumab (final concentration 10 μg / ml) for 1 hour in PBS at room temperature, and then washed before co-culture. The data showed a significant reduction in spheroid size in wells treated with imugatuzumab-loaded TCR / FcR transduced NK cells. Figure 14B shows the quantification of total cumulative green intensity over time observed in 14A, and the data showed a significant decrease in total cumulative green intensity (Figure 14C) when spheroids were treated with imugatuzumab-loaded TCR / FcR transduced NK cells. [Figure 14-2] Same as above. [Figure 14-3] Same as above.

[0144] [Figure 15-1] Figures 15A–15C show the binding of cetuximab, amibantamab, or imugatuzumab to TCR / FcR#2 transduced NK cells. NK cells were derived from umbilical cord blood and expanded in complete NK cell proliferation medium (Click's / RPMI) using irradiated (100 Gy) UAPC feeder cells (2:1 feeder cell:NK ratio) and recombinant human IL-2 (200 U / ml). Untransduced NK cells or TCR / FcR#2 transduced NK cells were treated with cetuximab (final concentration 250 μg / ml) (Figure 15A), amibantamab (final concentration 100 μg / ml) (Figure 15B), or imugatuzumab (final concentration 100 μg / ml) (Figure 15C) in Click's / RPMI medium at 37°C for 1 hour before washing the cells. NK cells were stained with Alexa-Fluor647 affinity purified F(ab')2 fragment goat anti-human IgG(H+L) antibody and analyzed by flow cytometry to confirm binding of cetuximab, amivantamab, or imugatuzumab. The results showed that the antibodies bound to TCR / FcR transduced NK cells with higher affinity compared to NT NK cells. Notably, unlike amivantamab or imugatuzumab, cetuximab (containing an Fc domain with low binding affinity to WT CD16) was found to bind only to CD16ha TCR / FcR transduced NK cells. [Figure 15-2] Same as above.

[0145] [Figure 16-1]Figures 16A–16C show uTNK15 and TCR / FcR#2 transdextrin NK cells exhibiting enhanced antitumor activity against PATC-148 (PDAC) cell spheroids compared to NT NK cells when loaded with cetuximab (Cetux), imugatuzumab (Imga), or amibantamab (Ami). Antibody-loaded TCR / FcR transdextrin NK cells showed enhanced PDAC cell death compared to unloaded TCR / FcR NK cells or loaded / unloaded non-transduced (NT) NK cells. Figure 16A is a representative image of GFP transdextrin PDAC (PATC-148) spheroids, left untreated, treated with unloaded or loaded NT NK cells, or treated with unloaded or loaded TCR / FcR#2 transdextrin NK cells. The loaded cells were prepared with imugatuzumab (final concentration 100 μg / ml), amibantamab (final concentration 100 μg / ml), and cetuximab (final concentration 250 μg / ml). A readily observable and significant decrease in spheroid size was observed in wells treated with antibody-loaded TCR / FcR transduced NK cells. Figure 16B is a graph quantification of total cumulative green intensity over time from the experiment performed in Figure 16A, and the data shows a significant decrease in total cumulative green intensity when spheroids were treated with antibody-loaded TCR / FcR transduced NK cells (Figure 16C). [Figure 16-2] Same as above.

[0146] [Figure 17-1]Figures 17A–17C describe uTNK15 and TCR / FcR#2 transdextrinsed NK cells that exhibit enhanced antitumor activity against WiDR (CRC) cell spheroids compared to NT NK cells when loaded with cetuximab (Cetux), imugatuzumab (Imga), or amibantamab (Ami). Antibody-loaded TCR / FcR transdextrinsed NK cells showed enhanced CRC cell death compared to unloaded TCR / FcR NK cells or loaded / unloaded non-transdextrinsed (NT) NK cells. Figure 17A shows representative images of GFP-transduced WiDR spheroids, either unloaded, treated with unloaded or loaded NT NK cells, or treated with unloaded or loaded TCR / FcR#2 transdextrinsed NK cells. Loaded cells were prepared with imugatuzumab (final concentration 100 μg / ml), amibantamab (final concentration 100 μg / ml), or cetuximab (final concentration 250 μg / ml). The data show a significant decrease in spheroid size in wells treated with antibody-loaded TCR / FcR transduced NK cells. Figure 17B is a graph quantification of total cumulative green intensity over time from the experiment performed in Figure 16A, and the data show a significant decrease in total cumulative green intensity when spheroids were treated with antibody-loaded TCR / FcR transduced NK cells (Figure 17C). [Figure 17-2] Same as above.

[0147] [Figure 18-1]Figures 18A-18F show uTNK15 and TCR / FcR#2 transduced NK cells that exhibited enhanced antitumor activity against various solid tumor cell lines when loaded with the anti-EGFR antibodies cetuximab, imugatuzumab, or amivantamab. TCR / FcR transduced NK cells loaded with the antibodies showed increased mortality compared to unloaded TCR / FcR NK cells or loaded / unloaded non-transduced (NT) NK cells. NK cells were either non-transduced (NT) or TCR / FcR#2 transduced, and were washed with cetuximab (final concentration 250 μg / ml), amivantamab (final concentration 100 μg / ml), or imugatuzumab (final concentration 100 μg / ml) in Click's / RPMI medium at 37°C for 1 hour, either unloaded or loaded. NK cells were co-cultured with tumor cells SKOV3 (Figure 18A), PATC-148 (Figure 18C), or WiDR (Figure 18E) in a 2:1 E:T ratio. Compared to antibody alone, loaded / unloaded non-transduced (NT) NK cells, or unloaded TCR / FcR#2 NK cells, antibody-loaded TCR / FcR#2 transduced NK cells showed significantly increased cytotoxic activity against EGFR+SKOV3 (Figure 18B), PATC-148 (Figure 18D), or WiDR (Figure 18F) cell lines. [Figure 18-2] Same as above. [Figure 18-3] Same as above. [Figure 18-4] Same as above. [Figure 18-5] Same as above. [Figure 18-6] Same as above.

[0148] [Figure 19]Figures 19A-19B show the binding of low-Fcγ receptor affinity antibodies (e.g., rituximab) or high-Fcγ receptor affinity antibodies (obinutuzumab) to TCR / FcR#2 transduced NK cells. NK cells were derived from umbilical cord blood and expanded in complete NK cell proliferation medium (Click's / RPMI) using irradiated (100 Gy) UAPC feeder cells (2:1 feeder cell:NK ratio) and recombinant human IL-2 (200 U / ml). Untransduced NK cells or TCR / FcR#2 transduced NK cells were loaded with rituximab (final concentration 100 μg / ml) (Figure 19A) or obinutuzumab (final concentration 100 μg / ml) (Figure 19B) in Click's / RPMI medium at 37°C for 1 hour, followed by washing. NK cells were stained with Alexa-Fluor647 affinity purified F(ab')2 fragment goat anti-human IgG(H+L) antibody and analyzed by flow cytometry. The results showed that antibodies with low and high Fcγ receptor affinity bound to TCR / FcR transduced NK cells at a higher rate compared to NT NK cells. Notably, unlike obinutuzumab, rituximab (containing an Fc domain with low binding affinity to WT CD16) was found to bind only to CD16ha TCR / FcR transduced NK cells.

[0149] [Figure 20]Figures 20A-20B describe the enhanced antitumor activity against Raji (B-cell lymphoma) cells compared to NT NK cells at various E:T ratios when loaded with the anti-CD20 antibody rituximab or obinutuzumab. NK cells were either untransduced (NT), transduced with TCR / FcR#2 and unloaded, or loaded with rituximab (final concentration 100 μg / ml) (Figure 20A) or obinutuzumab (100 μg / ml) in Click / RPMI medium at 37°C for 1 hour, followed by washing. NK cells were co-cultured with chromium-51 labeled Raji cells at various E:T ratios (5:1, 2:1, 1:1, or 1:2) for 4 hours, and chromium release (corresponding to cancer cell cytotoxicity) was measured. Compared to unloaded TCR / FcR NK cells or loaded NT NK cells, antibody-loaded TCR / FcR#2 transduced NK cells showed increased cytotoxicity against CD20+ Raji cells.

[0150] [Figure 21] Figure 21 describes an exemplary combination of therapeutic strategies described herein (e.g., antibody-loaded uTNK15 and TCR / FcR transduced NK cells).

[0151] [Figure 22-1]Figures 22A-22B show that erranatamab-loaded uTNK15 and TCR / FcR-transduced NK cells exhibited enhanced antitumor activity against BCMA+ multiple myeloma cells (e.g., MM.1S cells) and outline clinical trials including the combination of TCR / FcR-transduced NK cells and erranatamab. Figure 22A shows the results of bioluminescence imaging (BLI) experiments in which mice were irradiated, inoculated with 5 × 10⁵ MM.1S cells, and given a sham injection or a single injection (IV) of 1 × 10⁷ NK cells that were either untransduced or transduced with uTNK15 and TCR / FcR#2. Animals were imaged weekly. The results showed that mice administered with erranatamab-loaded TCR / FcR-transduced NK cells exhibited enhanced antitumor activity in vivo compared to controls. Figure 22B provides a schematic overview of an exemplary clinical trial demonstrating the binding of manipulated uTNK15 and TCR / FcR transduced NK cells (abbreviated as "TCR / FcR#2NK cells") to erlanatamab. [Figure 22-2] Same as above.

[0152] [Figure 23]Figure 23 shows the binding of T cell engagers (e.g., tecristamagb (anti-BCMA), erranatamab (anti-BCMA), and blinatumumab (anti-CD19)) to uTNK15 and TCR / FcR#2-transduced NK cells, but not to NT NK cells. Natural killer (NK) cells were extracted from umbilical cord blood and cultured in complete medium supplemented with irradiated (100 Gy) uAPC feeder cells in a 2:1 ratio (feeder cells:NK). Recombinant human IL-2 was added at a concentration of 200 U / ml to promote NK cell expansion. Five days after expansion, NK cells were either untransduced or transduced with uTNK15 and TCR / FcR#2. These cells were treated with tecristamagb (20 μg / ml), erlanatamab (10 μg / ml), or blinatumumab (10 μg / ml) in complete medium at 37°C for 1 hour. After incubation, the cells were washed and subjected to flow cytometry analysis to verify binding. Prior to flow cytometry, cells incubated with BCMA or His-tagged CD19 antigen were stained with APC anti-His-tagged antibody (catalog number: 362605, Biolegend). Flow cytometry analysis showed that all T cell engagers were able to efficiently bind to manipulated NK cells, but binding to NT NK cells was not observed.

[0153] [Figure 24-1]Figures 24A-24B show that uTNK15 and TCR / FcR#2 transduced NK cells loaded with the bispecific anti-CD19 / CD3 antibody blinatumomab (10 μg / ml in Click / RPMI medium at 37°C for 1 hour and washed before co-culture) exhibit enhanced antitumor activity against CD19+ tumor cells (e.g., Raji cells or B-LCL cells) in a short-term Cr51 assay at various E:T ratios compared to blinatumomab-loaded NT NK cells. Figure 24A shows the results for B-LCL cells, with significantly higher cell death by manipulated NK cells at E:T ratios of 5:1, 2.5:1, and 1.25:1. Figure 24B shows the results for Raji cells, with significantly higher cell death by manipulated NK cells at E:T ratios of 5:1 and 2.5:1. Compared to blinatumomab-loaded NT NK cells, blinatumomab-loaded engineered NK cells showed enhanced cytotoxicity against CD19+ tumor cells. [Figure 24-2] Same as above.

[0154] [Figure 25] Figure 25 shows that uTNK15 and TCR / FcR#2 transdextrinsed NK cells loaded with blinatumomab showed increased cell death compared to unloaded uTNK15 and TCR / FcR#2 transdextrinsed NK cells or loaded / unloaded non-transduced (NT) NK cells. NK cells were either NT (statistically abbreviated as NT or NT NK) or transduced with uTNK15 and TCR / FcR#2 (statistically abbreviated as "TCR / FcR#2"). NK cells were either unloaded or loaded with blinatumomab (10 μg / ml in Click / RPMI medium, 37°C for 1 hour, washed before co-culture) in a 1:1 effector-versus-target (E:T) ratio before co-culture with tumor cells. Compared to loaded or unloaded NT NK cells, unloaded engineered NK cells, or blinatumumab alone, engineered NK cells loaded with blinatumumab showed significantly increased cytotoxic activity against CD19-transduced SKOV3 cancer cells.

[0155] [Figure 26-1] Figures 26A-26B describe how uTNK15 and TCR / FcR#2 transduced NK cells loaded with the bispecific anti-BCMA / CD3 antibody teclistamag (20 μg / ml in Click / RPMI medium, 37°C for 1 hour, and washed before co-culture) showed enhanced antitumor activity against BCMA+ tumor cells (e.g., MM1s cells, H929 cells) at various E:T ratios in a short-term Cr51 assay compared to non-transduced (NT) NK cells loaded with teclistamag. Figure 26A shows the results for MM1s cells, with significantly higher cell death by manipulated NK cells at E:T ratios of 5:1, 2.5:1, and 1.25:1. Figure 26B shows the results for H929 cells, with significantly higher cell death by manipulated NK cells at E:T ratios of 5:1, 2.5:1, 1.25:1, and 1:2. Compared to teclistamagb-loaded NT NK cells, teclistamagb-loaded manipulated NK cells showed enhanced cytotoxicity against BCMA+ tumor cells. [Figure 26-2] Same as above.

[0156] [Figure 27-1] Figures 27A-27B describe how uTNK15 and TCR / FcR#2 transduced NK cells loaded with the bispecific anti-GPRC5D / CD3 antibody talketamab (20 μg / ml in Click / RPMI medium, 37°C for 1 hour, and washed before co-culture) showed enhanced antitumor activity against GPRC5D+ tumor cells (e.g., MM1s cells, H929 cells) at various E:T ratios in a short-term Cr51 assay compared to non-transduced (NT) NK cells loaded with talketamab. Figure 27A shows the results for MM1s cells. Figure 27B shows the results for H929 cells. Compared to talketamab-loaded NT NK cells, talketamab-loaded engineered NK cells showed enhanced cytotoxicity against GPRC5D+ tumor cells. [Figure 27-2] Same as above.

[0157] [Figure 28-1]Figures 28A-28B describe how uTNK15 and TCR / FcR#2 transducible NK cells loaded with the bispecific anti-BCMA / CD3 antibody erranatamab (10 μg / ml in Click / RPMI medium, 37°C for 1 hour, and washed before co-culture) showed enhanced antitumor activity against BCMA+ tumor cells (e.g., MM1s cells, H929 cells) at various E:T ratios in a short-term Cr51 assay compared to non-transduced (NT) NK cells loaded with erranatamab. Figure 28A shows the results for MM1s cells, where significantly more cell death was observed by manipulated NK cells at E:T ratios of 5:1, 2.5:1, 1.25:1, and 1:2. Figure 28B shows the results for H929 cells, with significantly higher cell death by engineered NK cells at E:T ratios of 5:1, 2.5:1, 1.25:1, and 1:2. Compared to elanatamab-loaded NT NK cells, elanatamab-loaded engineered NK cells showed enhanced cytotoxicity against BCMA+ tumor cells. [Figure 28-2] Same as above.

[0158] [Figure 29-1]Figures 29A-29B show that pertuzumab (an anti-HER2 agent that binds to wild-type CD16 with low affinity) binds to uTNK15 and TCR / FcR#2 transduced NK cells but not to non-transduced (NT) NK cells. Pertuzumab-loaded engineered NK cells showed superior antitumor activity against HER2+SKOV3 ovarian cancer cells in a long-term xCELLigence death assay. Figure 29A shows the binding of pertuzumab to engineered NK cells. NK cells were extracted from umbilical cord blood and cultured in complete medium supplemented with irradiated (100 Gy) uAPC feeder cells in a 2:1 ratio (feeder cells:NK). Recombinant human IL-2 was added at a concentration of 200 U / ml to further promote NK cell expansion. Five days after expansion, natural killer (NK) cells were transduced with uTNK15 and TCR / FcR#2 constructs, or left untransduced (NT). These cells were then loaded with pertuzumab (200 μg / ml in complete medium, 37°C for 1 hour). After incubation, the cells were washed and subjected to flow cytometry analysis to verify binding. Flow cytometry analysis revealed that pertuzumab could bind to the manipulated NK cells, but binding to NT NK cells was not observed. Figure 29B, pertuzumab-loaded uTNK15 and TCR / FcR#2 transduced NK cells showed increased cell death compared to unloaded uTNK15 and TCR / FcR#2 transduced NK cells, or loaded / unloaded untransduced (NT) NK cells. NK cells were transduced with NT (statistically abbreviated as NT or NT NK) or uTNK15 and TCR / FcR#2 (statistically abbreviated as "TCR / FcR#2"). NK cells were either unloaded or loaded with pertuzumab (200 μg / ml, Click / RPMI medium at 37°C for 1 hour, washed before co-culture) with tumor cells in a 1:1 effector-to-target (E:T) ratio. Compared to loaded or unloaded NTNK cells, unloaded engineered NK cells, or pertuzumab alone, pertuzumab-loaded engineered NK cells showed significantly increased cytotoxic activity against HER2+SKOV3 ovarian cancer cells. [Figure 29-2] Same as above.

[0159] [Figure 30-1]Figures 30A-30B show that trastuzumab (anti-HER2) bound to uTNK15 and TCR / FcR#2 transduced NK cells with higher affinity than non-transduced (NT) NK cells, and trastuzumab-loaded engineered NK cells showed superior antitumor activity against HER2+SKOV3 ovarian cancer cells in a long-term xCELLigence death assay. Figure 30A shows the binding of trastuzumab to engineered NK cells. NK cells were extracted from umbilical cord blood and cultured in complete medium supplemented with irradiated (100 Gy) uAPC feeder cells in a 2:1 ratio (feeder cells:NK). Recombinant human IL-2 was added at a concentration of 200 U / ml to promote NK cell expansion. Five days after expansion, natural killer (NK) cells were transduced with uTNK15 and TCR / FcR#2 constructs or left non-transduced (NT). Next, these cells were loaded with trastuzumab (200 μg / ml in complete medium at 37°C for 1 hour). After incubation, the cells were washed and subjected to flow cytometry analysis to verify binding. Flow cytometry analysis revealed that trastuzumab could bind to engineered NK cells with higher affinity than observed in NT NK cells. Figure 30B, uTNK15 and TCR / FcR#2 transduced NK cells loaded with trastuzumab showed increased cell death compared to unloaded uTNK15 and TCR / FcR#2 transduced NK cells or loaded / unloaded NT NK cells. NK cells were either NT cells (statistically abbreviated as "NT" or "NT NK") or transduced with uTNK15 and TCR / FcR#2 (statistically abbreviated as "TCR / FcR#2"). NK cells were either unloaded or loaded with trastuzumab (200 μg / ml, in Click / RPMI medium at 37°C for 1 hour, and washed before co-culture) in a 1:1 effector-versus-target (E:T) ratio before co-culture with tumor cells. Compared to loaded or unloaded NT NK cells, unloaded engineered NK cells, or trastuzumab alone, engineered NK cells loaded with trastuzumab showed significantly increased cytotoxic activity against HER2+SKOV3 ovarian cancer cells. [Figure 30-2] Same as above.

[0160] [Figure 31-1]Figures 31A-31B show that tafacitamab (anti-CD19) bound to uTNK15 and TCR / FcR#2 transduced NK cells with higher affinity than non-transduced (NT) NK cells, and tafacitamab-loaded engineered NK cells showed superior antitumor activity against transduced CD19+SKOV3 ovarian cancer cells in a long-term xCELLigence death assay. Figure 31A shows that NK cells were extracted from umbilical cord blood and cultured in complete medium supplemented with irradiated (100 Gy) uAPC feeder cells in a 2:1 ratio (feeder cells:NK). Recombinant human IL-2 was added at a concentration of 200 U / ml to promote NK cell expansion. Five days after expansion, natural killer (NK) cells remained untransduced or transduced with uTNK15 and TCR / FcR#2 constructs. These cells were treated with tafacitamab (10 μg / ml in complete medium at 37°C for 1 hour). After incubation, the cells were washed and subjected to flow cytometry analysis to verify binding. Flow cytometry analysis revealed that tafacitamab showed a higher and stronger binding affinity to engineered NK cells compared to NT NK cells. Figure 31B, uTNK15 and TCR / FcR#2 transduced NK cells loaded with tafacitamab showed increased cell death compared to unloaded uTNK15 and TCR / FcR#2 transduced NK cells or loaded / unloaded NT NK cells. NK cells were either NT cells (statistically abbreviated as "NT" or "NT NK") or transduced uTNK15 and TCR / FcR#2 (statistically abbreviated as "TCR / FcR#2"). NK cells were either unloaded or loaded with tafacitamab (200 μg / ml, Click / RPMI medium at 37°C for 1 hour, washed before co-culture) with tumor cells in a 1:1 effector-to-target (E:T) ratio. Compared to loaded or unloaded NT NK cells, unloaded engineered NK cells, or tafacitamab alone, engineered NK cells loaded with tafacitamab showed significantly increased cytotoxic activity against transduced CD19+SKOV3 ovarian cancer cells. [Figure 31-2] Same as above.

[0161] [Figure 32-1]Figures 32A-32B show that brentuximab (anti-CD30 antibody-drug conjugate) bound to uTNK15 and TCR / FcR#2 transduced NK cells with higher affinity than non-transduced (NT) NK cells, and brentuximab-loaded engineered NK cells showed superior antitumor activity against CD30+ Karpas tumor cells in the IncuCyte cytotoxicity assay. Figure 32A shows that NK cells were isolated from umbilical cord blood and expanded in complete medium in the presence of irradiated (100 Gy) uAPC feeder cells (feeder cell:NK ratio of 2:1) and recombinant human IL-2 (200 U / ml). Seven days after expansion, NK cells were either untransduced or transduced with uTNK15 and TCR / FcR#2 constructs. These cells were loaded with brentuximab (100 μg / ml, in complete medium, at 37°C for 1 hour) and washed before flow cytometry validation of brentuximab binding. Brentuximab bound to engineered NK cells with higher affinity compared to NT NK cells, as determined by staining with F(ab')2 anti-human antibody and flow cytometry analysis of NK cells. Figure 32B, brentuximab-loaded uTNK15 and TCR / FcR#2 transduced NK cells showed enhanced death of CD30+ (Karpas) tumor cells in the IncuCyte cytotoxicity assay compared to unloaded engineered NK cells or brentuximab-loaded NT NK cells. NK cells were derived from expanded umbilical cord blood as described in 31A and were either untransduced or transduced with uTNK15 and TCR / FcR#2 constructs. NT NK cells or engineered NK cells were loaded with brentuximab (100 μg / ml in complete medium, 37°C for 1 hour, washed before co-culture) and co-cultured with CD30+ Karpas tumor cell lines in a 1:1 effector-to-target ratio. Real-time cytotoxicity of effector cells against Karpas cells was measured every 2 hours over 24 hours. Compared to engineered NK cells or NT NK cells not loaded with brentuximab, engineered NK cells loaded with brentuximab showed increased cytotoxicity of CD30+ Karpas cells. [Figure 32-2] Same as above. [Modes for carrying out the invention]

[0162] Detailed explanation In accordance with long-standing patent law practice, in this specification, when used in conjunction with the word "comprising" to include the claims, the words "a" and "an" mean "one or more." Some embodiments of this disclosure may consist of, or essentially consist of, one or more elements, method steps, and / or methods of this disclosure. Any method or composition described herein may be carried out in relation to any other method or composition described herein, and different embodiments may be combined.

[0163] Throughout this specification, unless otherwise specified in the context, the terms “comprise,” “comprises,” and “comprising” are understood to mean including the steps or elements or groups of steps or elements described, but not to mean excluding other steps or elements or groups of steps or elements. “Consists of” means including and being limited to what follows the phrase “consists of.” Thus, the expression “consists of” indicates that the enumerated elements are required or essential, and other elements may not be present. “Essentially consists of” means including the elements enumerated after the phrase, and being limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the enumerated elements. Thus, the expression “essentially consists of” indicates that the enumerated elements are required or essential, but other elements are optional and may or may not be present depending on whether they affect the activity or action of the enumerated elements.

[0164] Throughout this specification, any reference to “one embodiment,” “embodiment,” “specific embodiment,” “related embodiment,” “certain embodiment,” “another embodiment,” or “further embodiment,” or any combination thereof, means that any particular feature, structure, or characteristic described in relation to an embodiment is included in at least one embodiment of the present invention. Therefore, although the aforementioned terms appear in various places throughout this specification, they do not necessarily all refer to the same embodiment. Furthermore, any particular feature, structure, or characteristic can be combined in any suitable manner in one or more embodiments.

[0165] As used herein, the terms “or” and “and / or” are used to describe multiple components in combination or mutually exclusive. For example, “x, y, and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” It is particularly intended that x, y, or z may be excluded from embodiments.

[0166] Throughout this application, the term “about” is used in accordance with its plain and common meaning in the fields of cell biology and molecular biology, indicating that the value includes the standard deviation of error in the apparatus or method employed to determine that value.

[0167] As used herein, the terms “CD3 receptor complex” or “CD3 coreceptor complex” refer to the protein complex that acts as a T cell coreceptor in nature and is composed of a CD3ζ chain, a CD3γ chain, a CD3δ chain, and two CD3ε chains (although in alternatives, only one CD3ε chain is used).

[0168] As used herein, the term “engineered” refers to human-generated entities, including cells, nucleic acids, polypeptides, vectors, and the like. In at least some cases, the engineered entities are synthetic and comprise elements that do not exist in nature or are not composed in the manner utilized herein. In specific embodiments, vectors are engineered by recombinant nucleic acid technology, and cells are engineered by transfection or transduction of the engineered vector. In specific embodiments, the engineered cell is a cell comprising one or more exogenous antigen receptors (e.g., chimeric antigen receptors, T cell receptors, etc.), suicide genes, cytokines and / or cytokine receptors (e.g., IL-15, IL-15R, etc.), chemokine / homing receptors, and / or modifications of one or more endogenous genes.

[0169] The phrase "pharmaceutically or pharmacologically acceptable" means, as appropriate, molecular entities and compositions that, when administered to animals such as humans, do not cause adverse reactions, allergic reactions, or other adverse reactions. The preparation of pharmaceutical compositions containing antibodies or additional active ingredients will be known to those skilled in the art in light of this disclosure. Furthermore, it will be understood that when administered to animals (e.g., humans), preparations should meet the standards of sterility, pyrogenicity, general safety, and purity required by the FDA's Office of Biological Standards.

[0170] As used herein, “pharmaceutically acceptable carriers” include any and all aqueous solvents (e.g., water, alcohol / aqueous solutions, saline, sodium chloride, ringer's dextrose, and other parenteral vehicles), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as etyloleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antimicrobial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption retarders, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, body fluids, and nutritional supplements, of which such materials and combinations thereof will be known to those skilled in the art. The pH and precise concentrations of the various components in the pharmaceutical composition are adjusted according to well-known parameters.

[0171] As used herein, the term “subject” generally refers to an individual having or suspected of having cancer. A subject may be any living organism or animal subject that is the subject of a method or material, including mammals, e.g., humans, laboratory animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cattle, sheep, goats, pigs, turkeys, and chickens), domestic pets (e.g., dogs, cats, and rodents), horses, and transgenic non-human animals. A subject may be a patient who has or is suspected of having a disease (sometimes called a medical condition), such as a benign or malignant neoplasm or cancer. A subject may be undergoing or have previously undergone treatment. A subject may be asymptomatic. A subject may be a healthy individual who desires cancer prevention. The term “individual” is used interchangeably, at least in some cases. As used herein, “subject” or “individual” may or may not be housed in a medical facility and may be treated as an outpatient of a medical facility. An individual may receive “one or more medical compositions” via the internet. Individuals may include all ages of human or non-human animals, and therefore include both adults and adolescents (i.e., children) and infants, as well as individuals in utero. Since this term does not imply a need for medical treatment, individuals may participate in experiments voluntarily or involuntarily, whether clinically or in support of basic scientific research.

[0172] As used herein, “treatment” or “procedure” includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may even include a minimal reduction in one or more measurable markers of the disease or condition being treated, such as cancer. Treatment may optionally include either the alleviation or improvement of one or more symptoms of the disease or condition, or the delay of the progression of the disease or condition. “Treatment” does not necessarily mean the complete eradication or cure of the disease or condition, or any symptoms associated therewith. Treatment may mean the relief of at least one symptom of the disease or condition.

[0173] As used herein, “TCR / CD3 complex” refers to a protein complex naturally found on the surface of T cells, comprising T cell receptor (TCR) α and β chains, invariant natural killer T cell receptor (iTCR) α and β chains, and / or T cell receptor γ and δ chains, in addition to CD3ζ, CD3γ, CD3δ, and CD3ε chains.

[0174] I. Embodiments of the Disclosure Natural killer (NK) cells, as well as solid tumors, represent a novel cellular immunotherapy for patients with malignant hematological disorders. This disclosure specifically relates to NK cells that have been modified to have enhanced function as immunotherapy compared to unmodified NK cells. This modification gives NK cells greater versatility when used in conjunction with other therapeutic agents and, in at least some embodiments, enables them to possess T-cell-like activity by utilizing the CD3 / TCR receptor complex. In specific embodiments, NK cells are modified to express either (i) a single CD3 chain (CD3 zeta, CD3 epsilon, CD3 delta, or CD3 gamma) or part or all of a human CD3 receptor complex (including any combination of CD3 zeta, epsilon (one or two copies of epsilon), gamma, and zeta), or (ii) a single CD3 chain or human CD3 receptor complex (including any combination of CD3 delta, epsilon (one or two molecules), gamma, and zeta) as a full-length or partial protein heterologously linked to one or more intracellular signaling domains; or (iii) the CD3 complex may or may not include a T cell receptor (αβ or γδ) and / or an iTCR receptor (αβ). The disclosure relates to the use of CD3-expressing NK cells in the diagnosis and treatment of diseases, and includes the use of cells in combination with a bispecific or multispecific antibody in which one epitope of the antibody binds to CD3 on the CD3-expressing NK cells. CD3-expressing NK cells can be pre-complexed with bispecific / multispecific antibodies ex vivo and / or combined in vivo to redirect their specificity to target antigens. In diagnostic embodiments, labeled NK cells can be loaded with any type of bispecific or multispecific antibody, including at least an anti-CD3 antibody, and loaded labeled NK cells can be monitored for transport to the site of the target antigen to which another antibody on the bispecific or multispecific antibody binds.

[0175] In certain embodiments of this disclosure, the TCR recognizes an antigen and / or epitope presented by a major histocompatibility complex (MHC). In certain embodiments, the antigen and / or epitope is a peptide, a lipid, and / or a glycolipid. In certain embodiments, the MHC is a class I MHC. In certain embodiments, the MHC is a class II MHC. In certain embodiments, the MHC is a non-classical MHC. In certain embodiments, the MHC is a class I-like MHC. In certain embodiments, the MHC is CD1d.

[0176] In certain embodiments, the TCR target antigen is not the primary agent conferring target antigen specificity to the transduced effector cells. In certain embodiments, the TCR primarily acts as a stabilizer of the CD3 coreceptor complex, while the antibody provides primary target antigen specificity to the transduced effector cells.

[0177] II. Compositions of the Disclosure This disclosure relates to compositions comprising at least modified NK cells expressing at least a portion of the TCR / CD3 complex. In some cases, the compositions also comprise monospecific antibodies, bispecific antibodies, and / or multispecific antibodies, including within the same formulation, although in alternative embodiments, the NK cells and antibodies are utilized as physically separate compositions.

[0178] A. TCR / CD3 modification of NK cells In certain embodiments, this specification provides NK cells that have been manually modified to express some or all of the CD3 / TCR receptor complex. In specific embodiments, the NK cells are modified to include all components of the CD3 complex, including CD3ζ, CD3ε, CD3γ, and CD3δ. In certain cases, the full length of CD3ζ, CD3ε, CD3γ, and CD3δ, including their extracellular, transmembrane, and intracellular domains, is utilized, while in alternative embodiments, only a portion of one or more of CD3ζ, CD3ε, CD3γ, and CD3δ is utilized, each of which may or may not be combined with one or more intracellular signaling domains, such as CD16, NKG2D, DAP10, DAP12, CD28, 41BB, 2B4, CD27, OX40, or any combination thereof.

[0179] In certain embodiments, the amino acid sequence (e.g., polypeptide) may include amino acids represented by the single letter "X" or the three-letter code "Xaa". In some embodiments, the amino acid represented by "X" or "Xaa" is any naturally occurring amino acid, for example, but not limited to, arginine (Arg, R), histidine (His, H), lysine (Lys, K), aspartic acid (Asp, D), glutamic acid (Glu, E), serine (Ser, S), threonine (Thr, T), asparagine (Asn, N), glutamine (Gln, Q), glycine (Gly, G), proline (Pro, P), cysteine ​​(Cys, C), alanine (Ala, A), valine (Val, V), isoleucine (Ile, I), leucine (Leu, L), methionine (Met, M), phenylalanine (Phe, F), tyrosine (Tyr, Y), or tryptophan (Trp, W).

[0180] In certain embodiments, specific sequences of any of the CD3 receptor components, including wild-type or mutant components, are utilized, insofar as the mutant CD3 receptor enables signaling via the CD3 complex that leads to activation and death of targets. In some cases, CD3 / TCR complex-related polypeptides, polynucleotides encoding them, and / or constructs comprising such polynucleotides are described in our International Patent Application Publication WO2023 / 004425A2 (PCT / US2022 / 074062), published on 26 January 2023, which is incorporated herein by reference in its entirety for the purposes described herein.

[0181] In some cases, the following CD3ε, CD3δ, CD3γ, and CD3ζ sequences are used for NK cell modification.

[0182] CD3 epsilon (CD3ε, CD3e) (UniProtKB - P07766 (CD3E_HUMAN))

[0183] Signal peptide MQSGTHWRVLGLCLLSVGVW (Sequence ID: 1)

[0184] Extracellular domain sp|P07766|23-126 DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD (Sequence ID: 2)

[0185] transmembrane domain sp|P07766|127-152 VMSVATIVIVDICITGGLLLLVYYWS (Sequence ID: 3)

[0186] intracellular domain sp|P07766|153-207 KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (Sequence ID: 4)

[0187] An example of the Homo sapiens CD3e molecule (CD3E), mRNA, can be found in the NCBI reference sequence: GENBANK® Accession number NM_000733.4 ATGCAGTCGGGCACTCACTGGAGAGTTCTGGGCCTCTGCCTCTTATCAGTTGGCGTTTGGGGGCAAGATGGTAATGAAGAAATGGGTGGTATTACACAGACACCATATAAAGTCTCCATCTCTGGAACCACAGTAATATTGACATGCCCTCAGTAT CCTGGATCTGAAATACTATGGCAACACAATGATAAAAACATAGGCGGTGATGAGGATGATAAAAACATAGGCAGTGATGAGGATCACCTGTCACTGAAGGAATTTTCAGAATTGGAGCAAAGTGGTTATTATGTCTGCTACCCCAGAGGAAGCAAA CCAGAAGATGCGAACTTTTATCCTACCTGAGGGCAAGAGTGTGTGAGAACTGCATGGAGATGGATGTGATGTCGGTGGCCACAATTGTCATAGTGGACATCTGCATCACTGGGGGCTTGCTGCTGCTGGTTTACTACTGGAGCAAGAATAGAAAG GCCAAGGCCAAGCCTGTGACACGAGGAGCGGGTGCTGGCGGCAGGCAAAGGGGACAAAACAAGGAGAGGCCACCACCTGTTCCCAACCCAGACTATGAGCCCATCCGGAAAGGCCAGCGGGACCTGTATTCTGGCCTGAATCAGAGACGCATCTGA (Sequence number: 5)

[0188] Examples of the complete CD3 epsilon sequences for each nucleic acid and amino acid are shown below (underlined indicates the signal peptide sequence, and in some embodiments, the signal peptide may be modified, omitted, and / or replaced with an alternative signal peptide): ATGCAGAGCGGCACCCACTGGAGAGTGCTGGGCCTGTGCCTGCTGAGCGTGGGCGTGTGGGGCCAG GACGGCAACGAGGAATGGGCGGCATCACCCAGACCCCCTACAAGGTGAGCATCAGCGGCACCACCGTGATCCTGACCTGCCCCCAGTACCCCGGCAGCGAGATCCTGTGGCAGCACAACGACAAGAACATCGGCGGC GACGAGGACGACAAGAACATCGGCAGCGACGAGGACCACCTGAGCCTGAAGGAGTTCAGCGAGCTGGAGCAGAGCGGCTACTACGTGTGCTACCCCAGAGGCAGCAAGCCCGAGGACGCCAACTTCTACCTGTACCTGA GAGCCAGAGTGTGCGAGAACTGCATGGAGATGGACGTGATGAGCGTGGCCACCATCGTGATCGTGGACATCTGCATCACCGGCGGCCTGCTGCTGCTGGTGTACTACTGGAGCAAGAACAGAAAGGCCAAGGCCAAGCC CGTGACCAGAGGCGCCGGCGCCGGCGGCAGACAGAGAGGCCAGAACAAGGAGAGACCCCCCCCCGTGCCCAACCCCGACTACGAGCCCATCAGAAAGGGCCAGAGAGACCTGTACAGCGGCCTGAACCAGAGAAGAATC (Sequence number: 6) MQSGTHWRVLGLCLLSVGVWGQ DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (Sequence ID: 7)

[0189] CD3 Delta (CD3δ, CD3d) (UniProtKB-P04234(CD3D_HUMAN))

[0190] Signal peptide MEHSTFLSGLVLATLLSQVS (Sequence ID: 8)

[0191] Extracellular domain sp|P04234|22-105 FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVA (Sequence ID: 9)

[0192] transmembrane domain sp|P04234|106-126 GIIVTDVIATLLLALGVFCFA (Sequence ID: 10)

[0193] intracellular domain sp|P04234|127-171 GHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK (Sequence ID: 11)

[0194] Homo sapiens CD3d molecule, delta (CD3-TCR complex), mRNA (cDNA clone MGC:88324 IMAGE:30412345), complete cd GENBANK®:BC070321.1 ATGGAACATAGCACGTTTCTCTCTGGCCTGGTACTGGCTACCCTTCTCTCGCAAGTGAGCCCCTTCAAGATACCTATAGAGGAACTTGAGGACAGAGTGTTTGTGAATTGCAATACCAGCATCACATGG GTAGAGGGAACGGTGGGAACACTGCTCTCAGACATTACAAGACTGGACCTGGGAAAACGCATCCTGGACCCACGAGGAATATATAGGTGTAATGGGACAGATATATACAAGGACAAAGAATCTACCGTG CAAGTTCATTATCGAATGTGCCAGAGCTGTGTGGAGCTGGATCCAGCCACCGTGGCTGGCATCATTGTCACTGATGTCATTGCCACTCTGCTCCTTGCTTTGGGAGTCTTCTGCTTTGCTGGACATGAG ACTGGAAGGCTGTCTGGGGCTGCCGACACACAAGCTCTGTTGAGGAATGACCAGGTCTATCAGCCCCTCCGAGATCGAGATGATGCTCAGTACAGCCACCTTGGAGGAAACTGGGCTCGGAACAAGTGA (Sequence number: 12)

[0195] Complete examples of the CD3 delta sequences for each nucleic acid and amino acid are shown below (underlined indicates the signal peptide sequence, and in some embodiments, the signal peptide may be modified, omitted, and / or replaced with an alternative signal peptide): ATGGAGCACAGCACCTTCCTGAGCGGCCTGGTGCTGGCCACCCTGCTGAGCCAGGTGAGCCCCTTCAAGATCCCCATCGAGGAGCTGGAGGACAGAGTGTTCGTGAACTGCAACACCAGCATCACCTGGGTGGAGGGCACCGTGGGCACCCTGCTGAGCGACATCACCAGACTGGACCTGGGCAAGAGAATCCTGGACCCCAGAGGCATCTACAGATGCAACGGCACCGACATCTACAAGGACAAGGAGAGCACCGTGCAGGTGCACTACAGAATGTGCCAGAGCTGC GTGGAGCTGGACCCCGCCACCGTGGCCGGCATCATCGTGACCGACGTGATCGCCACCCTGCTGCTGGCCCTGGGCGTGTTCTGCTTCGCCGGCCACGAGACCGGCAGACTGAGCGGCGCCGCCGACACCCAGGCCCTGCTGAGAAACGACCAGGTGTACCAGCCCCTGAGAGACAGAGACGACGCCCAGTACAGCCACCTGGGCGGCAACTGGGCCAGAAACAAG (Sequence number: 13) MEHSTFLSGLVLATLLSQVSP FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK (Sequence ID: 14)

[0196] CD3 gamma (CD3γ, CD3g) (T cell surface glycoprotein CD3 gamma chain gene CD3G P09693) Signal peptide MEQGKGLAVL ILAIILLQGTLA (Sequence ID: 15)

[0197] Extracellular domain sp|P09693|23-116 QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATIS (Sequence ID: 16)

[0198] transmembrane domain sp|P09693|117-137 GFLFAEIVSIFVLAVGVYFIA (Sequence ID: 17)

[0199] intracellular domain sp|P09693|138-182 GQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN (Sequence ID: 18)

[0200] Homo sapiens CD3g molecule (CD3G), mRNA; NM_000073.3:81-629 Homo sapiens CD3g molecule (CD3G), mRNA ATGGAACAGGGGAAGGGCCTGGCTGTCCTCATCCTGGCTATCATTCTTCTTCAAGGTACTTTGGCCCAGTCAATCAAAGGAAACCACTTGGTTAAGGTGTATGACTATCAAGAAGATGGTTCGGTACTTCTGACTTG TGATGCAGAAGCCAAAAATATCACATGGTTTAAAGATGGGAAGATGATCGGCTTCCTAACTGAAGATAAAAAAAAATGGAATCTGGGAAGTAATGCCAAGGACCCTCGAGGGATGTATCAGTGTAAAGGATCACAGA ACAAGTCAAAACCACTCCAAGTGTATTACAGAATGTGTCAGAACTGCATTGAACTAAATGCAGCCACCATATCTGGCTTTCTCTTTGCTGAAATCGTCAGCATTTTCGTCCTTGCTGTTGGGGTCTACTTCATTGCT GGACAGGATGGAGTTCGCCAGTCGAGAGCTTCAGACAAGCAGACTCTGTTGCCCAATGACCAGCTCTACCAGCCCCTCAAGGATCGAGAAGATGACCAGTACAGCCACCTTCAAGGAAACCAGTTGAGGAGGAATTGA (Sequence number: 19)

[0201] Examples of the complete CD3 gamma sequences of each nucleic acid and amino acid are shown below (underlined indicates the signal peptide sequence, and in some embodiments, the signal peptide may be modified, omitted, and / or replaced with an alternative signal peptide): ATGGAACAGGGGAAGGGCCTGGCTGTCCTCATCCTGGCTATCATTCTTCTTCAAGGTACTTTGGCCCAGTCAATCAAAGGAAACCACTTGGTTAAGGTGTATGACTATCAAGAAGATGGTTCGGTACTTCTGACTTGTGATGCAGAAGCCAAAAATATCACATGGTTTAAAGATGGGAAGATGATCGGCTTCCTAACTGAAGATAAAAAAAAATGGAATCTGGGAAGTAATGCCAAGGACCCTCGTGGGATGTATCAGTGTAAAGGATCACAGAACAAGTCAAAACCACTCCAAGTGTATTACAGA ATGTGTCAGAACTGCATTGAACTAAATGCAGCCACCATATCTGGCTTTCTCTTTGCTGAAATCGTCAGCATTTTCGTCCTTGCTGTTGGGGTCTACTTCATTGCTGGACAGGATGGAGTTCGCCAGTCGAGAGCTTCAGACAAGCAGACTCTGTTGCCCAATGACCAGCTCTACCAGCCCCTCAAGGATCGAGAAGATGACCAGTACAGCCACCTTCAAGGAAACCAGTTGAGGAGGAAT (Sequence number: 20) MEQGKGLAVLILAIILLQGTLA QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN (Sequence ID: 21)

[0202] CD3 zeta (CD3ζ, CD3z)

[0203] Signal peptide sp|P20963| SP MKWKALFTAAILQAQLPITEA (Sequence ID: 22)

[0204] Extracellular domain sp|P20963|22-30 ECD QSFGLLDPK (Sequence ID: 23)

[0205] Transmembrane domain sp|P20963|31-51 tmd LCYLLDGILFIYGVILTALFL (SEQ ID NO: 24)

[0206] Intracellular domain sp|P20963|52-164 ICD RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 25)

[0207] Examples showing the entire CD3 zeta sequences of each nucleic acid and amino acid are shown below (the underlines indicate signal peptide sequences, and in some embodiments, the signal peptide may be modified, omitted, and / or replaced with an alternative signal peptide): ATGAAGTGGAAGGCGCTTTTCACCGCGGCCATCCTGCAGGCACAGTTGCCGATTACAGAGGCA CAGAGCTTTGGCCTGCTGGATCCCAAACTCTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCCTTGTTCCTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO: 26) MKWKALFTAAILQAQLPITEA QSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 27)

[0208] Homo sapiens CD247 molecule (CD247; also called CD3 zeta), transcript variant 1, mRNA NCBI Reference Sequence NM_198053.3 NM_198053.3:65-559 Homo sapiens CD247 molecule (CD247), transcript variant 1, mRNA ATGAAGTGGAAGGCGCTTTTCACCGCGGCCATCCTGCAGGCACAGTTGCCGATTACAGAGGCACAGAGCTTTGGCCTGCTGGATCCCAAACTCTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCCTTGTTCCTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA (SEQ ID NO: 28)

[0209] In specific embodiments, NK cells are modified to express one or more of the TCRα, TCRβ, TCRγ, and TCRδ chains, and any combination thereof can be utilized. In certain embodiments, the TCR may be an invariant natural killer cell TCR (iTCR). In specific cases, NK cells are modified to express the T cell receptor (TCR)αβ chain, iTCRαβ chain, or TCRγδ chain. In certain cases, NK cells are modified to express only part or all of the constant regions of one or more of the TCRα, iTCRα, TCRβ, iTCRβ, TCRγ, and TCRδ chains. NK cells may be modified to express only part or all of the constant regions of the T cell receptor (TCR)αβ chain, TCRγδ chain, or iTCRαβ chain. If a portion of the constant region is used, that portion may be at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, or 400 amino acids, including consecutive amino acids of any constant region. The portion of the constant region may contain at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of the amino acids of the constant region, including consecutive amino acids of the constant region.

[0210] In specific cases, any sequence included herein may be used to modify NK cells, while in other cases, sequences related in identity may be used. For example, related sequences that are at least 80, 85, 90, 95, 96, 97, 98, or 99% identical to any sequence included herein may be used in this disclosure.

[0211] Specific constructs for the expression of various TCR / CD3 proteins in NK cells can be utilized in various configurations. In specific cases, NK cells may be transduced or transfected with one or more vectors for expressing any of the various proteins included herein, which contain at least one or more components of the TCR / CD3 complex. In specific cases, the one or more vectors themselves may or may not be multicistronic, by ultimately producing more than one distinct polypeptide. When one or more multicistronic vectors are employed, they may utilize one or more internal ribosome entry sites (IRESs) and / or one or more 2A self-cleaving peptide sites. In some embodiments, the 2A self-cleaving peptide sites are encoded by codon-optimized polynucleotides. When one or more 2A sequences are utilized, the following may be used, where GSG is an optional linker.

[0212] T2A (GSG) EGRGSLLTCGDVEENPGP (Sequence ID: 29)

[0213] P2A (GSG) ATNFSLLKQAGDVEENPGP (Sequence ID: 30)

[0214] E2A (GSG) QCTNYALLKLAGDVESNPGP (Sequence ID: 31)

[0215] F2A (GSG) VKQTLNFDLLKLAGDVESNPGP (Sequence ID: 32)

[0216] In situations where multiple protein components are expressed from a multicistronic vector, the 5'-to-3' order on the polynucleotide vector may be any order, although alternatively, they may be present on the vector in a specific order. A multicistronic vector may express multiple components of the TCR / CD3 receptor complex and not other heterologous proteins, or it may express multiple components of the TCR / CD3 receptor complex and one or more other heterologous proteins. In specific embodiments, two or more multicistronic vectors are provided, each encoding one or more components of the TCR / CD3 receptor complex, as well as one or more other heterologous proteins such as cytokines and / or engineered receptors (e.g., receptors containing the extracellular portion of Fc-binding proteins).

[0217] Figures 3A and 3C show an example of a multicistronic vector containing the full-length CD3ε, CD3δ, CD3γ, and CD3ζ molecules separated by the same or different 2A self-cleaving peptide sites ("CD3 complex"). The multicistronic vector may contain the respective signal peptides, extracellular domains, transmembrane domains, and intracellular domains of CD3ε, CD3δ, CD3γ, and CD3ζ.

[0218] Figure 3C provides examples of various iTCR and / or CD16 expression constructs for manipulating NK cells. In certain embodiments of this disclosure, the TCR / CD3 receptor complex component is expressed in NK cells from different vectors. In any case, the vector(s) may express a TCR that is directional to a specific antigen of interest, such as a cancer antigen or a viral antigen, or an undirectional TCR. The TCR may or may not contain at least a portion of CD3ζ, including the intracellular domain of CD3ζ, and furthermore, NK cells also express CD3ζ as a molecule separate from the TCR and as part of the CD3 receptor complex.

[0219] In specific embodiments, TCRs, such as iTCRs in modified NK cells, are utilized not necessarily as a therapeutic and / or targeted portion of the NK cell, but as a structural support or scaffold to promote the function or enhanced function of the CD3 receptor complex. That is, a TCR can be any TCR and is not necessarily utilized for its ability to target a specific antigen. In such cases, as a non-limiting example, a TCR that targets viral antigens, glycolipids, bacterial antigens, etc., can be employed in NK cells used for cancers that are not necessarily related to that particular virus, bacterium, and / or glycolipid. In other cases, a TCR is selected for its ability to target a specific antigen related to the target cancer (e.g., tumor-associated antigen).

[0220] In certain embodiments, this specification provides CD3 constructs comprising fusions having intracellular co-stimulatory domains derived from CD16, NKG2D, DAP10, DAP12, 2B4, 4-1BB, CD2, CD28, DNAM, or any combination thereof. In certain embodiments, the intracellular co-stimulatory domains are fused to CD3δ, CD3ε, CD3γ, and / or CD3ζ. In certain embodiments, such a CD3 fusion construct comprises CD3ζ fused to a DAP10 intracellular co-stimulatory domain. In certain embodiments, such a CD3 fusion construct comprises CD3ζ fused to a CD28 intracellular co-stimulatory domain. In certain embodiments, such a CD3 fusion construct comprises CD3ζ fused to both a DAP10 intracellular co-stimulatory domain and a CD28 intracellular co-stimulatory domain. In certain embodiments, CD3ζ fused to the DAP10 intracellular costimulatory domain is represented by a polynucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 33. In certain embodiments, CD3ζ fused to the CD28 intracellular costimulatory domain is represented by a polynucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 34. In certain embodiments, CD3ζ fused to the DAP10 intracellular costimulatory domain and the CD28 intracellular costimulatory domain is represented by a polynucleotide sequence that is at least or exactly identical to Sequence ID No. 35 by 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.In certain embodiments, CD3ζ fused to the DAP10 intracellular costimulatory domain is represented by an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 36. In certain embodiments, CD3ζ fused to the CD28 intracellular costimulatory domain is represented by an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 37. In certain embodiments, CD3ζ fused to the DAP10 intracellular costimulatory domain and the CD28 intracellular costimulatory domain is represented by an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 38. In certain embodiments, CD3ζ fused to the intracellular domain may not include the C-terminal 2A domain. In certain embodiments, CD3ζ fused to the intracellular domain may not include the N-terminal signal peptide domain. ATGAAGTGGAAGGCGCTTTTCACCGCGGCCATCCTGCAGGCACAGTTGCCGATTACAGAGGCCACAGAGCTTTGGCCTGCTGGATCCCAAACTCTGCTACCTGCTGGATGGAATCCTTTCATCTATGGTGTCATTCACTGCCTTGTTTCCTGTTCTGCTTTGCGCCAGCCCCGCAGAAGATGGCAAAGTCTACATCAACATGCCAGGGGCAGAAGTGTAAGTCCAGAGCGCCCCGGTACCAGAGCAGGCCCCGCTGACAAGCAGCCTTAACGAGCCTCAATGAGCTCAATGGAAGTCCAAGAAGATGGGGGAAAGCCGCAGAGAAGGAAGACCCTCAGGAAGGAAGCCTGTACAATGAACTGCCAGAAAGATAAGATGGCGGAGGCCTCACAGTGATTGGGATGAAAGCGCGCGGAGGGGCAAGGGCCAGAGGCCCTTACCAGGGTCTCAGTACAGCCACCAAGGCACACCTACGCAGCCCTCTCTCCAGAGCCAACTACGCCCTGCTGAAGCTGGCCCGGACGTGGAGAGAGACAACCCCGGCCCC (sequence number: 33) ATGAAGTGGAAGGCGCTTTCACCGCGGCCATCCTGCAGGCACAGTTGCCGATTACAGAGGCACAGAGCTTTGGCCTGCTGGATCCCAAACTCTGCTACCTGCTGGATGGAATCCTCTTCACTTATGGTGTCATTCACTGCCTTGTTCCTGAGGAGTAAGAGGAGC AGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCGCGCCCGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAAGAGTGAAGTTCAGGAGCGCAGACGCCCCCGGTACCAGGCAGGCCAGAACCAGCTCT ATAACGAGCTCAATCTAGGACGAAGAGAGGAGGAGTACGATGTTTTGGGACAAGAGACGTGGCGGGACCCTGAGATGGGGGAAAGCCGCGAGAAGGAAGGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCACAGGACACCTACGACGCCCTTCACATGCAGGCCTGCCCCTCGCGCGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC (sequence number: 34) ATGAAGTGGAAGGCGCTTTCACCGCGGCCATCCTGCAGGCACAGTTGCCGATTACAGAGGCACAGAGCTTTGGCCTGCTGGATCCCAAACTCTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCCTTGTTCCTGAGGGAGTAAGAGGAGCAGGCTCCTGCACAGTGAC TACATGAACATGACTCCCCGCCGCCCCGGGCCCACCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCACTTTGCGCACGCCCACGCGCCAGCCCCGCCCAAGAAGATGGCAAAGTCTACATCAACATGCCAGGCAGGGGCAGAGTGAAGTTCAGCAGGAGCGCAG ACGCCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCGGGACCCTGAGATGGGGGAAAGCCGCGAGAAGGAAGGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGATTGGGATGAAAGGCGAGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACAAGGACACCTACGACGCCCTTCACATGCAGCCCTGCCCCTCGCATGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC (sequence number: 35) MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLLCARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQCTNYALLKLAGDVESNPGP (Sequence ID: 36) MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQCTNYALLKLAGDVESNPGP (Sequence ID: 37) MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSLCARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQCTNYALLKLAGDVESNPGP (Sequence ID: 38)

[0221] In certain embodiments, the DAP10 intracellular costimulatory domain is represented by a polynucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 39. In certain embodiments, the CD28 intracellular costimulatory domain is represented by a polynucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 40. In certain embodiments, the DAP10 intracellular costimulatory domain and the CD28 intracellular costimulatory domain are represented by a polynucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 41. In certain embodiments, the DAP10 intracellular costimulatory domain is represented by an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 42. In certain embodiments, the CD28 intracellular costimulatory domain is represented by an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 43. In certain embodiments, the DAP10 intracellular costimulatory domain and the CD28 intracellular costimulatory domain are represented by an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 44. CTTTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGGCAAAGTCTACATCAACATGCCAGGCAGGGGC (SEQ ID: 39) AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCA(SEQ ID: 40) AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCACTTTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGGCAAAGTCTACATCAACATGCCAGGCAGGGGC (SEQ ID: 41) LCARPRRSPAQEDGKVYINMPGRG (SEQ ID: 42) RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID: 43) RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSLCARPRRSPAQEDGKVYINMPGRG (SEQ ID: 44)

[0222] UTNK15-DAP10 refers to a polynucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No. 45. In certain embodiments, the UTNK15-DAP10 amino acid sequence may be represented by an amino acid sequence that is at least or exactly identical to sequence number 46 by 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. (Array:46)

[0223] UTNK15-28 refers to a polynucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No. 47. In certain embodiments, the UTNK15-28 amino acid sequence may be represented by an amino acid sequence that is at least or exactly identical to SEQ ID NO: 48 by 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0224] UTNK15-28-DAP10 refers to a polynucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No. 49. In certain embodiments, the UTNK15-28-DAP10 amino acid sequence may be represented by an amino acid sequence that is at least or exactly identical to SEQ ID NO: 50 by 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0225] As described in Figure 3C and above, the term “linked” refers to being present on the same polynucleotide vector and does not necessarily mean that the two polypeptides are expressed as a single polypeptide. For example, cytokines generated from the vectors of this disclosure may ultimately be generated as molecules separate from any one or more TCR / CD3 receptor complex components. On the other hand, the term “fused” or “combined” refers to two polypeptides containing a peptide bond that connects the two molecules, i.e., two polypeptides covalently linked by an amide bond and not separated by a splitting element such as a 2A element.

[0226] In some embodiments, the TCR construct includes a human papillomavirus (HPV)-specific TCR chain. In some embodiments, the TCR construct including the HPV-specific TCR chain includes TCR alpha and TCR beta chains that target the HPV18 E6 protein and / or the HPV18 E7 protein. In some embodiments, the HPV18 E6 epitope is amino acids 121-135 and / or amino acids 77-91 of the HPV18 E6 protein. In some embodiments, the TCR construct including the HPV-specific TCR chain includes TCR alpha and TCR beta chains that target the HPV18 E7 protein. In some embodiments, the HPV18 E7 epitope is amino acids 11-19. In some embodiments, the HPV-specific TCR sequence, TCR variable domain sequence, CDR sequence, and / or TCR constant domain sequence are described in International Publication No. WO2015 / 009604A1, which is incorporated herein by reference for the purposes described herein.

[0227] In some embodiments, the TCR is an invariant natural killer T cell TCR (iTCR). In some embodiments, the iTCR may bind to certain CD1d-expressing cells, such as specific cancer cells, monocytes, and / or macrophages, that promote tumor growth. While not theoretically limited, the CD1d molecule is not polymorphic, and therefore, in certain embodiments, recognition of antigens presented by CD1d by NK cells with transgenic iTCRs does not result in host-graft mismatch. In certain embodiments, utilizing the iTCR instead of a standard T cell-derived TCR can reduce complications associated with MHC-1 / 2 mismatch.

[0228] In some embodiments, the iTCR complex includes an alpha(α) chain iTCR (iTCRα). In some embodiments, the construct encoding iTCRα includes a polynucleotide coding sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 51 (e.g., iTCRα associated with Vα24-Jα18 in humans). In some embodiments, iTCRα contains an amino acid sequence that is at least, or exactly, identical to SEQ ID NO: 52 by 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (for example, iTCRα associated with Vα24-Jα18 in humans).

[0229] iTCRα ATGAAGAAGCACCTGACCACCTTTCTGGTCATCCTGTGGCTGTACTTCTCAGAGGCAACGGCAAGAACCAGGTGGAACAGAGCCCTCAGAGCCTGATCATCCTGGAAGGCAAGAACTGCACCCTGCAGTGCAACTACACCGTGTCTCCCTTCAGCAACCTGCGGTGGTACAAGCAGGATACAGGCAGAGGCCCTGTGTCTCTGACCATCATGACCTTCAGCGAGAACACCAAGAGCAACGGCCGGTACACCGC CACACTGGATGCCGATACAAAGCAGAGCAGCCTGCACATCACAGCCAGCCAGCTGAGCGATAGCGCCAGCTACATCTGCGTGGTGTCCGATAGAGGCAGCACCCTGGGCAGACTGTACTTTGGCAGAGGCACCCAGCTGACCGTGTGGCCCGATATTCAGAACCCTGATCCTGCCGTGTACCAGCTGAGAG ACAGCAAGTCCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAAGCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGACAAGggcGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAATAGCGCCGTGGCCTGGTCAACAAGAGCGATTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGACACATTCTTCCCAAAGTCCTGAGAGCAGCTGCGACGTGGAAGCTGGTGGAAAAGAGCTTCGAGACACACCTGAACTTCCAGAACCTGAGCGTGATCGGCTTCGGATCCTGCTGCTGTAAAAGTGCCGGCTTCAACCTGCTGATCCGAGACTTGGTCTAGC (sequence number: 51) MKKHLTTFLVILWLYFYRGNGKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSLTIMTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSDRGSTLGRLYFGRGTQLTVWPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (Sequence ID: 52)

[0230] iTCRβ

[0231] In some embodiments, the iTCR complex includes a beta (β) chain iTCR (iTCRβ). In some embodiments, the construct encoding iTCRβ includes a polynucleotide coding sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs. In some embodiments, iTCRβ contains an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs. ATGAAGAAGCACCTGACCACCTTTCTGGTCATCCTGTGGCTGTACTTCTCAGAGGCAACGGCAAGAACCAGGTGGAACAGAGCCCTCAGAGCCTGATCATCCTGGAAGGCAAGAACTGCACCCTGCAGTGCAACTACACCGTGTCTCCCTTCAGCAACCTGCGGTGGTACAAGCAGGATACAGGCAGAGGCCCTGTGTCTCTGACCATCATGACCTTCAGCGAGAACACCAAGAGCAACGGCCGGTACACCGC CACACTGGATGCCGATACAAAGCAGAGCAGCCTGCACATCACAGCCAGCCAGCTGAGCGATAGCGCCAGCTACATCTGCGTGGTGTCCGATAGAGGCAGCACCCTGGGCAGACTGTACTTTGGCAGAGGCACCCAGCTGACCGTGTGGCCCGATATTCAGAACCCTGATCCTGCCGTGTACCAGCTGAGAG ACAGCAAGTCCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAAGCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGACAAGggcGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAATAGCGCCGTGGCCTGGTCAACAAGAGCGATTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGACACATTCTTCCCAAAGTCCTGAGAGCAGCTGCGACGTGGAAGCTGGTGGAAAAGACTTCGAGACAGACACCTGACTGCGACGTGATCGGGCTTCGGATCCTGCTGCTGAAAGTGCCGGCTTCAACCTGCTGATCCGAGACTTGGTCTAGC (sequence number: 53) MTIRLLCYMGFYFLGAGLMEADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELHLIHYSYGVNSTEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCASSELRALGPSSYNSPLHFGNGTRLTVTDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (Sequence ID: 54) ATGACCATCCGGCTGCTGTGCTATATGGGCTTCTACTTCCTCGGAGCCGGCCTGATGGAAGCCGACATCTACCAGACACCTAGATACCTGGTCATCGGCACCGGCAAAAGATCACCCTGGAATGCTCCCAGACCATGGGGCCACGACAAGATGTACTGGTATCAGCAGGACCCCGGCATGGAACTGCATCTGATCACTACAGCTACGGCGTGAACAGCACCGAGAAGGGCGAT CTGTCTAGCGAGAGCACCGTGTCCAGAATCCGGACCGAGCACTTCCCACTGACACTGGAAAGCGCCAGACCTAGCCACACCAGCCAGTACCTGTGTGCCTCTAGTGAAGGCGGAGGACTCAAGTTGGCGAAAAACATCCAATACTTTGGTGCTGGCACCCGGCTTTCTGTCCTGGACCTGAACAAGGTGTTCCCTCCAGAGGTGGCCGTTCGAGCCTTCGAGGCCGAGATC AGCCACACACAGAAAGCCACACTCGTGTGTCTGGCCACCGGCTTTTCCCCGATCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCCACAGCGGCGTCtGCACAGAGATCCCCGCTCTGAAAGAACAGCCCGCTCTGAACGACAGCCCGGTACTGTCTGAGCGAGACAGACTGAGAGAGTGCCCGCACCTTCTGGCAGAACCCCAGAAACACTTCAGATGCCAGGTGCAGTTTTACGCCTGGAGCGAGAACGACGAGTGGACCCAGGATAGAGCCAAGCCTGTGCTGTCGCGAAGCCTGGTGCGAAGCCTGGTGCGAACCAGATCGTGTGCCACATCCTTGTATGAGATTCTGCTGGGCAAAGCCACTCTTGTACGCCGTTGTCCTGCCACCTCTGTATGGGCCATGGTCAAGAGAAAAGACTTC (sequence number: 55) MTIRLLCYMGFYFLGAGLMEADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELHLIHYSYGVNSTEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCASSEGGGLKLAKNIQYFGAGTRLSVLDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (Sequence ID: 56) ATGACCATCCGGCTGCTGTGCTATATGGGCTTCTACTTCCTCGGAGCCGGCCTGATGGAAGCCGACATCTACCAGACACCTAGATACCTGGTCATCGGCACCGGCAAAAGATCACCCTGGAATGCTCCCAGACCATGGGGCCACGACAAGATGTACTGGTATCAGCAGGACCCCGGCATGGAACTGCATCTGATCACTACAGCTACGGCGTGAACAGCACCGAGAAGGGCGA TCTGTCTAGCGAGAGCACCGTGTCCAGAATCCGGACCGAGCACTTCCCACTGACACTGGAAAGCGCCAGACCTAGCCACACCAGCCAGTACCTGTGTGCCTCTAGTGAATTCGCCAGCTCCGTGCGAGGGAATACTATCTACTTTGGTGAGGGATCTTGGCTTACGGTAGTAGACCTGAACAAGGTGTTCCCTCCAGAGGTGGCCGTGTTCGAGCCTTCTGAGGCCGAGATC GCCACACACAGAAAGCCACACTCGTGTGTCTGGCCACCGGCTTTTCCCGATCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCCACAGCGGCGTCtGCACAGATCCCCAGCCCTCTGAAAGAACAGCCCGCTCTGAACGACAGCCCGGTACTGTCTGAGCGAGAACTGAGAGTGTCCGCCACCTCTGGCAGAACCCCAGAAACACTTCAGATGCCAGGTGCAGTTTTACGCCTGGACGGAAGGAACGACGAGTGGACCCAGGATAGAGCCAAGCCTGTGCTGGCGAAGCCTGGTGCGAAGCCTGGTGCGAACCAGATCGTGTGCCACATCCTTGTATGAGATTCTGCTGGGCAAAGCCACTCTTGTACGCCGTTGTCCTGCCACCTCTGTATGGGCCATGGTCAAGAGAAAAGACTTC (sequence number: 57) MTIRLLCYMGFYFLGAGLMEADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELHLIHYSYGVNSTEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCASSEFASSVRGNTIYFGEGSWLTVVDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (Sequence ID: 58)

[0232] In some embodiments, NK cells contain an iTCR complex beta (β) chain iTCR (iTCRβ). In some embodiments, the construct encoding iTCRβ includes a polynucleotide coding sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs. In some embodiments, iTCRβ contains an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs.

[0233] In some embodiments, the construct encoding iTCRβ includes a polynucleotide coding sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs. In some embodiments, iTCRβ includes an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs. ATGACCATCCGGCTGCTGTGCTATATGGGCTTCTACTTCCTCGGAGCCGGCCTGATGGAAGCCGACATCTACCAGACACCTAGATACCTGGTCATCGGCACCGGCAAAAGATCACCCTGGAATGCTCCCAGACCATGGGCCACGACAAGATGTACTGGTATCAGCAGGACCCCGGCATGGAACTGCATCTGATCACTACAGCTACGGCGTGAACAGCACCGAGAAGGGCG ATCTGTCTAGCGAGAGCACCGTGTCCAGAATCCGGACCGAGCACTTCCCACTGACACTGGAAAGCGCCAGACCTAGCCACACCAGCCAGTACCTGTGTGCCACTGGACAGGGGGCGCAAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAGGACCTCAAGAATGTGTTTCCGCCGAAGTCGCGGTTTTGAACCATCAGAAGCCGAGATCTCTCATACA CAAAAAGGCGACGCTCGTATGCCTCGCGACGGGATTTTATCCGGACCACGTCGAGCTTTCCTGGTGGGTTAAcGGAAAGGAGGTGCATTCCGGAGTTTGCACGGACCCTCAGCCATTGAAGGAACCCCGCACTGAACGACAGTAGGTATTGCCTTTCATCTGCCGCGGTGTCTGCGACATTTCTGGCAAAACCCAAGAAATCACTTCAGATGTCAAGTTCAGTTTCATCGGTTCAGGCGAGAATGATGAGTGGACACAAGATAGGGCTAACCCGTGACTCAAATAGTCTCTGCGAGGCCTGGGGAGGCGATTGCGGCTTTCACATCAGAATCATACCAACAACAAGGAGTATTGAGCGCGACAATTCTTTACGAAATTCTGCTTGGAAAGCGACTCTGTACGCGGTGTCCTGTTCCGCTTTTGGTTCTTATGGCAATGGTTAAAACGAAAGGATAGTAGGGGC (sequence number: 59; iTCRβ clone 3) MTIRLLCYMGFYFLGAGLMEADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELHLIHYSYGVNSTEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCATGQGAQDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (Sequence ID: 60; iTCRβ clone 3) ATGACCATCCGGCTGCTGTGCTATATGGGCTTCTACTTCCTCGGAGCCGGCCTGATGGAAGCCGACATCTACCAGACACCTAGATACCTGGTCATCGGCACCGGCAAAAAGATCACCCTGGAATGCTCCCAGACCATGGGCCACGACAAGATGTACTGGTATCAGCAGGACCCCGGCATGGAACTGCATCTGATCCACTACAGCTACGGCGTGAACAGCACCGAGAAGGGCGATCTGTCTAGCGAGAGCACCGTGTCCAGAATCCGGACCGAGCACTTCCCACTGACACTGGAAAGCGCCAGACCTAGCCACACCAGCCAGTACCTGTGTGCCAGCAGTGATGGGGTGGGGAGCAATCAGCCCCAGCATTTTGGTGATGGGACTCGACTCTCCATCCTAGAGGACCTCAATAAGGTGTTTCCGCCCGAAGTCGCGGTTTTTGAACCATCAGAAGCCGAGATCTCTCATACACAAAAGGCGACGCTCGTATGCCTcGCGACGGGATTTttcCCGGACCACGTCGAGCTTTCCTGGTGGGTTAAcGGAAAGGAGGTGCATTCCGGAGTTTGCACGGACCCTCAGCCATTGAAGGAACAGCCCGCACTGAACGACAGTAGGTATTGCCTTTCATCTCGCCTGCGCGTGTCTGCGACATTCTGGCAAAACCCAAGAAATCACTTCAGATGTCAAGTTCAGTTCTACGGTCTCAGCGAGAATGATGAGTGGACACAAGATAGGGCTAAACCCGTGACTCAAATAGTCTCTGCCGAGGCCTGGGGGAGGGCGGATTGCGGCTTCACATCAgtgTCATACCAACAAGGAGTATTGAGCGCGACAATTCTTTACGAAATTCTGCTTGGGAAAGCGACTCTGTACGCGGTGCTCGTGTCCGCTTTGGTTCTTATGGCAATGGTTAAACGAAAGGACTTC (SEQ ID NO: 61; iTCRβ clone 18) MTIRLLCYMGFYFLGAGLMEADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELHLIHYSYGVNSTEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCASSDGVGSNQPQHFGDGTRLSILEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (Sequence ID: 62; iTCRβ clone 18) ATGACCATCCGGCTGCTGTGCTATATGGGCTTCTACTTCCTCGGAGCCGGCCTGATGGAAGCCGACATCTACCAGACACCTAGATACCTGGTCATCGGCACCGGCAAAAGATCACCCTGGAATGCTCCCAGACCATGGGCCACGACAAGATGTACTGGTATCAGCAGGACCCCGGCATGGAACTGCATCTGATCACTACAGCTACGGCGTGAACAGCACCGAAGGGC GATCTGTCTAGCGAGACCACGTGTCCAGAATCCGGACCGAGCACTTCCCACTGACACTGGAAAGCGCCAGACCTAGCCACACCAGCCAGTACCTGTGTGCCAGCAGTGAGGGGCTGGAACACCATATATTTTGGAGAGGGAAGTTGGCTCACTGTTGTAGGGACCTCAATAAGGTGTTTCCGCCCGAAGTCGCGGTTTTGAACCATCAGAAGCCGAGATCTCTCAT ACACAAAAAGGCGACGCTCGTATGCCTCGCGCGAGCTTTCCTGGTGGGTTAAcGGAAAGGAGGTGCATTCCGGAGTTTGCACGGACCCTCAGCCATTGAAGGAACGCCCGCACTGAACGACAGTAGGTATTGCCTTTCATCTGCCGCGGTGTCTGCGACATTTCTGGCAAAACCCAAGAAATCACTTCAGATGTCAAGTTCAGTTTCATCGGTTCAGGCGAGAATGATGAGTGGACACAAGATAGGGCTAACCCGTGACTCAAATAGTCTCTGCGAGGCCTGGGGAGGCGGATTGCGGCTTCACATCAgtgTCATACCAACAAGGAGTATTGAGCGCGACAATTCTTCTTACGAAATTCTGCTTGGAAAGCGACTCTGTACGCGGTGCTCGGCTTTGGTTCTTATGGCAATGGTTAAAACGAAAAGGACTTC (sequence number: 63; iTCRβ clone 24) MTIRLLCYMGFYFLGAGLMEADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELHLIHYSYGVNSTEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCASSEGAGNTIYFGEGSWLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (Sequence ID: 64; iTCRβ clone 24) ATGACCATCCGGCTGCTGTGCTATATGGGCTTCTACTTCCTCGGAGCCGGCCTGATGGAAGCCGACATCTACCAGACACCTAGATACCTGGTCATCGGCACCGGCAAAAGATCACCCTGGAATGCTCCCAGACCATGGGCCACGACAAGATGTACTGGTATCAGCAGGACCCCGGCATGGAACTGCATCTGATCACTACAGCTACGGCGTGAACAGCACCGAAGGGC GATCTGTCTAGCGAGAGCACCGTGTCCAGAATCCGGACCGAGCACTTCCCACTGACACTGGAAAGCGCCAGACCTAGCCACACCAGCCAGTACCTGTGTGCCAGCAGTGACAGGGATAGCAATCAGCCCCAGCATTTTGGTGATGGGACTCGACTCTCCATCCTAGAGGACCTCAATAAGGTGTTTCCGCCCGAAGTCGCGGTTTTGAACCATCAGAAGCCGAGATCTCTC ATACACAAAAGGCGACGCTCGTATGCCTcGCGACGGGATTTttcCCGGACCACGTCGAGCTTTCCTGGTGGGTTAAcGGAAAGGAGGTGCATTCCGGAGTTTGCACGGACCCTCAGCCATTGAAGGAACCCCGCACTGAACGACAGTAGGTATTGCCTTTCATCTGCCGCGGTGTCTCGACATTCTGGCAAAACCCAAGAAATCACTTCAGATGTCAAGTTCAGTCTCAGGCGAGAATGATGAGTGGACACAAGATAGGGCTAACCCGTGACTCAAATAGTCTCTGCGAGGCCTGGGGAGGCGGATTGCGGCTTCACATCAgtgTCATACCAACAAGGAGTATTGAGCGCGACAATTCTTCTTACGAAATTCTGCTTGGAAAGCGACTCTGTACGCGGTGTCCTGTCCGCTTTGGTTCTTATGGCAATGGTTAAAACGAAAAGGACTTC (sequence number: 65; iTCRβ clone 51) MTIRLLCYMGFYFLGAGLMEADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELHLIHYSYGVNSTEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCASSDRDSNQPQHFGDGTRLSILEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (Sequence ID: 66; iTCRβ clone 51) ATGACCATCCGGCTGCTGTGCTATATGGGCTTCTACTTCCTCGGAGCCGGCCTGATGGAAGCCGACATCTACCAGACACCTAGATACCTGGTCATCGGCACCGGCAAAAGATCACCCTGGAATGCTCCCAGACCATGGGGCCACGACAAGATGTACTGGTATCAGCAGGACCCCGGCATGGAACTGCATCTGATCACTACAGCTACGGCGTGAACAGCACCGAGAAGGGCGAT CTGTCTAGCGAGAGCACCGTGTCCAGAATCCGGACCGAGCACTTCCCACTGACACTGGAAAGCGCCAGACCTAGCCACACCAGCCAGTACCTGTGTGCCAGCGTGGGTCCGGTACCCTCCTACAATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAGGACCTTCAAGAATGTGTTTCCGCCGAAGTCGCGGTTTTGAACCATCAGAAGCCGAGATCCTCTCAT ACACAAAAAGGCGACGCTCGTATGCCTCGCGACGGGATTTTATCCGGACCACGTCGAGCTTTCCTGGTGGGTTAAcGGAAAGGAGGTGCATTCCGGAGTTTGCACGGACCCTCAGCCATTGAAGGAACCCCGCACTGAACGACAGTAGGTATTGCCTTTCATCTGCCGCGGTGTCTCGACATTCTGGCAAAACCCAAGAAATCACTTCAGATGTCAAGTTCAGTTTCATCGGTTCAGGCGAGAATGATGAGTGGACACAAGATAGGGCTAACCCGTGACTCAAATAGTCTCTGCGAGGCCTGGGGAGGCGATTGCGGCTTTCACATCAGAATCATACCAACAACAAGGAGTATTGAGCGCGACAATTCTTTACGAAATTCTGCTTGGAAAGCGACTCTGTACGCGGTGTCCTGTCCGCTTTGGTTCTTATGGCAATGGTTAAAACGAAAGGATAGTAGGGGC (Sequence number: 67; iTCRβ clone 56) MTIRLLCYMGFYFLGAGLMEADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELHLIHYSYGVNSTEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCASVGPVPSYNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (Sequence ID: 68; iTCRβ clone 56) ATGACCATCCGGCTGCTGTGCTATATGGGCTTCTACTTCCTCGGAGCCGGCCTGATGGAAGCCGACATCTACCAGACACCTAGATACCTGGTCATCGGCACCGGCAAAAGATCACCCTGGAATGCTCCCAGACCATGGGGCCACGACAAGATGTACTGGTATCAGCAGGACCCCGGCATGGAACTGCATCTGATCACTACAGCTACGGCGTGAACAGCACCGAGAAGGGCGAT CTGTCTAGCGAGAGCACCGTGTCCAGAATCCGGACCGAGCACTTCCCACTGACACTGGAAAGCGCCAGACCTAGCCACACCAGCCAGTACCTGTGTGCCAGCAGTGGGGTGACTAGCGCCTCCTCAATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAGGACCTTCAAGAATGTGTTTCCGCCGAAGTCGCGGTTTTGAACCATCAGAAGCCGAGATCTCTC ATACACAAAAGGCGACGCTCGTATGCCTcGCGACGGGATTTTATCCGGACCACGTCGAGCTTTCCTGGTGGGTTAAcGGAAAGGAGGTGCATTCCGGAGTTTGCACGGACCCTCAGCCATTGAAGGAACCCCGCACTGAACGACAGTAGGTATTGCCTTTCATCTGCCGCGGTGTCTCGCACATTTCTGGCAAAACCCAAGAAATCACTTCAGATGTCAAGTTCAGTTTCATCGGTTCAGGCGAGAATGATGAGTGGACACAAGATAGGGCTAACCCGTGACTCAAATAGTCTCTGCGAGGCCTGGGGAGGCGATTGCGGCTTTCACATCAGAATCATACCAACAACAAGGAGTATTGAGCGCGACAATTCTTTACGAAATTCTGCTTGGAAAGCGACTCTGTACGCGGTGTCCTGTTCCGCTTTTGGTTCTTATGGCAATGGTTAAAACGAAAGGATAGTAGGGGC (Sequence number: 69; iTCRβ clone 76) MTIRLLCYMGFYFLGAGLMEADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELHLIHYSYGVNSTEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCASSGVTSASYNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (Sequence ID: 70; iTCRβ clone 76) ATGACCATCCGGCTGCTGTGCTATATGGGCTTCTACTTCCTCGGAGCCGGCCTGATGGAAGCCGACATCTACCAGACACCTAGATACCTGGTCATCGGCACCGGCAAAAGATCACCCTGGAATGCTCCCAGACCATGGGCCACGACAAGATGTACTGGTATCAGCAGGACCCCGGCATGGAACTGCATCTGATCACTACAGCTACGGCGTGAACAGCACCGAGAAGGGCG ATCTGTCTAGCGAGAGCACCGTGTCCAGAATCCGGACCGAGCACTTCCCACTGACACTGGAAAGCGCCAGACCTAGCCACACCAGCCAGTACCTGTGTGCCAGCAGTGGAGGGGGAGGAGACCCAGTACTTCGGGCCAGGCACGCGCTCCTGGTGCTCGAGGACCTCAAGAATGTGTTTCCGCCGAAGTCGCGGTTTTGAACCATCAGAAGCCGAGATCTCTCATACA CAAAAAGGCGACGCTCGTATGCCTCGCGAGGATTTTATCCGGACCACGTCGAGCTTTCCTGGTGGGTTAAcGGAAAGGAGGTGCATTCCGGAGTTTGCACGGACCCTCAGCCATTGAAGGAACCCCGCACTGAACGACAGTAGGTATTGCCTTTCATCTGCCGCTGCGCGTGTCTGCGACATTTCTGGCAAAACCCAAGAAATCACTTCAGATGTCAAGTTCAGTTTCATCGGTTCAGGCGAGAATGATGAGTGGACACAAGATAGGGCTAACCCGTGACTCAAATAGTCTCTGCGAGGCCTGGGGAGGCGATTGCGGCTTTCACATCAGAATCATACCAACAACAAGGAGTATTGAGCGCGACAATTCTTTACGAAATTCTGCTTGGAAAGCGACTCTGTACGCGGTGTCCTGTTCCGCTTTTGGTTCTTATGGCAATGGTTAAAACGAAAGGATAGTAGGGGC (Sequence number: 71; iTCRβ clone 93) MTIRLLCYMGFYFLGAGLMEADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELHLIHYSYGVNSTEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCASSGGGEETQYFGPGTRLLVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (Sequence ID: 72; iTCRβ clone 93) ATGACCATCCGGCTGCTGTGCTATATGGGCTTCTACTTCCTCGGAGCCGGCCTGATGGAAGCCGACATCTACCAGACACCTAGATACCTGGTCATCGGCACCGGCAAAAGATCACCCTGGAATGCTCCCAGACCATGGGCCACGACAAGATGTACTGGTATCAGCAGGACCCCGGCATGGAACTGCATCTGATCACTACAGCTACGGCGTGAACAGCACCGAAGGGC GATCTGTCTAGCGAGAGCACCGTGTCCAGAATCCGGACCGAGCACTTCCCACTGACACTGGAAAGCGCCAGACCTAGCCACACCAGCCAGTACCTGTGTGCCAGCAGTGCGCAGGGGTCAGGAAAAAACTGTTTTTTGCAGTGGAACCCAGCTCTCTGTCTTGGAGGACCTAATAAGGTGTTTCCGCCCGAAGTCGCGGTTTTGAACCATCAGAAGCCGAGATCTCTC ATACACAAAAGGCGACGCTCGTATGCCTcGCGACGGGATTTttcCCGGACCACGTCGAGCTTTCCTGGTGGGTTAAcGGAAAGGAGGTGCATTCCGGAGTTTGCACGGACCCTCAGCCATTGAAGGAACCCCGCACTGAACGACAGTAGGTATTGCCTTTCATCTGCCGCGGTGTCTCGACATTCTGGCAAAACCCAAGAAATCACTTCAGATGTCAAGTTCAGTCTCAGGCGAGAATGATGAGTGGACACAAGATAGGGCTAACCCGTGACTCAAATAGTCTCTGCGAGGCCTGGGGAGGCGGATTGCGGCTTCACATCAgtgTCATACCAACAAGGAGTATTGAGCGCGACAATTCTTCTTACGAAATTCTGCTTGGAAAGCGACTCTGTACGCGGTGCTCGGCTTTGGTTCTTATGGCAATGGTTAACGAAAAGGACTTC (sequence number: 73; iTCRβ clone 96) MTIRLLCYMGFYFLGAGLMEADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELHLIHYSYGVNSTEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCASSAQGVSEKLFFGSGTQLSVLEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (Sequence ID: 74; iTCRβ clone 96)

[0234] In some embodiments, the construct encoding the iTCRβ chain includes an iTCRβ Vβ-DJ region containing a polynucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs. ACTGGACAGGGGGCGCAAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAG (Sequence ID: 75; iTCRβ clone 3) AGCAGTGCCCCTGGAGGGTCTGAAGCTTTCTTTGGACAAGGCACCAGACTCACAGTTGTAGAG (Sequence ID: 76; iTCRβ clone 4) AGCAGTGAACTCGACAGGGAAGGAAACACCATATATTTTGGAGAGGGAAGTTGGCTCACTGTTGTAGAG (Sequence ID: 77; iTCRβ clone 7) AGCAGTGATATGGGACCCGTCTCCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (Sequence ID: 78; iTCRβ clone 9) AGCAGTGAGGAAGAGCGGGGCCCCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (Sequence ID: 79; iTCRβ clone 13) AGCAGTGATGGGGTGGGGAGCAATCAGCCCCAGCATTTTGGTGATGGGACTCGACTCTCCATCCTAGAG (Sequence ID: 80; iTCRβ clone 18) AGCAGTGACCCCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (Sequence ID: 81; iTCRβ clone 20) AGCAGTGAGGCCCCAACAGGAACCGGGGCCAACGTCCTGACTTTCGGGGCCGGCAGCAGGCTGACCGTGCTGGAG (Sequence ID: 82; iTCRβ clone 21) AGCAGTGATCCTCGACTAGCGGGGGGGGTCGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (Sequence ID: 83; iTCRβ clone 23) AGCAGTGAGGGGGCTGGAAACACCATATATTTTGGAGAGGGAAGTTGGCTCACTGTTGTAGAG (Sequence ID: 84; iTCRβ clone 24) AGCAGTTATGCTACAGGCTACAATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAG (Sequence ID: 85; iTCRβ clone 26) AGCAGTGAACGGCAGGGCTCCACAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAG (Sequence ID: 86; iTCRβ clone 28) AGCAGTGCTCCGACTAGCGGGAGGGACAATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAG (Sequence ID: 87; iTCRβ clone 29) AGCAGTGAATGGACTAGCGGGGGGCCCAACACCGGGGAGCTGTTTTTTGGAGAAGGCTCTAGGCTGACCGTACTGGAG (Sequence ID: 88; iTCRβ clone 30) AGCAGTGAACGGGGGGCTAGCACAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAG (Sequence ID: 89; iTCRβ clone 32) AGCAGTGAAGGGCTAGCGGGAGAACCTCTCTTAGGCAATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAG (Sequence ID: 90; iTCRβ clone 33) AGCAGTGAGGCAGGCGGCCACACAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAG (Sequence ID: 91; iTCRβ clone 34) AGCAGTGAATACCAAGAGACCCAGTACTTCGGGCCAGGCACGCGGCTCCTGGTGCTCGAG (Sequence ID: 92; iTCRβ clone 36) AGCACCGACAGGGGATCTTTCGGGGAGCTGTTTTTTGGAGAAGGCTCTAGGCTGACCGTACTGGAG (Sequence ID: 93; iTCRβ clone 37) GGAGGAGGGACATCTCAAGAGACCCAGTACTTCGGGCCAGGCACGCGGCTCCTGGTGCTCGAG (Sequence ID: 94; iTCRβ clone 38) AGCAGTCCGACTAGCGGGATGGGGGAGACCCAGTACTTCGGGCCAGGCACGCGGCTCCTGGTGCTCGAG (Sequence ID: 95; iTCRβ clone 39) AGCAGTGAGTTCGGGGCCAACGTCCTGACTTTCGGGGCCGGCAGCAGGCTGACCGTGCTGGAG (Sequence ID: 96; iTCRβ clone 41) AGCAGTGTCCGTAGCGGGAGAGGGGACAATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAG (Sequence ID: 97; iTCRβ clone 42) AGCAGTGTCCAGGAGGAAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAG (Sequence ID: 98; iTCRβ clone 43) AGCAGTGATAGTAGCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (Sequence ID: 99; iTCRβ clone 44) AGCAGTGGTACTACGGGACAGGAATCCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (Sequence ID: 100; iTCRβ clone 46) AGCAGTGTAAGGGGGAACCACAATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAG (Sequence ID: 101; iTCRβ clone 47) AGCAGTGAACTTCAGCGGGAGGGTTCTCCAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAG (Sequence ID: 102; iTCRβ clone 48) AGCAGTGTCCGGGACAGGGATGAAAAACTGTTTTTTGGCAGTGGAACCCAGCTCTCTGTCTTGGAG (Sequence ID: 103; iTCRβ clone 49) AGCAGTGAGGGTCAGGGAGGTTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (Sequence ID: 104; iTCRβ clone 50) AGCAGTGACAGGGATAGCAATCAGCCCCAGCATTTTGGTGATGGGACTCGACTCTCCATCCTAGAG (Sequence ID: 105; iTCRβ clone 51) AGCAGTGATCGGTCTAGCGGAGCCAAAAACATTCAGTACTTCGGCGCCGGGACCCGGCTCTCAGTGCTGGAG (Sequence ID: 106; iTCRβ clone 52) AGCAGTGCCACGACTAGCGGGAGGACAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAG (Sequence ID: 107; iTCRβ clone 53) AGCAGTGAATTTCGGCAGCGGGAGTCCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (Sequence ID: 108; iTCRβ clone 54) AGCAGTGAAATAGCGGGAGTGGCCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (Sequence ID: 109; iTCRβ clone 55) AGCGTGGGTCCGGTACCCTCCTACAATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAG (Sequence ID: 110; iTCRβ clone 56) AGCAGTGAACGGCGCGGGAGACGGGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAG (Sequence ID: 111; iTCRβ clone 57) AGCAGTGGGACAGGGTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (Sequence ID: 112; iTCRβ clone 58) AGCAGTGACCGTAGCGGGAGCTCCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (Sequence ID: 113; iTCRβ clone 59) AGCAGTGACAGCACCGGGGAGCTGTTTTTTGGAGAAGGCTCTAGGCTGACCGTACTGGAG (Sequence ID: 114; iTCRβ clone 60) AGCAGTGCTAGCGGGAGCAATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAG (Sequence ID: 115; iTCRβ clone 61) AGCAGTGACGGGACTAGCGGCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (Sequence ID: 116; iTCRβ clone 62) AGCAGTGAATATGAAAAACTGTTTTTTGGCAGTGGAACCCAGCTCTCTGTCTTGGAG (Sequence ID: 117; iTCRβ clone 63) AGCAGTGAGTCCGGCCCCCGCAATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAG (Sequence ID: 118; iTCRβ clone 64) AGCAGTGGCCGACTAGCGGGAGAGGAAGAGACCCAGTACTTCGGGCCAGGCACGCGGCTCCTGGTGCTCGAG (Sequence ID: 119; iTCRβ clone 66) AGCAGTGAGGGTGGCAGGGTCGATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAG (Sequence ID: 120; iTCRβ clone 67) AGCAGTGAGGCTAACTATGGCTACACCTTCGGTTCGGGGACCAGGTTAACCGTTGTAGAG (Sequence ID: 121; iTCRβ clone 68) AGCAGTCAGGACGGATTGGGATATGGCTACACCTTCGGTTCGGGGACCAGGTTAACCGTTGTAGAG (Sequence ID: 122; iTCRβ clone 69) AGCAGTGGGCGCCTCCACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (Sequence ID: 123; iTCRβ clone 70) AGCAGTGAATATAACAGCACAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAG (Sequence ID: 124; iTCRβ clone 71) AGCAGTGAACCCGGATTGGGGGAGCTGTTTTTTGGAGAAGGCTCTAGGCTGACCGTACTGGAG (Sequence ID: 125; iTCRβ clone 72) AGCATCCTGGGAGAGGGGCGGAATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAG (Sequence ID: 126; iTCRβ clone 73) AGCAGTGCCCCGGGACAGATCTATGGCTACACCTTCGGTTCGGGGACCAGGTTAACCGTTGTAGAG (Sequence ID: 127; iTCRβ clone 74) AGCAGTGACAACCAAGAGACCCAGTACTTCGGGCCAGGCACGCGGCTCCTGGTGCTCGAG (Sequence ID: 128; iTCRβ clone 75) AGCAGTGGGGTGACTAGCGCCTCCTACAATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAG (Sequence ID: 129; iTCRβ clone 76) AGCAGTCCTGAGCCCACCACCCTAGCGGGAGTCCACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (Sequence ID: 130; iTCRβ clone 77) AGCAGTGGGACACAGAGGGCTGAAAAACTGTTTTTTGGCAGTGGAACCCAGCTCTCTGTCTTGGAG (Sequence ID: 131; iTCRβ clone 78) AGCAGTGGGACTAGCGGGAGCACAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAG (Sequence ID: 132; iTCRβ clone 79) AGCAGTGAGGCGGGACAGGGTTCCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (Sequence ID: 133; iTCRβ clone 80) AGCACCTCTAGCCGCACCGGGGAGCTGTTTTTTGGAGAAGGCTCTAGGCTGACCGTACTGGAG (Sequence ID: 134; iTCRβ clone 83) AGCAGTGAACCGGGGGAGCGGAATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAG (Sequence ID: 135; iTCRβ clone 84) AGCAGTGAAGGTCGGGTTAACTATGGCTACACCTTCGGTTCGGGGACCAGGTTAACCGTTGTAGAG (Sequence ID: 136; iTCRβ clone 85) AGCAGTGAATCAGAAGGGGGCACAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAG (Sequence ID: 137; iTCRβ clone 86) AGCAGTCCCGGGGGGACTAGCGGGAGGGCACGTCCCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (Sequence ID: 138; iTCRβ clone 87) AGCAGTGGGAGGGAGGGGGACCCCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (Sequence ID: 139; iTCRβ clone 88) AGCAGTGGACTAGCGAACACCGGGGAGCTGTTTTTTGGAGAAGGCTCTAGGCTGACCGTACTGGAG (Sequence ID: 140; iTCRβ clone 89) AGCAGTGGGACGACAGGGGATACACGCACAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAG (Sequence ID: 141; iTCRβ clone 90) AGCAGTGAAGACCGGGACAGGGGTCACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (Sequence ID: 142; iTCRβ clone 91) AGCAGTGAACTAGCGAATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAG (Sequence ID: 143; iTCRβ clone 92) AGCAGTGGAGGAGGGGAGGAGACCCAGTACTTCGGGCCAGGCACGCGGCTCCTGGTGCTCGAG (Sequence ID: 144; iTCRβ clone 93) AGCAGTGAATATGCAGGGTGGGGCGGCAATCAGCCCCAGCATTTTGGTGATGGGACTCGACTCTCCATCCTAGAG (Sequence ID: 145; iTCRβ clone 94) AGCAGTGAATTGGACGGGACTAGCGCCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (Sequence ID: 146; iTCRβ clone 95) AGCAGTGCGCAGGGGGTCAGCGAAAAACTGTTTTTTGGCAGTGGAACCCAGCTCTCTGTCTTGGAG (Sequence ID: 147; iTCRβ clone 96) AGCAGTGAAGTGGCGGGAGCGGACACCGGGGAGCTGTTTTTTGGAGAAGGCTCTAGGCTGACCGTACTGGAG (Sequence ID: 148; iTCRβ clone 97) AGCAGCGGCAGGGGGCCAGGGGAAAGTGCAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAG (Sequence ID: 149; iTCRβ clone 98)

[0235] B. NK cell Fc binding modification As described herein, engineered NK cells can be modified to express transgenic constructs (heterogeneous constructs) encoding polypeptides containing an extracellular Fc-binding domain of an Fc receptor (e.g., an Fc-binding domain). The Fc-binding domain may be immobilized on the cell membrane, for example, via a transmembrane domain (TMD). The Fc-binding domain may be included as part of an Fc receptor or derived from an Fc receptor. The Fc receptor may be an Fcγ receptor, e.g., FcγRI(CD64); FcγRIIA, IIB, and IIC(CD32); FcγRIIIA and / or IIIB(CD16). In certain embodiments, the Fcγ receptor is FcγRIII. In certain embodiments, FcγRIII is FcγRIIIA, which may be the high-affinity variant CD16(CD16ha; e.g., F158V). In some embodiments, cells express polypeptides containing one or more transgenic Fc-binding domains.

[0236] As described herein, TCR / FcR constructs include a coding sequence for the extracellular binding domain of the Fc receptor. In exemplary embodiments described herein, the TCR / FcR construct may include a chimeric polypeptide containing a CD16-derived Fc-binding domain (e.g., FcγRIII). In some embodiments, the CD16-derived extracellular Fc-binding domain is a high-affinity polymorphism including an F158V substitution. In some embodiments, the polypeptide containing the CD16-derived Fc-binding domain is substantially devoid of an intracellular signaling domain (ICD). In some embodiments, the polypeptide containing the CD16 extracellular domain (e.g., the Fc-binding domain) includes any heterologous hinge, heterologous or CD16-derived TMDs, and / or heterologous and / or CD16-derived ICDs. In certain embodiments, the CD16-derived Fc-binding domain does not contain mutations that make the binding domain uncleavable and / or refractory.

[0237] In some embodiments, the polynucleotide encoding the transgenic Fc-binding domain comprises a signal peptide. In some embodiments, the signal peptide is a signal peptide that may be found in relation to an endogenous Fc receptor containing the Fc-binding domain of interest. In some embodiments, the signal peptide is a heterologous signal peptide that is not naturally associated with the Fc-binding protein.

[0238] In some embodiments, the TCR / FcR construct comprises an Fc receptor extracellular binding domain polypeptide encoded by a polynucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs. In certain embodiments, the TCR / FcR construct comprises an Fc receptor extracellular binding domain polypeptide that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs. In some embodiments, in the sequences described herein, underlines refer to signal peptide sequences, which in some embodiments may be modified, omitted, and / or substituted with alternative signal peptides.

[0239] As described herein, in some embodiments, the TCR / FcR construct comprises a chimeric Fc receptor extracellular binding domain polypeptide (e.g., comprising one or more sequences derived from a non-Fc receptor polypeptide and / or a different Fc receptor polypeptide). In some embodiments, the TCR / FcR construct comprises a human CD32-derived hinge region linking an Fc receptor extracellular domain (ECD) sequence to a transmembrane domain (TMD) sequence. In some embodiments, the TCR / FcR construct comprises a CD3ζ-derived TMD sequence and / or an intracellular domain (ICD) sequence. In some embodiments, the human CD32-derived hinge region is encoded by a polynucleotide sequence that is at least, or exactly, identical to SEQ ID NO: 160 by 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In certain embodiments, the hinge region derived from human CD32 includes a polypeptide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 161. In some embodiments, the TMD region derived from CD3ζ is encoded by a polynucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 162. In certain embodiments, the human CD3ζ-derived TMD region comprises a polypeptide sequence that is at least, or exactly, identical to sequence number 163 by 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.In some embodiments, the CD3ζ-derived ICD region is encoded by a polynucleotide sequence that is at least, or exactly, identical to SEQ ID NO: 164 by 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In certain embodiments, the human CD3ζ-derived ICD region includes a polypeptide sequence that is at least, or exactly, identical to SEQ ID NO: 165 by 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the TCR / FcR construct includes a CD16-derived TMD sequence and / or ICD sequence. In some embodiments, the human CD16-derived TMD region is encoded by a polynucleotide sequence that is at least, or exactly, identical to SEQ ID NO: 166 by 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In certain embodiments, the human CD16-derived TMD region includes a polypeptide sequence that is at least, or exactly, identical to sequence number 167 by 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the human CD16-derived ICD region is encoded by a polynucleotide sequence that is at least, or exactly, identical to sequence number 168 by 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In a particular embodiment, the human CD16-derived ICD region includes a polypeptide sequence that is at least, or exactly, identical to sequence number 169 by 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Polypeptide containing the extracellular domain (Fc-binding domain) of TCR / FcR#1 CD16: WT CD16, polynucleotide sequence (SEQ ID NO: 150) ATGTGGCAGCTGCTCCTCCCAACTGCTCTGCTACTTCTAGTTTCAGCT GGCATGCGGACTGAAGATCTCCCAAAGGCTGTGGTGTTCCTGGAGCCTCAATGGTACAGGGTGCTCGAGAAGGACAGTGTGACTCTGAAGTGCCAGGGAGCCTACTCCCCTGAGGACAATTCCACACAGTGGTTTCACAATGAGAGCCTCATCTCAAGCCAGGCCTCGAGCTACTTCAT TGACGCTGCCACAGTCGACGACAGTGGAGAGTACAGGTGCCAGACAAACCTCTCCACCCTCAGTGACCCGGTGCAGCTAGAAGTCCATATCGGCTGGCTGTTGCTCCAGGCCCCTCGGTGGGTGTTCAAGGAGGAAGACCCTATTCACCTGAGGTGTCACAGCTGGAAGAACACTGCTC TGCATAAGGTCACATATTTACAGAATGGCAAAGGCAGGAAGTATTTTCATCATAATTCTGACTTCTACATTCCAAAAGCCACACTCAAAGACAGCGGCTCCTACTTCTGCAGGGGGCTTTTTGGGAGTAAAAATGTGTCTTCAGAGACTGTGAACATCACCATCACTCAAGGTTTGGCA GTGTCAACCATCTCATCATTCTTTCCACCTGGTACCAAgtctctttctgcttggtgatggtactcctttttgcagtggacacaggactatatttctctgtgaagacaaacattcgaagctcaacaagagactggaaggaccataaatttaaatggagaaaggaccctcaagacaaaTAA (Sequence number: 150) Polypeptide containing the TCR / FcR#2 CD16 extracellular domain (Fc-binding domain): WT high affinity CD16, polynucleotide sequence (CD16ha, F158V) (SEQ ID NO: 151) ATGTGGCAGCTGCTCCTCCCAACTGCTCTGCTACTTCTAGTTTCAGCT GGCATGCGGACTGAAGATCTCCCAAAGGCTGTGGTGTTCCTGGAGCCTCAATGGTACAGGGTGCTCGAGAAGGACAGTGTGACTCTGAAGTGCCAGGGAGCCTACTCCCCTGAGGACAATTCCACACAGTGGTTTCACAATGAGAGCCTCATCTCAAGCCAGGCCTCGAGCTACTTCAT TGACGCTGCCACAGTCGACGACAGTGGAGAGTACAGGTGCCAGACAAACCTCTCCACCCTCAGTGACCCGGTGCAGCTAGAAGTCCATATCGGCTGGCTGTTGCTCCAGGCCCCTCGGTGGGTGTTCAAGGAGGAAGACCCTATTCACCTGAGGTGTCACAGCTGGAAGAACACTGCTC TGCATAAGGTCACATATTTACAGAATGGCAAAGGCAGGAAGTATTTTCATCATAATTCTGACTTCTACATTCCAAAAGCCACACTCAAAGACAGCGGCTCCTACTTCTGCAGGGGGCTTgTTGGGAGTAAAAATGTGTCTTCAGAGACTGTGAACATCACCATCACTCAAGGTTTGGCA GTGTCAACCATCTCATCATTCTTTCCACCTGGTACCAAgtctctttctgcttggtgatggtactcctttttgcagtggacacaggactatatttctctgtgaagacaaacattcgaagctcaacaagagactggaaggaccataaatttaaatggagaaaggaccctcaagacaaaTAA (Sequence number: 151) Polypeptide containing the TCR / FcR#3 CD16 extracellular domain (Fc-binding domain): CD16ha ECD, CD32 hinge, CD16 TMD, and CD16 ICD, polynucleotide sequence (SEQ ID NO: 152) ATGTGGCAGCTGCTCCTCCCAACTGCTCTGCTACTTCTAGTTTCAGCT GGCATGCGGACTGAAGATCTCCCAAAGGCTGTGGTGTTCCTGGAGCCTCAATGGTACAGGGTGCTCGAGAAGGACAGTGTGACTCTGAAGTGCCAGGGAGCCTACTCCCCTGAGGACAATTCCACACAGTGGTTTCACAATGAGAGCCTCATCTCAAGCCAGGCCTCGAGCTAC TTCATTGACGCTGCCACAGTCGACGACAGTGGAGAGTACAGGTGCCAGACAAACCTCTCCACCCTCAGTGACCCGGTGCAGCTAGAAGTCCATATCGGCTGGCTGTTGCTCCAGGCCCCTCGGTGGGTGTTCAAGGAGGAAGACCCTATTCACCTGAGGTGTCACAGCTGGAAGA ACACTGCTCTGCATAAGGTCACATATTTACAGAATGGCAAAGGCAGGAAGTATTTTCATCATAATTCTGACTTCTACATTCCAAAAGCCACACTCAAAGACAGCGGCTCCTACTTCTGCAGGGGGCTTgTTGGGAGTAAAAATGTGTCTTCAGAGACTGTGAACATCACCGTCCA AGTGCCCAGCATGGGCAGCTCTTCACCAATGGGGtctcttttctgcttggtgatggtactcctttttgcagtggacacaggactatatttctctgtgaagacaaacattcgaagctcaacaagagactggaaggaccataaatttaaatggagaaaggaccctcaagacaaaTAA (Sequence number: 152) Polypeptide containing the TCR / FcR#4 CD16 extracellular domain (Fc-binding domain): CD16ha ECD, CD32 hinge, CD3ζ TMD, and CD3ζ ICD, polynucleotide sequence (SEQ ID NO: 153) ATGTGGCAGCTGCTCCTCCCAACTGCTCTGCTACTTCTAGTTTCAGCT GGCATGCGGACTGAAGATCTCCCAAAGGCTGTGGTGTTCCTGGAGCCTCAATGGTACAGGGTGCTCGAGAAGGACAGTGTGACTCTGAAGTGCCAGGGAGCCTACTCCCCTGAGGACAATTCCACACAGTGGTTTCACAATGAGAGCCTCATCTCAAGCCAGGCCTCGAGCTACTTCATTGACGCTGCCACAGTGACGACAGTGGAGAGTACAGGTGCCAGACAAACCTCTCCACCCTC AGTGACCCGGTGCAGCTAGAAGTCCATATCGGCTGGCTGTTGCTCCAGGCCCCTCGGTGGGTGTTCAAGGAGGAAGACCCTTACCTGAGGTGTCACAGCTGGAAGAACACTGCTCTGCATAAGGTCACATATTTACAGAATGGCAAAGGCAGGAAGTATTTTCATCATAATTCTGACTTCTACATTCCAAAAGCCACACTCAAAAGACAGCGGCTCCCTACTTCTGCAGGGGGCTTgTT GGAGTAAAAAATGTGTCTTCAGAGACTGTGAACATCACCGTCCAAGTGCCCAGCATGGGCAGCTCTTCACCAATGGGGCTCTGTTACCTTTTCTGACGGTATTCTTTATTACGGCGTCATCCTCACTGCCCTCTTTTTGAGGGGTCAAGTTTTCAAGATCCCGCGACGCACCTGCTTACCAGCAAGGACAAAATCAACTTTATAACGAACTCAACCTTGGTCGGCGGGAAGAATATGATGTACTCGACAAAAGAAGAGGCCCGCGACCGGAGATGGTGCAAACCACAGCGGAGGAAAACCCCCCAGGAGGTCTCTATAACGAATTGCAGAAAGATAAAATGGCCGAGGCTATTCAGAGAATCGGCATGAAAGGAGAGGCGACGAGGAAAAGGGCATGATGGTCTGTATCAAGGGCTCAGCACGCAACAAAGGACACATATGATGGCCTTGCATAGCAGCGCGTTCCGCGCTAA (sequence number: 153) Polypeptide containing the TCR / FcR#5 CD16 extracellular domain (Fc-binding domain): CD16ha ECD, CD32 hinge, CD16 TMD, polynucleotide sequence (SEQ ID NO: 154) ATGTGGCAGCTGCTCCTCCCAACTGCTCTGCTACTTCTAGTTTCAGCT GGCATGCGGACTGAAGATCTCCCAAAGGCTGTGGTGTTCCTGGAGCCTCAATGGTACAGGGTGCTCGAGAAGGACAGTGTGACTCTGAAGTGCCAGGGAGCCTACTCCCCTGAGGACAATTCCACACAGTGGTTTCACAATGAGAGCCTCATCTCAA GCCAGGCCTCGAGCTACTTCATTGACGCTGCCACAGTCGACGACAGTGGAGAGTACAGGTGCCAGACAAACCTCTCCACCCTCAGTGACCCGGTGCAGCTAGAAGTCCATATCGGCTGGCTGTTGCTCCAGGCCCCTCGGTGGGTGTTCAAGGAGGAA GACCCTATTCACCTGAGGTGTCACAGCTGGAAGAACACTGCTCTGCATAAGGTCACATATTTACAGAATGGCAAAGGCAGGAAGTATTTTCATCATAATTCTGACTTCTACATTCCAAAAGCCACACTCAAAGACAGCGGCTCCTACTTCTGCAGGG GGCTTgTTGGGAGTAAAAATGTGTCTTCAGAGACTGTGAACATCACCGTCCAAGTGCCCAGCATGGGCAGCTCTTCACCAATGGGGgtctctttctgcttggtgatggtactcctttttgcagtggacacaggactatatttctctgtgaagacaTAA (Sequence number: 154) Polypeptide containing the extracellular domain (Fc-binding domain) of TCR / FcR#1 CD16: WT CD16, amino acid sequence (SEQ ID NO: 155) MWQLLLPTALLLLVSA GMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLFGSKNVSSETVNITITQGLAVSTISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK (Sequence ID: 155) Polypeptide containing the TCR / FcR#2 CD16 extracellular domain (Fc-binding domain): WT high affinity CD16, amino acid sequence (CD16ha, F158V) (SEQ ID NO: 156) MWQLLLPTALLLLVSA GMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK (Sequence ID: 156) Polypeptide containing the TCR / FcR#3 CD16 extracellular domain (Fc-binding domain): CD16ha ECD, CD32 hinge, CD16 TMD, and CD16 ICD, amino acid sequence (SEQ ID NO: 157) MWQLLLPTALLLLVSA GMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITVQVPSMGSSSPMGVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK (Sequence ID: 157) Polypeptide containing the TCR / FcR#4 CD16 extracellular domain (Fc-binding domain): CD16ha ECD, CD32 hinge, CD3ζ TMD, and CD3ζ ICD, amino acid sequence (SEQ ID NO: 158) MWQLLLPTALLLLVSA GMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITVQVPSMGSSSPMGLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (Sequence ID: 158) Polypeptide containing the TCR / FcR#5 CD16 extracellular domain (Fc-binding domain): CD16ha ECD, CD32 hinge, CD16 TMD, amino acid sequence (SEQ ID NO: 159) MWQLLLPTALLLLVSA GMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITVQVPSMGSSSPMGVSFCLVMVLLFAVDTGLYFSVKT (Sequence ID: 159) CD32 hinged polynucleotide (SEQ ID NO: 160) GTCCAAGTGCCCAGCATGGGCAGCTCTTCACCAATGGGG (Sequence ID: 160) CD32 hinge polypeptide (SEQ ID NO: 161) VQVPSMGSSSPMG (Sequence ID: 161) CD3ζ transmembrane domain polynucleotide (SEQ ID NO: 162) CTCTGTTACCTTCTTGACGGTATTCTTTTTATTTACGGCGTCATCCTCACTGCCCTCTTTTTG (Sequence ID: 162) CD3ζ transmembrane domain polypeptide (SEQ ID NO: 163) LCYLLDGILFIYGVILTALFL (Sequence ID: 163) CD3ζ intracellular signaling domain polynucleotide (SEQ ID NO: 164) AGGGTCAAGTTTTCAAGATCCGCCGACGCACCTGCTTACCAGCAAGGACAAAATCAACTTTATAACGAACTCAACCTTGGTCGGCGGGAAGAATATGATGTACTCGACAAAAGAAGAGGCCGCGACCCGGAGATGGGTGGCAAACCACAGCGGAGGAAAAACCCCCAGGAA GGTCTCTATAACGAATTGCAGAAAGATAAAATGGCCGAGGCTTATTCAGAGATCGGCATGAAAGGAGAGCGACGACGAGGAAAGGGGCATGATGGTCTGTATCAAGGGCTCAGCACGGCAACAAAGGACACATATGATGCCTTGCATATGCAGGCGCTTCCGCCGCGCTAA (Sequence number: 164) CD3ζ intracellular signal transduction domain polypeptide (SEQ ID NO: 165) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (Sequence ID: 165) CD16 transmembrane domain polynucleotide (SEQ ID NO: 166) GTCTCTTTCTGCTTGGTGATGGTACTCCTTTTTGCAGTGGACACAGGACTATATTTCTCTGTG (Sequence ID: 166) CD16 transmembrane domain polypeptide (SEQ ID NO: 167) VSFCLVMVLLFAVDTGLYFSV (Sequence ID: 167) CD16 intracellular signaling domain polynucleotide (SEQ ID NO: 168) AAGACAAACATTCGAAGCTCAACAAGAGACTGGAAGGACCATAAATTTAAATGGAGAAAGGACCCTCAAGACAAATAA (SEQ ID NO: 168) CD16 intracellular signaling domain polypeptide (SEQ ID NO: 169) KTNIRSSTRDWKDHKFKWRKDPQDK (Sequence ID: 169)

[0240] C. Exemplary transgenic open reading frame and vector NK cells are modified to express heterologous (i.e., transgenic) polynucleotide vectors (i.e., constructs). In some embodiments, the TCR / FcR construct comprises a multicistronic open reading frame polynucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs. In a particular embodiment, the TCR / FcR construct includes a transgenic vector sequence booked by an LTR that is at least or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence numbers 177-181. In some embodiments, in the sequences described herein, underlines indicate signal peptide sequences, and in some embodiments, the signal peptides may be modified, omitted, and / or replaced with alternative signal peptides.

[0241] In some embodiments, this specification provides heterogeneous polynucleotide vectors that include a pair of iTCR chains but do not include a CD16 extracellular domain (Fc-binding domain) containing a polypeptide (e.g., an "iTCR" construct, e.g., construct iTCR3). In some embodiments, the iTCR construct includes a multicistronic open reading frame polynucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 170. In a particular embodiment, the iTCR construct includes a transgenic vector sequence booked by an LTR that is at least or exactly identical to sequence number 176 by 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. iTCR3 Open Reading Frame (SEQ ID NO: 170) ATGAAGAAGCACCTGACCACCTTTCTGGTCATCCTGTGGCTGTACTTCTACAGAGGCAACGGC TCR / FcR# 1 Open Reading Frame (ORF) (SEQ ID NO: 171) ATGAAGAAGCACCTGACCACCTTTCTGGTCATCCTGTGGCTGTACTTCTACAGAGGCAACGGC ATGTGGCAGCTGCTCCTCCCAACTGCTCTGCTACTTCTAGTTTCAGCT GGCATGCGGACTGAAGATCTCCCAAAGGCTGTGGTGTTCCTGGAGCCTCAATGGTACAGGGTGCTCGAGAAGGACAGTGTGACTCTGAAGTGCCAGGGAGCCTACTCCCCTGAGGACAATTCCACACAGTGGTTTCACAATGAGAGCCTCATCTCAAGCCAGGCCTCGAGCTACTTCATTGACGCTGCCACAGTCGACGACAGTGGAGAGTACAGGTGCCAGACAAACCTCTCCACCCTCAGTGACCCGGTGCAGCTAGAAGTCCATATCGGCTGGCTGTTGCTCCAGGCCCCTCGGTGGGTGTTCAAGGAGGAAGACCCTATTCACCTGAGGTGTCACAGCTGGAAGAACACTGCTCTGCATAAGGTCACATATTTACAGAATGGCAAAGGCAGGAAGTATTTTCATCATAATTCTGACTTCTACATTCCAAAAGCCACACTCAAAGACAGCGGCTCCTACTTCTGCAGGGGGCTTTTTGGGAGTAAAAATGTGTCTTCAGAGACTGTGAACATCACCATCACTCAAGGTTTGGCAGTGTCAACCATCTCATCATTCTTTCCACCTGGGTACCAAgtctctttctgcttggtgatggtactcctttttgcagtggacacaggactatatttctctgtgaagacaaacattcgaagctcaacaagagactggaaggaccataaatttaaatggagaaaggaccctcaagacaaaTAA (SEQ ID NO: 171) TCR / FcR#2 Open Reading Frame (SEQ ID NO: 172) ATGAAGAAGCACCTGACCACCTTTCTGGTCATCCTGTGGCTGTACTTCTACAGAGGCAACGGC ATGTGGCAGCTGCTCCTCCCAACTGCTCTGCTACTTCTAGTTTCAGCT GGCATGCGGACTGAAGATCTCCCAAAGGCTGTGGTGTTCCTGGAGCCTCAATGGTACAGGGTGCTCGAGAAGGACAGTGTGACTCTGAAGTGCCAGGGAGCCTACTCCCCTGAGGACAATTCCACACAGTGGTTTCACAATGAGAGCCTCATCTCAAGCCAGGCCTCGAGCTACTTCATTGACGCTGCCACAGTCGACGACAGTGGAGAGTACAGGTGCCAGACAAACCTCTCCACCCTCAGTGACCCGGTGCAGCTAGAAGTCCATATCGGCTGGCTGTTGCTCCAGGCCCCTCGGTGGGTGTTCAAGGAGGAAGACCCTATTCACCTGAGGTGTCACAGCTGGAAGAACACTGCTCTGCATAAGGTCACATATTTACAGAATGGCAAAGGCAGGAAGTATTTTCATCATAATTCTGACTTCTACATTCCAAAAGCCACACTCAAAGACAGCGGCTCCTACTTCTGCAGGGGGCTTgTTGGGAGTAAAAATGTGTCTTCAGAGACTGTGAACATCACCATCACTCAAGGTTTGGCAGTGTCAACCATCTCATCATTCTTTCCACCTGGGTACCAAgtctctttctgcttggtgatggtactcctttttgcagtggacacaggactatatttctctgtgaagacaaacattcgaagctcaacaagagactggaaggaccataaatttaaatggagaaaggaccctcaagacaaaTAA (SEQ ID NO: 172) TCR / FcR#3 Open Reading Frame (SEQ ID NO: 173) ATGAAGAAGCACCTGACCACCTTTCTGGTCATCCTGTGGCTGTACTTCTACAGAGGCAACGGC ATGTGGCAGCTGCTCCTCCCAACTGCTCTGCTACTTCTAGTTTCAGCT GGCATGCGGACTGAAGATCTCCCAAAGGCTGTGGTGTTCCTGGAGCCTCAATGGTACAGGGTGCTCGAGAAGGACAGTGTGACTCTGAAGTGCCAGGGAGCCTACTCCCCTGAGGACAATTCCACACAGTGGTTTCACAATGAGAGCCTCATCTCAAGCCAGGCCTCGAGCTACTTCATTGACGCTGCCACAGTCGACGACAGTGGAGAGTACAGGTGCCAGACAAACCTCTCCACCCTCAGTGACCCGGTGCAGCTAGAAGTCCATATCGGCTGGCTGTTGCTCCAGGCCCCTCGGTGGGTGTTCAAGGAGGAAGACCCTATTCACCTGAGGTGTCACAGCTGGAAGAACACTGCTCTGCATAAGGTCACATATTTACAGAATGGCAAAGGCAGGAAGTATTTTCATCATAATTCTGACTTCTACATTCCAAAAGCCACACTCAAAGACAGCGGCTCCTACTTCTGCAGGGGGCTTgTTGGGAGTAAAAATGTGTCTTCAGAGACTGTGAACATCACCGTCCAAGTGCCCAGCATGGGCAGCTCTTCACCAATGGGGgtctctttctgcttggtgatggtactcctttttgcagtggacacaggactatatttctctgtgaagacaaacattcgaagctcaacaagagactggaaggaccataaatttaaatggagaaaggaccctcaagacaaaTAA (SEQ ID NO: 173) TCR / FcR#4 Open Reading Frame (SEQ ID NO: 174) ATGAAGAAGCACCTGACCACCTTTCTGGTCATCCTGTGGCTGTACTTCTACAGAGGCAACGGC ATGTGGCAGCTGCTCCTCCCAACTGCTCTGCTACTTCTAGTTTCAGCT GGCATGCGGACTGAAGATCTCCCAAAGGCTGTGGTGTTCCTGGAGCCTCAATGGTACAGGGTGCTCGAGAAGGACAGTGTGACTCTGAAGTGCCAGGGAGCCTACTCCCCTGAGGACAATTCCACACAGTGGTTTCACAATGAGAGCCTCATCTCAAGCCAGGCCTCGAGCTACTTCATTGACGCTGCCACAGTGACGACAGTGGAGAGTACAGGTGCCAGACAAACCTCTCCACCCTC AGTGACCCGGTGCAGCTAGAAGTCCATATCGGCTGGCTGTTGCTCCAGGCCCCTCGGTGGGTGTTCAAGGAGGAAGACCCTTACCTGAGGTGTCACAGCTGGAAGAACACTGCTCTGCATAAGGTCACATATTTACAGAATGGCAAAGGCAGGAAGTATTTTCATCATAATTCTGACTTCTACATTCCAAAAGCCACACTCAAAAGACAGCGGCTCCCTACTTCTGCAGGGGGCTTgTT GGAGTAAAAAATGTGTCTTCAGAGACTGTGAACATCACCGTCCAAGTGCCCAGCATGGGCAGCTTCTCACCAATGGGGCTCTGTTACCTTTTCTGACGGTATTCTTTATTTACGGCGTCATCCTCACTGCCCTCTTTTTGAGGGGTCAAGTTTTCAAGATCCCGCGACGCACCTGCTTACCAGCAAGGACAAAATCAACTTTATAACGAACTCAACCTTGGTCGGCGGGAAGAATATGATGTACTCGACAAAAGAAGAGGCCCGCGACCGGAGATGGTGCAAACCACAGCGGAGGAAAACCCCCCAGGAGGTCTCTATAACGAATTGCAGAAAGATAAAATGGCCGAGGCTATTCAGAGAATCGGCATGAAAGGAGAGGCGACGAGGAAAAGGGCATGATGGTCTGTATCAAGGGCTCAGCACGCAACAAAGGACACATATGATGGCCTTGCATATGCCATGCAGCGCGTTCCGCGCTAA (sequence number: 174) TCR / FcR#5 Open Reading Frame (SEQ ID NO: 175) ATGAAGAAGCACCTGACCACCTTTCTGGTCATCCTGTGGCTGTACTTCTACAGAGGCAACGGC ATGTGGCAGCTGCTCCTCCCAACTGCTCTGCTACTTCTAGTTTCAGCT GGCATGCGGACTGAAGATCTCCCAAAGGCTGTGGTGTTCCTGGAGCCTCAATGGTACAGGGTGCTCGAGAAGGACAGTGTGACTCTGAAGTGCCAGGGAGCCTACTCCCCTGAGGACAATTCCACACAGTGGTTTCACAATGAGAGCCTCATCTCAAGCCAGGCCTCGAGCTACTTCATTGACGCTGCCAGATCGACGACAGTGGAGAGTACAGGTGCCAGACAAACCTCTCCACCCTCAGTGACCCGGTGCAGCTAGAAGTCCATATCGGCTGGCTGTTGCTCCAGGCCCTCGGTGGGTGTTCAAGGGAGA GACCCTATTCACCTGAGGTGTCACAGCTGGAAGAACACTGCTCTGCATAAGGTCACATATTTACAGAATGGCAAAGGCAGGAAGTATTTTCATCATAATTCTGACTTCTACATTCCAAAAGCCACACTCAAAGACAGCGGCTCCTACTTCTGCAGGGGGCTTgTTGGGAGTAAAAATGTGTCTTCAGAGACTGTGAACATCACCGTCCAAGTGCCCAGCATGGGCAGCTTCACCAATGGGGGgtctctttctgcttggtgatggtactcctttttgcagtggacaggactatattctctgtgaagacaTAA (sequence number: 175) iTCR3 transmissor ATGAAGAAGCACCTGACCACCTTTCTGGTCATCCTGTGGCTGTACTTCTACAGAGGCAACGGC TCR / FcR#1 transgenic vector (SEQ ID NO: 177) ATGAAGAAGCACCTGACCACCTTTCTGGTCATCCTGTGGCTGTACTTCTACAGAGGCAACGGC ATGTGGCAGCTGCTCCTCCCAACTGCTCTGCTACTTCTAGTTTCAGCT TCR / FcR#2 transgenic vector (SEQ ID NO: 178) ATGAAGAAGCACCTGACCACCTTTCTGGTCATCCTGTGGCTGTACTTCTACAGAGGCAACGGC ATGTGGCAGCTGCTCCTCCCAACTGCTCTGCTACTTCTAGTTTCAGCT TCR / FcR#3 transgenic vector (SEQ ID NO: 179) ATGAAGAAGCACCTGACCACCTTTCTGGTCATCCTGTGGCTGTACTTCTACAGAGGCAACGGC ATGTGGCAGCTGCTCCTCCCAACTGCTCTGCTACTTCTAGTTTCAGCT TCR / FcR#4 transgenic vector (SEQ ID NO: 180) ATGAAGAAGCACCTGACCACCTTTCTGGTCATCCTGTGGCTGTACTTCTACAGAGGCAACGGC ATGTGGCAGCTGCTCCTCCCAACTGCTCTGCTACTTCTAGTTTCAGCT TCR / FcR#5 transgenic vector (SEQ ID NO: 181) ATGAAGAAGCACCTGACCACCTTTCTGGTCATCCTGTGGCTGTACTTCTACAGAGGCAACGGC ATGTGGCAGCTGCTCCTCCCAACTGCTCTGCTACTTCTAGTTTCAGCT

[0242] D.NK cells NK cells modified to express the TCR / CD3 receptor complex can be obtained from any suitable source, including fresh or frozen sources. In certain embodiments, the NK cells are not NK cells obtained from iPSC differentiation. In certain embodiments, the NK cells are not derived from an NK cell line (e.g., NK-92). In certain embodiments, the NK cells are obtained from human peripheral blood mononuclear cells (PBMCs), unstimulated leukocyte products (PBSCs), NK cell lines (e.g., NK-92), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow, or umbilical cord blood by methods known in the art. Specifically, NK cells can be isolated from umbilical cord blood (CB), peripheral blood (PB), bone marrow, stem cells, NK cell lines, or mixtures thereof. In certain embodiments, NK cells are isolated from pooled CBs. CBs can be pooled from 2, 3, 4, 5, 6, 7, 8, 9, 10, or more units. The NK cells can be autologous or allogeneic with respect to the recipient individual. Isolated NK cells may or may not be haplotype-matched to the target of cell therapy. NK cells can be detected by specific surface markers, such as CD16 and CD56 in humans, for example. In some cases, the source of NK cells is umbilical cord blood, where NK cells exist in a heterogeneous mixture of cells, and certain cells expressing CD3 may be depleted. Alternatively, umbilical cord blood can be used to induce NK cells by isolating CD34+ cells.

[0243] NK cells may or may not be pre-activated and augmented with one or more inflammatory cytokines. In some cases, NK cells are pre-activated either before or after modification (e.g., manipulation). In specific embodiments, pre-activating NK cells may involve culturing isolated NK cells in the presence of one or more cytokines. NK cells can be stimulated with IL-2 or other cytokines that bind to the common gamma chain (e.g., IL-7, IL-12, IL-15, IL-18, IL-21, and others). In certain embodiments, the cytokines to be pre-activated may be selected from the group consisting of IL-12, IL-15, IL-18, and combinations thereof. One or more additional cytokines may be used in the pre-activation step. Pre-activation may be for a short period of time, such as 5 to 72 hours, e.g., 10 to 50 hours, particularly 10 to 20 hours, e.g., 12, 13, 14, 15, 16, 17, 18, 19, or 20 hours, specifically about 16 hours. The pre-activated culture may contain IL-12 at a concentration of 0.1–150 ng / mL, for example 0.5–50 ng / mL, particularly 1–20 ng / mL, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 ng / mL, specifically about 10 ng / mL. The pre-activated culture may contain IL-18 and / or IL-15 at a concentration of 10–100 ng / mL, for example 40–60 ng / mL, particularly 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55 ng / mL, specifically about 50 ng / mL.

[0244] In some cases, NK cells are expanded before being modified to express constructs described herein (e.g., uTNK15 and / or TCR / FcR constructs). Pre-activated NK cells can be expanded in the presence of artificial antigen-presenting cells (aAPCs) and / or feeders / fragments or NK-activating beads. Pre-activated NK cells may be washed two, three, four, or five times, specifically three times, before expansion. aAPCs may be engineered to express CD137 ligand and / or membrane-bound cytokines. Membrane-bound cytokines may be membrane-bound IL-21 (mIL-21) or membrane-bound IL-15 (mIL-15). In certain embodiments, aAPCs are engineered to express CD137 ligand and mRNA. aAPCs may be derived from cancer cells, such as leukemia cells. aAPCs may not express endogenous HLA class I, II, or CD1d molecules. They may express ICAM-1 (CD54) and LFA-3 (CD58). In particular, aAPC may be K562 cells, such as K562 cells engineered to express CD137 ligand and mIL-21. aAPC may be irradiated. In some embodiments, fragments of APC may be used to expand NK cells. The manipulation may be carried out by any method known in the art, such as retroviral transduction. Retroviral transduction may be carried out at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after co-culturing NK with antigen-presenting cells. In some embodiments, retroviral transduction may include co-transduction of one or more constructs. In some embodiments, retroviral transduction may occur after co-culturing with antigen-presenting cells or about 5 days later. In some embodiments, co-culture with antigen-presenting cells is continued after transduction of NK cells. Expansion can last for approximately 2 to 30 days, for example 3 to 20 days, particularly 12 to 16 days, for example 12, 13, 14, 15, 16, 17, 18, or 19 days, specifically about 14 days. Pre-activated NK cells and aAPCs can be present in a ratio of approximately 3:1 to 1:3, for example 2:1, 1:1, 1:2, specifically about 1:2.The expansion culture may further contain cytokines that promote expansion, such as IL-2. IL-2 may be present at concentrations of approximately 10–500 U / mL, for example, 100–300 U / mL, and particularly around 200 U / mL. IL-2 can be replenished into the expansion culture, for example, every 2–3 days. aAPC can be added to the culture at least twice, for example, around day 7 of expansion.

[0245] In certain embodiments, NK cells are transfected or transduced with one or more membrane-bound cytokines, including IL-21, IL-12, IL-18, IL-23, IL-7, or IL-15, which are secreted by the NK cells or immobilized on the NK cell membrane. In such cases, the membrane-bound cytokines may be immobilized on the NK cell membrane by specific transmembrane domains, such as the transmembrane domains of CD8, CD28, CD27, B7H3, IgG1, IgG4, CD4, DAP10, and DAP12.

[0246] After preparation, modified NK cells can be immediately injected (e.g., with an effective amount of one or more bispecific or multispecific antibodies), or the NK cells can be stored by cryopreservation or other means. In some cases, when NK cells are supplied from cryopreservation, they were inactivated before cryopreservation using an inactivator (e.g., kinase inhibitors, e.g., dasatinib, nilotinib, rapamycin, etc.). In certain embodiments, the cells can be grown ex vivo as a bulk population for days, weeks, or months within about 1, 2, 3, 4, or 5 days.

[0247] ENK cell load In certain embodiments, NK cells are loaded with an antibody before use. NK cells can be loaded in any particular way, including immediately before culture (e.g., incubation) or injection, to form a complex of NK cells and the antibody. The culture (e.g., incubation) conditions are sufficiently suitable for an effective amount of antibody to bind to the surface of the NK cells. When using a monospecific antibody, the Fc region of the monospecific antibody binds to the NK cells, while the antigen-binding domain of the monospecific antibody binds freely to the target antigen. In certain embodiments of using a multispecific antibody, one or more antigen-binding domains of the antibody can bind to the surface of the NK cells, for example, via an antigen on the surface of the NK cells (e.g., NKp30, NKp44, NKp46, CD16, CD32, CD64, KIR, etc.), while other antigen-binding domains can bind freely to their target antigen. In certain embodiments of using a multispecific antibody, one or more antigen-binding domains of the antibody can bind to one or more target antigens. In certain embodiments where multispecific antibodies are used, the Fc region of the antibody binds to NK cells, and the antigen-binding domain of the antibody binds freely to the target antigen. In certain embodiments where multispecific antibodies are used, the Fc region of the antibody binds to NK cells via a transgenic construct (heterogeneous construct) encoding a polypeptide containing an extracellular Fc-binding domain of the Fc receptor (e.g., an Fc-binding domain).

[0248] The culture conditions under which NK cells are loaded may or may not be of a specific type having one or more specific parameters. In certain embodiments, NK cell loading occurs during culture at a specific temperature such as 37°C, while in alternative embodiments, the temperature may be 36°C or 38°C, or lower or higher. The duration of the loading process may be any suitable time, such as in the range of 1 minute to 24 hours or more. For example, it may be in the range of 1 minute to 24 hours, 1 minute to 18 hours, 1 minute to 12 hours, 1 minute to 6 hours, 1 minute to 1 hour, 30 minutes to 24 hours, 30 minutes to 18 hours, 30 minutes to 12 hours, 30 minutes to 6 hours, 30 minutes to 1 hour, 1 to 24 hours, 1 to 18 hours, 1 to 12 hours, 1 to 6 hours, 6 to 24 hours, 6 to 18 hours, 6 to 12 hours, 12 to 24 hours, 12 to 18 hours, or 18 to 24 hours. In some embodiments, the duration of the loading step can be approximately 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 hours or longer, or any range derivable therefrom. In certain embodiments, the cell culture medium is a basal medium or a compound medium. In some cases, the culture may or may not contain one or more reagents used during the pre-activation and / or growth step. In certain embodiments, the culture contains one or more cytokines, for example, one or more of IL-12, IL-15, IL-2, and IL-18. In some embodiments, the culture contains all kinds of APCs.

[0249] In some embodiments, NK cell loading is carried out so that the antibody remains bound to the NK cell surface after washing the NK cells. In some embodiments, NK cells are loaded with antibody under conditions suitable for retaining at least a detectable fraction of the antibody on the NK cell surface for at least or exactly 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, 120 hours, 132 hours, 144 hours, 156 hours, 168 hours, 180 hours, 192 hours, 204 hours, 216 hours, 228 hours, 240 hours, 252 hours, 264 hours, 276 hours, 288 hours, 300 hours, 312 hours, or more than 312 hours, or any range that can be derived therefrom. In some embodiments, NK cells are loaded with antibodies under conditions suitable for antibody retention on the NK cell surface for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days, or longer than 15 days, or any range that can be derived therefrom. In some embodiments, NK cells are loaded with antibodies under conditions suitable for antibody retention on the NK cell surface after cryopreservation and / or thawing. In some embodiments, confirmation of antibody loading on the NK cell surface can be determined using flow cytometry.

[0250] In some embodiments, NK cells are at least, exactly or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 The antibody is loaded at a final concentration of 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 μg / ml, or a final concentration greater than 100 μg / ml, or any range that can be derived therefrom. In some embodiments, NK cells are at least, exactly or about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 5 The antibody is loaded at a final concentration of 80, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780, 800, 820, 840, 860, 880, 900, 920, 940, 960, 980, or 1000 μg / ml, or a final concentration greater than 1000 μg / ml, or any range that can be derived from there.

[0251] In some embodiments, NK cells and antibodies are present in amounts of at least or exactly about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, NK cells are incubated to be conjugated with antibodies at a percentage of 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range that can be derived from there.

[0252] In some embodiments, NK cells and antibodies are present in amounts of at least or exactly about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, and 3%. 7%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79 Antibodies at %, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range that can be derived from there, are incubated with NK cells for at least 1, 2, 3, 4, 5, or 6 hours after incubation. They are incubated to remain coupled for 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, or any range that can be derived from there.

[0253] In certain embodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or more than 20%, or any range that can be derived therefrom, of the amount of antibody conjugated with the engineered NK cells as measured about 1 hour after incubation and washing, is bound to the NK cell surface at least 3 days after incubation and washing. In certain embodiments, at least about 9% of the antibody conjugated with the engineered NK cells as measured about 1 hour after incubation and washing is bound to the NK cell surface at least 3 days after incubation and washing.

[0254] In certain embodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or more than 20%, or any range that can be derived therefrom, of the amount of antibody conjugated with the engineered NK cells as measured about 1 hour after incubation and washing, is bound to the NK cell surface at least 5 days after incubation and washing. In certain embodiments, at least about 6% of the antibody conjugated with the engineered NK cells as measured about 1 hour after incubation and washing is bound to the NK cell surface at least 5 days after incubation and washing.

[0255] In certain embodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or more than 20%, or any range that can be derived therefrom, of the amount of antibody conjugated with the engineered NK cells as measured about 1 hour after incubation and washing, is bound to the NK cell surface at least 7 days after incubation and washing. In certain embodiments, at least about 4% of the antibody conjugated with the engineered NK cells as measured about 1 hour after incubation and washing is bound to the NK cell surface at least 7 days after incubation and washing.

[0256] In certain embodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or more than 20%, or any range that can be derived therefrom, of the amount of antibody conjugated with the engineered NK cells as measured about 1 hour after incubation and washing, is bound to the NK cell surface at least 12 days after incubation and washing. In certain embodiments, at least about 3% of the amount of antibody conjugated with the engineered NK cells as measured about 1 hour after incubation and washing, is bound to the NK cell surface at least 12 days after incubation and washing.

[0257] In certain embodiments, the composition containing engineered NK cells and antibodies is cryopreserved. In certain embodiments, the composition containing engineered NK cells and antibodies is thawed from cryopreservation, and at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of engineered NK cells are conjugated with antibodies.

[0258] In certain embodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90%, or any range that can be derived therefrom, of the amount of antibody conjugated with non-cryostored engineered NK cells as measured about 1 hour after incubation and washing, is bound to the NK cell surface after thawing from cryopreservation. In certain embodiments, at least about 30% of the amount of antibody conjugated with non-cryostored engineered NK cells as measured about 1 hour after incubation and washing, is bound to the NK cell surface after thawing from cryopreservation. In certain embodiments, at least about 40% of the antibodies bound to the NK cell surface after thawing from cryopreservation are bound to the NK cell surface, compared to the amount of antibodies bound to the uncryopreserved engineered NK cells, as measured about 1 hour after incubation and washing. In certain embodiments, at least about 50% of the antibodies bound to the NK cell surface after thawing from cryopreservation are bound to the NK cell surface, compared to the amount of antibodies bound to the uncryopreserved engineered NK cells, as measured about 1 hour after incubation and washing. In certain embodiments, at least about 60% of the antibodies bound to the NK cell surface after thawing from cryopreservation are bound to the NK cell surface, compared to the amount of antibodies bound to the uncryopreserved engineered NK cells, as measured about 1 hour after incubation and washing.

[0259] In some embodiments, incubation of NK cells and antibodies is carried out in any suitable NK cell medium known to those skilled in the art. In certain embodiments, incubation of NK cells and antibodies is carried out in a medium comprising, consisting of, or essentially derived from Click's / RPMI medium. In certain embodiments, incubation of NK cells and antibodies is carried out in a medium comprising, consisting of, or essentially derived from Click's medium. In certain embodiments, incubation of NK cells and antibodies is carried out in a medium comprising, consisting of, or essentially derived from RPMI medium. In certain embodiments, incubation of NK cells and antibodies is carried out in a medium comprising, consisting of, or essentially derived from SCGM medium. In certain embodiments, incubation of NK cells and antibodies is carried out in vivo, such as in the blood, lymph, and / or tumor of the subject.

[0260] In certain embodiments, NK cell loading may include in vivo loading of NK cells. In some embodiments, subjects may be administered one or more doses of antibody before administration of NK cells. In some embodiments, subjects may be administered one or more doses of antibody after administration of NK cells. In some embodiments, subjects may be administered one or more doses of antibody before administration of loaded NK cells, and subjects may be administered one or more doses of antibody after administration of loaded NK cells.

[0261] In some embodiments, the NK cell load is at least, exactly, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 5 This may include providing 2, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 mg of antibody once or more before administration of NK cells. In some embodiments, the NK cell load is at least, exactly, or about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, This may include providing 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780, 800, 820, 840, 860, 880, 900, 920, 940, 960, 980, or 1000 mg of antibody once or more before NK cell administration.

[0262] In some embodiments, the NK cell load is at least, exactly, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 5 This may include providing 2, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 mg of antibodies once or twice after administration of NK cells. In some embodiments, the NK cell load is at least, exactly, or about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, This may include providing 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780, 800, 820, 840, 860, 880, 900, 920, 940, 960, 980, or 1000 mg of antibodies once or more after NK cell administration.

[0263] In some embodiments, the NK cell load is at least, exactly, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, This may include providing 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 mg of antibodies simultaneously with the administration of NK cells. In some embodiments, loading NK cells may include providing the subject with at least, exactly, or about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780, 800, 820, 840, 860, 880, 900, 920, 940, 960, 980, or 1000 mg of antibody simultaneously with the administration of NK cells.

[0264] The antibody of this composition is supplied to an effective amount of the NK cells of this disclosure, thereby generating a complex that is "chimeric antigen receptor-like." In particular, the antigen-binding domain of the antibody binds to the NK cells via an antigen, such as a cell surface protein. Multiple antibodies may be supplied to multiple NK cells so that multiple cell / antibody complexes exist. The antibody may be of any type, such as monospecific, bispecific, or multispecific, and in certain cases, the antibody engages both the NK cells and the target antigen via the antigen-binding domain of the antibody (such as the engager of the art, which is a fusion protein consisting of two single-chain variable fragments (scFv) of different antibodies). In the case of a monospecific antibody, the antigen-binding domain of the antibody binds to a target antigen, such as a cancer antigen, and another part of the antibody, such as the Fc region of the antibody, binds to the NK cell. If the antibody is multispecific, one or more antigen-binding domains of the antibody may bind to the NK cell (e.g., via an NK cell surface antigen), and one or more antigen-binding domains of the antibody may bind to one or more target antigens. In certain embodiments where multispecific antibodies are used, one or more antigen-binding domains and / or Fc regions of the antibody can bind to NK cells. In certain embodiments, one or more Fc regions of the antibody can bind to NK cells via polypeptides encoded by a transgenic construct. The multispecific antibody may be, for example, dispecific, trispecific, or tetraspecific. If the antibody is trispecific or tetraspecific, additional antigen-binding domains may bind to other cells, such as stem cells.

[0265] In certain embodiments, the antibody can bind to type c lectins such as CD16 (including CD16a or CD16b), CD32, CD56, CD64, NKG2D, NKG2C, costimulatory molecules such as CS1, DNAM, 2B4, CD2, and any NK cell surface antigen (which may or may not be a receptor) on NK cells such as NCR, NKp30, NKp44, NKp46, or KIR, thereby inducing NK cells to target and enhance their responsiveness and specificity to different tumors.

[0266] In some embodiments, the antibody may bind to any suitable antigen (antigens described herein, such as those described as targets, such as TCRs). In certain embodiments, the antibody targets CD123. In certain embodiments, the antibody targets EGFR. In certain embodiments, the antibody targets EGFR2. In certain embodiments, the antibody is bispecific and targets both EGFR and c-MET. In certain embodiments, the antibody is imugatuzumab, amivantamab, and / or margetuximab.

[0267] Complex formation can be carried out by any suitable means such that the conditions are sufficient for the appropriate region of the antibody to bind to the appropriate surface region of the NK cell. In some cases, specific culture media may be used. In certain cases, Plasma-Lyte A and / or human serum albumin may be used, but not always. Once the complex is formed in culture, it may or may not be washed before administration to a target, such as by infusion. In some embodiments, the NK cells and antibody are administered separately and the complex is formed in vivo. In certain embodiments, the NK cells and antibody are administered separately and together and the complex is formed in vitro and in vivo. In certain embodiments, the composition containing the NK cells and antibody is washed with PBS to remove unbound antibody. In certain embodiments, the composition containing the NK cells and antibody is washed at least 1, 2, 3, 4, or 5 times, or more than 5 times, to remove unbound antibody. In certain embodiments, the composition containing NK cells and antibodies is washed for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes, or longer, to remove unbound antibodies. In certain embodiments, the composition containing NK cells and antibodies is washed twice to remove unbound antibodies. In certain embodiments, the washing is exactly or about 5 minutes. In certain embodiments, the washing includes, for example, stirring the composition using a cell shaker.

[0268] F. Pre-activation In some embodiments, NK cells are pre-activated before being administered to a recipient organism. The pre-activation step may or may not be performed before any proliferation step. In specific embodiments, NK cells are pre-activated with one or more cytokines, and in specific embodiments, NK cells are pre-activated with one or more of IL-12, IL-15, IL-2, and IL-18, including two, three, or more. If all three of IL-12, IL-15, IL-2, and IL-18 are not available, it may be IL-12 and IL-15 but not IL-18; or IL-12 and IL-18 but not IL-15; or IL-15 and IL-18 but not IL-12. IL-2 may or may not be used instead of IL-15.

[0269] In certain embodiments, the pre-activating cytokines may be IL-12, IL-15, and IL-18. One or more additional cytokines may be used in the pre-activation step. Pre-activation may be short, such as 5 to 72 hours, e.g., 10 to 50 hours, particularly 10 to 20 hours, e.g., 12, 13, 14, 15, 16, 17, 18, 19, or 20 hours, and may specifically be about 16 hours in some cases. The pre-activating culture may contain IL-18 and / or IL-15 at concentrations of 10 to 100 ng / mL, e.g., 40 to 60 ng / mL, particularly 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 ng / mL, specifically about 50 ng / mL. In some cases, the pre-activation culture may contain IL-12 at concentrations of 0.1 to 150 ng / mL, including concentrations of 1 to 20 ng / mL, e.g., 10 ng / mL. In alternative embodiments, NK cells may be stimulated with IL-2, or other cytokines that bind to a common gamma chain (e.g., IL-7, IL-21, and others), which may be in addition to, or as a substitute for, IL-12, IL-15, and IL-18. In such cases, the pre-activation culture may contain IL-12 at concentrations of 0.1 to 150 ng / mL, e.g., 0.5 to 50 ng / mL, particularly 1 to 20 ng / mL, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ng / mL, particularly about 10 ng / mL.

[0270] G. Proliferation In certain embodiments, NK cells are grown to increase their quantity before being administered to an individual in need. The grown cells may or may not originate from pre-activated NK cells, so that a pre-activation step may occur before the growth step. The NK cell growth step may be any suitable step in which an NK cell population is grown, but in certain embodiments, the growth step utilizes one or more specific reagents, such as in the culture, to enhance its growth. In certain embodiments, NK cells may not be grown. IL-2, IL-15, IL-18, or any combination of these cytokines may be added to the growth culture before or during growth. In specific embodiments, NK cells can be grown ex vivo in a flask or in one of several different bioreactor configurations with continuous perfusion of medium / additives.

[0271] In specific embodiments, NK cells (whether pre-activated or not) may be washed before and / or after proliferation (e.g., with PBS or Plasma Lyte or human serum albumin or culture medium or a combination thereof) once, twice, three times, four times, or five times, particularly three times. In some embodiments, the cells are washed three times in particular. In certain embodiments, NK cells are grown in the presence of artificial antigen-presenting cells (aAPCs). In certain embodiments, NK cells are grown in the presence of a fragment of aAPC. The aAPC can be engineered to express CD137 ligand and / or membrane-bound cytokines. The membrane-bound cytokine may be membrane-bound IL-21 (mIL-21) or membrane-bound IL-15 (mIL-15). In certain embodiments, the aAPC is engineered to express CD137 ligand and mRNA. The aAPC may be derived from cancer cells, such as leukemia cells. aAPCs may not express endogenous HLA class I, II, or CD1d molecules. They may express ICAM-1 (CD54) and LFA-3 (CD58) or CD48. In particular, aAPCs may be K562 cells, such as K562 cells manipulated to express CD137 ligand and mIL-21. Manipulation may be performed using methods known in the art, such as retroviral transduction, but any viral or non-viral vector can be used. aAPCs may or may not be irradiated. Growth may take place over a specific period, e.g., about 2 to 30 days, e.g., 3 to 20 days, particularly 12 to 16 days, e.g., 12, 13, 14, 15, 16, 17, 18 or 19 days, particularly about 14 days. Pre-activated NK cells and aAPCs may be present in a ratio of about 3:1 to 1:3, e.g., 2:1, 1:1, 1:2, particularly about 1:2. The growth culture may further contain one or more cytokines to promote growth, such as IL-2. IL-2 may be present at concentrations of approximately 10-500 U / mL, for example, 100-300 U / mL, and particularly around 200 U / mL. IL-2 can be replenished into the growth culture at regular intervals, for example, every 2-3 days.aAPC can be added to the culture at least twice, for example, around day 7. The cytokines used in the pre-activation and / or growth steps may be recombinant human cytokines.

[0272] In some embodiments, after proliferation, NK cells may be immediately used by any means, such as conjugating them with one or more antibodies, or they may be stored by methods such as cryopreservation. In some embodiments, the cells can be grown ex vivo as a bulk population for several days, weeks, or months within approximately 1, 2, 3, 4, or 5 days.

[0273] Activated and / or proliferated NK cells secrete type I cytokines such as interferon-γ, tumor necrosis factor-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF), activating both innate and adaptive immune cells, as well as other cytokines and chemokines. By measuring these cytokines, the activation state of NK cells can be determined. In addition, other methods known in the art to determine NK cell activation can be used for the characterization of NK cells in this disclosure.

[0274] Accordingly, with respect to specific pre-activation and proliferation modes of this disclosure, in certain embodiments, NK cells pre-activated with any combination of IL-12, IL-15, and / or IL-18, and then proliferated in aAPCs such as K562 cells expressing mIL-21 and CD137 ligand, provide a very potent cell product. Thus, methods of using the NK cells of the present invention for the treatment of various diseases, such as immunotherapy for cancer patients, are provided. In an exemplary method, isolated NK cells may be subjected to a short period, such as about 16 hours, for pre-activation with a combination of cytokines such as interleukin-12 (IL-12), IL-15, and / or IL-18, and then subjected to proliferation with artificial antigen-presenting cells (aAPCs) such as K562 feeder cells expressing membrane-bound IL-21 and CD137 ligand, and / or exogenous IL-2. IL-2, IL-15, IL-18, or combinations thereof, may be added to the proliferation culture at least twice.

[0275] H. Cryopreservation In certain cases, the NK cells and / or antibodies of this disclosure are stored in a cryopreservation medium composition comprising at least one cryoprotective agent, serum (human or animal serum) or a non-serum substitute (not human or animal serum), and at least one cytokine and / or at least one growth factor. In some cases, the cryoprotective agent is dimethyl sulfoxide (DMSO), glycerin, glycerol, hydroxyethyl starch, or a combination thereof. The non-serum substitute may be any kind, comprising at least platelet lysate and / or blood product lysate (e.g., human serum albumin). In embodiments of compositions utilizing one or more (including two or more) cytokines, the cytokines may be natural, recombinant, or synthetic proteins. At least one of the cytokines may be a Food and Drug Administration (FDA) approved cytokine. Examples of cytokines and growth factors include at least IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-22, interferon, tumor necrosis factor, stem cell factor, FLT3-ligand, APRIL, thrombopoietin, erythropoietin, or combinations thereof. In the serum embodiment, the serum may be human serum (including human AB serum) or animal-derived serum such as bovine serum. DMSO and other cryoprotective agents, if used, may constitute 4-10%, 4-6%, 4-8%, 5-10%, 5-8%, 6-10%, 6-8%, 8-10%, etc., of the composition.In embodiments where serum is used, the serum is available in the following concentrations: 5-99%, 5-95%, 5-90%, 5-85%, 5-80%, 5-75%, 5-70%, 5-65%, 5-60%, 5-55%, 5-50%, 5-45%, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-99%, 10-95%, 10-90%, 10-85%, 10-80%, 10-75%, 10-70%, 10-65%, and 10-60%. %, 10-55%, 10-50%, 10-45%, 10-40%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 20-99%, 20-95%, 20-90%, 20-85%, 20-80%, 20-75%, 20-70%, 20-65%, 20-60%, 20-55%, 20-50%, 20-45%, 20-40%, 20-35%, 20-30%, 20-25%, 30-99%, 30-95%, 30-90 %, 30-85%, 30-80%, 30-75%, 30-70%, 30-65%, 30-60%, 30-55%, 30-50%, 30-45%, 30-40%, 30-35%, 40-99%, 40-95%, 40-90%, 40-85%, 40-80%, 40-75%, 40-70%, 40-65%, 40-60%, 40-55%, 40-50%, 40-45%, 50-99%, 50-95%, 50-90%, 50-85%, 50-80 The composition may contain %, 50-75%, 50-70%, 50-65%, 50-60%, 50-55%, 60-99%, 60-95%, 60-90%, 60-85%, 60-80%, 60-75%, 60-70%, 60-65%, 70-99%, 70-95%, 70-90%, 70-85%, 70-80%, 70-75%, 80-99%, 80-95%, 80-90%, 80-85%, 90-99%, 90-95%, or 95-99%. The composition may contain at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or less of serum.In certain embodiments, the composition comprises platelet lysate, which may be at any concentration in the composition, but in certain embodiments, the platelet lysate is 5-99%, 5-95%, 5-90%, 5-85%, 5-80%, 5-75%, 5-70%, 5-65%, 5-60%, 5-55%, 5-50%, 5-45%, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-99%, 10-95%, 10-90%, 10-85% , 10-80%, 10-75%, 10-70%, 10-65%, 10-60%, 10-55%, 10-50%, 10-45%, 10-40%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 20-99%, 20-95%, 20-90%, 20-85%, 20-80%, 20-75%, 20-70%, 20-65%, 20-60%, 20-55%, 20-50%, 20-45%, 20-40%, 20-35%, 20-30%, 20- 25%, 30-99%, 30-95%, 30-90%, 30-85%, 30-80%, 30-75%, 30-70%, 30-65%, 30-60%, 30-55%, 30-50%, 30-45%, 30-40%, 30-35%, 40-99%, 40-95%, 40-90%, 40-85%, 40-80%, 40-75%, 40-70%, 40-65%, 40-60%, 40-55%, 40-50%, 40-45%, 50-99%, 50-95%, 50-90% The composition contains 50-85%, 50-80%, 50-75%, 50-70%, 50-65%, 50-60%, 50-55%, 60-99%, 60-95%, 60-90%, 60-85%, 60-80%, 60-75%, 60-70%, 60-65%, 70-99%, 70-95%, 70-90%, 70-85%, 70-80%, 70-75%, 80-99%, 80-95%, 80-90%, 80-85%, 90-99%, 90-95%, or 95-99%. The composition may contain at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or less of platelet lysates.

[0276] The composition may contain components at specific concentrations, including cytokines and / or growth factors. In specific cases, any cytokine, including, for example, IL-2, IL-21, and / or IL-15, may be present in the composition at specific concentrations. IL-2 may be present at concentrations of, for example, 1-5000, 1-1000, 1-500, 1-100, 100-5000, 100-5000, 500-5000, 500-1000, or 1000-5000 U / mL. In specific cases, IL-2 may be present in the composition at concentrations of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 U / mL, or less. In a particular embodiment, IL-21 is present in the composition at concentrations of 10-3000, 10-2000, 10-1000, 10-500, 10-100, 100-3000, 100-2000, 100-1000, 500-3000, 500-2000, 500-1000, 1000-3000, 1000-2000, or 2000-3000 ng / mL. IL-21 may be present in the composition at concentrations of at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, or 3000 ng / mL, or lower. IL-15 may be present in the composition at concentrations of 1-2000, 1-1000, 1-500, 1-100, 100-2000, 100-1000, 100-500, 500-2000, 500-1000, or 1000-2000 ng / mL. IL-15 may be present in the composition at concentrations of at least 10, 50, 100, 500, 1000, 1500, or 2000 ng / mL, or less.

[0277] A composition as encompassed herein, comprising at least one cryoprotective agent, serum or a non-serum substitute for serum, and at least one cytokine and / or at least one growth factor, may further comprise any number of immune cells and / or stem cells of any kind. In specific embodiments, the cells are NK cells, T cells, B cells, NKT cells derived from mature bone marrow or peripheral blood cells; cell lines such as tumor cell lines (e.g., NK92 or other NK lines) which may be derived from bone marrow, peripheral blood, skin, adipose tissue, or a combination thereof; hematopoietic stem cells, induced pluripotent stem cells, MSCs (cell populations also referred to in the literature as “mesenchymal stem cells” and “mesenchymal stromal cells”), or mixtures thereof. In embodiments utilizing NK cells, the NK cells may be proliferating NK cells or not. Embodiments of the Disclosure also encompass pharmaceutical compositions comprising any composition of the Disclosure and a suitable pharmaceutically acceptable carrier.

[0278] In certain embodiments, cells and / or antibodies are treated with one or more inactivators (e.g., kinase inhibitors, e.g., dasatinib, nilotinib, rapamycin, etc.) before cryopreservation.

[0279] In some embodiments, the techniques described herein include inactivating NK cells, which involves treating NK cells with an effective amount of one or more inactivators under conditions that produce inactivated NK cells. In some embodiments, the inactivator is a kinase inhibitor. In some embodiments, the inactivator is a mechanical target of a rapamycin (mTOR) inhibitor. In some embodiments, the mTOR inhibitor is rapamycin, everolimus, and / or temsirolimus. In some embodiments, the mTOR inhibitor is rapamycin. In some embodiments, the inactivator is a tyrosine kinase (TK) inhibitor. In some embodiments, TK inhibitors include lorlatinib, brigatinib, ceritinib, alectinib, crizotinib, bosutinib, ponatinib, nilotinib, dasatinib, imatinib, zanubrutinib, acalabrutinib, ibrutinib, capmatinib, pexidartinib, dacomitinib, osimertinib, erlotinib, gefitinib, lapatinib, afatinib, pemigatinib, erdafitinib, nintedanib, gilteritinib, midos These include taurine, tucatinib, neratinib, baricitinib, ruxolitinib, fedratinib, tofacitinib, ripretinib, selumetinib, binimetinib, cobimetinib, trametinib, upadacitinib, avapritinib, serpercatinib, cabozantinib, fostamatinib, lalotrectinib, entrectinib, axitinib, regorafenib, pazopanib, sorafenib, lenvatinib, vandetanib, and / or sunitinib. In some embodiments, the TK inhibitor is a BCR-Abl inhibitor. In some embodiments, the TK inhibitor is bosutinib, ponatinib, nilotinib, dasatinib, and / or imatinib. In some embodiments, the TK inhibitor is dasatinib and / or nilotinib.

[0280] In some embodiments, treatment with the inactivator is performed at any point during the culture of NK cells. In some embodiments, the treatment is performed for approximately 24 hours to approximately 96 hours, approximately 36 hours to approximately 84 hours, or approximately 48 hours to approximately 72 hours. In some embodiments, the treatment is performed for approximately 24 hours, approximately 48 hours, or approximately 72 hours. In some embodiments, NK cells are treated with an inactivator at a concentration of approximately 1 to approximately 1000 nM. In some embodiments, NK cells are treated with an inactivator at a concentration of approximately 5 to approximately 500 nM. In some embodiments, NK cells are treated with an inactivator at a concentration of approximately 20 to approximately 200 nM. In some embodiments, NK cells are treated with an inactivator at a concentration of approximately 30 to approximately 100 nM. In some embodiments, inactivated NK cells have increased expression of one or more C-kit, CCR-5, CD62L, and / or CXCR4, and / or decreased expression of one or more NKG2D, DNAM, OX-40, TRAIL, HLA-DR, CD2, CD25, ICOS, and / or CD95, compared to activated NK cells. In some embodiments, the techniques described herein include a method for maintaining the viability of a population of cells at least 50% after cryopreservation of the population, the method including the steps of: subjecting the population to an effective amount of one or more inactivators (e.g., tyrosine kinase inhibitors) to inactivate the cells before cryopreservation; cryopreserving the cells; and thawing the population, wherein the viability of the population at thawing is at least 50%. In some cases, at thawing the cells, the viability of the population of cells after cryopreservation of the population is at least 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%.

[0281] III. Heterogeneous Proteins and Mutations In certain embodiments, NK cells are modified to express not only one or more components of the TCR / CD3 complex and Fc-binding proteins, but also one or more other heterologous proteins. These heterologous proteins can promote the activity of NK cells in any way, including at least their activation, persistence, expansion, homing, and / or cytotoxicity.

[0282] A. Rhone-specific antibodies, bispecific antibodies, or multispecific antibodies Aspects of this disclosure relate to the use of antibodies or functional fragments thereof in compositions also comprising certain NK cells. The term “antibody” means any isotype of intact immunoglobulin, or a fragment thereof that can compete with an intact antibody for specific binding to a target antigen, such as via an antigen-binding domain, and includes chimeric antibodies, humanized antibodies, fully human antibodies, monospecific antibodies, and multispecific antibodies (including at least bispecific and trispecific antibodies). As used herein, the terms “antibody” and “immunoglobulin” are used interchangeably and refer to any of several classes of structurally related proteins that function as part of the immune response of animals, including IgG, IgD, IgE, IgA, IgM, and related proteins, as well as polypeptides containing an antibody CDR domain that retains antigen-binding activity. In certain embodiments, the antibody includes scFv. In certain embodiments, the antibody may be any antibody or antibody-like structure known in the art, including antibody fragments, single-domain antibodies, scFv, bispecific antibodies, bispecific diabodies, triplicate antibodies, scFv-Fc, and other antibody constructs and engagers.

[0283] The term "antigen" refers to a molecule or part of a molecule that can be bound by a selective binder, such as an antibody. An antigen may have one or more epitopes that can interact with different antibodies.

[0284] The term "epitope" refers to any region or portion of a molecule that can trigger an immune response by binding to an immunoglobulin or T cell receptor. Epitope determinants may include chemically active surface groups such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and may have specific three-dimensional structural properties and / or specific charge properties. Generally, antibodies specific to a particular target antigen preferentially recognize epitopes on the target antigen within the complex mixture.

[0285] The epitope region of a given polypeptide can be identified using many different epitope mapping techniques known in the art, including X-ray crystallography, nuclear magnetic resonance spectroscopy, site-directed mutagenesis mapping, and protein display arrays, for example, see Epitope Mapping Protocols (Johan Rockberg and Johan Nilvebrant, Ed., 2018) Humana Press, New York, NY. Such techniques are publicly known in the art and are described, for example, in U.S. Patent No. 4,708,871; Geysen et al. Proc. Natl. Acad. Sci. USA 81:3998-4002 (1984); Geysen et al. Proc. Natl. Acad. Sci. USA 82:178-182 (1985); Geysen et al. Molec. Immunol. 23:709-715 (1986). For example, see the epitope mapping protocol (see above). Furthermore, the antigenic region of a protein can also be predicted and identified using standard antigenicity plots and hydroxyl plots.

[0286] Intact antibodies generally consist of two full-length heavy chains and two full-length light chains, but in some cases they may contain fewer chains, such as naturally occurring antibodies in camelids, which may contain only heavy chains. The antibodies disclosed herein may originate from a single source or they may be “chimeras,” meaning that different parts of the antibody originate from two different antibodies. For example, the variable region or CDR region may originate from a rat or mouse source, while the constant region originates from a different animal source, such as a human. Antibodies or conjugated fragments can be produced by hybridoma, recombinant DNA technology, or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise specified, the term “antibody” includes its derivatives, variants, fragments, and mutaines, examples of which are given below (Sela-Culang et al. Front Immunol. 2013; 4: 302; 2013).

[0287] The term "light chain" includes the full-length light chain and its fragments that have a variable region sequence sufficient to confer binding specificity. The full-length light chain has a molecular weight of approximately 25,000 daltons and contains a variable region domain (abbreviated herein as VL) and a constant region domain (abbreviated herein as CL). There are two classifications of light chains, called κ (kappa) and λ (lambda). The term "VL fragment" refers to a fragment of a monoclonal antibody light chain that contains all or part of the light chain variable region, including the CDR. The VL fragment may further contain the light chain constant region sequence. The variable region domain of the light chain is located at the amino terminus of the polypeptide.

[0288] The term "heavy chain" includes the full-length heavy chain and its fragments that have a variable region sequence sufficient to confer binding specificity. The full-length heavy chain has a molecular weight of approximately 50,000 daltons and contains a variable region domain (abbreviated herein as VH) and three constant region domains (abbreviated herein as CH1, CH2, and CH3). The term "VH fragment" means a fragment of the heavy chain of a monoclonal antibody that contains all or part of the heavy chain variable region, including the CDR. The VH fragment may further contain the heavy chain constant region sequence. The number of heavy chain constant region domains depends on the isotype. The VH domain is located at the amino terminus of the polypeptide, the CH domain is at the carboxy terminus, and CH3 is closest to the -COOH terminus. The isotype of an antibody can be IgM, IgD, IgG, IgA, or IgE, defined by the presence of a heavy chain, each of which has five classifications: mu (μ) chain, delta (δ) chain, gamma (γ) chain, alpha (α) chain, or epsilon (ε) chain. IgG has several subtypes, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM subtypes include IgM1 and IgM2. IgA subtypes include IgA1 and IgA2.

[0289] Antibodies can be whole immunoglobulins of any isotype or classification, chimeric antibodies, or hybrid antibodies with specificity for two or more antigens. They can also be fragments containing hybrid fragments (e.g., F(ab')2, Fab', Fab, Fv, etc.). Furthermore, immunoglobulins include natural, synthetic, or genetically modified proteins that act like antibodies by binding to specific antigens and forming complexes. The term antibody includes genetically modified or otherwise altered forms of immunoglobulins, such as:

[0290] The term "monomer" refers to an antibody containing only one Ig unit. Monomers are the basic functional units of antibodies. The term "dimer" refers to an antibody containing two Ig units linked to each other via the constant domain (Fc, or fragment crystallizable region) of the antibody heavy chain. This complex can be stabilized by a connecting (J) chain protein. The term "multimer" refers to an antibody containing more than two Ig units linked to each other via the constant domain (Fc region) of the antibody heavy chain. This complex can be stabilized by a connecting (J) chain protein.

[0291] The term "bivalent antibody" refers to an antibody that contains two antigen-binding sites. These two sites may either have the same antigen specificity, or they may have bispecificity, meaning the two antigen-binding sites have different antigen specificities.

[0292] Bispecific antibodies are a class of antibodies having two paratopes, each having different binding sites to two or more different epitopes. In some embodiments, bispecific antibodies may be biparatopic, and bispecific antibodies may specifically recognize different epitopes from the same antigen. In some embodiments, bispecific antibodies may be constructed from a pair of different single-domain antibodies called "nanobodies." Single-domain antibodies are sourced and modified from cartilaginous fish and camels. Nanobodies can be linked together by linkers using techniques typical to those skilled in the art. Such methods for the selection and linking of nanobodies are described in PCT applications published WO2015 / 044386A1, WO2010 / 037838A2, and Bever et al., Anal Chem. 86:7875-7882 (2014), each of which is specifically incorporated herein by reference in whole.

[0293] Bispecific antibodies can be constructed as whole IgG, Fab'2, Fab'PEG, diabody, or scFv. Diabody and scFv can be constructed using only variable domains without an Fc region, potentially reducing the influence of anti-idiotype reactions. Bispecific antibodies can be generated by various methods, including, but not limited to, hybridoma fusion or Fab' fragment linking. See, for example, Songsivilai and Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148:1547-1553 (1992), where each of these is specifically incorporated by reference.

[0294] In certain embodiments, the antigen-binding domain may be multispecific or heterospecific by multimerizing with VH and VL region pairs that bind to different antigens. For example, an antibody may bind to or interact with (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, or (c) at least one other component. Thus, embodiments may include, but are not limited to, bispecific, triplicate, quadruplicate, and other multispecific antibodies or their antigen-binding fragments that are directed to other targets such as epitopes and Fc receptors on effector cells.

[0295] In some embodiments, multispecific antibodies can be used via short, flexible polypeptide chains and directly linked using routine methods known in the art. An example of this is a diabody, a bivalent bispecific antibody in which VH and VL domains are expressed on a single polypeptide chain. A linker too short to allow pairing between domains on the same chain is used to pair the domains with complementary domains on another chain, forming two antigen-binding sites. The functionality of the linker is applicable to triabodies, tetrabodies, and higher-order antibody multimer embodiments (see, e.g., Hollinger et al., Proc Natl. Acad. Sci. USA 90:6444-6448 (1993); Polijak et al., Structure 2:1121-1123 (1994); Todorovska et al., J. Immunol. Methods 248:47-66 (2001)).

[0296] In contrast to bispecific whole antibodies, bispecific diabodies may also be advantageous because they can be readily constructed and expressed in E. coli. Diabodies (and other polypeptides, such as antibody fragments) with appropriate binding specificity can be readily selected from a library using phage display (International Publication No. WO94 / 13804). By keeping one arm of the diabody constant, for example, to have specificity directed towards a protein, libraries can be constructed with the other arms altered, allowing for the selection of antibodies with appropriate specificity. Bispecific whole antibodies can be prepared by alternative methods described in Ridgeway et al., (Protein Eng., 9:616-621, 1996) and Krah et al., (N Biotechnol. 39:167-173, 2017), each of which is incorporated herein by reference in whole.

[0297] A heteroconjugate antibody consists of two covalently linked monoclonal antibodies with different specificities. See, for example, U.S. Patent No. 6,010,902, which is incorporated herein by reference in its entirety.

[0298] In this specification, the portion of the antibody molecule's Fv fragment that binds with high specificity to the antigen's epitope is referred to as the "paratope." The paratope consists of amino acid residues that contact the antigen's epitope to facilitate antigen recognition. Each of the two Fv fragments of an antibody is composed of two variable domains, VH and VL, in a dimerized structure. The primary structure of each variable domain contains three hypervariable loops separated and flanked by a framework region (FR). The hypervariable loops are the region with the highest primary sequence variability in any mammalian antibody molecule. The term hypervariable loop is sometimes used interchangeably with the term "complementarity-determining region (CDR)." The length of the hypervariable loops (or CDRs) varies among antibody molecules. The framework regions of all given mammalian antibody molecules have high primary sequence similarity / consensus. The consensus of the framework regions can be used by those skilled in the art to identify both the framework regions and the hypervariable loops (or CDRs) scattered between them. Hypervariable loops are given identifying names that distinguish their position within the polypeptide and the domain in which they reside. The CDRs in the VL domain are identified as L1, L2, and L3, with L1 located at the most distal end and L3 closest to the CL domain. CDRs are also sometimes named CDR-1, CDR-2, and CDR-3. L3 (CDR-3) is generally the most variable region in all antibody molecules produced by a given organism. CDRs are linearly aligned regions in the primary structure of the polypeptide chain, separated from each other by framework regions. The amino terminus (N-terminus) of the VL chain is named FR1. The region identified as FR2 lies between the L1 and L2 hypervariable loops. FR3 lies between the L2 and L3 hypervariable loops, and the FR4 region is closest to the CL domain. This structure and nomenclature are repeated for the VH chain, which contains three CDRs identified as H1, H2, and H3. The majority of amino acid residues in the variable domain or Fv fragments (VH and VL) are part of the framework region (approximately 85%).The three-dimensional or tertiary structure of an antibody molecule is such that the framework region is more abundant inside the molecule and occupies the majority of the structure, while the CDR is located on the outer surface of the molecule.

[0299] Several methods have been developed to identify the precise amino acids that make up each of these regions, and these can be used by those skilled in the art. This can be done using any of the many multiple sequence alignment methods and algorithms that identify the conserved amino acid residues that make up the framework regions, and thus identify the CDRs located between the framework regions, although their lengths may vary. Three commonly used methods have been developed to identify antibody CDRs: Kabat (TT Wu and E.A. Kabat, "AN ANALYSIS OF THE SEQUENCES OF THE VARIABLE REGIONS OF BENCE JONES PROTEINS AND MYELOMA LIGHT CHAINS AND THEIR IMPLICATIONS FOR ANTIBODY COMPLEMENTARITY," J Exp Med, vol. 132, no. 2, pp. 211-250, Aug. 1970); Chothia (C. Chothia et al., "Conformations of immunoglobulin hypervariable regions," Nature, vol. 342, no. 6252, pp. 877-883, Dec. 1989); and IMGT (M.-P. Lefranc et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Developmental & Comparative). (Described in Immunology, vol. 27, no. 1, pp. 55-77, Jan. 2003). Each of these methods includes its own numbering system for identifying the amino acid residues that make up the variable region. In most antibody molecules, the amino acid residues that actually come into contact with the antigen epitope are located in the CDR, but in some cases residues within the framework region contribute to antigen binding.

[0300] Those skilled in the art can use one of several methods to determine the paratopes of antibodies. These methods include: 1) computational prediction of the tertiary structure of antibody / epitope binding interactions based on the amino acid sequence of the antibody variable region and the chemical properties of the epitope composition; 2) hydrogen-deuterium exchange and mass spectrometry; 3) polypeptide fragmentation and peptide mapping approaches that generate multiple overlapping peptide fragments from the full length of a polypeptide and evaluate the binding affinity of these peptides to the epitope; and 4) antibody phage display library analysis in which antibody Fab fragments encoding mammalian genes are expressed by bacteriophages so that they are incorporated into the coat of phages. A population of these Fab-expressing phages can then be made to interact with an immobilized antigen or expressed in another exogenous expression system. Unbound Fab fragments are washed away, leaving only the specifically bound Fab fragments that have bound to the antigen. The bound Fab fragments can be easily isolated, and the gene encoding them can be determined. This approach can also be used as appropriate for Fv fragments or smaller regions of Fab fragments containing specific VH and VL domains.

[0301] In certain embodiments, affinity-mature antibodies are enhanced by one or more modifications in one or more CDRs of their own, resulting in improved affinity for a target antigen compared to parent antibodies without those modifications. Certain affinity-mature antibodies have nanomolar or picomolar affinity for a target antigen. Affinity-mature antibodies are produced by methods known in the art. For example, Marks et al., Bio / Technology 10:779 (1992) describes affinity maturation by shuffling of VH and VL domains, and random mutagenesis of CDRs and / or framework residues employed in phage display is described by Rajpal et al., PNAS. 24: 8466-8471 (2005) and Thie et al., Methods Mol Biol. 525:309-22 (2009), in combination with the computational method demonstrated by Tiller et al., Front. Immunol. 8:986 (2017).

[0302] Chimeric immunoglobulins are the products of fusion genes derived from different species. "Humanized" chimeras typically have a framework region (FR) derived from human immunoglobulin, while one or more CDRs are derived from non-human sources.

[0303] In certain embodiments, a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence from another specific species or belonging to a specific antibody class or subclass, and the remainder of the chain is identical or homologous to a corresponding sequence in an antibody from another species or belonging to another antibody class or subclass, as well as a fragment of such an antibody, insofar as it exhibits the desired biological activity. See, for example, U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851 (1984). For methods relating to chimeric antibodies, see, for example, U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1985). Each of these is incorporated herein by reference in its entirety. CDR grafting is described, for example, in U.S. Patents 6,180,370, 5,693,762, 5,693,761, 5,585,089, and 5,530,101, which are incorporated herein by reference for all purposes.

[0304] In some embodiments, the function of chimeric antibodies is optimized and immunogenicity is reduced by minimizing the non-human species-derived antibody polypeptide sequence. Specific amino acid residues in the non-antigen-recognition region of a non-human antibody are modified to be homologous to corresponding residues in a human antibody or isotype. An example is a "CDR-grafted" antibody, where the antibody contains one or more CDRs that are either from a specific species or belong to a specific antibody class or subclass, but the rest of the antibody chain(s) is either from a different species or is identical or homologous to corresponding sequences in antibodies belonging to a different antibody class or subclass. For human use, the V region, consisting of CDR1, CDR2, and partial CDR3 from both the light and heavy chain dispersion regions of a non-human immunoglobulin, is grafted onto the human antibody framework region, replacing the naturally occurring antigen receptors in the human antibody with non-human CDRs. In some cases, the corresponding non-human residues are replaced with framework region residues from the human immunoglobulin. Furthermore, humanized antibodies may contain residues not found in the recipient or donor antibody to further enhance performance. Humanized antibodies may also contain the constant region (Fc) of immunoglobulins, typically at least a portion of the constant region of human immunoglobulins. See, for example, Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Presta, Curr. Op. Struct. Biol. 2:593 (1992); Vaswani and Hamilton, Ann. Allergy, Asthma and Immunol. 1:105 (1998); Harris, Biochem. Soc. Transactions 23; 1035 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428 (1994); Verhoeyen et al., Science 239:1534-36 (1988).

[0305] Intrabodies are immunoglobulins that are localized within cells and bind to intracellular antigens, in contrast to secretory antibodies that bind to antigens in the extracellular lumen.

[0306] Polyclonal antibody preparations typically contain different antibodies against different determinants (epitopes). To produce polyclonal antibodies, a host such as a rabbit or goat is immunized with an antigen or antigen fragment, typically using an adjuvant and, if necessary, a carrier. The antibodies against the antigen are then recovered from the host's serum. Polyclonal antibodies can be made monospecific by affinity purification against the antigen.

[0307] A monoclonal antibody, or "mAb," refers to an antibody obtained from a homogeneous population of antibodies derived from an exclusive parent cell. For example, the population is identical except for trace amounts of naturally occurring mutations. Each monoclonal antibody is directed to a single antigenic determinant.

[0308] Functional antibody fragments and antigen-binding fragments can be utilized. Certain embodiments relate to antibody fragments such as antibody fragments that bind to and / or neutralize inflammatory mediators. The term functional antibody fragment includes antigen-binding fragments of antibodies that retain the ability to specifically bind to an antigen. These fragments consist of various sequences of variable region heavy chain (VH) and / or light chain (VL); in some embodiments, they include constant region heavy chain 1 (CH1) and light chain (CL). In some embodiments, they delete the Fc region, which consists of heavy chain 2 (CH2) and 3 (CH3) domains. Embodiments of antigen-binding fragments and their modifications may include: (i) Fab fragments consisting of VL, VH, CL, and CH1 domains; (ii) Fd fragments consisting of VH and CH1 domains; (iii) Fv fragments consisting of VH and VL domains; (iv) single-domain fragments consisting of a single VH or VL domain, dAb (Ward, 1989; McCafferty et al., 1990; Holt et al., 2003); and (v) isolated complementarity-determining regions (CDRs). Such terminology is found, for example, in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, NY (1989); Molec. Biology and Biotechnology: A Comprehensive Desk Reference (Myers, RA (ed.), New York: VCH Publisher, Inc.); Huston et al., Cell Biophysics, 22:189-224 (1993); Pluckthun and Skerra, Meth. Enzymol., 178:497-515 (1989) and in Day, ED, Advanced Immunochemistry, 2d ed., Wiley-Liss, Inc. New York, NY (1990); Antibodies, 4:259-277 (2015). All citations in this paragraph are incorporated by reference.

[0309] Antigen-binding fragments also include antibody fragments that precisely retain at least one, two, or three complementarity-determining regions (CDRs) from the light chain variable region. Fusion of a CDR-containing sequence to an Fc region (or its CH2 or CH3 region) is included within this definition, and for example, scFv fused directly or indirectly to an Fc region is included herein.

[0310] The term Fab fragment refers to a monovalent antigen-binding fragment of an antibody containing the VL, VH, CL, and CH1 domains. The term Fab' fragment refers to a monovalent antigen-binding fragment of a monoclonal antibody that is larger than a Fab fragment. For example, a Fab' fragment contains all or part of the VL, VH, CL, and CH1 domains as well as the hinge region. The term F(ab')2 fragment refers to a bivalent antigen-binding fragment of a monoclonal antibody containing two Fab' fragments linked by a disulfide crosslink at the hinge region. An F(ab')2 fragment may, for example, contain all or part of two VH and VL domains and further contain all or part of two CL and CH1 domains.

[0311] The term Fd fragment refers to a fragment of the heavy chain of a monoclonal antibody containing all or part of the VH, including the CDR. The Fd fragment may further contain the CH1 region sequence.

[0312] The term Fv fragment refers to a monovalent antigen-binding fragment of a monoclonal antibody that includes all or part of the VL and VH regions but does not include the CL and CH1 domains. The VL and VH regions include, for example, the CDR. A single-chain antibody (sFv or scFv) is an Fv molecule in which the VL and VH regions are linked by a flexible linker to form a single polypeptide chain that constitutes the antigen-binding fragment. Single-chain antibodies are discussed in detail in International Patent Application Publication WO88 / 01649 and U.S. Patents 4,946,778 and 5,260,203, the disclosures of which are incorporated herein by reference. The term (scFv)2 refers to a divalent or bispecific sFv polypeptide chain containing an oligomeric domain separated from sFv by a hinge region at its C-terminus (Pack et al. 1992). The oligomeric domain may include a self-associating α-helix, e.g., a leucine zipper, and may be further stabilized by additional disulfide bonds. The (scFv)2 fragment is also known as a "mini-antibody" or "mini-body".

[0313] A single-domain antibody is an antigen-binding fragment containing only a VH or VL domain. In some cases, two or more VH regions are covalently linked by a peptide linker to create a bivalent domain antibody. The two VH regions of a bivalent domain antibody can target the same or different antigens.

[0314] In some cases, the Fc region, which is a fragment crystallizable region, is utilized. The Fc region contains two heavy chain fragments, each containing the CH2 and CH3 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and hydrophobic interactions of the CH3 domain. As used herein, the term “Fc polypeptide” includes the native and mutaine forms of polypeptides derived from the Fc region of the antibody. This includes cleavage forms of such polypeptides that contain hinge regions that promote dimerization. In some embodiments, antibodies incorporating cytokines (including those mentioned herein) can be utilized (e.g., TRIKE).

[0315] In some embodiments, the antibody is an antibody-drug conjugate. In some embodiments, the antibody is specifically one or more of the following, but is not limited to: [fam]-trastuzumab deruxtecan, absiximab, adalimumab, ad-trastuzumab emtansine, aducanumab, alemtuzumab, alirocumab, amivantamab, aniflorumab, anisuvimab, atezolizumab, atorutibimab and maftibimab and odesibimab-ebgn (also known as immazeb), avelumab, basiliximab, verantamab mahodotin, belimumab, benralizumab, bevacizumab, bezlotoxumab, bimekizumab, blinatumomab, brentuximab Vedotin, Brodalumab, Brolucizumab, Brosumab, Canakinumab, Caplacizumab, Casiribimab + Imdevimab, Catumaxomab, Semiprimab, Certolizumab Pegol, Cetuximab, Cebostamab, Chryzanlizumab, Daclizumab, Daratumumab, Denosumab, Dinutuximab, Donanemab, Dostarimab, Dupilumab, Durvalumab, Eculizumab, Edrecolomab, Efalizumab, Elotuzumab, Emapalmab, Emicizumab, Enfortzumab Vedotin, eptinezumab, erenumab, evinacumab, evolocumab, falisimab, fremanezumab, galcanezumab, gemtuzumab, gemtuzumab-ozogamicin, golimumab, guselkumab, ibalizumab, ibritumomab, tiucetan, idarucizumab, imugatuzumab, inebilizumab, infliximab, inorimomab, inotuzumab, inotuzumab-ozogamicin, IPH61, ipilimumab, isatuximab, ixekizumab, lanadelmab, lecanemab, ronkastuximab, tesirin, margetuximab, mepolizumab, milbetuximab, sorabtansine, mogamulizumab, mosnetuzumab, moxetumomab Pasdotox, Muromonab-CD3, Narsoprimab, Natalizumab, Naxitamab, Nevacumab, Necitumumab, Nilsevimab, Nivolumab, Oviltoxaximab, Obinutuzumab, Ocrelizumab, Ofatumumab, Oraratumab, Omalizumab, Omblutamab, Oportuzumab Monatox, Palivizumab, Panitumumab, Pembrolizumab, Penprimab, Pertuzumab, PolatuzumabVedotin, Ramucirumab, Ranibizumab, Rabrizumab, Laxibakumab, Regdanvimab, Relatrimab, Reslizumab, Letifanlimab, Risankizumab, Rituximab, Romosozumab, Sacituzumab Govitecan, Sarilumab, Satralizumab, Secukinumab, Siltuximab, Syntilimab, Sotrovimab, Spesolimab, Stimulimab, Tafacitamab, Teventafusp, Tecristamab, Teprizumab, Teprotumumab, Tezeperumab, Childraquizumab, Tislerizumab, Tisotumab Vedotin, tixagevimab, silgabimab, tocilizumab, tripalimab, tocitumomab-I131, tralokinumab, trastuzumab, tremelimumab, ubrituximab, ustekinumab, vedolizumab, or any combination thereof. In some embodiments, the antibody is erranatamab. In some embodiments, the antibody is imugatuzumab. In some embodiments, the antibody is margetuximab. In some embodiments, the antibody is amibantamab. In some embodiments, the antibody is blinatumomab. In some embodiments, the antibody is obinutuzumab. In some embodiments, the antibody is IPH61 (also known as IPH6101 or SAR443579). In some embodiments, the antibody is tecristamag. In some embodiments, the antibody is cetuximab. In some embodiments, the antibody is rituximab.

[0316] In some embodiments, NK cells are modified to express one or more monospecific, bispecific, and / or multispecific antibodies, while in other embodiments, NK cells do not express antibodies, but antibodies are utilized in conjunction with the NK cells (i.e., NK cells are loaded with antibodies and / or utilized in combination therapy regimens with antibodies).

[0317] In some embodiments, the antibody may be an engager that crosslinks specific immune effector cells with specific target cells in order to destroy those target cells. This disclosure enables the use of modified NK cells with a standard T cell engager (BiTE), which is often modified to express CD3, a T cell antigen to which the BiTE engager binds. In such cases, the BiTE used in the present invention may also target cancer antigens or viral antigens that can be adapted to the disease state of the intended recipient individual. For example, the BiTE may be adapted to bind to cancer antigens characteristic of cancer cells in an individual, and / or to bind to antigens characteristic of cells associated with another disorder (e.g., autoimmune disorder). The anti-CD3 antibody of the BiTE may target the CD3γ, CD3δ, CD3ε, or CD3ζ chains. In some embodiments, the BiTE may target other T cell-associated proteins, but is not limited to CD28.

[0318] In some embodiments, the antibody is a BiTE, which may be one or more of the following, but is not limited to: AMG 160 / akapatamab, AMG 199 / TNB 585, AMG 330, AMG 427 / emirodatamab, AMG 509, AMG 701, AMG 910, APVO414 / ES414 / MOR209, APVO436, Blinatumomab / Blincyto, Katsumakisomab / Removab, CC-1, CC-93269 / EM801, Sibisatamab / RG7802 / RO6958688, CLN-049, Elranatamab / PF-06863135, EMB-06, Epcolitamab / GEN3013, ERY974, Flotetuzumab / MGD006, Grofitamab / RG6026 / RO7082859, ISB 1342 / GBR 1342, JNJ-63709178, JNJ-63898081, JNJ-67571244, JNJ-75348780, Limbocertamab / REGN 5458, M701, M802, MGD007, Mosnetuzumab / RG7828, Nivatrotamab / Hu3F8-BsAb, Odronextamab / REGN1979, REGN4018, REGN5459, REGN7075, REGN5678, Talcetamab / JNJ-64407564, Tarlatamab / AMG 757, Teclistamag / JNJ-64007957, Tepozitamab / MCLA-117, TNB-383B, TNB-486, TNB-585, XmAb13676 / Pramotamab, XmAb14045 / Vibecotamab, and / or XmAb18087 / Chizutamab.

[0319] In some embodiments, BiTE may target CD30, PSMA, MUC17, CD33, FLT3, STEAP1, BCMA, CLDN18.2, CD123, CD19, CD20, EpCAM, CEA, GPC3, CD38, CD33, CD22, HER2, GPA33, GD2, MUC16, GPRC5D, DLL-3, CLEC12A, FcRH5, and / or SSTR.

[0320] In some cases, in addition to expressing a CD3 / TCR complex that makes NK cells available as a BiTE-based therapy, NK cells may be modified to be conjugated with and / or express one or more bispecific NK engagers (BiKEs). A BiKE comprises an antibody that binds to a surface protein on NK cells, including, but not limited to, NKp30, NKp44, NKp46, CD16, CD32, CD64, KIR, and further comprises an antibody that binds to a desired target antigen. A BiKE can target NK cells via an antibody-binding domain directed to NK surface proteins such as, for example, CD16, CS1, CD32, CD64, CD56, NKG2D, NKG2C, DNAM, 2B4, CD2, NCR, NKp30, NKp44, NKp46, or KIR. In such cases, the BiKE used in the present invention may also target cancer antigens or viral antigens that can be adapted to the disease state of the intended recipient individual. For example, BiKE can be adapted to bind to cancer antigens characteristic of cancer cells in an individual, and / or to bind to antigens characteristic of cells associated with another disorder (e.g., autoimmune disorder). In some embodiments, BiKE is AFM13, which targets CD16 and CD30.

[0321] In certain embodiments, the antibody is blinatumomab. In certain embodiments, the antibody is teventafusp. In certain embodiments, the antibody is mosnetuzumab. In certain embodiments, the antibody is tecristamag. In certain embodiments, the antibody is grofitamab. In certain embodiments, the antibody is epcolitamab. In some embodiments, the antibody is flotetuzumab. In some embodiments, the antibody is APV0436. In some embodiments, the antibody is TNB383B. In certain embodiments where a multispecific antibody may be used, one or more antigen-binding domains of the antibody can bind to one or more target antigens.

[0322] In embodiments in which NK cells express the CD3 / TCR complex (including iTCR) and one or more other heterologous proteins (e.g., antibodies), one or more vectors can be used to transfect or transduce the cells with the CD3 / TCR complex components and one or more other heterologous proteins. In some cases, one or more CD3 / TCR complex components and one or more heterologous proteins may or may not be on the same multi-cistronic vector.

[0323] In certain embodiments, the antibody includes an Fc domain derived from IgG1. In certain embodiments, the antibody includes an Fc domain derived from IgG4. In certain embodiments, the antibody includes a glycosylated (e.g., afucosylated) Fc domain. In certain embodiments, the antibody includes an unglycosylated Fc domain. In certain embodiments, the antibody includes an Fc domain with or without Fc domain modification, such as that described in Xinhua Wang et al., "IgG Fc engineering to modulate antibody effector functions," Protein Cell, 2018 Jan; 9(1): 63-73, which is incorporated herein by reference for the purposes described herein.

[0324] B. Manipulated receptors In specific embodiments, NK cells are engineered to express one or more engineered receptors. In some cases, the engineered receptors are engineered antigen receptors that target any type of cancer antigen or viral antigen. The receptors can be adapted to target a desired antigen based on the disease state of the intended recipient individual.

[0325] In some embodiments, engineered NK cells may be modified to express one or more chemokines, chemokine receptors, cytokines, cytokine receptors, and / or suicide genes. In certain embodiments, NK cells are engineered to express CD16, CD32, CD64, or other Fc-region binding proteins. In certain embodiments, NK cells are engineered to express the CD16 gene and / or its characteristic Fc-binding region. In certain embodiments, NK cells are engineered to express the wild-type CD16 gene and / or its characteristic Fc-binding region. In certain embodiments, NK cells are engineered to express the high-affinity wild-type human CD16 gene and / or its characteristic Fc-binding region. In certain embodiments, NK cells are engineered to express the CD32 gene and / or its characteristic Fc-binding region. In certain embodiments, NK cells are engineered to express the CD64 gene and / or its characteristic Fc-binding region. In certain embodiments, NK cells are engineered to express a transgenic receptor that is a target of a multispecific antibody.

[0326] C. cytokines In some embodiments, cells expressing NK cells are engineered to express one or more heterologous cytokines and / or to upregulate the normal expression of one or more heterologous cytokines. The cells may or may not be transfected with one or more cytokines on the same vector as other genes. In certain embodiments, NK cells may be modified to express one or more cytokines, cytokine receptors, chemokines, chemokine receptors, and / or suicide genes.

[0327] One or more cytokines may be co-expressed from a vector containing a polypeptide other than the engineered antigen receptor and / or suicide gene. For example, interleukin-15 (IL-15) is tissue-limited and is observed only in serum or systemically at any level under pathological conditions. IL-15 possesses several desirable properties for adoptive therapy. IL-15 is a homeostatic cytokine that promotes the eradication of established tumors by inducing the development and proliferation of natural killer cells and mitigating the suppression of tumor resident cell function, and inhibiting activation-induced cell death (AICD). In addition to IL-15, other cytokines are also considered. These include, but are not limited to, cytokines (e.g., IL-2, IL-12, IL-18, and / or IL-21), chemokines, and other molecules that contribute to the activation and proliferation of cells used for human application. NK cells expressing IL-15 enable sustained supportive cytokine signaling, which is useful for survival after injection. In certain embodiments, NK cells expressing IL-21 are capable of continuing supportive cytokine signaling, which is useful for survival after injection. In certain embodiments, cytokines are expressed as part of a multicistronic construct with one or more functional proteins and / or marker proteins.

[0328] In some embodiments, cells express one or more exogenously provided engineered receptors, where the engineered receptors include chemokine receptors and / or cytokine receptors. In some embodiments, the cytokine receptor is the IL-15 receptor. In some embodiments, the cytokine receptor is a non-naturally occurring variant of the cytokine receptor. In some embodiments, the cytokine receptor is IL-15, IL-12, IL-2, IL-18, IL-21, IL-23, or GMCSF receptors, or a combination thereof.

[0329] In specific embodiments, cells express one or more exogenously supplied cytokines. For example, cytokines may be IL-15, IL-12, IL-2, IL-18, IL-21, IL-23, GMCSF, or a combination thereof. Cytokines can be exogenously supplied to NK cells because they are expressed from intracellular expression vectors. In other cases, intracellular endogenous cytokines are upregulated by manipulation of endogenous cytokine expression control, such as genetic recombination at the cytokine promoter site. When cytokines are supplied to cells on an expression construct, the cytokines may be encoded from the same vector as one or more components of a polypeptide containing the CD3 / TCR complex and / or the Fc receptor extracellular domain.

[0330] In some embodiments, specific sequences of IL-15 are utilized, such as the following (underlined signals may be included, modified, or omitted): ATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCC GGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGC AGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGC (Sequence number: 182) MRISKPHLRSISIQCYLCLLLNSHFLTEA GIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence ID: 183)

[0331] In some embodiments, specific sequences of IL-21 are utilized, such as the following (underlined signals may or may not be included): An example codon-optimized polynucleotide sequence containing SEQ ID NO: 184-IL-21 (signal peptide is underlined). ATGAGGAGCAGTCCAGGCAATATGGAGCGGATAGTAATTTGTCTCATGGTAATATTCCTCGGTACTCTGGTA CATAAATCTTCCTCTCAAGGTCAGGACCGCCATATGATTCGAATGCGGCAGCTGATTGACATAGTCGATCAACTGAAGAACTATGTGAATGATCTTGTGCCCGAGTTTTTGCCAGCCCCTGAAGACGTAGAAACTAATTGTGAGTGGAGTGCCTTTTCCTGCTTTCAAAAGGCACAGCTGAAATCCGCCAACACGGGCAATAACGAA CGGATAATTAACGTATCCATTAAGAAGCTGAAGCGGAAGCCGCCCTCAACCAATGCGGGACGGCGGCAAAAGCATCGCTTGACCTGTCCGTCATGCGACAGCTACGAGAAAAAAGCCCCCGAAGGAGTTCTTGGAACGCTTCAAGAGTCTCCTTCAGAAAATGATTCACCAGCACCTGTCCTCACGGACGCACGGAAGCGAGGACAGT (Sequence number: 184) An example codon-optimized amino acid sequence including SEQ ID NO: 185-IL-21 (signal peptide is underlined). MRSSPGNMERIVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (Sequence ID: 185) An exemplary polynucleotide sequence including SEQ ID NO: 186-IL-21. GGCCAGGACCGGCACATGATCCGGATGAGACAGCTGATCGACATCGTGGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTCCTGCCTGCCCCCGAGGACGTGGAAACAAACTGCGAGTGGAGCGCCTTCAGCTGCTTCCAGAAGGCCCAGCTGAAAAGCGCCAACACCGGCAACAACGAGCGGATCATC AACGTGTCCATCAAGAAGCTGAAGCGGAAGCCCCCCAGCACCAACGCCGGAAGAAGGCAGAAGCACAGACTGACCTGCCCCAGCTGCGACAGCTACGAGAAGAAGCCCCCTAAAGAGTTCCTGGAACGGTTCAAGAGCCTGCTGCAGAAGATGATCCACCAGCACCTGAGCAGCCGGACCCACGGCTCTGAGGACAGC (Sequence number: 186) An example amino acid sequence including SEQ ID NO: 187-IL-21. GQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (Sequence ID: 187)

[0332] D. Antigen The modified NK cells described herein are used in conjunction with bispecific or multispecific antibodies that target one or more specific antigens. In addition, NK cells may be modified with engineered antigen receptors that target one or more specific antigens. When NK cells are modified with one or more engineered antigen receptors, the antigen targeted by the bispecific or multispecific antibody and the antigen targeted by the one or more engineered antigen receptors may or may not be the same antigen. In some cases, the antigen targeted by the bispecific or multispecific antibody and the antigen targeted by the one or more engineered antigen receptors may be different antigens but are associated with the same type of cancer.

[0333] Some antigens targeted by antibodies and / or genetically engineered antigen receptors are expressed in the context of diseases, conditions, or cell types targeted via adoptive cell therapy. Diseases and conditions include proliferative, neoplastic, and malignant diseases and disorders, and include hematological cancers, lymphomas, leukemias, and / or cancers of the immune system, such as myelomas, including B, T, and myeloid leukemias, lymphomas, and multiple myeloma. In some embodiments, antigens are selectively expressed or overexpressed on disease or condition cells, e.g., tumor or pathogenic cells, compared to normal or non-targeted cells or tissues. In other embodiments, antigens are expressed on normal cells and / or engineered cells.

[0334] This method allows for the targeting of any suitable antigen. Antigens may, in some cases, be associated with specific cancer cells but not with non-cancer cells. In some embodiments, antigens may be associated with cells characteristic of autoimmune disorders but not with healthy / non-disordered cells. Exemplary antigens include, but are not limited to, infectious agent-derived antigen molecules, auto / autoantigens, tumor / cancer-associated antigens, and neoplastic tumor antigens (Linnemann et al., 2015). In certain embodiments, antigens may include NY-ESO, CD19, EBNA, CD123, HER2, CA-125, TRAIL / DR4, CD20, CD22, CD70, CD38, CD123, CLL1, carcinoembryonic antigens, alpha-fetoprotein, CD56, AKT, Her3, epithelial tumor antigens, CD319 (CS1), ROR1, folate-binding proteins, HIV-1 envelope glycoprotein gp120, and HIV. -1 Envelope glycoproteins gp41, CD5, CD23, CD30, HERV-K, IL-11Rα, κ chain, λ chain, CSPG4, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, FcRH5, CD99, p53, mutant p53, Ras, mutant ras, c-Myc, cytoplasmic serine / threonine kinases (e.g., A-Raf, B-Raf, and C-Raf), cyclin-dependent kinases Tyrosinase, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MART-1, Melanoma-related antigen, BAGE, DAM-6, -10, GAGE-1, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, MC1R, mda-7, gp75, Gp100, PSA, PSM, Tyrosinase, Tyrosinase-related protein, TRP-1, T RP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, phosphoinositide 3-kinase (PI3K), TRK receptor, PRAME, P15, RU1, RU2, SART-1, SART-3, Wilms tumor antigen (WT1), AFP, -catenin / m, caspase-8 / m, CDK-4 / m, ELF2M, GnT-V, G250, HAGE, HSP70-2M, HST-2,KIAA0205, MUM-1, MUM-2, MUM-3, Myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP, Annexin II, CDC27 / m, TPI / mbcr-abl, BCR-ABL, Interferon Regulator 4 (IRF4), ETV6 / AML, LDLR / FUT, Pml / RAR, Tumor-Associated Calcium Signal Transducer 1 (TACSTD1) TACSTD2, Receptor Tyrosine Kinase (e.g., Epidermal Growth Factor Receptor (EGFR)) In particular, EGFRvIII), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), VEGFR2, cytoplasmic tyrosine kinases (e.g., src family, syk-ZAP70 family), integrin-binding kinase (ILK), transcriptional signaling and activating factors STAT3, STATS, and STATE, hypoxia-inducible factors (e.g., HIF-1 and HIF-2), nuclear factor-κB (NF-B), Notch receptors (e.g., Notch1-4), NY ESO 1, c-Met, mammalian targeted rapamycin (mTOR), WNT, extracellular signal-regulated kinase (ERK), and their regulatory subunits, PMSA, PR-3, MDM2, mesoserine, renal cell carcinoma-5T4, SM22α, carbonic anhydrase I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL / AML1, GD2, proteinase 3, hTERT, sarcoma translocation breakpoint, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2) This includes ETS fusion genes, NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, GD3, fucosyl GM1, mesocerian, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SAGE, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos-related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNC1, and LRRN1. Examples of antigen sequences are:For example, in the GenBank® database: CD19 (accession number NG_007275.1), EBNA (accession number NG_002392.2), WT1 (accession number NG_009272.1), CD123 (accession number NC_000023.11:1336785-1382689), NY-ESO (accession number: NC_000023.11), EGFRvIII (accession number: NG_007726.3), MUC1 (accession number: NG_029383.1), HER2 (accession number: NG_007503.1), CA-125 (accession number NG_055257.1), WT1 (accession number NG_009272.1), Mage-A3 (accession number NG_013244.1), Mage-A4 (accession number NG_013245.1), Mage-A10 (accession number NC_000023.11), TRAIL / DR4 (accession number NC_000003.12), and / or CEA (accession number NC_000019.10) are known in the art.

[0335] Tumor-associated antigens may originate, for example, from prostate cancer, breast cancer, colorectal cancer, lung cancer, pancreatic cancer, kidney cancer, mesothelioma, ovarian cancer, liver cancer, brain tumor, bone cancer, gastric cancer, spleen cancer, testicular cancer, cervical cancer, anal cancer, gallbladder cancer, thyroid cancer, or melanoma cancer. Exemplary tumor-associated antigens or tumor cell-derived antigens include MAGE1, 3, and MAGE4 (or other MAGE antigens such as those disclosed in International Patent Publication WO99 / 40188); PRAME; BAGE; RAGE, Lage (also known as NY ESO 1); SAGE; and HAGE or GAGE. These non-exclusive examples of tumor antigens are expressed in a wide range of tumor types, such as melanoma, lung cancer, sarcoma, and bladder cancer. See, for example, U.S. Patent No. 6,544,518. Examples of prostate cancer tumor-associated antigens include prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), prostatic acid phosphate, NKX3.1, and prostate 6-stage membrane epithelial antigen (STEAP).

[0336] Other tumor-associated antigens include Plu-1, HASH-1, HasH-2, Cripto, and Criptin. Furthermore, tumor antigens can also be autopeptide hormones; for example, gonadotrophin hormone-releasing hormone (GnRH), a short peptide with a total length of 10 amino acids, is useful in the treatment of many cancers.

[0337] Antigens may include genes mutated in tumor cells, such as telomerase enzymes, survivorin, mesoserine, mutant ras, bcr / abl rearrangements, Her2 / neu, mutant or wild-type p53, cytochrome P450 1B1, and abnormally expressed intron sequences like N-acetylglucosaminyltransferase-V; clonal rearrangements of immunoglobulin genes that generate unique idiotypes in myeloma and B-cell lymphoma; tumor antigens containing epitopic regions or epitopic peptides derived from oncoviral processes, such as human papillomavirus proteins E6 and E7; Epstein-Barr virus protein LMP2; and epitope regions or epitope peptides derived from genes transcribed at different levels in tumor cells compared to normal cells, such as carcinoembryonic antigens and unmutated oncophetal proteins with tumor-selective expression, such as α-fetoprotein.

[0338] E. Suicide gene In certain embodiments, suicide genes are used in conjunction with NK cell therapy to control their use and enable termination of cell therapy at a desired event and / or time. Suicide genes are employed in transdextrins for the purpose of inducing the death of transdextrins when needed. Cells of this disclosure, modified to possess one or more vectors incorporated herein, may contain one or more suicide genes. In some embodiments, the term “suicide gene” as used herein is defined as a gene that, upon administration of a prodrug or other agent, results in the transfer of a gene product to a compound that kills a host cell. In other embodiments, the suicide gene optionally encodes a gene product that is targeted by a drug (such as an antibody) that targets the suicide gene product.

[0339] In some cases, cell therapy may be subject to the use of one or more suicide genes of any kind if the individual receiving and / or having received cell therapy is considered to be at risk of exhibiting or being imminent of exhibiting one or more symptoms of one or more adverse events, including cytokine release syndrome, neurotoxicity, anaphylaxis / allergy, and / or on-target / off-tumor toxicity (for example). The use of suicide genes may be part of a planned protocol for treatment, or may be used only if the need for such use is recognized. In some cases, cell therapy may be terminated with a drug that targets a suicide gene or its gene product because treatment is no longer needed.

[0340] The use of suicide genes can be initiated when at least one adverse event occurs in the individual, and this adverse event can be recognized by any means, including during periodic monitoring, whether continuous or not, from the initiation of cell therapy. Adverse events can be detected by examinations and / or tests. If the individual is experiencing cytokine release syndrome (sometimes called a cytokine storm), the individual may exhibit elevated levels of inflammatory cytokines (one or more) (for example: interferon-γ, granulocyte-macrophage colony-stimulating factor, IL-10, IL-6, and TNF-α), fever, fatigue, hypotension, hypoxia, tachycardia, nausea, capillary leakage, cardiac / renal / hepatic dysfunction, or a combination thereof. If the individual is neurotoxic, the individual may exhibit confusion, delirium, aplasia, and / or seizures. In some cases, the individual may be tested for markers associated with the development and / or severity of cytokine release syndrome, such as C-reactive protein, IL-6, TNF-α, and / or ferritin.

[0341] Examples of suicide genes include engineered non-secretory (including membrane-bound) tumor necrosis factor (TNF)-α mutant polypeptides (see PCT / US19 / 62009, the whole of which is incorporated herein by reference), which may be affected by the delivery of antibodies that bind to TNF-α variants. Examples of suicide gene / prodrug combinations that may be used include herpes simplex virus-thymidine kinase (HSV-tk) and ganciclovir, acyclovir, or FIAU; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase-thymidylate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside. Escherichia coli purine nucleoside phosphorylase is a so-called suicide gene that converts the prodrug 6-methylpurine deoxyriboside to the toxic purine 6-methylpurine and may be used. Other suicide genes include CD20, CD52, inducible caspase 9, purine nucleoside phosphorylase (PNP), cytochrome p450 enzyme (CYP), carboxypeptidase (CP), carboxylesterase (CE), nitroreductase (NTR), guanine ribosyltransferase (XGRTP), glycosidase enzyme, methionine-α,γ-lyase (MET), and thymidine phosphorylase (TP).

[0342] F. Knockout or knockdown of endogenous genes In certain embodiments, the NK cells of this disclosure may include gene editing of NK cells to remove 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more endogenous genes in the NK cells. In some cases, gene editing is performed in NK cells expressing one or more xenotransfer genes (e.g., CD3, TCR, etc.), while in other cases, gene editing is performed in at least some cases in NK cells that do not express xenotransfer genes but will eventually express one or more xenotransfer genes. In certain embodiments, the NK cells to be gene edited are proliferating NK cells.

[0343] In certain embodiments, one or more endogenous genes in NK cells are modified, such as by disruption of expression, which reduces some or all of their expression. In certain cases, one or more genes are knocked down or knocked out using the process of this disclosure. In certain cases, multiple genes are knocked down or knocked out using the same process as the process of this disclosure. The genes edited in NK cells may be any, but in specific embodiments, the genes are those whose gene product inhibits the activity and / or proliferation of NK cells. In certain cases, the genes edited in NK cells enable NK cells to function more effectively in the tumor microenvironment. In specific cases, the genes are one or more of the following: NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, TDAG8, CD5, CD7, SLAMF7, CD38, LAG3, TCR, β2-microglobulin, HLA, CD73, CREB, CREM, ICER, and CD39. In specific embodiments, the TGFBR2 gene is knocked out or knocked down in NK cells. In specific embodiments, the CISH gene is knocked out or knocked down in NK cells. In specific embodiments, the CD38 gene is knocked out or knocked down in NK cells. In specific embodiments, the glucocorticoid receptor (GR) gene is knocked out or knocked down in NK cells.

[0344] In some embodiments, gene editing is carried out using one or more DNA-binding nucleic acids, such as modifications mediated by RNA-guided endonucleases (RGENs). For example, modifications can be carried out using clustered regularly spaced short palindromic repeats (CRISPR) and CRISPR-related (Cas) proteins. Generally, the “CRISPR system” refers collectively to transcripts and other elements involved in directing the expression or activity of CRISPR-related (“Cas”) genes, and includes the sequence encoding the Cas gene, the tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or active partial tracrRNA), the tracr-mate sequence (including “direct repeats” and tracrRNA-processed partial direct repeats in the context of endogenous CRISPR systems), the guide sequence (also called “spacers” in the context of endogenous CRISPR systems), and / or other sequences and transcripts from the CRISPR locus. Methods utilizing the CRISPR system are well known in the art.

[0345] IV. Method of administration and use of therapeutic compositions In some embodiments, TCR / FcRs containing NK cells are administered to individuals in need. In some embodiments, NK cells containing TCR / FcRs are administered to individuals in need in combination with monospecific antibodies, bispecific antibodies, and / or multispecific antibodies. In some embodiments, co-administration to individuals in need is carried out in such a manner that the TCR / FcR NK cells and antibodies are in close proximity, and therefore the antibodies and NK cells can interact via one or more receptors (e.g., CD3 / TCR complex, CD16, etc.). In some cases, the two components are administered to the individual separately, while in others, the two components are complexed together before administration by ex vivo methods or similar means (i.e., one, two, or more components are loaded onto the NK cells). In another embodiment, the NK cells express antibodies. In some cases, the two components are not pre-complexed before administration but are co-administered by any suitable route of administration, such as co-infusion to the patient. In some embodiments, one or more components (e.g., monospecific antibodies, bispecific antibodies, and / or multispecific antibodies) may be loaded onto NK cells ex vivo, while other components, which may be the same or different components, may be co-administered.

[0346] In some embodiments, the use of NK cells containing TCR / FcR conjugated with an antibody results in a synergistic effect. In some embodiments, the combined use of NK cells containing TCR / FcR and blinatumomab results in a synergistic effect, increasing the cytotoxicity of NK cells against cells with CD19 (e.g., cancer cells). In some embodiments, the combined use of NK cells containing TCR / FcR and obinutuzumab results in a synergistic effect, increasing the cytotoxicity of NK cells against cells with CD20 (e.g., cancer cells). In some embodiments, the combined use of NK cells containing TCR / FcR and rituximab results in a synergistic effect, increasing the cytotoxicity of NK cells against cells with CD19 (e.g., cancer cells). In some embodiments, the combined use of NK cells containing TCR / FcR and amivantamab results in a synergistic effect, increasing the cytotoxicity of NK cells against cells with EGFR and / or c-MET (e.g., cancer cells). In some embodiments, the combination of NK cells containing TCR / FcR and imugatuzumab produces a synergistic effect, increasing the cytotoxicity of NK cells against cells with EGFR (e.g., cancer cells). In some embodiments, the combination of NK cells containing TCR / FcR and cetuximab produces a synergistic effect, increasing the cytotoxicity of NK cells against cells with EGFR (e.g., cancer cells). In some embodiments, the combination of NK cells containing TCR / FcR and tecristamag produces a synergistic effect, increasing the cytotoxicity of NK cells against cells with BCMA (e.g., cancer cells). In some embodiments, the combination of NK cells containing TCR / FcR and erlanatamab produces a synergistic effect, increasing the cytotoxicity of NK cells against cells with BCMA (e.g., cancer cells). In some embodiments, the combination of NK cells containing TCR / FcR and talketamab produces a synergistic effect, increasing the cytotoxicity of NK cells against cells with GPRC5D (e.g., cancer cells). In some embodiments, the combination of NK cells containing TCR / FcR and pertuzumab produces a synergistic effect, increasing the cytotoxicity of NK cells against HER2-containing cells (e.g., cancer cells).In some embodiments, the combination of NK cells containing TCR / FcR and trastuzumab produces a synergistic effect, increasing the cytotoxicity of NK cells against HER2-containing cells (e.g., cancer cells). In some embodiments, the combination of NK cells containing TCR / FcR and tafacitamab produces a synergistic effect, increasing the cytotoxicity of NK cells against CD19-containing cells (e.g., cancer cells). In some embodiments, the combination of NK cells containing TCR / FcR and brentuximab produces a synergistic effect, increasing the cytotoxicity of NK cells against CD30-containing cells (e.g., cancer cells).

[0347] Embodiments of this disclosure relate to methods of using compositions comprising NK cells and antibodies provided herein for treating or preventing medical diseases or disorders. In some embodiments, the method comprises administering a therapeutically effective amount of TCR / FcR-modified NK cells having one or more antibodies to a subject, thereby treating or preventing a disease in the subject, for example, reducing the risk of disease, reducing the severity of disease, and / or delaying the onset of disease. In certain embodiments of this disclosure, cancer or infection is treated by transfer of a composition comprising an NK cell population and corresponding antibodies. In certain embodiments of this disclosure, autoimmune disorders are treated by transfer of a composition comprising an NK cell population and corresponding antibodies. In at least some cases, NK cells may enhance the adaptive immune response by reversing the anti-inflammatory tumor microenvironment due to the release of inflammatory cytokines and promoting the differentiation, activation, and / or recruitment of accessory immune cells to malignant tumor sites. In certain embodiments, the providing step may include culturing NK cells with antibody molecules for a specific time (e.g., about 5 minutes to about 24 hours or more), and storing the NK cells and antibody molecules for a certain period of time before infusion / administration (e.g., about 1, 2, 3, 4, 5 days, or a period exceeding 5 days).

[0348] In certain embodiments of this disclosure, autoimmune disorders include disorders characterized by abnormal B cells. In certain embodiments, autoimmune disorders include B cell-associated autoimmunity. In certain embodiments, autoimmune disorders include systemic lupus erythematosus (SLE). In certain embodiments, autoimmune disorders include systemic sclerosis (SSc). In certain embodiments, autoimmune disorders include multiple sclerosis (MS). In certain embodiments, autoimmune disorders include Graves' disease. In certain embodiments, autoimmune disorders include rheumatoid arthritis. In certain embodiments, autoimmune disorders include myositis. In certain embodiments, autoimmune disorders include dermatomyositis. In certain embodiments, autoimmune disorders include myasthenia gravis. In certain embodiments, autoimmune disorders include Sjögren's syndrome. In certain embodiments, autoimmune disorders include diffuse sclerosis. In certain embodiments, autoimmune disorders include inflammatory myopathy. In certain embodiments, autoimmune disorders include ANCA-associated systemic vasculitis. In certain embodiments, the autoimmune disorder includes antiphospholipid syndrome. In certain embodiments, the autoimmune disorder includes immune nephritis. In certain embodiments, the autoimmune disorder includes immune thrombocytopenia (ITP). In certain embodiments, the autoimmune disorder includes refractory POEMS (polyneuritis, organomegaly, endocrine disorders / edema, monoclonal protein, skin changes) syndromes. In certain embodiments, the autoimmune disorder includes amyloidosis. In certain embodiments, the autoimmune disorder includes autoimmune hemolytic anemia. In certain embodiments, the autoimmune disorder includes vasculitis.

[0349] The cancers for which the therapeutic method of the present invention is useful include all malignant cell types, such as those found in solid tumors or hematological malignancies. Exemplary solid tumors include, but are not limited to, tumors of organs selected from the group consisting of the pancreas, colon, cecum, stomach, brain, head, neck, ovaries, kidneys, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. Exemplary hematological malignancies include tumors of the bone marrow, T-cell or B-cell malignancies, leukemia, lymphoma, blastoma, and myeloma. Further examples of cancers that can be treated using the methods provided herein include, but are not limited to, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, gastric cancer or gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, various head and neck cancers, and melanoma.

[0350] Cancer can be, but is not limited to, the following histological types: neoplasm, malignant; carcinoma; undifferentiated carcinoma; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; dermal papillary carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; cavernous adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma of adenomatous polyp; adenocarcinoma, familial adenomatous polyposis; solid tumor; malignant carcinoid tumor; lobulated-alveolar adenocarcinoma; papillary adenocarcinoma; chromatocarcinoma; Acidophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; unencapsulated sclerotic carcinoma; adrenal cortical carcinoma; endometrial carcinoma; adnexal carcinoma; apocrine gland carcinoma; sebaceous gland carcinoma; keratinized gland carcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary gland; acinar cell carcinoma; adenosquamous cell carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; sarcoma, malignant; granulosa cell tumor Malignant ulcer; malignant androblastoma; Sertoli cell carcinoma; malignant Leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; angiosarcoma; malignant melanoma; achromatic melanoma; superficial spreading melanoma; lentigo malignant melanoma; lentigo genitalia; nodular melanoma; malignant melanoma of giant pigmented nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonic rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; Müller's mixed tumor; nephroblastoma; hepatoblastoma; Carcinosarcoma; Mesenchymal tumor, malignant; Brenner tumor, malignant; Philodes tumor, malignant; Synovial sarcoma; Mesothelioma, malignant; Embryonic disorders; Embryonic carcinoma; Teratoma, malignant; Ovarian goiter, malignant; Choriocarcinoma; Mesonephroma, malignant; Angiosarcoma; Hemangioendothelioma, malignant; Kaposi's sarcoma; Hemangioexiformoma, malignant; Lymphangiosarcoma; Osteosarcoma; Soft cortical osteosarcoma; Chondrosarcoma; Chondroblastoma, malignant; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma; Odontogenic tumor, malignant; Ameloepithelial gingivoma; Ameloepithelioma, malignant; Ameloepithelial fibrosarcoma; Pineal glandoma, malignant; Chordoma; Glioma, malignant; Ependymoma; Astrocytoma; Protoplasmic astrocytoma;Fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectodermoma; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; schwannoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin; paragranuloma; malignant lymphoma, microlymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specific non-Hodgkin lymphomas; B-cell lymphoma; low-grade / follicular non-Hodgkin lymphoma (NHL); microlymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL HL; high-grade lymphoblastic NHL; high-grade small non-necrotic cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-associated lymphoma; Waldenström macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative bowel disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythrocyte leukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryocytic leukemia; myelosarcoma; pilocytic cell leukemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); and chronic myeloblastic leukemia.

[0351] Therapies provided herein may include the administration of a combination of therapeutic agents, such as a first cancer therapeutic agent and a second cancer therapeutic agent. The therapy can be administered in any suitable method known in the art. For example, the first and second cancer therapies can be administered sequentially (at different times) or simultaneously (at the same time). In some embodiments, the first and second cancer therapies are administered in separate compositions. In some embodiments, the first and second cancer therapies are in the same composition. Embodiments of this disclosure relate to compositions and methods comprising therapeutic compositions. Different therapies may be administered in one composition or in two or more compositions, such as two, three, or four compositions. Various combinations of drugs may be employed. Examples of therapies other than those disclosed include surgery, chemotherapy, drug therapy, radiotherapy, hormone therapy, immunotherapy (other than those disclosed), or combinations thereof.

[0352] The treatments disclosed herein may be administered via the same route of administration or via different routes of administration. In some embodiments, cancer treatments may be administered intravenously, intramuscularly, subcutaneously, topically, orally, percutaneously, intraperitoneally, or orbitally, by implantation, by inhalation, intrathecally, intraventricularly, or nasally. In some embodiments, antibiotics may be administered intravenously, intramuscularly, subcutaneously, topically, orally, percutaneously, intraperitoneally, or orbitally, by implantation, by inhalation, intrathecally, intraventricularly, or nasally. The appropriate dosage may be determined based on the type of disease being treated, the severity and course of the disease, the individual's clinical condition, the individual's clinical history and response to treatment, and the discretion of the attending physician.

[0353] Treatment may include various "unit doses." A unit dose is defined as containing a predetermined amount of the therapeutic composition. The amount administered, as well as the specific route and prescription, are within the scope of the clinical practitioner's judgment. A unit dose does not need to be administered as a single injection, but may include continuous infusions over a period of time. In some embodiments, a unit dose includes a single-dose dose.

[0354] The dosage, both in terms of the number of treatments and the unit dose, depends on the desired therapeutic effect. The effective dose is understood to refer to the amount required to achieve a particular effect. In practice, in certain embodiments, it is intended that doses ranging from 10 mg / kg to 200 mg / kg may affect the protective capacity of these drugs. Therefore, possible doses include approximately 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 μg / kg, mg / kg, μg / day, or mg / day, or any range derived from there. Furthermore, such doses can be administered multiple times a day and / or over multiple days, weeks, or months.

[0355] In certain embodiments, an effective amount of the pharmaceutical composition may provide a blood level of about 1 μM to 150 μM. In other embodiments, an effective amount may provide a blood level of about 4 μM to 100 μM; or about 1 μM to 100 μM; or about 1 μM to 50 μM; or about 1 μM to 40 μM; or about 1 μM to 30 μM; or about 1 μM to 20 μM; or about 1 μM to 10 μM; or about 10 μM to 150 μM; or about 10 μM to 100 μM; or about 10 μM to about 50 μM; or about 25 μM to about 150 μM; or about 25 μM to about 100 μM; or about 25 μM to about 50 μM; or about 50 μM to about 150 μM; or about 50 μM to about 100 μM (or any range from which it can be derived). In other embodiments, the dose is the following blood levels of the drug derived from the therapeutic agent administered to the subject: approximately, at least, or at most approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, The concentrations may be 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 μM, or any range derived therefrom. In certain embodiments, the therapeutic agent administered to a subject is metabolized in the body to become a metabolized therapeutic agent, in which case the blood concentration may refer to the amount of that therapeutic agent. Alternatively, in the range in which the therapeutic agent is not metabolized by the subject, the blood concentration discussed herein may refer to the unmetabolized therapeutic agent.

[0356] The precise amount of therapeutic composition is also dependent on the practitioner's judgment and is specific to each individual. Factors influencing the dosage include the patient's physical and clinical condition, route of administration, intended treatment objective (symptom relief vs. cure), the potency, stability, and toxicity of the particular therapeutic substance or any other therapies the subject may be receiving.

[0357] Those skilled in the art will understand and recognize that dose units of μg / kg body weight or mg / kg body weight can be converted to equivalent concentration units of μg / ml or mM (blood concentration), such as 4 μM to 100 μM. It will also be understood that uptake is species and organ / tissue dependent. Applicable conversion factors and physiological assumptions regarding uptake and concentration measurements are well known, and those skilled in the art will be able to convert one concentration measurement to another and make reasonable comparisons and draw conclusions regarding the doses, efficacy, and results described herein.

[0358] Embodiments of this disclosure relate to methods of using compositions comprising NK cells (e.g., engineered NK cells) and antibodies provided herein for treating or preventing medical diseases or disorders. The methods involve administering therapeutically charged, optionally pre-activated, optionally enlarged NK cells and antibodies to a subject, thereby treating or preventing a disease in the subject, including reducing the risk of disease, reducing the severity of disease, and / or delaying the onset of disease. In certain embodiments of this disclosure, cancer or infection is treated by the transfer of a composition comprising an NK cell population and antibodies. In at least some cases, NK cells release inflammatory cytokines, which can increase the adaptive immune response by reversing the anti-inflammatory tumor microenvironment and promoting the differentiation, activation, and / or recruitment of accessory immune cells to malignant tumor sites.

[0359] In certain embodiments, cancers for which the compositions and methods described herein are useful in treating, preventing, and / or improving symptoms include cancers expressing at least CD19, CD20, CD30, HER2, GPRC5D, EGFR, EGFR2, BCMA, and / or c-MET. In certain embodiments, cancers for which the compositions and methods described herein are useful in treating, preventing, and / or improving symptoms include cancers expressing at least CD19. In certain embodiments, cancers for which the compositions and methods described herein are useful in treating, preventing, and / or improving symptoms include cancers expressing at least CD20. In certain embodiments, cancers for which the compositions and methods described herein are useful in treating, preventing, and / or improving symptoms include cancers expressing at least EGFR. In certain embodiments, cancers for which the compositions and methods described herein are useful in treating, preventing, and / or improving symptoms include cancers expressing at least BCMA. In certain embodiments, cancers for which the compositions and methods described herein are useful in treating, preventing, and / or improving symptoms include cancers expressing at least c-MET. In certain embodiments, cancers for which the compositions and methods described herein are useful in treating, preventing, and / or improving symptoms include cancers expressing at least CD70 and / or TROP2.

[0360] In certain embodiments, the compositions and methods described herein are used for the treatment, prevention, and / or improvement of symptoms associated with PDAC. In certain embodiments, the compositions and methods described herein are used for the treatment, prevention, and / or improvement of symptoms associated with CRC. In certain embodiments, the compositions and methods described herein are used for the treatment, prevention, and / or improvement of symptoms associated with ovarian cancer. In certain embodiments, the compositions and methods described herein are used for the treatment, prevention, and / or improvement of symptoms associated with leukemia. In certain embodiments, the compositions and methods described herein are used for the treatment, prevention, and / or improvement of symptoms associated with kidney cancer. In certain embodiments, the compositions and methods described herein are used for the treatment, prevention, and / or improvement of symptoms associated with glioblastoma. In certain embodiments, the compositions and methods described herein are used for the treatment, prevention, and / or improvement of symptoms associated with breast cancer. In certain embodiments, the compositions and methods described herein are used for the treatment, prevention, and / or improvement of symptoms associated with RCC. In certain embodiments, the compositions and methods described herein are used for the treatment, prevention, and / or improvement of symptoms associated with myeloma.

[0361] Cancer may, but is not limited to, the following histological types: neoplasm, malignant; carcinoma; undifferentiated carcinoma; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; dermal papillary carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; cavernous adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma of adenomatous polyp; adenocarcinoma, familial adenomatous polyposis; solid tumor; malignant carcinoid tumor; lobulated-alveolar adenocarcinoma; papillary adenocarcinoma; chromatocarcinoma; Eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; unencapsulated sclerotic carcinoma; adrenal cortical carcinoma; endometrial carcinoma; cutaneous adnexal carcinoma; apocrine gland carcinoma; sebaceous gland carcinoma; keratinized gland carcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary gland; acinar cell carcinoma; adenosquamous cell carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; sarcoma, malignant; granulosa cell malignant tumors; malignant an...

Claims

1. A polynucleotide containing a sequence that is at least approximately 80%, 85%, 90%, 95%, 98%, or 100% identical to a transcription reading frame containing sequence numbers 171–175.

2. The polynucleotide according to claim 1, wherein the polynucleotide is contained in a vector having at least about 80%, 85%, 90%, 95%, 98%, or 100% sequence identity with SEQ ID NOs. 177-181.

3. A polynucleotide comprising sequences encoding T cell receptor (TCR) alpha and TCR beta polypeptides, and / or TCR gamma and TCR delta polypeptides, and polypeptides containing a CD16-derived Fc-binding domain.

4. The polynucleotide according to claim 3, wherein the TCR polypeptide is an invariant TCR (iTCR) polypeptide.

5. The polynucleotide according to claim 4, wherein the iTCRα and iTCRβ polypeptides and / or the polynucleotide encoding them include a sequence that is at least about 80%, 85%, 90%, 95%, 98%, or 100% identical to one or more of the sequence numbers 51 to 149.

6. The polynucleotide according to claim 4, wherein the iTCRβ polypeptide comprises a polynucleotide encoding a Vβ-DJ region which is at least about 80%, 85%, 90%, 95%, 98%, or 100% identical to one or more of the sequence numbers 75 to 149.

7. The polynucleotide according to claim 4, wherein the iTCRβ polypeptide and / or the polynucleotide encoding it comprises a sequence that is at least about 80%, 85%, 90%, 95%, 98%, or 100% identical to one or more of sequence numbers 59 to 74.

8. The polynucleotide according to claim 4, wherein the encoded iTCRβ polypeptide is at least about 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO:

60.

9. The polynucleotide according to claim 8, wherein the polynucleotide encoding the iTCRβ polypeptide is at least about 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO:

59.

10. The polynucleotide according to claim 4, wherein the iTCRα polypeptide and / or the polynucleotide encoding it comprises a sequence that is at least about 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NOs. 51-52.

11. The polynucleotide according to claim 3, wherein the polypeptide containing the CD16-derived Fc-binding domain contains a human CD16A Fc-binding domain.

12. The polynucleotide according to claim 3, further comprising the Fc-binding domain fused in an N-terminus-to-C-terminus order to any hinge domain, a transmembrane domain (TMD), and one or more any intracellular signaling domains (ICD).

13. The polynucleotide according to claim 12, wherein the TMD is derived from CD16 or CD3ζ.

14. The polynucleotide according to claim 12, wherein the TMD comprises or consists of a sequence having at least about 90% identity with sequence number 163 or 167.

15. The polynucleotide according to claim 12, wherein the hinge domain is derived from CD32.

16. The polynucleotide according to claim 12, comprising a hinge domain having at least about 90% identity with sequence number 161.

17. The polynucleotide according to claim 12, comprising an ICD derived from CD16 and / or CD3ζ.

18. The polynucleotide according to claim 3, wherein the polypeptide comprising the CD16-derived Fc-binding domain does not contain a mutation that makes the CD16-derived Fc-binding domain resistant to cleavage.

19. The polynucleotide according to claim 3, comprising a coding sequence that is at least approximately 80%, 85%, 90%, 95%, 98%, or 100% identical to one or more of sequence numbers 150 to 154.

20. The polynucleotide according to claim 3, wherein the polynucleotide further encodes one or more cytokine sequences.

21. The polynucleotide according to claim 20, wherein the cytokine comprises IL-15 and / or IL-21.

22. Engineered NK cells comprising the polynucleotide described in any one of claims 1 to 21.

23. A method for treating a disease in an individual, comprising administering the manipulated NK cells described in claim 22 to an individual in need of treatment.

24. a) CD3 protein complexes comprising all or part of a single-strand or any combination of CD3δ, CD3ε, CD3γ, or CD3ζ, b) At least one cytokine of any choice, c) at least one TCRα chain and TCRβ chain, and / or a TCRγ chain and TCRδ chain, d) Polypeptide containing a CD16-Fc binding domain Engineered immune cells containing one or more transgenic polynucleotides encoding a specific character.

25. The manipulated immune cell according to claim 24, wherein one or more transgenic polynucleotides include a multi-cistronic transcription open reading frame.

26. The engineered immune cells according to claim 24, wherein the cells are modified to express some or all of CD3δ, and two of CD3ε, CD3γ, and / or CD3ζ.

27. The manipulated immune cell according to claim 24, wherein one or more of CD3δ, CD3ε, CD3γ, and / or CD3ζ are linked to one or more heterologous intracellular signaling domains.

28. The manipulated immune cell according to claim 27, wherein the heterologous intracellular signaling domain is selected from the group consisting of CD16, NKG2D, DAP10, DAP12, 2B4, 4-1BB, CD2, CD28, and combinations thereof.

29. The manipulated immune cell according to claim 27, wherein the heterologous intracellular signaling domain includes the DAP10 intracellular signaling domain.

30. The manipulated immune cell according to claim 29, wherein the heterologous intracellular signaling domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO:

42.

31. The manipulated immune cell according to claim 27, wherein the heterologous intracellular signaling domain includes a CD28 intracellular signaling domain.

32. The manipulated immune cell according to claim 31, wherein the heterologous intracellular signaling domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO:

43.

33. The manipulated immune cell according to claim 27, wherein the heterologous intracellular signaling domain comprises the DAP10 and CD28 intracellular signaling domains.

34. The manipulated immune cell according to claim 33, wherein the heterologous intracellular signaling domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO:

44.

35. The engineered immune cell according to claim 24, wherein the coding sequence of a CD3 protein complex and at least one cytokine is contained in a first multi-cistronic construct, and the coding sequence of the polypeptide comprising at least one TCRα and TCRβ chain, and / or TCRγ and TCRδ chain, and a CD16 Fc-binding domain is encoded by a second multi-cistronic construct.

36. The engineered immune cell according to claim 24, comprising a cytokine coding sequence, wherein the cytokine comprises IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, and / or GMCSF.

37. The manipulated immune cells according to claim 36, wherein the cells are modified to express a polynucleotide sequence that is at least 85% identical to UT-NK15-DAP10 (SEQ ID NO: 45), UT-NK15-28 (SEQ ID NO: 47), or UTNK15-28-DAP10 (SEQ ID NO: 49).

38. The engineered immune cell according to claim 37, wherein the cytokine comprises IL-15 and / or IL-21.

39. The manipulated immune cell according to claim 38, wherein the cytokine comprises IL-15 and includes a polypeptide sequence and / or a polynucleotide sequence encoding it that is at least 85% identical to one or more of the sequence numbers 182 to 183.

40. The manipulated immune cell according to claim 38, wherein the cytokine comprises IL-21 and includes a polypeptide sequence and / or a polynucleotide sequence encoding it that is at least 85% identical to one or more of the sequence numbers 184 to 187.

41. The manipulated immune cell according to claim 24, wherein the TCR polypeptide is an invariant TCR (iTCR) polypeptide, and the iTCRα and iTCRβ polypeptides and / or the polynucleotides encoding them include sequences that are at least about 80%, 85%, 90%, 95%, 98%, or 100% identical to one or more of SEQ ID NOs. 51 to 149.

42. The manipulated immune cell according to any one of claims 24 to 41, wherein the TCR polypeptide is an invariant TCR (iTCR) polypeptide.

43. The manipulated immune cell according to claim 42, wherein the iTCRα and iTCRβ polypeptides and / or the polynucleotides encoding them include a sequence that is at least about 80%, 85%, 90%, 95%, 98%, or 100% identical to one or more of the sequence numbers 51 to 149.

44. The manipulated immune cell according to claim 42, wherein the iTCRβ polypeptide comprises a polynucleotide encoding a Vβ-DJ region which is at least about 80%, 85%, 90%, 95%, 98%, or 100% identical to one or more of SEQ ID NOs. 75 to 149.

45. The manipulated immune cell according to claim 42, wherein the iTCRβ polypeptide and / or the polynucleotide encoding it comprises a sequence that is at least about 80%, 85%, 90%, 95%, 98%, or 100% identical to one or more of the sequence numbers 59 to 74.

46. The engineered immune cell according to claim 42, wherein the encoded iTCRβ polypeptide is at least about 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO:

60.

47. The manipulated immune cell according to claim 46, wherein the polynucleotide encoding the iTCRβ polypeptide is at least about 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO:

59.

48. The manipulated immune cell according to claim 42, wherein the iTCRα polypeptide and / or the polynucleotide encoding it comprises a sequence that is at least about 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NOs. 51-52.

49. The manipulated immune cell according to claim 24, wherein the polypeptide containing the CD16 Fc-binding domain contains a human CD16-derived Fc-binding domain.

50. The manipulated immune cell according to claim 24, wherein the polypeptide containing the CD16-derived Fc-binding domain contains a human CD16A Fc-binding domain.

51. The manipulated immune cell according to claim 24, wherein the Fc-binding domain is fused to any hinge domain, a transmembrane domain (TMD), and one or more any intracellular signaling domains (ICD) in an N-terminus to C-terminus order.

52. The manipulated immune cell according to claim 51, wherein the TMD is derived from CD16 or CD3ζ.

53. The manipulated immune cell according to claim 51, wherein the TMD comprises or consists of a sequence having at least about 90% identity with sequence number 163 or 167.

54. The manipulated immune cell according to claim 51, wherein the hinge domain is derived from CD32.

55. The manipulated immune cell according to claim 51, wherein the hinge domain comprises or consists of a sequence having at least about 90% identity with sequence number 161.

56. The manipulated immune cells according to claim 51, comprising ICDs derived from CD16 and / or CD3ζ.

57. The manipulated immune cell according to claim 24, comprising a coding sequence that is at least approximately 80%, 85%, 90%, 95%, 98%, or 100% identical to one or more of sequence numbers 171 to 175.

58. The manipulated immune cells according to any one of claims 24 to 41, wherein the cells are natural killer (NK) cells.

59. The manipulated NK cells according to claim 58, wherein the NK cells are derived from umbilical cord blood (CB), peripheral blood (PB), bone marrow, stem cells, or a combination thereof.

60. The engineered NK cells according to claim 58, wherein the NK cells are primary NK cells and are not derived from stem cells and / or induced pluripotent stem cells (iPSCs).

61. The manipulated NK cells according to claim 58, wherein the NK cells are loaded with (and complexed with) one or more antibodies.

62. The manipulated NK cells according to claim 61, wherein the one or more antibodies are one or more monospecific antibodies, bispecific antibodies, or multispecific antibodies.

63. The engineered NK cell according to claim 61, comprising at least one antibody containing a glycosylated Fc domain having high affinity for wild-type CD16.

64. The manipulated NK cell according to claim 61, wherein at least one of the antibodies comprises an unglycosylated Fc domain having low affinity for the wild-type CD16 Fc-binding domain.

65. The manipulated NK cell according to claim 64, wherein the unglycosylated Fc domain is loaded (complexed) with a transgenic polypeptide containing a CD16 Fc-binding domain.

66. The manipulated NK cells according to claim 61, wherein one or more antibodies comprise IgG1 and / or IgG4 Fc domains.

67. The engineered NK cells according to claim 61, wherein one or more antibodies target the antigens CD3, CD16, CD28, EGFR, c-MET, CD30, PSMA, MUC17, CD33, FLT3, STEAP1, BCMA, CLDN18.2, CD123, CD19, CD20, EpCAM, CEA, GPC3, CD38, CD33, CD22, HER2, GPA33, GD2, MUC16, GPRC5D, DLL-3, CLEC12A, FcRH5, and / or SSTR.

68. The engineered NK cells according to claim 61, wherein one or more antibodies target CD3, CD16, CD28, CD19, CD20, CD30, HER2, GPRC5D, EGFR, c-MET, and / or BCMA.

69. The one or more antibodies mentioned above include [fam]-trastuzumab deruxtecan, absiximab, adalimumab, ad-trastuzumab emtansine, aducanumab, alemtuzumab, alirocumab, amivantamab, aniflorumab, ansuvimab, atezolizumab, atorutibimab and maftibimab and odesibimab-ebgn (also known as immazeb), avelumab, basiliximab, verantamab mahodotin, belimumab, benralizumab, bevacizumab, bezlotoxumab, bimekizumab, blinatumomab, and brentuximab. Vedotin, Brodalumab, Brolucizumab, Brosumab, Canakinumab, Caplacizumab, Casiribimab + Imdevimab, Catumaxomab, Semiprimab, Certolizumab Pegol, Cetuximab, Cebostamab, Chryzanlizumab, Daclizumab, Daratumumab, Denosumab, Dinutuximab, Donanemab, Dostarimab, Dupilumab, Durvalumab, Eculizumab, Edrecolomab, Efalizumab, Elotuzumab, Emapalmab, Emicizumab, Enfortzumab Vedotin, eptinezumab, erenumab, evinacumab, evolocumab, falisimab, fremanezumab, galcanezumab, gemtuzumab, gemtuzumab-ozogamicin, golimumab, guselkumab, ibalizumab, ibritumomab, tiucetan, idarucizumab, imugatuzumab, inebilizumab, infliximab, inorimomab, inotuzumab, inotuzumab-ozogamicin, IPH61, ipilimumab, isatuximab, ixekizumab, lanadelmab, lecanemab, ronkastuximab, tesirin, margetuximab, mepolizumab, milbetuximab, sorabtansine, mogamulizumab, mosnetuzumab, moxetumomab Pasdotox, Muromonab-CD3, Narsoprimab, Natalizumab, Naxitamab, Nevacumab, Necitumumab, Nilsevimab, Nivolumab, Ovirtoxakimab, Obinutuzumab, Ocrelizumab, Ofatumumab, Oraratumab, Omalizumab, Omblutamab, Oportuzumab Monatox, Palivizumab, Panitumumab, Pembrolizumab, Penprimab, Pertuzumab, PolatuzumabVedotin, Ramucirumab, Ranibizumab, Rabrizumab, Laxibakumab, Regdanvimab, Relatrimab, Reslizumab, Letifanlimab, Risankizumab, Rituximab, Romosozumab, Sacituzumab Govitecan, Sarilumab, Satralizumab, Secukinumab, Siltuximab, Syntilimab, Sotrovimab, Spesolimab, Stimulimab, Tafacitamab, Teventafusp, Tecristamab, Teprizumab, Teprotumumab, Tezeperumab, Childraquizumab, Tislerizumab, Tisotumab Vedotin, tixagevimab, silgabimab, tocilizumab, tripalimab, tositumomab-I131, tralokinumab, trastuzumab, tremelimumab, ubrituximab, ustekinumab, vedolizumab, AMG 160 / akapatamab, AMG 199 / TNB 585, AMG 330, AMG 427 / emirodatamab, AMG 509, AMG 701, AMG 910, APVO414 / ES414 / MOR209, APVO436, Katsumakisomab / Removab, CC-1, CC-93269 / EM801, Sibisatamab / RG7802 / RO6958688, CLN-049, Elranatamab / PF-06863135, EMB-06, Epcolitamab / GEN3013, ERY974, Flotetuzumab / MGD006, Grofitamab / RG6026 / RO7082859, ISB 1342 / GBR 1342, JNJ-63709178, JNJ-63898081, JNJ-67571244, JNJ-75348780, Limbocertamab / REGN 5458, M701, M802, MGD007, Mosnetuzumab / RG7828, Nivatrotamab / Hu3F8-BsAb, Odronectamab / REGN1979, REGN4018, REGN5459, REGN7075, REGN5678, Talketamab / JNJ-64407564, Tarulatamab / AMG Manipulated NK cells according to claim 61, comprising 757, tepozitamab / MCLA-117, TNB-383B, TNB-486, TNB-585, XmAb13676 / pramotamab, XmAb14045 / vibecotamab, XmAb18087 / tidutamab, and / or AFM13.

70. The engineered NK cells according to claim 61, wherein the one or more antibodies include erranatamab, imugatuzumab, margetuximab, amibantamab, blinatumomab, obinutuzumab, IPH61 (also known as IPH6101 or SAR443579), tecristamag, cetuximab, talketamab, pertuzumab, trastuzumab, tafacitamab, brentuximab, and / or rituximab.

71. The manipulated NK cells according to claim 70, wherein the one or more antibodies include or consist of erlanatamab.

72. The manipulated NK cells according to claim 70, wherein the one or more antibodies include or consist of imugatuzumab.

73. The manipulated NK cells according to claim 70, wherein the one or more antibodies include or consist of margetuximab.

74. The manipulated NK cells according to claim 70, wherein the one or more antibodies include or consist of amivantamab.

75. The manipulated NK cells according to claim 70, wherein the one or more antibodies include or consist of blinatumomab.

76. The manipulated NK cells according to claim 70, wherein the one or more antibodies include or consist of obinutuzumab.

77. The manipulated NK cells according to claim 70, wherein the one or more antibodies include or consist of IPH61.

78. The manipulated NK cells according to claim 70, wherein the one or more antibodies include or consist of teclistamag.

79. The manipulated NK cells according to claim 70, wherein the one or more antibodies include or consist of cetuximab.

80. The manipulated NK cells according to claim 70, wherein the one or more antibodies include or consist of rituximab.

81. The manipulated NK cells according to claim 70, wherein the one or more antibodies include or consist of talketamab.

82. The manipulated NK cells according to claim 70, wherein the one or more antibodies include or consist of pertuzumab.

83. The manipulated NK cells according to claim 70, wherein the one or more antibodies include or consist of trastuzumab.

84. The manipulated NK cells according to claim 70, wherein the one or more antibodies include or consist of tafacitamab.

85. The manipulated NK cells according to claim 70, wherein the one or more antibodies include or consist of brentuximab.

86. The manipulated NK cells according to claim 61, wherein the NK cells express one or more antibodies.

87. The engineered NK cells according to claim 58, wherein the NK cells are further modified to express one or more additional heterologous proteins selected from the group consisting of antigen receptors, cytokines, homing receptors, chemokine receptors, and combinations thereof.

88. The manipulated NK cells according to claim 58, wherein the NK cells are pre-activated with one or more cytokines.

89. The manipulated NK cell according to claim 88, wherein the cytokine is IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, or a combination thereof.

90. The manipulated NK cells according to claim 58, wherein the NK cells further comprise one or more manipulated mutations in endogenous genes.

91. The engineered NK cell according to claim 90, wherein the endogenous gene is GR, TGFBR2, CISH, and / or CD38.

92. A composition comprising manipulated NK cells according to any one of claims 58 to 91.

93. The composition according to claim 92, further comprising a pharmaceutically acceptable excipient.

94. The composition according to claim 92 or 93, wherein the composition is contained in a delivery device.

95. A method for treating a disease in an individual, comprising the step of administering to the individual a therapeutically effective amount of any one of the cells described in claims 24 to 41.

96. The method according to claim 95, wherein the disease is an autoimmune disease, an infectious disease, and / or cancer.

97. The method according to claim 96, wherein the disease is an autoimmune disease.

98. The method according to claim 97, wherein the autoimmune disease includes B cell-associated autoimmunity, systemic lupus erythematosus (SLE), systemic sclerosis (SSc), multiple sclerosis (MS), Graves' disease, rheumatoid arthritis (RA), myositis, dermatomyositis, myasthenia gravis, Sjögren's syndrome, diffuse sclerosis, inflammatory myopathy, ANCA-associated systemic vasculitis, antiphospholipid syndrome, immune nephritis, ITP, refractory POEMS syndrome, amyloidosis, autoimmune hemolytic anemia, and / or vasculitis.

99. The method according to claim 95, wherein the disease is cancer.

100. The method according to claim 99, wherein the cancer expresses CD19, CD20, CD30, HER2, GPRC5D, EGFR, c-MET, and / or BCMA.

101. The method according to claim 99, wherein the cancer is pancreatic cancer, colorectal cancer, ovarian cancer, kidney cancer, glioblastoma, breast cancer, renal cancer, myeloma, and / or leukemia.

102. The method according to claim 98 or 101, further comprising administering one or more monospecific antibodies, bispecific antibodies, and / or multispecific antibodies to an individual simultaneously or at different times.

103. The method according to claim 102, wherein the one or more antibodies include erranatamab, imugatuzumab, margetuximab, amibantamab, blinatumomab, obinutuzumab, IPH61 (also known as IPH6101 or SAR443579), tecristamab, cetuximab, talketamab, pertuzumab, trastuzumab, tafacitamab, brentuximab, and / or rituximab.

104. The method according to claim 102, wherein one or more antibodies are administered simultaneously, and / or NK cells manipulated with one or more antibodies are complexed before administration to an individual.

105. The method according to claim 102, wherein the one or more antibodies are administered more than once, including at least once at a time after the administration of the manipulated NK cells.

106. The method according to claim 102, wherein the one or more antibodies are administered more than once, including at least once at the time prior to the administration of the manipulated NK cells.

107. The method according to claim 102, wherein the one or more antibodies are administered more than once, including at least once before administration of the manipulated NK cells and at least once after administration of the manipulated NK cells.

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