WO2023006120A1 - 通用型t细胞及其应用 - Google Patents

通用型t细胞及其应用 Download PDF

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WO2023006120A1
WO2023006120A1 PCT/CN2022/109558 CN2022109558W WO2023006120A1 WO 2023006120 A1 WO2023006120 A1 WO 2023006120A1 CN 2022109558 W CN2022109558 W CN 2022109558W WO 2023006120 A1 WO2023006120 A1 WO 2023006120A1
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刘凌峰
钟文婷
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St Phi Therapeutics Co Ltd
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St Phi Therapeutics Co Ltd
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Priority to EP22848724.5A priority Critical patent/EP4378952A4/en
Priority to US18/293,291 priority patent/US20240335535A1/en
Priority to CN202280053071.0A priority patent/CN118103392A/zh
Priority to CA3227400A priority patent/CA3227400A1/en
Priority to KR1020247007047A priority patent/KR20240042483A/ko
Priority to AU2022317972A priority patent/AU2022317972A1/en
Priority to IL310433A priority patent/IL310433A/en
Priority to JP2024505358A priority patent/JP2024528077A/ja
Publication of WO2023006120A1 publication Critical patent/WO2023006120A1/zh
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Definitions

  • the present disclosure relates to the field of cell therapy, more specifically, to a universal T cell and its application, especially a chimeric natural killer receptor universal T cell (CNK-UT) and its application.
  • CNK-UT chimeric natural killer receptor universal T cell
  • CD19-targeted CAR-T cells achieved the first targeted elimination of tumor cells in patients with B-cell leukemia in the history of human medicine, becoming another treatment technology that can truly cure leukemia after bone marrow stem cell transplantation treatment technology, and opened up precision medicine cell therapy new era. This technology is expected to be applied to the treatment of various blood tumors and solid tumors. However, the current clinical efficacy of conventional CAR-T in the treatment of solid tumors is not good.
  • the immunosuppressive microenvironment of solid tumors has a large number of immunosuppressive cells, such as TAM, Treg, and MDSC, and it is difficult for traditional CAR-T designs to break through the tumor (4)
  • Traditional CAR-T is a personalized cell therapy, and the source of T cells is the patient itself. If the patient has received a large amount of radiotherapy and chemotherapy, the immune system is impaired, and it is difficult to separate from the peripheral blood of the patient. Even if a sufficient number of T cells are isolated and transformed, the proliferation and killing functions of T cells are still very weak, so it is difficult to exert a therapeutic effect.
  • TCR immunoglobulin and T cell receptor
  • the TCR is a transmembrane heterodimer consisting of ⁇ and ⁇ chains or ⁇ and ⁇ chains linked by disulfide bonds. Within these chains, complementarity determining regions (CDRs) determine which antigens the TCR will bind to.
  • CDRs complementarity determining regions
  • the human MHC class I gene region includes alleles of HLA-A, B, and C sites, which encode classic class I antigens (molecules) such as HLA-A antigen, B antigen, and C antigen, called HLA-A, HLA-B and HLA-C.
  • HLA-A antigen, B antigen, and C antigen called HLA-A, HLA-B and HLA-C.
  • These antigenic molecules exist on the surface of all body cells and bind to intracellular protein epitope peptides for recognition by the immune system. If cells produce mutated proteins, or are invaded by foreign bacteria or viruses, after cells present these mutated proteins or heterologous protein epitopes, immune cells will carry out immune attack and killing after recognition, thereby eliminating diseased cells, bacteria and virus invasion cell.
  • MHC molecules are a key factor in presenting viral antigens and triggering the immune system's attack against viral pathogens
  • viruses have gradually formed a strategy of targeting and inhibiting MHC molecules during millions of years of evolution to avoid immune surveillance and clearance.
  • a very typical example is human cytomegalovirus (HCMV), which evolves together with its host and achieves immune escape through many functional proteins in the genome.
  • the unique short (US) genomic region of HCMV encodes at least five glycoproteins (US2, US3, US6, US10, US11) that use specialized mechanisms to downregulate MHC class I molecules, thereby hindering presentation of viral antigens to cells Toxic T lymphocytes (CTL).
  • CTL Toxic T lymphocytes
  • CMV US glycoprotein can hijack the endoplasmic reticulum-associated degradation (ERAD) mechanism to inhibit the presentation of viral antigens mediated by MHC molecules, thereby evading the immune surveillance system.
  • ER-associated degradation is a protein clearance system. Misfolded, misassembled or metabolically regulated proteins in the ER are selectively dislocated from the ER into the cytosol through a specific membrane penetration mechanism, where they are subsequently absorbed by the cytoplasmic ubiquitin proteasome system (UPS). )degradation.
  • UPS cytoplasmic ubiquitin proteasome system
  • Both HCMV US2 and US11 proteins are ER-resident type I integral membrane glycoproteins that co-select this ERAD pathway to promote the degradation of MHC class I heavy chains, thereby inhibiting MHC class I antigen presentation. Expression of either protein results in rapid degradation of newly synthesized MHC class I heavy chains. US2 and US11 bind to the heavy chain through their luminal domains and recruit host cell proteins that extract polypeptides from the ER membrane by "pulling" the cytoplasmic tail of the heavy chain. After translocation into the cytoplasm, MHC class I molecules are ubiquitinated and degraded by the proteasome.
  • US2 In addition to class I molecules, US2 also leads to the degradation of two proteins of the class II pathway, DR- ⁇ and DM- ⁇ , and HFE, a non-canonical major histocompatibility complex (MHC) class I protein involved in iron regulation .
  • MHC major histocompatibility complex
  • the luminal domain of US2 is responsible for binding MHC class I and class II molecules, and the transmembrane domain (TM) and cytoplasmic domain (CT) interact with cellular components of ER-associated degradation pathways and contribute to translocation and promotion of class I and class II proteins.
  • the cytoplasmic tail of US2 is sufficient to interact with signal peptide peptidase (SPP), an essential component of the US2-dependent MHC I dislocation complex.
  • SPP signal peptide peptidase
  • US2 interacts with the endoplasmic reticulum-resident RING-type E3 ligase TRC8 through its TM domain, which also contributes to the ubiquitination and proteasomal degradation of US2 tail-anchored MHC I and II molecules.
  • US11-induced degradation of MHC-I molecules requires Derlin1 but not SPP.
  • the ER cavity domain of US11 interacts with the cavity domain of the MHC-I heavy chain, while the TM domain of US11 binds Derlin-1. Therefore, the main function of US11 may be to deliver MHC-I molecules to Derlin-1 and then induce their mislocalization to the cytosol for proteasomal degradation. Furthermore, US11 activates unfolded proteins. Through Derlin-1, US11 associates with TMEM129 as an ERAD RING E3 ligase and recruits Ube2J2 to ubiquitinate MHC-I prior to US11-induced degradation.
  • US3 glycoproteins physically associate with peptide-loaded MHC class I heterodimers, resulting in retention of the class I complex in the ER and inhibition of binding of the invariant chain to class II DR- ⁇ in the ER. Binding of aggregates, leading to mislocalization of the class II complex and reduced peptide loading.
  • US3 is able to interfere with the intracellular trafficking and maturation of MHC class I molecules during the early stages of HCMV infection.
  • US3 is an endoplasmic reticulum-resident membrane protein. Domain swapping experiments revealed that the luminal domain of US3 is sufficient for US3 to maintain the ER by itself, whereas both the luminal and transmembrane domains are required for the retention of MHC class I molecules in the ER.
  • US2 and US3 glycoproteins inhibit class II antigen presentation by disrupting or eliminating the function of class II proteins.
  • US2 binds to class II DRs and leads to rapid and efficient proteasome-mediated degradation of only the ⁇ -chain of the class II DR ⁇ complex.
  • US2 also leads to the degradation of the ⁇ -chain of DM, an MHC class II complex required for loading antigenic peptides onto class II DR complexes.
  • HCMV US3 binds to class II DR ⁇ heterodimers, inhibiting the association of the invariant chain (Ii), leading to intracellular mislocalization and reduced peptide loading of the DR complex.
  • HCMV US10 encodes an endoplasmic reticulum membrane glycoprotein that also interacts with components of MHC class I antigen presentation. US10 binds free class I heavy chains and delays their transport from the ER. However, US10 does not affect US2 or US11.
  • E19 The adenoviral gene product E3/19K (E19) can also retain class I molecules in the secretory pathway and interfere with antigen presentation.
  • E19 is also an endoplasmic reticulum-resident glycoprotein that abrogates cell surface transport of major histocompatibility complex class I (MHC-I) and MHC-I-associated chain A and B (MICA/B) molecules.
  • MHC-I major histocompatibility complex class I
  • MICA/B MHC-I-associated chain A and B
  • E3/19K contains three functional modules: a luminal domain for interaction with MHC-I and MICA/B molecules, a transmembrane domain, and a dilysine motif in the cytoplasmic tail that returns the Golgi apparatus to endoplasmic reticulum. Studies have shown that transmembrane domains (TMDs) and ER return signals are required to ensure efficient ER localization, transport inhibition of MHC-I and MICA/B molecules, and proteasomal degradation.
  • the HCMV L protein US6 affects antigen presentation through a completely different strategy.
  • US6 inhibits translocation of cytoplasmic peptides through the TAP complex (TAP1/2).
  • TAP1/2 TAP complex
  • US6 binds to the luminal side of the ER of TAP1 and causes a conformational change that prevents ATP binding.
  • Residues 89–108 in the ER-luminal domain of US6 contribute to the binding of US6 to TAP and are both necessary and sufficient for this repression.
  • This inhibition of TAP activity affects not only the expression of the classical MHC class I alleles, but also the expression of the nonclassical alleles HLA-C and HLA-G in fetal cytotrophoblast cells.
  • HSV ICP47 As the HCMV US6 protein, HSV ICP47 is expressed early in the infection cycle and is dispensable for in vitro replication, the same strategy could be applied to prevent class I assembly. ICP47 blocks TAP-mediated peptide transport and binds tightly to the TAP1-TAP2 complex. A clue to the mechanism by which ICP47 blocks TAP is that it exhibits high species selectivity. Both HSV1 and HSV2I CP47 inhibited simian, monkey, pig, dog and bovine TAP and had little effect on mouse, rat, guinea pig or rabbit TAP. ICP47 has an approximately 100-fold higher affinity for human TAP than for mouse TAP. ICP47 inhibits peptide binding to TAP but does not affect ATP binding. ICP47 has a 10-1000-fold higher affinity for TAP than most peptides, acts as a competitive inhibitor of peptide binding to TAP, and is thought to bind directly to the peptide-binding site.
  • ⁇ / ⁇ T lymphocytes recognize peptide-MHC ligands through a multimeric protein assembly known as the ⁇ T cell antigen receptor (TCR) CD3 complex.
  • TCR ⁇ T cell antigen receptor
  • the structure consists of a variable ⁇ TCR dimer that binds antigen and three invariant dimers (CD3 ⁇ , ⁇ , and ⁇ ) involved in TCR.CD3 surface trafficking, stabilization and signal transduction.
  • the ⁇ T-cell receptor ( ⁇ TCR) is expressed on the majority (approximately 95%) of T cells and plays a key role in T-cell activation by recognizing major histocompatibility complex (MHC)-anchored antigens.
  • MHC major histocompatibility complex
  • TCR-mediated T cell activation is a critical step in the pathogenesis of graft-versus-host disease (GVHD) during allogeneic hematopoietic cell transplantation (allo-HCT) and allogeneic CAR-T cell therapy.
  • GVHD graft-versus-host disease
  • allo-HCT allogeneic hematopoietic cell transplantation
  • allogeneic CAR-T cell therapy allogeneic CAR-T cell therapy.
  • the human leukocyte antigen (HLA) system or complex is a group of related proteins encoded by the human major histocompatibility complex (MHC) gene complex. These cell surface proteins are responsible for regulating the immune system.
  • HLA mismatch occurs when the donor's HLA on the allograft is different from the recipient's.
  • the HLA mismatch leads to the activation of alloreactive T cells, which causes acute cellular rejection (ACR) within six months of transplantation.
  • Mismatched donor HLA antigens are also targets for the development of neonatal donor-specific HLA antibodies (dnDSA), which play a potentiating role in acute and chronic transplant T-cell rejection.
  • graft-versus-host disease GVHD
  • HLA expression on allogeneic T cells is necessary to reduce rejection of recipient immune system against allogeneic T cell TCR ⁇ and/or HLA class I.
  • the purpose of this disclosure is to overcome the problems of tumor target diversity, tumor immunosuppressive environment, personalized treatment cell source, inability to standardize and scale production, and low killing efficiency in existing tumor cell therapy.
  • the aim is to provide a universal T (UT) module that can hijack the ERAD machinery to block TCR, MHC molecules in the ER, prevent their trafficking and facilitate their translocation into the cytoplasm for ubiquitination by the proteasome and degradation.
  • This design can target any protein (including endogenous or exogenous protein) to effectively inhibit expression and rapidly degrade for therapeutic purposes.
  • the concept of the UT assembly is based on the discovery that viral ER-resident glycoproteins can hijack ERAD regulatory machinery to inhibit/block MHC molecule assembly, trafficking or facilitate their ubiquitination and proteasomal degradation, thereby inhibiting MHC-mediated viral antigen presentation to evade immune surveillance.
  • Efficient downregulation of TCR will significantly suppress TCR-mediated immune attack and reduce GVHD during T cell allogeneic transfusion.
  • Inclusion of native viral ER-resident glycoproteins can further inhibit MHC molecules, thereby preventing peptide presentation to recipient CD8+ T cells and inhibiting immune recognition by allogeneic T cells.
  • UT components can improve the compatibility and long-term persistence of allogeneic T cells after infusion.
  • general T cells deficient in surface expression of TCR ⁇ and MHC molecules can be further genetically engineered for therapeutic purposes.
  • Another object of the present disclosure is to provide the introduction of NK elements, especially optimized recombinant NK elements, so that T cells can recognize all virus-infected cells and tumor cells as efficiently and broadly as NK cells.
  • CNK- T cells can efficiently activate and kill tumor cells.
  • NK targets include family member proteins such as MICA, MICB, and ULBP1–6, which can be widely expressed in various tumor cells and cover different stages of tumor progression, CNK technology can effectively solve the off-target effect of a single CAR-T, eliminate Chances of tumor immune escape.
  • the complex adapter (Chimeric Adaptor) introduced by CNK in the design can effectively transduce and amplify NK signals, overcome the limitations of immune checkpoints such as PD1 signals, efficiently activate T cells, and achieve tumor cell protection. kill.
  • the composite adapter (Chimeric Adapter) can amplify the signal of CNK-T cells, resist the immunosuppression in the tumor environment, realize the activation of T cells, and realize the killing of tumor cells; in addition, CNK-T cells recognize the target through NK Point, but also clear MDSC and other immunosuppressive cells. After the virus infects cells, it expresses a specific functional protein, the assembly or transport of MHCI or directly promotes the directional degradation of MHCI molecules, thereby inhibiting the presentation of viral antigenic epitopes and generating immune escape.
  • the present disclosure by redesigning viral elements and targeting TCR molecules, TCR molecular endoplasmic reticulum retention and directional degradation are realized. At the same time, the present disclosure also realizes the expression inhibition or directional degradation of MHCI molecules by introducing specific viral elements, thereby realizing the universality of T cells. type transformation.
  • CNK-UT has broad-spectrum tumor recognition and killing ability.
  • This disclosure further designs the compound specific target CAR/CNK-UT product, and demonstrates that the CAR/CNK-UT product has more powerful killing and activation functions on tumor cells than conventional CAR-T; in animal experiments, the CAR/CNK-UT product It also has a more efficient tumor clearance ability.
  • UT technology realizes allogeneic universal transformation, and T cells are derived from healthy donors, thereby realizing the standardized and large-scale production of CNK-UT products, which can be prepared in advance and guarantee the function of T cells to kill and activate tumor cells.
  • Fig. 1 shows the CNK-UT multi-functional complex (multi-functional complex) of four kinds of structures, wherein, Fig. 1A is basic CNK-UT multi-functional complex; Fig. 1 B is to increase targeting MHC I binding protein molecular domain The CNK-UT multifunctional complex; Figure 1C is the CNK-UT multifunctional complex that increases the binding protein molecular domain targeting MHC I and the complex adapter; Figure 1D increases the binding protein molecule targeting MHC I CNK-UT multifunctional complexes targeting tumor cell-killing receptors such as domains and CARs or TCRs.
  • Figure 2 shows the structures of four CNK-UT elements expressing CNK-UT multifunctional complexes, wherein, as shown in Figure 2A, a single lentiviral EF1 ⁇ promoter expression vector promotes the expression of one of the combinations of CNK-UT elements : DAP10-DAP12 ICD-T2A-NKG2D-p2A-anti-TCR-AdE3 ERAD; as shown in Figure 2B, use a single lentiviral EF1 ⁇ promoter expression vector to promote the expression of one of the combinations of CNK-UT elements: DAP10-DAP12 ICD -T2A-NKG2D-p2A-anti-TCR-US2 ERAD; as shown in Figure 2C, using a single lentiviral expression vector, using EF1 ⁇ and CMV promoters, respectively regulating DAP10-CD3 ⁇ -T2A-NKG2D-p2A-anti-TAA scFv -DAP10 and anti-TCR-AdE3 ERAD
  • Figure 3 shows the flow cytometric detection results of a basic phenotype of CNK-UT (DAP10-CD3 ⁇ -T2A-NKG2D-p2A-anti-TCR scFv-AdE3) cells.
  • Figure 4 shows the results of recognition and specific killing of CNK-UT cells on the human colon adenoma cell line HT29, wherein Figure 4A shows the expression of different NK target proteins in the human colon adenoma cell line HT29; Figure 4B It shows that CNK-UT cells have efficient killing and activating functions on HT29.
  • Figure 5 shows the recognition and specific killing of MDA-MB453 by CNK-UT cells, wherein Figure 5A shows the expression of different NK target proteins in the triple negative breast cancer cell line MDA-MB453; Figure 6B shows CNK-UT cells showed efficient killing and activating functions on THP1.
  • Figure 6 shows the recognition and specific killing of THP1 by CNK-UT cells, in which Figure 6A shows the expression of different NK targets of THP1 in acute myeloid leukemia cells; Figure B shows CAR/CNK-UT cells Compared with conventional CAR-T cells, it shows more efficient killing and activation functions on HepG2 cells.
  • Figure 7 shows that CNK-UT cells can upgrade conventional CAR-T technology to achieve more powerful targeted killing and activation capabilities, in which Figure 7A shows the GPC3, PD-L1 and different Expression of NK targets; Figure 7B shows
  • Figure 8 shows that CNK-UT cells can upgrade conventional CAR-T technology to achieve targeted killing and activation of tumor cells with high expression of PD-L1, wherein Figure 8A shows the GPC3 of hepatocellular carcinoma (HCC) PLC , PD-L1 and the expression of different NK targets; Figure 8B shows that CNK-UT cells show more efficient killing and activation functions on HepG2 cells than conventional CAR-T cells.
  • HCC hepatocellular carcinoma
  • FIG. 9 shows that GPC3 CAR/CNK-UT cells have more efficient tumor clearance ability in mice than GPC3 CAR-T cells.
  • Figure 10 shows that GPC3 CAR/CNK-UT cells have a more efficient tumor clearance ability than GPC3 CAR-T.
  • FIG 11 shows that GPC3 CAR/CNK-UT can specifically recognize and kill triple-negative breast cancer cell MDA-MB453.
  • FIG. 12 shows that GPC3 CAR/CNK-UT can specifically recognize and kill acute myeloid leukemia cell line THP1.
  • Figure 13 shows the recognition, specific killing and activation ability of CNK-UT cells to renal cell carcinoma 786-O cells.
  • Figure 14 shows the ability of CNK-UT cells to recognize, specifically kill and activate renal cell carcinoma ACHN cells.
  • Figure 15 shows the ability of CNK-UT cells to recognize, specifically kill and activate lung epithelial adenoma A549 cells.
  • Figure 16 shows the ability of CNK-UT cells to recognize, specifically kill and activate the AML cell line UL60.
  • Figure 17 shows the ability of CNK-UT cells to recognize, specifically kill and activate AML cell line U937 (purchased from U937, Cat. No. CL-0239).
  • Figure 18 shows the ability of CNK-UT cells to recognize, specifically kill and activate AML cell line U937.
  • Figure 19 shows the ability of CNK-UT cells to recognize, specifically kill and activate the pancreatic cancer cell line PANC-1 (purchased from Proceed, Cat. No. CL-0184).
  • chimeric natural killer receptor refers to a composition comprising a chimeric NK activating receptor component and a chimeric NK signal transduction component.
  • CNK T cell refers to a T cell containing a CNK construct.
  • chimeric natural killer receptor-universal T cell refers to the expression of NK activation receptor component constructs, CNK signal transduction component constructs and UT component constructs of T cells.
  • CAR/CNK-UT is a chimeric antigen receptor and chimeric natural killer receptor universal T cell (chimeric antigen receptor&chimeric natural killer receptor-Universal T cell, CNK-UT), which refers to the expression of specific tumor antigen CAR structure, T cells of NK activating receptor module construct, CNK signal transduction module construct and UT module construct.
  • the term "about” refers to a measurable value, such as amount, duration, etc., and includes ⁇ 20%, ⁇ 10%, ⁇ 5%, ⁇ 1%, ⁇ 0.5% or ⁇ 0.1% variation mode.
  • antibody refers to an immunoglobulin molecule that specifically binds to an antigen.
  • Antibodies can be intact immunoglobulins derived from natural or recombinant sources and can be immunoreactive portions of intact immunoglobulins.
  • Antibodies are generally tetramers of immunoglobulin molecules.
  • Antibodies in the present disclosure can exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab), as well as single chain antibodies and humanized antibodies.
  • costimulatory ligand includes molecules on antigen-presenting cells (such as APCs, dendritic cells, B cells and other immune cells) that specifically bind co-stimulatory cognates on T cells Molecules, thereby providing signals that mediate T cell responses including, but not limited to, proliferation, activation, differentiation, etc., in addition to primary signals provided by, for example, TCR/CD3 complex binding to peptide-loaded MHC molecules.
  • APCs antigen-presenting cells
  • dendritic cells dendritic cells
  • B cells and other immune cells
  • Costimulatory ligands may include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L) , intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, and Toll ligand receptor The bound agonist or antibody and the ligand that specifically binds to B7-H3.
  • Costimulatory ligands also include, inter alia, antibodies that specifically bind to costimulatory molecules present on T cells, such as but not limited to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function Associated antigen-1 (LFA-1), CD2, CD7LIGHT, NKG2C, B7-H3 and ligands that specifically bind to CD83.
  • antibodies that specifically bind to costimulatory molecules present on T cells such as but not limited to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function Associated antigen-1 (LFA-1), CD2, CD7LIGHT, NKG2C, B7-H3 and ligands that specifically bind to CD83.
  • LFA-1 lymphocyte function Associated antigen-1
  • costimulatory molecule or “costimulatory receptor” refers to an associated binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of the T cell, such as but not limited to proliferation.
  • Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, Toll ligand receptors. Costimulatory molecules also include non-naturally engineered proteins.
  • a "co-stimulatory signal” as used herein refers to a signal that combines with a primary signal, such as TCR/CD3 linkage, resulting in T cell proliferation and/or up- or down-regulation of key molecules.
  • stimulation refers to a primary response induced by binding of a stimulatory molecule (such as a TCR/CD3 complex) to its cognate ligand, thereby mediating a signal transduction event, such as, but not limited to, signal transduction through a TCR/CD3 complex guide.
  • a stimulatory molecule such as a TCR/CD3 complex
  • Stimulation may mediate altered expression of certain molecules, such as downregulation of TGF-B, and/or reorganization of cytoskeletal structures, among others.
  • stimulation molecule refers to a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen presenting cell.
  • a "stimulatory ligand” as used herein refers to a cognate binding partner (herein referred to as T-cell) that can bind to a cognate binding partner on a T-cell when present on an antigen-presenting cell (e.g., APC, dendritic cell, B-cell, etc.) "Stimulatory molecules”) specifically bind to ligands that mediate primary responses of T cells, including but not limited to activation, initiation of immune responses, proliferation, and the like.
  • Stimulatory ligands are well known in the art and include, inter alia, peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.
  • a “vector” is a composition of matter that contains an isolated nucleic acid and that can be used to deliver the isolated nucleic acid to the interior of a cell.
  • Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids and viruses.
  • the term “vector” includes autonomously replicating plasmids or viruses. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like.
  • viral vectors include, but are not limited to, lentiviruses, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, and the like.
  • non-viral vectors include, but are not limited to, CRISPR vector systems, Sleeping Beauty transposon systems, and the like.
  • Activated refers to the state of T cells that have been stimulated sufficiently to induce detectable cell proliferation. Activation can also be associated with induced cytokine production and detectable effector functions.
  • the term "activated T-cell” especially refers to a T-cell that is undergoing cell division.
  • the present disclosure provides a multi-functional complex comprising the following components:
  • NK cell activation receptor module which at least comprises NK cell activation receptor or its functional variant, and said NK cell activation receptor comprises: (a) NK cell activation receptor extracellular domain (ED) or its function Variants, (b) NK cell activating receptor transmembrane domain (TMD) or functional variant thereof, and (c) NK cell activating receptor intracellular domain (ICD) or functional variant thereof; optionally,
  • ED NK cell activation receptor extracellular domain
  • TMD NK cell activating receptor transmembrane domain
  • ICD NK cell activating receptor intracellular domain
  • the NK cell activating receptor extracellular domain or its functional variant, the NK cell activating receptor transmembrane domain or its functional variant and/or the NK cell activating receptor intracellular domain or its function contain hinges or joints between variants;
  • CNK signal transduction component which at least comprises (i) NK cell signal converter (adaptor) or its functional variant
  • described NK cell signal converter comprises: (a) NK cell signal converter extracellular domain (ED) or its functional variant, (b) NK cell signal converter transmembrane domain (TMD) or its functional variant, and (c) NK cell signal converter intracellular domain (ICD) or its functional variant
  • ED extracellular domain
  • TMD NK cell signal converter transmembrane domain
  • ICD NK cell signal converter intracellular domain
  • the NK cell signal converter extracellular domain or its functional variant, the NK cell signal converter transmembrane domain or its functional variant and/or the NK cell signal converter contains hinges or linkers between endodomains or functional variants thereof; and
  • (3) UT assembly which at least comprises (i) a recombinant protein molecule or a functional variant thereof targeting degradation of TCR, MHC and/or NK cell targets, said targeting degradation of TCR, MHC and/or NK cell targets
  • the recombinant protein molecule contains: (a) binding protein molecular domains or functional variants targeting TCR, MHC and/or NK cell targets, (b) transmembrane transmembrane of viral endoplasmic reticulum (ER) resident glycoproteins domain or its functional variant, and (c) the cytoplasmic domain of the viral endoplasmic reticulum resident glycoprotein or its functional variant; the transmembrane domain of the viral endoplasmic reticulum resident glycoprotein or its functional variant
  • the cytoplasmic domain of the endoplasmic reticulum resident glycoprotein of body and virus or its functional variant forms the ERAD degradation domain;
  • hinges or linkers are included between the NK-activating receptor module, the CNK signal transduction module and/or the UT module.
  • the NK cell activating receptor in the NK activating receptor module is selected from NKG2D, NKG2C, NKG2E, NKG2F, NKG2H, CD94, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS4, KIR3DS1, and natural cytotoxicity is regulated by body, TRAIL, DNAM-1, CD16a, 2B4, NTB-A, CRACC and NKp80; preferably, the natural cytotoxicity receptor is selected from NKp46, NKp44 and NKp30.
  • the NK cell activating receptor is a mammalian NK cell activating receptor; preferably, the mammal is selected from the group consisting of humans, primates, mice, horses, cattle, sheep, Goats, cats, pigs, dogs, llamas, alpacas, elephants, squirrels, guinea pigs.
  • the NK cell activating receptor is a recombinant NK cell activating receptor comprising NK cell activating receptor domains from different sources.
  • the NK cell activating receptor is a human NK cell activating receptor; preferably, the NK cell activating receptor is a recombinant human NK cell activating receptor domain comprising different NK cell activating receptor.
  • the NK cell activating receptor is a murine NK cell activating receptor; preferably, the NK cell activating receptor is a recombinant comprising different murine NK cell activating receptor domains NK cell activating receptor.
  • the NK cell activating receptor is a recombinant NK cell activating receptor comprising human and murine NK cell activating receptor domains.
  • the extracellular domain of the NK cell-activating receptor is the extracellular domain of a human or murine NK cell-activating receptor.
  • the transmembrane domain of the NK cell-activating receptor is the transmembrane domain of human or mouse NK cell-activating receptor.
  • the intracellular domain of the NK cell-activating receptor is the intracellular domain of a human or murine NK cell-activating receptor.
  • the functional variant of the NK cell activating receptor is selected from mutants of NK cell activating receptors, wild-type fusion proteins, or fusion proteins of wild-type and mutant types.
  • the extracellular domain of human NKG2D comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.1, preferably having 85%, 90%, 95%, 96%, 97% %, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the extracellular domain of human NKG2D is shown in SEQ ID NO.1.
  • the full-length sequence of human NKG2D comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.2, preferably having 85%, 90%, 95%, 96%, An amino acid sequence with 97%, 98%, 99% or more identity, more preferably an amino acid sequence with 98% or more 99% identity; the amino acid sequence of the full-length sequence of human NKG2D is shown in SEQ ID NO.2.
  • the extracellular domain of mouse NKG2D comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.3, preferably having 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, 99% or more identity, more preferably 98% or 99% or more identity amino acid sequence; the amino acid sequence of the extracellular domain of mouse NKG2D is shown in SEQ ID NO.3.
  • the full-length sequence of mouse NKG2D comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 4, preferably having 85%, 90%, 95%, 96% , 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the full-length sequence of mouse NKG2D is shown in SEQ ID NO.4.
  • the full-length sequence of human-mouse recombinant NKG2D comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 5, preferably having 85%, 90%, 95%, 96% %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the full-length sequence of human-mouse recombinant NKG2D is shown in SEQ ID NO.5 Show.
  • the full-length sequence of human NKG2C comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.6, preferably having 85%, 90%, 95%, 96%, An amino acid sequence with an identity of 97%, 98%, or 99%, more preferably an amino acid sequence with an identity of 98% or more; the amino acid sequence of the full-length sequence of human NKG2C is shown in SEQ ID NO.6.
  • the full-length sequence of human NKG2E comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 7, preferably having 85%, 90%, 95%, 96%, An amino acid sequence with an identity of 97%, 98%, or 99%, more preferably an amino acid sequence with an identity of 98% or more; the amino acid sequence of the full-length sequence of human NKG2E is shown in SEQ ID NO.7.
  • the full-length sequence of human NKG2F comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 8, preferably having 85%, 90%, 95%, 96%, An amino acid sequence with 97%, 98%, or 99% or more identity, more preferably an amino acid sequence with 98% or more 99% identity; the amino acid sequence of the full-length sequence of human NKG2F is shown in SEQ ID NO.8.
  • the full-length sequence of human CD94 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.9, preferably having 85%, 90%, 95%, 96%, An amino acid sequence with an identity of 97%, 98%, or 99%, more preferably an amino acid sequence with an identity of 98% or more; the amino acid sequence is shown in SEQ ID NO.9.
  • the full-length sequence of human KIR2DL4 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 10, preferably having 85%, 90%, 95%, 96%, An amino acid sequence with an identity of 97%, 98%, or 99% or more, more preferably an amino acid sequence with an identity of 98% or more than 99%; the amino acid sequence of the full-length sequence of human KIR2DL4 is shown in SEQ ID NO.10.
  • the full-length sequence of human KIR2DS1 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 11, preferably having 85%, 90%, 95%, 96%, An amino acid sequence with an identity of 97%, 98%, or 99%, more preferably an amino acid sequence with an identity of 98% or more; the amino acid sequence of the full-length sequence of human KIR2DS1 is shown in SEQ ID NO.11.
  • the full-length sequence of human KIR2DS2 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 12, preferably having 85%, 90%, 95%, 96%, An amino acid sequence with an identity of 97%, 98%, or 99%, more preferably an amino acid sequence with an identity of 98% or more; the amino acid sequence of the full-length sequence of human KIR2DS2 is shown in SEQ ID NO.12.
  • the full-length sequence of human KIR2DS4 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 13, preferably having 85%, 90%, 95%, 96%, An amino acid sequence with 97%, 98%, 99% or more identity, more preferably an amino acid sequence with 98% or more 99% identity; the amino acid sequence of the full-length sequence of human KIR2DS4 is shown in SEQ ID NO.13.
  • the full-length sequence of human KIR3DS1 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 14, preferably having 85%, 90%, 95%, 96%, An amino acid sequence with 97%, 98%, or 99% or more identity, more preferably an amino acid sequence with 98% or more 99% identity; the amino acid sequence of the full-length sequence of human KIR3DS1 is shown in SEQ ID NO.14.
  • the full-length sequence of human NKp46 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.15, preferably having 85%, 90%, 95%, 96%, An amino acid sequence with 97%, 98%, or 99% or more identity, more preferably an amino acid sequence with 98% or more 99% identity; the amino acid sequence of the full-length sequence of human NKp46 is shown in SEQ ID NO.15.
  • the full-length sequence of human NKp44 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.16, preferably having 85%, 90%, 95%, 96%, An amino acid sequence with 97%, 98%, 99% or more identity, more preferably an amino acid sequence with 98% or more 99% identity; the amino acid sequence of the full-length sequence of human NKp44 is shown in SEQ ID NO.16.
  • the full-length sequence of human NKp30 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.17, preferably having 85%, 90%, 95%, 96%, An amino acid sequence with an identity of 97%, 98%, or 99%, more preferably an amino acid sequence with an identity of 98% or more; the amino acid sequence of the full-length sequence of human NKp30 is shown in SEQ ID NO.17.
  • the full-length sequence of human DNAM1 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 18, preferably having 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, 99% or more identity, more preferably 98% or 99% or more identity amino acid sequence; the amino acid sequence of the full-length sequence of human DNAM1 is shown in SEQ ID NO.18.
  • the full-length sequence of human TRAIL comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 19, preferably having 85%, 90%, 95%, 96%, An amino acid sequence with 97%, 98%, or 99% or more identity, more preferably an amino acid sequence with 98% or more 99% identity; the amino acid sequence of the full-length sequence of human TRAIL is shown in SEQ ID NO.19.
  • the full-length sequence of human CD16a comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 20, preferably having 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, 99% or more identity, more preferably 98% or 99% or more identity amino acid sequence; the amino acid sequence of the full-length sequence of human CD16a is shown in SEQ ID NO.20.
  • the full-length sequence of human 2B4 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 21, preferably having 85%, 90%, 95%, 96%, An amino acid sequence with an identity of 97%, 98%, or 99%, more preferably an amino acid sequence with an identity of 98% or more; the amino acid sequence of the full-length sequence of human 2B4 is shown in SEQ ID NO.21.
  • the full-length sequence of human NTB-A comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 22, preferably having 85%, 90%, 95%, 96 %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the full-length sequence of human NTB-A is shown in SEQ ID NO.22 Show.
  • the full-length sequence of human CRACC comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 23, preferably having 85%, 90%, 95%, 96%, An amino acid sequence with an identity of 97%, 98%, or 99%, more preferably an amino acid sequence with an identity of 98% or more; the amino acid sequence of the full-length sequence of human CRACC is shown in SEQ ID NO.23.
  • the full-length sequence of human NKp80 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 24, preferably having 85%, 90%, 95%, 96%, An amino acid sequence with an identity of 97%, 98%, or 99% or more, more preferably an amino acid sequence with an identity of 98% or more than 99%; the amino acid sequence of the full-length sequence of human NKp80 is shown in SEQ ID NO.24.
  • the NK cell signal transducer in the CNK signal transduction module is DAP10 or DAP12.
  • the NK cell signal converter is a mammalian-derived NK cell signal converter; preferably, the mammal is selected from the group consisting of humans, primates, mice, horses, cattle, sheep, Goats, cats, pigs, dogs, llamas, alpacas, elephants, squirrels, guinea pigs.
  • the NK cell signal converter is a recombinant NK cell signal converter comprising NK cell signal converter domains from different sources.
  • the NK cell signal converter is a human NK cell signal converter; preferably, the NK cell signal converter is a recombinant comprising different human NK cell signal converter domains NK cell signal converter.
  • the NK cell signal transducer is a murine NK cell signal transducer; preferably, the NK cell signal transducer is a recombinant comprising different murine NK cell signal transducer domains NK cell signal converter.
  • the NK cell signal transducer is a recombinant NK cell signal transducer comprising human and murine NK cell signal transducer domains.
  • the extracellular domain of the NK cell signal converter is the extracellular domain of a human or mouse NK cell signal converter.
  • the transmembrane domain of the NK cell signal converter is the transmembrane domain of a human or mouse NK cell signal converter.
  • the intracellular domain of the NK cell signal converter is the intracellular domain of a human or mouse NK cell signal converter
  • the CNK cell signal converter functional variant is selected from a mutant of DAP10 or DAP12, or a fusion protein of DAP10 and DAP12, or a fusion of wild-type DAP10 or DAP12 and mutant DAP10 or DAP12 fusion protein.
  • the CNK signal transduction component further comprises (ii) an immunoreceptor activation signaling domain (ITAM) and/or (iii) a T cell co-stimulatory signaling domain.
  • ITAM immunoreceptor activation signaling domain
  • the NK cell signal converter or its functional variant, the immunoreceptor activation signaling domain (ITAM) and/or the T cell co-stimulatory signaling domain comprise A hinge or linker; preferably, said NK cell signal transducer or a functional variant thereof is fused to said immunoreceptor activation signaling domain (ITAM) domain.
  • the immunoreceptor activation signaling domain is derived from the intracellular activation signaling domain of an immune receptor; preferably, the immune receptor is selected from the group consisting of TCR ⁇ , CD2, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, FcR ⁇ , CD66d, Fc ⁇ RI, Fc ⁇ RI, Fc ⁇ RII, Fc ⁇ RIII, Dectin-1, CLEC-1, CD72, CD79A, CD79B; preferably, the immune receptor activation signaling domain (ITAM ) is fused to an NK cell signal transducer or a functional variant thereof; preferably, the immune receptor is CD3 ⁇ .
  • the immune receptor is CD3 ⁇ .
  • the T cell co-stimulatory signaling domain is derived from the intracellular signaling domain of a co-stimulatory molecule; preferably, the co-stimulatory molecule is selected from the group consisting of MHC class I molecules, TNF receptor proteins, immune Globulin-like protein, cytokine receptor, integrin, signaling molecule for lymphocyte activation (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor, OX40, CD2, CD7, CD16, CD27, CD28, CD30, CD40, CD38, CD35, CD79A, CD79B, CDS, ICAM-1, LFA-1, (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46,
  • the full-length sequence of human DAP10 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.25, preferably having 85%, 90%, 95%, 96%, An amino acid sequence with 97%, 98%, or 99% or more identity, more preferably an amino acid sequence with 98% or more 99% identity; the amino acid sequence of the full-length sequence of human DAP10 is shown in SEQ ID NO.25.
  • the full-length sequence of human DAP10 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.26, preferably having 85%, 90%, 95%, 96%, An amino acid sequence with 97%, 98%, or 99% or more identity, more preferably an amino acid sequence with 98% or more 99% identity; the amino acid sequence of the full-length sequence of human DAP10 is shown in SEQ ID NO.26.
  • the transmembrane domain of human DAP10 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.27, preferably having 85%, 90%, 95%, 96% , 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the transmembrane domain of human DAP10 is shown in SEQ ID NO.27.
  • the full-length sequence of human DAP12 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.28, preferably having 85%, 90%, 95%, 96%, An amino acid sequence with 97%, 98%, or 99% or more identity, more preferably an amino acid sequence with 98% or more 99% identity; the amino acid sequence of the full-length sequence of human DAP12 is shown in SEQ ID NO.28.
  • the transmembrane domain of human DAP12 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.29, preferably having 85%, 90%, 95%, 96% , 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the transmembrane domain of human DAP12 is shown in SEQ ID NO.29.
  • the transmembrane domain fusion protein of human DAP10 and human DAP12 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.30, preferably having 85%, 90%, Amino acid sequences with 95%, 96%, 97%, 98%, 99% or more identity, more preferably amino acid sequences with 98% or more 99% identity; the amino acid sequences of the transmembrane domains of human DAP10 and human DAP12 are as follows: Shown in SEQ ID NO.30.
  • the human DAP10-DAP12 fusion protein sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.31, preferably having 85%, 90%, 95%, 96% , 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the human DAP10-DAP12 fusion protein sequence is shown in SEQ ID NO.31.
  • the human CD3zeta intracellular signaling domain sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.32, preferably having 85%, 90%, 95%, Amino acid sequences with 96%, 97%, 98%, 99% or more identity, more preferably amino acid sequences with 98% or more 99% identity; the amino acid sequence of human CD3zeta intracellular signaling domain sequence is as SEQ ID NO. 32.
  • the human DAP10-CD3zeta sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.33, preferably having 85%, 90%, 95%, 96%, 97% %, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the human DAP10-CD3zeta sequence is shown in SEQ ID NO.33.
  • the human DAP12-CD3zeta sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.34, preferably having 85%, 90%, 95%, 96%, 97% %, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the human DAP12-CD3zeta sequence is shown in SEQ ID NO.34.
  • the human DAP10-DAP12-CD3zeta sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.35, preferably having 85%, 90%, 95%, 96% , 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the human DAP10-DAP12-CD3zeta sequence is shown in SEQ ID NO.35.
  • the human 41BB intracellular signaling domain sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.36, preferably having 85%, 90%, 95%, Amino acid sequences with 96%, 97%, 98%, 99% or more identity, more preferably amino acid sequences with 98% or more 99% identity; the amino acid sequence of human 41BB intracellular signaling domain sequence is as SEQ ID NO. 36.
  • the human DAP10-41BB sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.37, preferably having 85%, 90%, 95%, 96%, 97% %, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the human DAP10-41BB sequence is shown in SEQ ID NO.37.
  • the human DAP10-41BB-CD3zeta sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.38, preferably having 85%, 90%, 95%, 96% , 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the human DAP10-41BB-CD3zeta sequence is shown in SEQ ID NO.38.
  • the human CD28 intracellular signaling domain sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.39, preferably having 85%, 90%, 95%, Amino acid sequences with 96%, 97%, 98%, 99% or more identity, more preferably amino acid sequences with 98% or more 99% identity; the amino acid sequence of human CD28 intracellular signaling domain sequence is as SEQ ID NO. 39.
  • the human DAP10-CD28 sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.40, preferably having 85%, 90%, 95%, 96%, 97% %, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the human DAP10-CD28 sequence is shown in SEQ ID NO.40.
  • the human DAP10-CD28-CD3zeta sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.41, preferably having 85%, 90%, 95%, 96% , 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the human DAP10-CD28-CD3zeta sequence is shown in SEQ ID NO.41.
  • the human DAP12-41BB sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.42, preferably having 85%, 90%, 95%, 96%, 97% %, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the human DAP12-41BB sequence is shown in SEQ ID NO.42.
  • the human DAP12-41BB-CD3zeta sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.43, preferably having 85%, 90%, 95%, 96% , 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the human DAP12-41BB-CD3zeta sequence is shown in SEQ ID NO.43.
  • the human DAP12-CD28 sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.44, preferably having 85%, 90%, 95%, 96%, 97% %, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the human DAP12-CD28 sequence is shown in SEQ ID NO.44.
  • the human DAP12-CD28-CD3zeta sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.45, preferably having 85%, 90%, 95%, 96% , 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the human DAP12-CD28-CD3zeta sequence is shown in SEQ ID NO.45.
  • the TCR-targeting binding protein molecule domain or its functional variant is from a TCR antibody or a functional fragment thereof or a combination thereof.
  • the antibody is selected from TCR ⁇ antibody, TCR ⁇ antibody, TCR ⁇ antibody, TCR ⁇ antibody, TCR ⁇ antibody, TCR ⁇ antibody, TCR V ⁇ 2 antibody, TCR C ⁇ 1 antibody;
  • the functional fragment of the antibody is selected from Fd, Fv , Fab, Fab', F(ab') 2, Fv (scFv), single chain antibody (scFv) or nanobody (nanobody), diabody, triabody and four chain antibody;
  • the TCR antibody It is a TCR single-chain antibody;
  • the amino acid sequence of the TCR single-chain antibody comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.116, preferably having 85%, 90%, 95% , 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences;
  • the amino acid sequence of the full-length sequence of the TCR single-chain antibody is as shown in SEQ ID Show
  • the ERAD degradation domain in the UT module is from HCMV glycoprotein US2, US3, US11 or US10, adenovirus E3-19K or HHV-7US21.
  • the full-length sequence of the HCMV glycoprotein US2 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.46, preferably having 85%, 90%, 95% , 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the full-length sequence of the HCMV glycoprotein US2 is as SEQ ID Shown in NO.46.
  • the HLA binding domain of the HCMV glycoprotein US2 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.47, preferably having 85%, 90%, 95% %, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the HLA binding domain of the HCMV glycoprotein US2 is as follows Shown in SEQ ID NO.47.
  • the ERAD degradation domain of the HCMV glycoprotein US2 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.48, preferably having 85%, 90%, 95% %, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the ERAD degradation domain of the HCMV glycoprotein US2 is as follows Shown in SEQ ID NO.48.
  • the full-length sequence of the HCMV glycoprotein US3 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.49, preferably having 85%, 90%, 95% , 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the full-length sequence of the HCMV glycoprotein US3 is as SEQ ID Shown in NO.49.
  • the HLA binding domain of the HCMV glycoprotein US3 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.50, preferably having 85%, 90%, 95% %, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the HLA binding domain of the HCMV glycoprotein US3 is as follows Shown in SEQ ID NO.50.
  • the ERAD degradation domain of the HCMV glycoprotein US3 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.51, preferably having 85%, 90%, 95% %, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the ERAD degradation domain of the HCMV glycoprotein US3 is as follows Shown in SEQ ID NO.51.
  • the full-length sequence of the HCMV glycoprotein US11 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.52, preferably having 85%, 90%, 95% , 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the full-length sequence of the HCMV glycoprotein US11 is as SEQ ID Shown in NO.52.
  • the MHC binding domain of the HCMV glycoprotein US11 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.53, preferably having 85%, 90%, 95% %, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the MHC binding domain of the HCMV glycoprotein US11 is as follows Shown in SEQ ID NO.53.
  • the ERAD degradation domain of the HCMV glycoprotein US11 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.54, preferably having 85%, 90%, 95% %, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the ERAD degradation domain of the HCMV glycoprotein US11 is as follows Shown in SEQ ID NO.54.
  • the full-length sequence of the HCMV glycoprotein US10 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.55, preferably having 85%, 90%, 95% , 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the full-length sequence of the HCMV glycoprotein US10 is as SEQ ID Shown in NO.55.
  • the HLA binding domain of the HCMV glycoprotein US10 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.56, preferably having 85%, 90%, 95% %, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the HLA binding domain of the HCMV glycoprotein US10 is as follows Shown in SEQ ID NO.56.
  • the ERAD degradation domain of the HCMV glycoprotein US10 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.57, preferably having 85%, 90%, 95% %, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the ERAD degradation domain of the HCMV glycoprotein US10 is as follows Shown in SEQ ID NO.57.
  • the full-length sequence of the adenovirus E3-19K comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.58, preferably having 85%, 90%, 95% %, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences;
  • the amino acid sequence of the full-length sequence of the adenovirus E3-19K is as follows Shown in SEQ ID NO.58.
  • the MHC binding domain of the adenovirus E3-19K comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.59, preferably having 85%, 90%, An amino acid sequence with 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence with 98% or more identity; the amino acid of the MHC binding domain of the adenovirus E3-19K The sequence is shown in SEQ ID NO.59.
  • the ERAD degradation domain of the adenovirus E3-19K comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.60, preferably having 85%, 90%, An amino acid sequence with 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence with 98% or more 99% identity; the amino acid of the ERAD degradation domain of the adenovirus E3-19K The sequence is shown in SEQ ID NO.60.
  • the full-length sequence of HHV-7US21 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.61, preferably having 85%, 90%, 95%, Amino acid sequences with 96%, 97%, 98%, 99% or more identity, more preferably amino acid sequences with 98% or more 99% identity; the amino acid sequence of the full-length sequence of HHV-7US21 is as SEQ ID NO. 61.
  • the MHC binding domain of HHV-7US21 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.62, preferably having 85%, 90%, 95% , 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the MHC binding domain of HHV-7US21 is as SEQ ID Shown in NO.62.
  • the ERAD degradation domain of HHV-7US21 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.63, preferably having 85%, 90%, 95% , 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the ERAD degradation domain of HHV-7US21 is as SEQ ID Shown in NO.63.
  • the UT module further comprises (ii) a binding protein molecular domain targeting MHC I and/or MHC II or a functional variant thereof.
  • the binding protein molecular domain targeting MHC I and/or MHC II or a functional variant thereof is an HLA-targeting binding protein molecular domain or a functional variant thereof.
  • the binding protein molecular domains or functional variants thereof targeting MHC I and/or MHC II are from viral endoplasmic reticulum proteins that inhibit the expression of MHC molecules or promote their degradation; preferably, the The viral endoplasmic reticulum glycoprotein is selected from HCMV US6, HSV ICP47, CPXV012, HPV E6/E7, EBV BNFL2a or BHV UL49.5; preferably, the binding protein molecular domain targeting MHC I and/or MHC II or a functional variant thereof comprising a TAP binding domain.
  • the full-length sequence of HCMV US6 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.64, preferably having 85%, 90%, 95%, 96% %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the full-length sequence of HCMV US6 is shown in SEQ ID NO.64 Show.
  • the TAP binding domain of HHV-7US6 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.65, preferably having 85%, 90%, 95% , 96%, 97%, 98%, 99% or more of an amino acid sequence of identity, more preferably an amino acid sequence of 98% or more of 99% identity; the amino acid sequence of the TAP binding domain of HHV-7US6 is as SEQ ID Shown in NO.65.
  • the full-length sequence of the HSV ICP47 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.66, preferably having 85%, 90%, 95%, 96 %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the full-length sequence of the HSV ICP47 is shown in SEQ ID NO.66 Show.
  • the TAP binding domain of the HSV ICP47 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.67, preferably having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the TAP binding domain of the HSV ICP47 is as SEQ ID NO. 67.
  • the full-length sequence of CPXV012 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.68, preferably having 85%, 90%, 95%, 96% , 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the full-length sequence of CPXV012 is shown in SEQ ID NO.68.
  • the TAP binding domain of CPXV012 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.69, preferably having 85%, 90%, 95%, 96% %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the TAP binding domain of CPXV012 is as shown in SEQ ID NO.69 Show.
  • the full-length sequence of EBV BNFL2a comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.70, preferably having 85%, 90%, 95%, 96% %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the full-length sequence of EBV BNFL2a is as shown in SEQ ID NO.70 Show.
  • the TAP binding domain of the EBV BNFL2a comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.71, preferably having 85%, 90%, 95%, Amino acid sequences with 96%, 97%, 98%, 99% or more identity, more preferably amino acid sequences with 98% or more 99% identity; the amino acid sequence of the TAP binding domain of EBV BNFL2a is as SEQ ID NO. 71.
  • the full-length sequence of BHV UL49.5 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.72, preferably having 85%, 90%, 95% , 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the full-length sequence of BHV UL49.5 is shown as SEQ ID Shown in NO.72.
  • the TAP binding domain of BHV UL49.5 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.73, preferably having 85%, 90%, 95% %, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the TAP binding domain of BHV UL49.5 is as follows Shown in SEQ ID NO.73.
  • the binding protein molecular domains or functional variants thereof targeting MHC I and/or MHC II are derived from viral glycoproteins that degrade MHC and/or MHC II molecules; preferably, viral glycoproteins selected from HCMV glycoprotein US2, US3, US11 or US10, adenovirus E3-19K or HHV-7US21.
  • the full-length sequence of US2 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.74, preferably having 85%, 90%, 95%, 96% , 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the full-length sequence of US2 is shown in SEQ ID NO.74.
  • the HLA binding domain of US2 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.75, preferably having 85%, 90%, 95%, 96% %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the HLA binding domain of US2 is shown in SEQ ID NO.75 Show.
  • the ERAD degradation domain of US2 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.76, preferably having 85%, 90%, 95%, 96 %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the ERAD degradation domain of US2 is shown in SEQ ID NO.76 Show.
  • the full-length sequence of US3 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.77, preferably having 85%, 90%, 95%, 96% , 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the full-length sequence of US3 is shown in SEQ ID NO.77.
  • the HLA binding domain of US3 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.78, preferably having 85%, 90%, 95%, 96% %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the HLA binding domain of US3 is shown in SEQ ID NO.78 Show.
  • the ERAD degradation domain of US3 comprises an amino acid sequence with 80% or more identity to the amino acid sequence shown in SEQ ID NO.79, preferably 85%, 90%, 95%, 96% %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the ERAD degradation domain of US3 is as shown in SEQ ID NO.79 Show.
  • the full-length sequence of US11 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.80, preferably having 85%, 90%, 95%, 96% , 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the full-length sequence of US11 is shown in SEQ ID NO.80.
  • the HLA binding domain of US11 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.81, preferably having 85%, 90%, 95%, 96 %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the HLA binding domain of US11 is shown in SEQ ID NO.81 Show.
  • the ERAD degradation domain of US11 comprises an amino acid sequence with 80% or more identity to the amino acid sequence shown in SEQ ID NO.82, preferably 85%, 90%, 95%, 96% %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the ERAD degradation domain of US11 is shown in SEQ ID NO.82 Show.
  • the binding protein molecular domains targeting MHC I and/or MHC II or functional variants thereof further comprise directed inhibition or degradation of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 or A viral protein of the NK target protein of ULBP6; preferably, the viral protein is selected from HCMV UL16, UL141, UL142 or adenovirus E3-19K.
  • the full-length sequence of HCMV UL16 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.83, preferably having 85%, 90%, 95%, 96% %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the full-length sequence of HCMV UL16 is shown in SEQ ID NO.83 Show.
  • the NK target protein binding domain of HCMV UL16 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.84, preferably having 85%, 90%, An amino acid sequence with 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence with 98% or more 99% identity; the amino acid of the NK target protein binding domain of HCMV UL16 The sequence is shown in SEQ ID NO.84.
  • the ERAD degradation domain of HCMV UL16 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.85, preferably having 85%, 90%, 95%, Amino acid sequences with 96%, 97%, 98%, 99% or more identity, more preferably amino acid sequences with 98% or more 99% identity; the amino acid sequence of the ERAD degradation domain of HCMV UL16 is as SEQ ID NO. 85.
  • the full-length sequence of HCMV UL141 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.86, preferably having 85%, 90%, 95%, 96% %, 97%, 98%, 99% or more identical amino acid sequences, more preferably having 98% or more than 99% identical amino acid sequences; the amino acid sequence of the full-length sequence of HCMV UL141 is shown in SEQ ID NO.86 Show.
  • the NK target protein binding domain of HCMV UL141 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.87, preferably having 85%, 90%, An amino acid sequence with 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence with 98% or more 99% identity; the amino acid of the NK target protein binding domain of HCMV UL141 The sequence is shown in SEQ ID NO.87.
  • the ERAD degradation domain of the HCMV UL141 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.88, preferably having 85%, 90%, 95%, Amino acid sequences with 96%, 97%, 98%, 99% or more identity, more preferably amino acid sequences with 98% or more 99% identity; the amino acid sequence of the ERAD degradation domain of HCMV UL141 is as SEQ ID NO. 88.
  • the full-length sequence of HCMV UL142 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.89, preferably having 85%, 90%, 95%, 96% %, 97%, 98%, 99% or more identical amino acid sequences, more preferably having 98% or more than 99% identical amino acid sequences; the amino acid sequence of the full-length sequence of HCMV UL142 is shown in SEQ ID NO.89 Show.
  • the MICA and ULBP3 binding domains of the HCMV UL142 comprise an amino acid sequence with 80% or more identity to the amino acid sequence shown in SEQ ID NO.90, preferably 85%, 90%, 95% %, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences;
  • the amino acid sequences of the MICA and ULBP3 binding domains of HCMV UL142 are as follows Shown in SEQ ID NO.90.
  • the Golgi resident domain of HCMV UL142 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.91, preferably having 85%, 90%, 95% , 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the Golgi resident domain of HCMV UL142 is as shown in SEQ ID Shown in NO.91.
  • the binding protein molecular domains or functional variants thereof targeting MHC I and/or MHC II also include viral proteins that transport MHC I molecules from the Golgi apparatus to lysosomes for degradation;
  • the viral protein is selected from HIV Nef, HIV Vpu, HHV-7 U21, HHV-8 KK3, HHV-8 KK5, MHV-68 MK3 and HTLV-1 p12.
  • the HIV Nef comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.92, preferably having 85%, 90%, 95%, 96%, 97% , 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of HIV Nef is shown in SEQ ID NO.92.
  • the HIV Vpu comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.93, preferably having 85%, 90%, 95%, 96%, 97% , 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the HIV Vpu is shown in SEQ ID NO.93.
  • the HHV-8 KK3 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.94, preferably having 85%, 90%, 95%, 96%, An amino acid sequence with an identity of 97%, 98%, or 99%, more preferably an amino acid sequence with an identity of 98% or above; the amino acid sequence of the HHV-8 KK3 is shown in SEQ ID NO.94.
  • the HHV-8 KK5 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.95, preferably having 85%, 90%, 95%, 96%, An amino acid sequence with an identity of 97%, 98%, or 99%, more preferably an amino acid sequence with an identity of 98% or above; the amino acid sequence of the HHV-8 KK5 is shown in SEQ ID NO.95.
  • the MHV-68 MK3 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.96, preferably having 85%, 90%, 95%, 96%, An amino acid sequence with 97%, 98%, 99% or more identity, more preferably an amino acid sequence with 98% or more 99% identity; the amino acid sequence of the MHV-68 MK3 is shown in SEQ ID NO.96.
  • the HTLV-1 p12 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.97, preferably having 85%, 90%, 95%, 96%, An amino acid sequence with 97%, 98%, 99% or more identity, more preferably an amino acid sequence with 98% or more 99% identity; the amino acid sequence of the HTLV-1 p12 is shown in SEQ ID NO.97.
  • the binding protein molecular domains or functional variants thereof targeting MHC I and/or MHC II also include a function that mediates MHC-polypeptide molecules returning to the endoplasmic reticulum from the Golgi apparatus and promoting its degradation.
  • Viral protein preferably, the viral protein comprises an MHC binding structure and a KDEL receptor binding domain; preferably, the viral protein is the Cowpox Virus protein CPXV203.
  • the full-length sequence of the vaccinia virus protein CPXV203 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.98, preferably having 85%, 90%, 95% , 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the full-length sequence of the vaccinia virus protein CPXV203 is as shown in SEQ ID Shown in NO.98.
  • the MHC binding domain of the vaccinia virus protein CPXV203 comprises an amino acid sequence with 80% or more identity to the amino acid sequence shown in SEQ ID NO.99, preferably 85%, 90%, 95% %, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences;
  • the amino acid sequence of the MHC binding domain of the vaccinia virus protein CPXV203 is as follows Shown in SEQ ID NO.99.
  • the KDEL receptor binding domain of the vaccinia virus protein CPXV203 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.100, preferably having 85%, 90% , 95%, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the KDEL receptor binding domain of the vaccinia virus protein CPXV203 The amino acid sequence is shown in SEQ ID NO.100.
  • the full-length sequence of the vaccinia virus protein CPXV203 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.101, preferably having 85%, 90%, 95% , 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the full-length sequence of the vaccinia virus protein CPXV203 is as shown in SEQ ID Shown in NO.101.
  • the MHC binding domain of the vaccinia virus protein CPXV203 comprises an amino acid sequence with 80% or more identity to the amino acid sequence shown in SEQ ID NO.102, preferably 85%, 90%, 95% %, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences;
  • the amino acid sequence of the MHC binding domain of the vaccinia virus protein CPXV203 is as follows Shown in SEQ ID NO.102.
  • the KDEL receptor binding domain of the vaccinia virus protein CPXV203 comprises an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO.103, preferably having 85%, 90% , 95%, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the KDEL receptor binding domain of the vaccinia virus protein CPXV203 The amino acid sequence is shown in SEQ ID NO.103.
  • the multifunctional complex further comprises component (4) complex adapter (Chimeric adapter) component and/or targeting and killing tumor cell receptor component;
  • the (4) composite adapter comprises: (i) an extracellular recognition domain targeting tumors; (ii) a transmembrane domain; and (iii) an intracellular signaling domain; optionally, the A hinge or linker is included between the tumor-targeting extracellular recognition domain, transmembrane domain and/or intracellular signaling domain;
  • the tumor-targeting extracellular recognition domain of the composite adapter component is selected from tumor antigen-specific binding domains, tumor microenvironment target antigen binding domains and/or chemotactic receptors targeting tumor microenvironment body.
  • the tumor-targeting extracellular recognition domain is selected from antibodies or functional fragments thereof, TCR or combinations thereof that can target and recognize tumor-associated antigens; the functional fragments of the antibodies are selected from Fd, Fv, Fab, Fab', F(ab')2, Fv (scFv), single chain antibody (scFv) or nanobody (nanobody), diabody, triabody and tetrabody.
  • the transmembrane domain of the composite adapter assembly is selected from the group consisting of NK cell activation receptor transmembrane domain, DAP10 transmembrane domain, DAP12 transmembrane domain, CD8 transmembrane domain , CD28 transmembrane domain, CD4 transmembrane domain, 4-1BB transmembrane domain, OX40 transmembrane domain, ICOS transmembrane domain, CTLA-4 transmembrane domain, PD-1 transmembrane domain, LAG -3 transmembrane domain, 2B4 transmembrane domain and BTLA transmembrane domain and combinations thereof; preferably, the NK cell activation receptor is selected from NKG2D, NKG2C, NKG2E, NKG2F, NKG2H, CD94, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS4, KIR3DS1, natural cytotoxicity receptors, TRAIL, DNAM-1, CD16
  • the intracellular signaling domain of the complex adapter module includes the intracellular signaling domain and/or co-stimulatory signaling domain of NK cell activating receptors.
  • the NK cell activating receptors are selected from NKG2D, NKG2C, NKG2E, NKG2F, NKG2H, CD94, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS4, KIR3DS1, natural cytotoxicity receptors, TRAIL, DNAM-1 , CD16a, 2B4, NTB-A, CRACC, and NKp80.
  • the intracellular signaling domain also includes a co-stimulatory signaling domain; preferably, the co-stimulatory signaling domain is selected from a T cell co-stimulatory signaling domain; including, but not Limited to those derived from MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte activation signaling molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors Body, OX40, CD2, CD7, CD16, CD27, CD28, CD30, CD40, CD38, CD35, CD79A, CD79B, CDS, ICAM-1, LFA-1, (CD11a/CD18), 4-1BB (CD137), B7 -H3, CDS, ICAM-1, ICOS(CD278), GITR, BAFFR, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80(KLRF1), NKp44,
  • the targeting and killing tumor cell receptor component comprises (i) an extracellular recognition domain targeting tumor antigens; (ii) a transmembrane domain; and (iii) an intracellular co-stimulatory signaling domain; (iv) T Cell activation signaling domain (ITAM); optionally, the extracellular recognition domain targeting tumor antigen, transmembrane domain, intracellular costimulatory signaling domain and/or T cell activation signaling domain (ITAM) containing hinges or joints between them;
  • the transmembrane domain targeting and killing tumor cell receptor components is selected from CD8 transmembrane domain, ⁇ and/or ⁇ chain transmembrane domain of T cell receptor, CD28 transmembrane domain, CD3 ⁇ transmembrane domain , CD45 transmembrane domain, CD4 transmembrane domain, CD5 transmembrane domain, CD8 transmembrane domain, CD9 transmembrane domain, CD16 transmembrane domain, CD22 transmembrane domain, CD33 transmembrane domain, CD37 Transmembrane domains, CD64 transmembrane domains, CD80 transmembrane domains, CD86 transmembrane domains, CD134 transmembrane domains, CD137 transmembrane domains, CD154 transmembrane domains, GITR transmembrane domains, and combinations thereof;
  • the T cell activation signaling domain is derived from CD3 ⁇ , common FcR ⁇ (FCER1G), Fc ⁇ RIIa, FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, CD278 ("ICOS"), Fc ⁇ RI CD66d, DAP10 and DAP12 and other intracellular signaling domains.
  • the linker is a flexible linker; preferably, the flexible linker comprises the amino acid sequence shown (Gly(x)Ser(y))n, wherein n is an integer from 1 to 10, and x and y are independently integers from 0 to 10, provided that x and y are not all 0; more preferably, the linker comprises the amino acid sequence shown in SEQ ID NO.105 or the amino acid shown in SEQ ID NO.106 sequence.
  • the linker is a hinge; preferably, the hinge is an IgG1 hinge or an IgG4 hinge.
  • the IgG1 hinge comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.106, preferably having 85%, 90%, 95%, 96%, 97% , 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the IgG1 hinge is shown in SEQ ID NO.106.
  • the IgG4 hinge comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.107, preferably having 85%, 90%, 95%, 96%, 97% , 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the IgG4 hinge is shown in SEQ ID NO.107.
  • cleavable peptides are included between the NK activating receptor module, CNK signal transduction module and/or UT module; for example, T2A peptide, GSG-T2A peptide, E2A peptide, GSG-E2A peptide , F2A peptide, GSG-F2A peptide, P2A peptide or GSG-P2A peptide.
  • the T2A comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.108, preferably having 85%, 90%, 95%, 96%, 97%, An amino acid sequence with an identity of 98% or more than 99%, more preferably an amino acid sequence with an identity of 98% or more; the amino acid sequence of T2A is shown in SEQ ID NO.108.
  • the amino acid sequence of the GSG-T2A peptide is shown in SEQ ID NO.109.
  • the P2A comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.110, preferably having 85%, 90%, 95%, 96%, 97%, An amino acid sequence with an identity of 98% or more than 99%, more preferably an amino acid sequence with an identity of 98% or more; the amino acid sequence of the P2A is shown in SEQ ID NO.110.
  • the amino acid sequence of the GSG-P2A peptide is shown in SEQ ID NO.111.
  • the E2A comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.112, preferably having 85%, 90%, 95%, 96%, 97%, An amino acid sequence with an identity of 98% or more than 99%, more preferably an amino acid sequence with an identity of 98% or more; the amino acid sequence of E2A is shown in SEQ ID NO.112.
  • the amino acid sequence of the GSG-E2A peptide is shown in SEQ ID NO.113.
  • the F2A comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.114, preferably having 85%, 90%, 95%, 96%, 97%, An amino acid sequence with an identity of 98% or more than 99%, more preferably an amino acid sequence with an identity of 98% or more; the amino acid sequence of the F2A is shown in SEQ ID NO.114.
  • amino acid sequence of the GSG-F2A peptide is shown in SEQ ID NO.115.
  • the multifunctional complex comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.116, preferably having 85%, 90%, 95%, 96% , 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the multifunctional complex comprises the amino acid sequence shown in SEQ ID NO.116 TCR antibody single chain antibody.
  • the multifunctional complex comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.117, preferably having 85%, 90%, 95%, 96% , 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the multifunctional complex is shown in SEQ ID NO.117.
  • the multifunctional complex comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.121, preferably having 85%, 90%, 95%, 96% , 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the multifunctional complex is shown in SEQ ID NO.121.
  • the multifunctional complex comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.123, preferably having 85%, 90%, 95%, 96% , 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the multifunctional complex is shown in SEQ ID NO.123.
  • the multifunctional complex comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.125, preferably having 85%, 90%, 95%, 96% , 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the multifunctional complex is shown in SEQ ID NO.125.
  • the multifunctional complex comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.12, preferably having 85%, 90%, 95%, 96% , 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the multifunctional complex is shown in SEQ ID NO.126.
  • the multifunctional complex comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.127, preferably having 85%, 90%, 95%, 96% , 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more than 99% identical amino acid sequences; the amino acid sequence of the multifunctional complex is shown in SEQ ID NO.127.
  • the multifunctional complex comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.128, preferably having 85%, 90%, 95%, 96% , 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the multifunctional complex is shown in SEQ ID NO.128.
  • the multifunctional complex comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.129, preferably having 85%, 90%, 95%, 96% , 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the multifunctional complex is shown in SEQ ID NO.129.
  • the multifunctional complex comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.130, preferably having 85%, 90%, 95%, 96% , 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the multifunctional complex is shown in SEQ ID NO.130.
  • the multifunctional complex comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.131, preferably having 85%, 90%, 95%, 96% , 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the multifunctional complex is shown in SEQ ID NO.131.
  • the present disclosure provides a nucleic acid molecule encoding the multifunctional complex.
  • the nucleic acid molecule is DNA or RNA.
  • the RNA is mRNA.
  • the nucleic acid molecule comprises a nucleotide sequence with 80% or more identity to the nucleotide sequence shown in SEQ ID NO.118, preferably 85%, 90%, 95%, Nucleotide sequences with 96%, 97%, 98%, 99% or more identity, more preferably amino acid sequences with 98% or more 99% identity; the nucleotide sequence of the nucleic acid molecule shown in SEQ ID NO.118 Show.
  • the nucleic acid molecule comprises a nucleotide sequence with 80% or more identity to the nucleotide sequence shown in SEQ ID NO.122, preferably 85%, 90%, 95%, Nucleotide sequences with 96%, 97%, 98%, 99% or more identity, more preferably amino acid sequences with 98% or 99% or more identity; the nucleotide sequence of the nucleic acid molecule shown in SEQ ID NO.122 Show.
  • the nucleic acid molecule comprises a nucleotide sequence with 80% or more identity to the nucleotide sequence shown in SEQ ID NO.124, preferably 85%, 90%, 95%, Nucleotide sequences with 96%, 97%, 98%, 99% or more identity, more preferably amino acid sequences with 98% or more 99% identity; the nucleotide sequence of the nucleic acid molecule is represented by SEQ ID NO.124 Show.
  • the present disclosure provides an expression vector comprising the nucleic acid.
  • the vector is selected from plasmids, cosmids, viral vectors, RNA vectors or linear or circular DNA or RNA molecules.
  • the viral vector is selected from the group consisting of retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated virus), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza virus), elastic Symptoviruses (e.g., Rabies and Vesicular Stomatitis Viruses), Paramyxoviruses (e.g., Myoviruses and Sendai), Positive-Strand RNA Viruses such as Picornaviruses and Alphaviruses, and Double-Stranded DNA Viruses, including Adenoviruses, herpesviruses (eg, herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus) and poxviruses (eg, vaccinia, fowlpox, and canarypox), Norwalk virus , togaviruses, flavivirus
  • the retrovirus is selected from avian leukoproliferative-sarcoma, mammalian C-type, B-type virus, D-type virus, HTLV-BLV collection, lentivirus, foamy virus.
  • the lentiviral vector is selected from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV or sheep demyelinating leukoencephalitis lentivirus.
  • the NK activating receptor module, CNK signal transduction module and/or UT module can be regulated and expressed under the same promoter of the same vector, or under different promoters, or expressed in multiple vectors.
  • the vector is a lentiviral vector
  • the gene encoding the NK activation receptor module, the CNK signal transduction module and/or the UT module contains a gene encoding a cleavable peptide; preferably, the The cleavable peptide is a 2A linker; the 2A linker is selected from T2A, P2A, E2A and F2A.
  • the vector further comprises a promoter; preferably, the promoter is an EF1 ⁇ promoter or a CMV promoter.
  • the present disclosure provides an immune cell comprising the nucleic acid or the expression vector.
  • the immune cells are selected from T cells, NKT cells, NK cells, B cells, monocytes, macrophages, etc.
  • the present disclosure provides a method for producing immune cells, which includes introducing the nucleic acid or the expression vector into the cells by a method selected from electroporation, sonication, gene gun (for example, with gene gun of Au-particles), lipofection, polymer transfection, nanoparticles or multimeric complexes.
  • a method for producing immune cells which includes introducing the nucleic acid or the expression vector into the cells by a method selected from electroporation, sonication, gene gun (for example, with gene gun of Au-particles), lipofection, polymer transfection, nanoparticles or multimeric complexes.
  • the present disclosure provides a pharmaceutical composition comprising the multifunctional complex, the nucleic acid, the expression vector, the immune cell and/or the immune cell produced by the method, and pharmaceutically acceptable carrier.
  • the present disclosure provides a multifunctional complex, the nucleic acid, the expression vector, the immune cell, the immune cell produced by the method and/or the pharmaceutical composition in Use in the preparation of medicines for treating diseases.
  • the present disclosure provides a method for treating a disease, comprising administering to a subject the multifunctional complex, the nucleic acid, the expression vector, the immune cell, and/or the pharmaceutical composition.
  • the diseases include various solid tumors and blood tumors, viral infectious diseases, and autoimmune diseases.
  • the solid tumor is selected from nervous system tumors, head and neck tumors, thoracic tumors, digestive system tumors, genitourinary system tumors, soft tissue and skin tumors, bone tumors and the like.
  • nervous system tumors include diffuse glioma, diffuse astrocytoma and anaplastic astrocytoma, glioblastoma, oligodendroglioma, oligoastrocytic tumors, diffuse glioma of childhood, other astrocytomas, ependymomas, neuronal and mixed neuronal-glioma tumors, medulloblastoma, other embryonal tumors, schwannomas, meningiomas, Solitary fibrous tumor and hemangiopericytoma, etc.
  • head and neck tumors include malignant tumors of the nasal cavity and sinuses, nasopharyngeal cancer, oral cavity cancer, laryngeal cancer, salivary gland tumors, intracranial tumors, thyroid cancer, tongue cancer, and the like.
  • thoracic tumors include lung cancer, esophageal cancer, cardia cancer, breast cancer, mediastinal tumors, and the like.
  • the digestive system tumors include gastric cancer, colorectal cancer, sigmoid colon and rectal cancer, liver cancer, pancreatic cancer and periampullary cancer, biliary tract cancer, malignant tumors of the small intestine, and the like.
  • urogenital tumors include renal cancer, prostate cancer, bladder cancer, testicular malignancy, penile cancer, cervical cancer, endometrial cancer, ovarian cancer, and the like.
  • soft tissue and skin tumors include malignant fibrous histiocytoma, rhabdomyosarcoma, synovial sarcoma, malignant melanoma of the skin, and the like.
  • bone tumors include osteosarcoma, Ewing's sarcoma, and the like.
  • the colon cancer is colon adenoma.
  • the breast cancer is triple negative breast cancer cells.
  • the liver cancer is hepatocellular carcinoma.
  • the disease is a hematological tumor selected from leukemia, lymphoma (HL), multiple myeloma (MM), myelodysplastic syndrome (MDS), etc.
  • HL lymphoma
  • MM multiple myeloma
  • MDS myelodysplastic syndrome
  • the leukemia is B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, acute myeloid leukemia, and the like.
  • viral infectious diseases include: viral diseases of the respiratory tract, viral diseases of the gastrointestinal tract, viral diseases of the liver, viral diseases of the skin and mucous membranes, viral diseases of the eye, viral diseases of the central nervous system, lymphatic Cellular viral diseases, insect-borne viral diseases, lentivirus infection diseases, etc.
  • respiratory viral diseases include infections by rhinovirus, adenovirus, respiratory syncytial virus, parainfluenza virus, and coronavirus; influenza; mumps, and the like.
  • gastrointestinal viral diseases include polio; Cooksackie virus infection; ECHO virus infection; viral gastroenteritis: including rotavirus gastroenteritis, norovirus Gastroenteritis, adenovirus gastroenteritis, astrovirus gastroenteritis, coronavirus gastroenteritis and calicivirus gastroenteritis, etc.
  • viral diseases of the liver include viral hepatitis A, viral hepatitis B, viral hepatitis C, viral hepatitis D, viral hepatitis E, Epstein-Barr viral hepatitis and giant cell viral hepatitis etc.
  • viral diseases of the skin and mucous membranes include measles, rubella, acute rash, chickenpox and herpes zoster, smallpox, herpes simplex virus infection, rabies, foot-and-mouth disease, and the like.
  • ocular viral diseases include epidemic keratoconjunctivitis, follicular conjunctivitis, herpetic keratoconjunctivitis, and the like.
  • viral diseases of the central nervous system include Japanese encephalitis, Western equine encephalitis, Eastern equine encephalitis, St. Louis encephalitis, Venezuelan equine encephalitis, Murray Valley encephalitis, California encephalitis encephalitis, encephalitis, and lymphocytic choroid plexus meningitis.
  • lymphocytic viral diseases include infectious mononucleosis, cytomegalovirus infection, acquired immunodeficiency syndrome, and the like.
  • the insect-borne viral diseases include viral hemorrhagic fevers: including epidemic hemorrhagic fever, yellow fever, Crimean-Congo hemorrhagic fever, Rift Valley fever, Argentine hemorrhagic fever, Venezuelan hemorrhagic fever, Lassa fever, Omsk hemorrhagic fever, Marburg disease, Ebola hemorrhagic fever, etc.; dengue fever and dengue hemorrhagic fever; West Nile fever; Colorado tick heat transfer; sandfly fever, etc.
  • lentiviral infection diseases include subacute sclerosing panencephalitis, Kuru's disease, progressive multifocal leukoencephalopathy and subacute spongiform encephalopathy (corticostriatal spinal cord degeneration) and the like.
  • autoimmune diseases include organ-specific autoimmune diseases and systemic autoimmune diseases
  • organ-specific autoimmune diseases include chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhage nephritic syndrome, vulgaris Pemphigus, pemphigoid, primary biliary cirrhosis, multiple sclerosis, acute idiopathic polyneuritis, etc.
  • systemic autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune Hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, etc.
  • the present disclosure provides a method of stimulating an immune response in a subject, the method comprising administering to the subject an effective amount of the multifunctional complex, the nucleic acid, the expression vector, the The immune cells, the immune cells produced by the method and/or the pharmaceutical composition.
  • the disclosure also provides cells, populations of cells, and compositions (including pharmaceuticals and therapeutic compositions) comprising said cells and populations, such as cells and populations produced by the methods provided, and methods, such as for administering said cells and compositions to methods of treatment of subjects such as patients.
  • compositions comprising cells for administration, including pharmaceutical compositions and formulations, such as compositions in unit dosage form comprising a number of cells for administration at a given dose or fraction thereof.
  • Pharmaceutical compositions and formulations generally comprise one or more optional pharmaceutically acceptable carriers or excipients.
  • the composition includes at least one additional therapeutic agent.
  • pharmaceutical formulation refers to a preparation in a form that permits the biological activity of the active ingredients contained therein to be effective, and that contains no additional components that would be unacceptably toxic to a subject to which the formulation will be administered.
  • “Pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation other than the active ingredient, which is nontoxic to the subject.
  • Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
  • the choice of vector is determined in part by the particular cell and/or method of administration. Accordingly, a variety of suitable formulations exist.
  • pharmaceutical compositions may contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. Preservatives or mixtures thereof are generally present in an amount of from about 0.0001% to about 2% by weight of the total composition.
  • Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives agents (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol, or benzyl alcohol; alkylparabens Esters such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues ) polypeptides; proteins such as serum albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine,
  • a buffer is included in the composition.
  • Suitable buffers include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate and various other acids and salts.
  • a mixture of two or more buffers is used. Buffering agents or mixtures thereof are generally present in an amount of from about 0.001% to about 4% by weight of the total composition. Methods for the preparation of administrable pharmaceutical compositions are known.
  • Formulations may include aqueous solutions.
  • a formulation or composition may also contain more than one active ingredient useful for the particular indication, disease or condition being treated with the cells, preferably those with complementary activities to the cells, where the respective activities do not adversely affect each other.
  • active ingredients are suitably present in combination in amounts effective for the intended purpose.
  • the pharmaceutical composition further comprises other pharmaceutically active agents or drugs, such as chemotherapeutic agents such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil , gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and/or vincristine.
  • chemotherapeutic agents such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil , gemcitabine, hydroxyurea, methotrexate,
  • a pharmaceutical composition comprises cells in an amount effective to treat or prevent a disease or condition, eg, a therapeutically effective amount or a prophylactically effective amount.
  • therapeutic or prophylactic efficacy is monitored by periodically assessing the treated subject.
  • the desired dose can be delivered by administering the cells as a single bolus, by administering the cells as multiple boluses, or by administering the cells as a continuous infusion.
  • Cells and compositions can be administered using standard administration techniques, formulations and/or devices. Administration of cells can be autologous or allogeneic. For example immune response cells or progenitor cells can be obtained from one subject and administered to the same subject or to a different, compatible subject.
  • the peripheral blood-derived immune response cells or their progeny eg, derived in vivo, ex vivo or in vitro
  • a therapeutic composition eg, a pharmaceutical composition comprising genetically modified immune response cells
  • it is usually formulated as a unit dose injectable form (solution, suspension, emulsion).
  • Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual or suppository administration.
  • the population of cells is administered parenterally.
  • parenteral as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal and intraperitoneal administration.
  • the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal or subcutaneous injection administration.
  • compositions are provided as sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which in some aspects can be buffered to a selected pH.
  • Liquid preparations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within an appropriate viscosity range to provide a longer contact time with a particular tissue.
  • Liquid or viscous compositions can comprise a carrier, which can be a solvent or dispersion medium comprising, for example, water, saline, phosphate buffered saline, polyol (eg glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.
  • a carrier which can be a solvent or dispersion medium comprising, for example, water, saline, phosphate buffered saline, polyol (eg glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.
  • Sterile injectable solutions can be prepared by introducing the cells into a solvent, for example mixed with a suitable carrier, diluent or excipient such as sterile water, physiological saline, glucose, dextrose and the like.
  • a suitable carrier diluent or excipient
  • the composition may contain auxiliary substances such as wetting, dispersing or emulsifying agents (e.g. methylcellulose), pH buffering agents, gelling or viscosity-increasing additives, preservatives, flavorings and/or colors, depending on the Route of administration and desired preparation.
  • auxiliary substances such as wetting, dispersing or emulsifying agents (e.g. methylcellulose), pH buffering agents, gelling or viscosity-increasing additives, preservatives, flavorings and/or colors, depending on the Route of administration and desired preparation.
  • suitable preparations for example mixed with a suitable carrier, diluent or excipient such as sterile water,
  • additives can be added to enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffering agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol and sorbic acid. Prolonged absorption of the injectable pharmaceutical form is brought about by the use of agents which delay absorption, for example, aluminum monostearate and gelatin.
  • Formulations for in vivo administration are generally sterile. Sterility is readily achieved by, for example, filtration through sterile membrane membranes.
  • cells, populations, and compositions are administered to a subject or patient suffering from a particular disease or condition to be treated, for example, by adoptive cell therapy (eg, adoptive T cell therapy).
  • adoptive cell therapy eg, adoptive T cell therapy
  • cells and compositions prepared by provided methods are administered to a subject, e.g., suffering from a disease or disorder or A subject at risk for a disease or condition.
  • methods thereby treat, eg, one or more symptoms of a disease or disorder, eg, by reducing tumor burden in cancers expressing antigens recognized by engineered T cells.
  • Methods of cell administration for adoptive cell therapy are known and can be used in conjunction with the provided methods and compositions.
  • methods of adoptive T cell therapy are described in, e.g., U.S. Patent Application Publication No. 2003/0170238 to Gruenberg et al; U.S. Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85) .
  • a "subject" as used herein is a mammal, such as a human or other animal, and typically a human.
  • the subject eg, patient, to whom the cell, cell population or composition is administered is a mammal, typically a primate such as a human.
  • the primate is a monkey or an ape.
  • a subject can be male or female, and can be of any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects.
  • the subject is a non-primate mammal, such as a rodent.
  • Treatment means to ameliorate or alleviate, in whole or in part, a disease or condition or disorder, or symptoms, adverse effects or consequences associated therewith or Phenotype. Desired therapeutic effects include, but are not limited to, prevention of occurrence or recurrence of the disease, alleviation of symptoms, alleviation of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction of the rate of disease progression, remission or alleviation of the disease state, and remission or improvement of prognosis. These terms do not imply a complete cure of the disease or complete elimination of any symptoms or effects of all symptoms or consequences.
  • delaying the development of a disease refers to delaying, hindering, slowing, retarding, stabilizing, inhibiting and/or delaying the development of a disease (eg, cancer). This delay can be of varying lengths of time depending on the history of the disease and/or the individual being treated. It will be apparent to those skilled in the art that a sufficient or significant delay may actually include prevention, since the individual does not develop the disease. For example, the development of advanced cancers such as metastases may be delayed.
  • Preventing includes providing prophylaxis against the occurrence or recurrence of a disease in a subject who may be predisposed to the disease but has not yet been diagnosed with the disease.
  • provided cells and compositions are used to delay the development of a disease or slow the progression of a disease.
  • “Inhibiting” a function or activity as used herein means reducing the function or activity when compared to the same conditions (except the condition or parameter of interest), or when compared to another condition. For example, a cell that inhibits tumor growth reduces the growth rate of the tumor compared to the growth rate of the tumor in the absence of the cell.
  • an "effective amount" of an agent means, at a dose/amount and for a period of time necessary, effective to achieve the desired result For example the amount of treatment or prevention results.
  • a “therapeutically effective amount” of an agent refers to a dosage and period of time necessary to achieve a desired therapeutic effect (e.g., for the treatment of a disease, condition, or disorder, and/or the pharmacokinetics of the treatment). or pharmacodynamic effects).
  • a therapeutically effective amount can vary depending on factors such as the disease state, age, sex, and weight of the subject, and the cell population administered.
  • provided methods comprise administering cells and/or compositions in an effective amount, eg, a therapeutically effective amount.
  • prophylactically effective amount refers to an amount effective to achieve the desired prophylactic result at the dosage and time period necessary. Usually, but not necessarily, a prophylactically effective amount will be less than a therapeutically effective amount because prophylactic doses are used in a subject prior to or early in the illness. In the case of lower tumor burdens, the prophylactically effective amount will in some aspects be higher than the therapeutically effective amount.
  • cells or individual populations or subtypes of cells are administered to a subject in the range of about 1 million to about 100 billion cells, e.g., 1 million to about 50 billion cells (e.g., about 500 billion cells) 10,000 cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or any two of the above range of values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or any range defined by any two of the above values ), and in some cases, from about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells cells, about 800 million cells, about 900 million cells,
  • the dose of total cells and/or the dose of individual subpopulations of cells is between at or about 10 cells/kg (kg) body weight to at or about 10 cells/kg (kg) body weight In the range of, for example, between 10 5 and 10 6 cells/kg body weight, for example, at least or at least about or at or about 1 ⁇ 10 5 cells/kg, 1.5 ⁇ 10 5 cells/kg, 2 ⁇ 10 5 cells/kg or 1 ⁇ 10 6 cells/kg body weight.
  • the cells are administered at or between at or about 104 and at or about 109 T cells per kilogram (kg) body weight or within a certain error range thereof, such as between 105 and 106 T cells between/kg body weight, for example at least or at least about or at or about 1 ⁇ 10 5 T cells/kg, 1.5 ⁇ 10 5 T cells/kg, 2 ⁇ 10 5 T cells/kg, or 1 ⁇ 10 6 T cells/kg body weight.
  • the cells may be administered by any suitable means, such as by bolus injection, by injection, such as intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, Intrachoroidal, intracameral, subperineal, subconjunctival, sub-Tenon, retrobulbar, peribulbar, or posterior juxtascleral delivery.
  • they are administered parenterally, intrapulmonarily, and intranasally, and if local treatment is desired, intralesional.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.
  • a given dose is administered by a single bolus administration of the cells. In some embodiments, it is administered by multiple boluses of the administered cells, eg, over a period not exceeding 3 days, or by continuous infusion of the administered cells.
  • a repeated dose approach wherein a first dose of cells is administered followed by one or more second consecutive doses.
  • the timing and size of multiple doses of cells is typically designed to increase the potency and/or activity and/or function of antigen-expressing T cells (eg, CAR-expressing T cells).
  • repeated administration reduces the downregulation or inhibitory activity that can occur when an inhibitory immune molecule, such as PD-1 and/or PD-L1, is upregulated on antigen-expressing, eg, CAR-expressing T cells.
  • the method involves administering a first dose, usually followed by one or more consecutive doses, with a specified time frame between doses.
  • administration of a given "dose” includes administration of a given amount or number of cells as a single composition and/or as a single uninterrupted administration (e.g., as a single injection or continuous infusion), Also included is the administration of a given amount or number of cell numbers as divided doses provided in multiple individual compositions or infusions over a specified period of time (not exceeding 3 days).
  • the first or consecutive dose is a single or consecutive administration of a specified number of cells administered or initiated at a single time point.
  • the first or successive doses are administered as multiple injections or infusions over a period not exceeding three days, for example three or two days, once a day, or as multiple infusions over a one-day period .
  • Embodiment 1 the design of CNK-UT multifunctional complex
  • CNK-UT multifunctional complex (multi-functional complex) with four structures is designed.
  • the first structure is the basic CNK-UT multifunctional complex
  • the other three structures are CNK-UT multifunctional complexes with more functions formed by further adding other modules on the basis of the first structure. Complex.
  • a basic CNK-UT multifunctional complex is used, the schematic diagram of which is shown in Figure 1A, which contains three components: (1) NK activation receptor component; (2) CNK signal transduction component; and ( 3) UT components.
  • hinges or linkers are included between the NK-activating receptor module, the CNK signal transduction module and/or the UT module.
  • NK cell activation receptor module which at least comprises NK cell activation receptor or its functional variant, and said NK cell activation receptor comprises: (a) NK cell activation receptor extracellular domain (ED) or its function Variants, (b) NK cell activating receptor transmembrane domain (TMD) or functional variant thereof, and (c) NK cell activating receptor intracellular domain (ICD) or functional variant thereof; optionally,
  • ED NK cell activation receptor extracellular domain
  • TMD NK cell activating receptor transmembrane domain
  • ICD NK cell activating receptor intracellular domain
  • the NK cell activating receptor extracellular domain or its functional variant, the NK cell activating receptor transmembrane domain or its functional variant and/or the NK cell activating receptor intracellular domain or its function contain hinges or joints between variants;
  • the NK cell activation receptors are selected from NKG2D, NKG2C, NKG2E, NKG2F, NKG2H, CD94, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS4, KIR3DS1, natural cytotoxicity receptors (Natural Cytotoxicity Receptors, NCR), TRAIL, signaling lymphoid activation molecules (signaling lymphocytic activation molecule, SLAM) family molecule 2B4 (also known as CD244), DNAX accessory molecule 1 (DNAM-1, also known as CD226), CD16a, 2B4, NTB-A, CRACC (CS1) and NKp80; wherein, the Natural cytotoxicity receptors include NKp46 (aka NCR1 or CD335), NKp44 (aka NCR2 or CD336), and NKp30 (aka NCR3 or CD337)
  • the CNK multifunctional complex comprises amino acid sequences selected from SEQ ID NO.1-24.
  • CNK signal transduction component which at least comprises (i) NK cell signal converter (adaptor) or its functional variant
  • described NK cell signal converter comprises: (a) NK cell signal converter extracellular domain (ED) or its functional variant, (b) NK cell signal converter transmembrane domain (TMD) or its functional variant, and (c) NK cell signal converter intracellular domain (ICD) or its functional variant
  • ED extracellular domain
  • TMD NK cell signal converter transmembrane domain
  • ICD NK cell signal converter intracellular domain
  • the NK cell signal converter extracellular domain or its functional variant, the NK cell signal converter transmembrane domain or its functional variant and/or the NK cell signal converter Contains hinges or linkers between endodomains or functional variants thereof
  • the NK cell signal converter in the CNK signal conversion component is DAP10 or DAP12.
  • the CNK cell signal converter functional variant is selected from a mutant of DAP10 or DAP12, or a fusion protein of DAP10 and DAP12, or a fusion protein of wild-type DAP10 or DAP12 and mutant DAP10 or DAP12 .
  • the CNK cell signal converter functional variant is selected from DAP10 mutants or DAP12 mutants, or fusion proteins of DAP10 and DAP12, or fusion proteins of wild-type DAP10 or DAP12 and mutant DAP10 or DAP12.
  • the CNK signal transduction component further comprises (ii) an immunoreceptor activation signaling domain (ITAM) and/or (iii) a T cell co-stimulatory signaling domain.
  • ITAM immunoreceptor activation signaling domain
  • the NK cell signal converter or its functional variant, the immunoreceptor activation signaling domain (ITAM) and/or the T cell co-stimulatory signaling domain comprise A hinge or linker; preferably, said NK cell signal transducer or a functional variant thereof is fused to said immunoreceptor activation signaling domain (ITAM) domain.
  • the immunoreceptor activation signaling domain is derived from the intracellular activation signaling domain of an immune receptor; preferably, the immune receptor is selected from the group consisting of TCR ⁇ , CD2, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, FcR ⁇ , CD66d, Fc ⁇ RI, Fc ⁇ RI, Fc ⁇ RII, Fc ⁇ RIII, Dectin-1, CLEC-1, CD72, CD79A, CD79B; preferably, the immune receptor activation signaling domain (ITAM ) is fused to an NK cell signal transducer or a functional variant thereof; preferably, the immune receptor is CD3 ⁇ ;
  • the T cell costimulatory signaling domain is from the intracellular signaling domain of a costimulatory molecule.
  • the CNK signal transduction component comprises an amino acid sequence selected from SEQ ID NO.25-45.
  • (3) UT assembly which at least comprises (i) a recombinant protein molecule or a functional variant thereof targeting degradation of TCR, MHC and/or NK cell targets, said targeting degradation of TCR, MHC and/or NK cell targets
  • the recombinant protein molecule contains: (a) binding protein molecular domains or functional variants targeting TCR, MHC and/or NK cell targets, (b) transmembrane transmembrane of viral endoplasmic reticulum (ER) resident glycoproteins domain or its functional variant, and (c) the cytoplasmic domain of the viral endoplasmic reticulum resident glycoprotein or its functional variant; the transmembrane domain of the viral endoplasmic reticulum resident glycoprotein or its functional variant
  • the cytoplasmic domain of the endoplasmic reticulum resident glycoprotein of body and virus or its functional variant forms the ERAD degradation domain;
  • the TCR-targeted binding protein molecular domain or its functional variant is from a TCR antibody or its functional fragment or a combination thereof.
  • the antibody is selected from TCR ⁇ antibody, TCR ⁇ antibody, TCR ⁇ antibody, TCR ⁇ antibody, TCR ⁇ antibody, TCR ⁇ antibody, TCR V ⁇ 2 antibody, TCR C ⁇ 1 antibody;
  • the functional fragment of the antibody is selected from Fd, Fv , Fab, Fab', F(ab') 2, Fv (scFv), single chain antibody (scFv) or nanobody (nanobody), diabody, triabody and four chain antibody;
  • the TCR antibody It is a TCR single-chain antibody.
  • the TCR-targeting binding protein molecule is a TCR ⁇ antibody.
  • the amino acid sequence of the TCR single-chain antibody comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.116, preferably having 85%, 90%, 95% , 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the full-length sequence of the TCR single-chain antibody is as shown in SEQ ID Shown in NO.116.
  • the ERAD degradation domain in the UT module is from HCMV glycoprotein US2, US3, US11 or US10, adenovirus E3-19K or HHV-7US21.
  • the ERAD degradation domain in the UT module comprises an amino acid sequence selected from SEQ ID NO.46-63.
  • Fragments of genetic elements expressing the various components of the basic CNK-UT multifunctional complex can be synthesized by gene synthesis techniques. Using a polycistronic expression scheme, using self-cleaving 2A peptide (2A: T2A, p2A, E2A, F2A) and other elements to connect the gene element fragments of different components to obtain the basic CNK-UT multifunctional complex Gene fragments: Cloning the synthesized basic CNK-UT multifunctional complex gene fragments into lentiviral vectors by molecular cloning technology, transfecting T cells, and realizing the simultaneous expression of multiple different elements, so as to realize the specificity of T cells to NK targets sexual recognition and killing, but also effectively inhibit and degrade TCR with the help of UT elements.
  • different functional elements can be placed under different promoters of the same lentiviral vector to transfect T cells to achieve simultaneous expression of multiple CNK-UT elements; or, different functional elements can be placed in two lentiviruses Simultaneously transfecting T cells on the vector can also realize the simultaneous expression of multiple CNK-UT elements.
  • a CNK-UT multifunctional complex that simultaneously degrades or inhibits TCR and MHC I and/or MHC II is used, the schematic diagram of which is shown in Figure 1B, which also includes three components: (1) NK activation is controlled by (2) CNK signal transduction module; and (3) UT module: comprising a recombinant protein molecular domain or its functional variant targeting degradation of TCR and a binding protein molecular structure targeting MHC I and/or MHC II Domain or its functional variants, compared with the basic CNK-UT multifunctional complex, the difference is that (3) the UT component increases the binding protein molecular domain or its functional variants targeting MHC I.
  • the NK activating receptor component, the CNK signal transduction component, and the UT component are connected by hinges or linkers.
  • the binding protein molecule domain or functional variant thereof targeting MHC I and/or MHC II is an HLA-targeting binding protein molecular domain or functional variant thereof.
  • the binding protein molecular domain targeting MHC I may be from a viral endoplasmic reticulum protein that inhibits MHC I molecules.
  • the viral endoplasmic reticulum glycoprotein that inhibits MHC I molecules is selected from human cytomegalovirus (human cytomegalovirus, HCMV) US6, herpes simplex virus (herpes simplex virus, HSV) ICP47, vaccinia virus (cowpox virus) , CPXV) CPXV12, bovine herpesvirus (BHV) UL49.5, or Epstein Barr virus (Epstein Barr virus, EBV) BNFL2a, etc.
  • the binding protein molecular domain or functional variant thereof targeting MHC I and/or MHC II comprises a TAP binding domain.
  • the TAP binding domain comprises an amino acid sequence selected from SEQ ID NO.64-73.
  • the MHC I-targeting binding protein molecular domain can also be derived from a viral glycoprotein that degrades MHC I molecules.
  • the viral glycoprotein is selected from HCMV glycoproteins US2, US3, US11, US10, adenovirus E19, and the like.
  • the binding protein molecular domain or functional variant thereof targeting MHC I and/or MHC II comprises an MHC binding domain.
  • the MHC binding domain comprises an amino acid sequence selected from SEQ ID NO.74-82.
  • the molecular domain of the binding protein targeting MHC I can also be derived from a viral protein that directedly inhibits or degrades NK target proteins such as MICA, MICB, and ULBP1-6.
  • the viral protein is selected from HCMV UL16, UL141, UL142 or adenovirus E3-19K9 and the like.
  • the amino acid sequence of the viral protein is as shown in SEQ ID NO.83-91.
  • the molecular domain of the binding protein targeting MHC I can also be derived from a viral protein that transports MHC I molecules from the Golgi apparatus to the lysosome for degradation.
  • the viral protein is selected from HIV Nef, HIV Vpu, HHV-7 U21, HHV-8 KK3, HHV-8 KK5, MHV-68 MK3 and HTLV-1 p12 and the like.
  • the viral protein comprises an amino acid sequence selected from SEQ ID NO.92-97.
  • the binding protein molecular domains or functional variants thereof targeting MHC I and/or MHC II further comprise a viral protein that mediates the return of MHC-polypeptide molecules from the Golgi apparatus to the endoplasmic reticulum and facilitates its degradation ;
  • the viral protein comprises an MHC binding structure and a KDEL receptor binding domain.
  • the viral protein is Cowpox Virus protein CPXV203.
  • the viral protein comprises an amino acid sequence selected from SEQ ID NO.98-103.
  • the CNK-UT multifunctional complex gene fragments that target the MHC I binding protein molecular domain were obtained; the synthetic CNK-UT was synthesized by molecular cloning technology
  • the gene fragment of the multifunctional complex is cloned into a lentiviral vector and transfected into T cells to realize the simultaneous expression of various elements, thereby realizing the specific recognition and killing of NK targets by T cells, and effectively inhibiting and degrading TCR with the help of UT elements and MHC I expression.
  • a multifunctional complex that simultaneously degrades TCR and MHC I and realizes the recognition and activation of CNK-T cells specific to tumor antigens is used, as shown in Figure 1C, which includes four components: (1 ) NK activating receptor module; (2) CNK signal transduction module; (3) UT module; and (4) composite adapter (Chimeric adapter) module, which is combined with the increased targeting MHC I binding protein molecular domain Compared with the CNK-UT multifunctional complex, the difference lies in the addition of (4) complex adapter components.
  • the NK activating receptor component, CNK signal transition component, UT component and/or compound adapter component are connected by hinges or joints.
  • the (4) composite adapter comprises: (i) an extracellular recognition domain targeting tumors; (ii) a transmembrane domain; and (iii) an intracellular signaling domain; optionally, the A hinge or linker is included between the tumor-targeting extracellular recognition domain, transmembrane domain and/or intracellular signaling domain;
  • the tumor-targeting extracellular recognition domain of the composite adapter assembly is selected from tumor antigen-specific binding domains, tumor microenvironment target antigen binding domains and/or tumor microenvironment-targeting Chemotactic receptors;
  • the tumor-targeting extracellular recognition domain is selected from antibodies or functional fragments thereof, TCR or combinations thereof that can target and recognize tumor-associated antigens;
  • the functional fragments of the antibodies are selected from Fd, Fv, Fab, Fab', F(ab')2, Fv (scFv), single-chain antibody (scFv) or nanobody (nanobody), diabody, triabody and tetrabody;
  • the transmembrane domain of the composite adapter assembly is selected from the group consisting of NK cell activation receptor transmembrane domain, DAP10 transmembrane domain, DAP12 transmembrane domain, CD8 transmembrane domain, CD28 Transmembrane domain, CD4 transmembrane domain, 4-1BB transmembrane domain, OX40 transmembrane domain, ICOS transmembrane domain, CTLA-4 transmembrane domain, PD-1 transmembrane domain, LAG-3 Transmembrane domain, 2B4 transmembrane domain and BTLA transmembrane domain and combinations thereof; preferably, the NK cell activation receptor is selected from NKG2D, NKG2C, NKG2E, NKG2F, NKG2H, CD94, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS4, KIR3DS1, natural cytotoxic receptors, TRAIL, DNAM-1, CD16a, 2
  • the intracellular signaling domain of the complex adapter assembly comprises the intracellular signaling domain and/or the co-stimulatory signaling domain of an NK cell activating receptor.
  • the NK cell activating receptor is selected from NKG2D, NKG2C, NKG2E, NKG2F, NKG2H, CD94, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS4, KIR3DS1, natural cytotoxicity receptor, TRAIL, DNAM-1, CD16a , 2B4, NTB-A, CRACC, and NKp80;
  • the intracellular signaling domain also includes a co-stimulatory signaling domain; preferably, the co-stimulatory signaling domain is selected from a T cell co-stimulatory signaling domain; including, but not limited to derivatives From MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte activation signaling molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD16, CD27, CD28, CD30, CD40, CD38, CD35, CD79A, CD79B, CDS, ICAM-1, LFA-1, (CD11a/CD18), 4-1BB (CD137), B7-H3 , CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NK
  • CNK-UT multifunctional complex gene fragments that increase the binding protein molecular domain targeting MHC I and the complex adapter; through molecular cloning technology Cloning the synthetic CNK-UT multifunctional complex gene fragment into a lentiviral vector, transfecting T cells, and realizing the simultaneous expression of four different elements, so as to realize the specific recognition and killing of NK targets by T cells, also with the help of UT
  • the element effectively inhibits and degrades the expression of TCR and MHC I; and realizes the specific recognition and activation of CNK-T cells to tumor antigens through a composite adapter.
  • a multifunctional complex that simultaneously degrades TCR and MHC I and achieves specific recognition and killing effects on tumor cells.
  • Its schematic diagram is shown in Figure 1D, which includes four components: (1) NK activation Receptor module; (2) CNK signal transduction module; (3) UT module; and (4) target killing tumor cell receptor module, it is multifunctional with the CNK-UT of increasing targeting MHC I binding protein molecular domain Compared with the complex, the difference lies in the addition of (4) targeting and killing tumor cell receptor components.
  • the NK activating receptor module, the CNK signal transduction module, the UT module and/or the tumor cell targeting and killing receptor module are connected by hinges or linkers.
  • the (4) targeting and killing tumor cell receptor component comprises (i) an extracellular recognition domain targeting a tumor antigen; (ii) a transmembrane domain; and (iii) an intracellular co-stimulatory signaling domain; ( iv) T cell activation signaling domain (ITAM); optionally, the extracellular recognition domain, transmembrane domain, intracellular co-stimulatory signaling domain and/or T cell activation signal targeting tumor antigen
  • T cell activation signaling domain T cell activation signaling domain
  • the transduction domains (ITAMs) contain hinges or linkers between them.
  • the transmembrane domain targeting and killing tumor cell receptor components is selected from CD8 transmembrane domain, ⁇ and/or ⁇ chain transmembrane domain of T cell receptor, CD28 transmembrane domain, CD3 ⁇ transmembrane domain , CD45 transmembrane domain, CD4 transmembrane domain, CD5 transmembrane domain, CD8 transmembrane domain, CD9 transmembrane domain, CD16 transmembrane domain, CD22 transmembrane domain, CD33 transmembrane domain, CD37 Transmembrane domain, CD64 transmembrane domain, CD80 transmembrane domain, CD86 transmembrane domain, CD134 transmembrane domain, CD137 transmembrane domain, CD154 transmembrane domain, GITR transmembrane domain, and combinations thereof
  • the T cell activation signaling domain is derived from CD3 ⁇ , common FcR ⁇ (FCER1G), Fc ⁇ RIIa, FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, CD278 ("ICOS"), Fc ⁇ RI CD66d, DAP10 and DAP12 intracellular signaling domain
  • the (4) targeting and killing tumor cell receptor component is CAR or TCR targeting and killing tumor cell receptors.
  • Embodiment 2 the design of the CNK-UT element that expresses CNK-UT multifunctional complex
  • a single lentiviral EF1 ⁇ promoter expression vector is used, and elements such as self-cleaving polypeptide 2A are used to connect one of the combinations of CNK-UT elements: DAP10-DAP12 ICD-T2A-NKG2D- p2A-anti-TCR-AdE3 ERAD, enabling the expression of multiple functional elements on a single vector.
  • a single lentiviral EF1 ⁇ promoter expression vector is used, and elements such as self-cleaving polypeptide 2A are used to connect one of the combinations of CNK-UT elements: DAP10-DAP12 ICD-T2A-NKG2D- p2A-anti-TCR-US2 ERAD, enabling the expression of multiple functional elements on a single vector.
  • two different lentiviral expression vectors are used to regulate anti-TAA scFv-CD28/4-1BB-CD3 ⁇ -T2A-DAP10-CD3 ⁇ -T2A-NKG2D and anti-TCR-
  • the expression of AdE3 ERAD-T2A-AdE3, after preparing different lentiviruses and co-transfecting T cells, can express multiple functional elements on the same T cell.
  • the nucleotide sequence of the whole gene synthesis CNK-UT (its nucleotide sequence is shown in SEQ ID NO.118, and its encoded protein is shown in SEQ ID NO.117, wherein the functional fragment in the chimeric antigen receptor is as follows Sequential connection DAP10-CD3 ⁇ -T2A-NKG2D-p2A-anti-TCR scFv-AdE3), connected to the lentiviral vector pCDH-CMV-MCS-EF1 ⁇ -Puro (purchased from Youbao Biology) or pLVX-EF1 ⁇ by molecular cloning -AcGFP1-C1 Vector (Takara), construct the CNK-UT lentiviral target vector, and confirm the correctness by sequencing, transform DH5 ⁇ competent cells (Thermo Fisher) with the plasmid, pick single-clonal colonies, and use the plasmid after large-scale culture
  • PureLink TM HiPure Plasmid Maxiprep Kit (
  • Embodiment 4 the virus preparation of CNK-UT
  • the CNK-UT lentiviral plasmid obtained in Example 3, as well as the packaging plasmids psPAX2 and pMD2.G (purchased from Addgene, catalog number: 12259 & 12260), were transfected with polyethyleneimine according to the ratio of 1.64pmol: 1.3pmol: 0.72pmol. (408727, Sigma) was transfected at the ratio of DNA: ⁇ g PEI 1:3, and co-transfected 293T cells (ATCC product, product number is CRL3216TM); for the specific packaging plasmid preparation method, refer to PureLink TM HiPure Plasmid Maxiprep Kit (K210006, Thermo) instructions; for specific transfection procedures, refer to Sigma transfection instructions.
  • Embodiment 5 Preparation of CNK-UT cells
  • CD3 sorting magnetic beads MS separation column and MiniMACS TM separation device (Miltenyi Biotec ) for positive sorting and elution of CD3 + T cells, and the isolated CD3 + T cells were passed through magnetic beads coupled with anti-CD3 antibody and anti-CD28 antibody (Human T-Activator CD3/CD28, Invitrogen, Cat.
  • the specific steps are as follows: the peripheral blood mononuclear cells were diluted to a concentration of 1 ⁇ 106 single cells/ml, spread in a 24-well plate, and then the magnetic beads were added to the cells at a ratio of 1:1 and Mix well, resuspend in culture medium (OpTmizerTM T-Cell Expansion SFM, A1048503, Life Technologies), add IL2 50U/ml and IL15 5ng/ml, culture in 37°C, 5% CO2 incubator for 1 day before loading
  • the lentivirus prepared in Example 4 with CNK-UT elements was transfected.
  • Fig. 3 is a flow cytometric detection result of a basic phenotype of CNK-UT (DAP10-CD3 ⁇ -T2A-NKG2D-p2A-anti-TCR scFv-AdE3) cells.
  • CNK-UT DAP10-CD3 ⁇ -T2A-NKG2D-p2A-anti-TCR scFv-AdE3 cells.
  • 5 ⁇ 10 5 untransfected T cells and T cells transfected with CNK-UT virus vector were collected respectively, and treated with antibodies CD8-Pacific Blue, CD4-APC, NKG2D-PE- Cy7, TCR- ⁇ -APC-Cy7 (all antibodies were purchased from Biolegend) were stained, and then the cell phenotype was identified by using a flow cytometer (BD FACSCanto II).
  • human colonic adenoma cell line HT29 was purchased from Procell (CL-0118); triple-negative breast cancer cell line MDA-MB453 was purchased from Procell (CL-0152); acute myeloid leukemia cell line THP1 was purchased from Procell (CL-0233) ; Hepatocellular carcinoma (HCC) HepG2 was purchased from Procell (CL-0103); Hepatocellular carcinoma (HCC) PLC was purchased from Procell (CL-0415).
  • the CNK-UT cells used in this experiment are the cells prepared in Example 5.
  • Figure 4 shows the recognition and specific killing of the human colon adenoma cell line HT29 by CNK-UT cells. in:
  • Human colonic adenoma cell line HT29 0.5 ⁇ 10 6 cells were digested with trypsin, stained with MICA-APC, MICB-APC, ULBP1-APC, ULBP2-APC, ULBP3-APC, and IgG isotype (IgG isotype) antibody was used as a control (purchased from R&D Systems), and then identify the cell phenotype by using a flow cytometer (BD FACSCanto II). The results showed that HT29 cells highly expressed ULBP2, and weakly expressed MICA, MICB, ULBP1 and ULBP3.
  • T cells and tumor cells For the co-culture experiment of T cells and tumor cells, take a 24-well plate and count 0.5 ⁇ 106 cells/well of the human colon adenoma cell line HT29 for plating. When the growth density of tumor cells reaches more than 80%, the effect-to-target ratio is 1:5.
  • CNK-UT cells have a good killing function on HT29 cells, 69.5% of the cells in the co-culture system of the control group are tumor cells, and T cells do not express CD25; while the co-culture system of CNK-UT cells Among them, it can be seen from the FSC/SSC diagram that most tumor cells have been eliminated, and the proportion of T cells has increased significantly, accounting for 84.1%, and 36.345% of CD8+T cells up-regulate CD25 expression, and 19.08% of CD4+T cells up-regulate Expression of CD25 (Fig. 4B).
  • the CNK-UT cells used in this experiment are the cells prepared in Example 5.
  • Figure 5 shows the recognition and specific killing of MDA-MB453 by CNK-UT cells.
  • MDA-MB453 0.5 ⁇ 10 6 cells were digested with trypsin, stained with MICA-APC, MICB-APC, ULBP1-APC, ULBP2-APC, ULBP3-APC, IgG isotype antibody as a control (purchased from R&D Systems), and then passed Cell phenotype identification was performed by flow cytometry (BD FACSCanto II). The results showed that MDA-MB453 cells highly expressed MICA and ULBP2, and weakly expressed MICB, ULBP1 and ULBP3.
  • the experimental data shows that 83.7% of MDA-MB453 cells up-regulate the expression of MICA, 33.6% of the cells up-regulate the expression of MICB, 31.2% of the cells up-regulate the expression of ULBP1, 95.0% of the cells up-regulate the expression of ULBP2, and 6.23% of the cells up-regulate the expression of ULBP3 (Fig.
  • CNK-UT cells have a good killing function to MDA-MB453 cells, 48.9% of the cells in the co-culture system of the control group are tumor cells, and T cells do not express CD25; while the co-culture of CNK-UT cells In the system, only 6.74% were tumor cells, and 14.8% of CD8+T cells up-regulated the expression of CD25, and 28.1% of CD4+T cells up-regulated the expression of CD25 (Fig. 5B).
  • the experimental results showed that CNK-UT cells had a good killing function on MDA-MB453 cells and achieved specific activation.
  • the CNK-UT cells used in this experiment are the cells prepared in Example 5.
  • FIG. 6 shows the recognition and specific killing of THP1 by CNK-UT cells
  • THP1 cells 0.5 ⁇ 10 6 THP1 cells were centrifuged, stained with MICA-APC, MICB-APC, ULBP1-APC, ULBP2-APC, ULBP3-APC, IgG isotype antibody as a control (purchased from R&D Systems), and then passed through flow cytometry (BD FACSCanto II) for cell phenotype identification.
  • the results showed that THP1 cells highly expressed ULBP1, ULBP2 and ULBP3, and weakly expressed MICA and MICB.
  • T cells and tumor cells For the co-culture experiment of T cells and tumor cells, take a 24-well plate, count AML cell line THP1 0.5 ⁇ 10 6 cells/well for plating, and add untransfected T cells and CNK-UT cells respectively according to the effect-to-target ratio of 1:5 (DAP10-CD3 ⁇ -T2A-NKG2D-p2A-anti-TCR scFv-AdE3), after 24h, all cells were harvested by centrifugation, and CD45-PerCP-Cy5.5, CD8-Pacific Blue, CD4-APC, CD137-PE- Cy7, CD25-APC-Cy7 (purchased from Biolegend) were stained, and then detected by flow cytometry.
  • the experimental data showed that 27.9% of THP1 cells up-regulated the expression of MICB, 99.0% of the cells up-regulated the expression of ULBP1, 95.8% of the cells up-regulated the expression of ULBP2, and 99.1% of the cells up-regulated the expression of ULBP3 (Fig. 6A); The cells have a good killing function.
  • the CNK-UT cells used in this experiment are the cells prepared in Example 5.
  • FIG. 7 shows that CNK-UT cells can upgrade conventional CAR-T technology to achieve more powerful targeted killing and activation capabilities.
  • HepG2 cells were digested with trypsin, stained with GPC3-APC, PD-L1-APC, MICA-APC, MICB-APC, ULBP1-APC, ULBP2-APC, ULBP3-APC, and IIgG isotype antibody as a control ( purchased from R&D Systems), and the cell phenotype was identified by flow cytometry (BD FACSCanto II).
  • the results showed that HepG2 cells highly expressed GPC3 and weakly expressed PD-L1, MICA, MICB, ULBP1 and ULBP2.
  • CAR/CNK-UT cells show more efficient killing and activation of HepG2 cells than conventional CAR-T cells
  • GPC3 CAR-T cells Referring to Example 3-Example 5, the whole gene synthesis anti-GPC3 41BB-CD3 ⁇ nucleotide sequence (SEQ ID NO.120, the amino acid sequence of its encoded protein is shown in SEQ ID NO.119 ), connected to the lentiviral vector pCDH-CMV-MCS-EF1 ⁇ -Puro by molecular cloning, and after preparing the plasmid and lentivirus, transfected T cells to obtain conventional GPC3 CAR-T cells.
  • SEQ ID NO.120 the whole gene synthesis anti-GPC3 41BB-CD3 ⁇ nucleotide sequence
  • CD137 up-regulate CD137 expression
  • CD4+T cells up-regulate CD137 expression
  • in the CNK-UT co-culture system only 8.01% of tumor cells, 57.9% CD8+T cells remained Up-regulate the expression of CD137
  • 33.1% CD4+T cells up-regulate the expression of CD137
  • in the GPC3 CAR/CNK-UT co-culture system only 4.86% of tumor cells remained, and 50.3% CD8+T cells up-regulated the expression of CD137; 35.42% CD4+T The cells upregulate the expression of CD137.
  • CNK-UT cells have a good killing function on HepG2 cells, and achieve specific activation, up-regulate the expression of CD137;
  • GPC3 CAR/CNK-UT cells integrated with GPC3 41BB-Z CAR and CNK-UT elements UT cells have more efficient tumor killing and activation functions.
  • the CNK-UT cells used in this experiment are the cells prepared in Example 5.
  • FIG. 8 shows that CNK-UT cells can upgrade conventional CAR-T technology to achieve targeted killing and activation of tumor cells with high expression of PD-L1.
  • PLC cells 0.5 ⁇ 10 6 PLC cells were digested with trypsin, stained with GPC3-APC, PD-L1-APC, MICA-APC, MICB-APC, ULBP1-APC, ULBP2-APC, ULBP3-APC, and IgG isotype antibody as a control ( purchased from R&D Systems), and the cell phenotype was identified by flow cytometry (BD FACSCanto II). The results showed that PLC cells highly expressed PD-L1 and ULBP1, and weakly expressed GPC3, MICA, MICB and ULBP2.
  • CNK-UT cells show more efficient killing and activation of HepG2 cells than conventional CAR-T cells
  • T cells and tumor cells Co-cultivation experiment of T cells and tumor cells, effect-to-target ratio 1:5, untransfected T cells, conventional GPC3 CAR-T cells, CNK-UT cells and GPC3 CAR/CNK-UT cells were added to PLC cells, co-cultured After 24 hours, all the cells were digested and detected by flow cytometry.
  • the tumor cells and T cells were distinguished by CD45, and the expression of CD25 and CD137, the activation markers on the surface of CD8/CD4 cells, was analyzed.
  • immunodeficiency mice (NSG mice) (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., Cat. No. T001475) were used as tumor-bearing models to conduct pharmacodynamic studies in vivo.
  • Negative control group give control T cells, T cells from the same donor, non-transfected T cells stimulated and expanded by magnetic beads;
  • Isotype control group Administer conventional GPC3 CAR-T cells: Prepare GPC3 CAR-T cells: Refer to Example 3-Example 5, synthesize the anti-GPC3 41BB-CD3 ⁇ nucleotide sequence from the whole gene, and link it to the slow
  • the virus vector pCDH-CMV-MCS-EF1 ⁇ -Puro after preparing the plasmid and lentivirus, was transfected into T cells to obtain conventional GPC3 CAR-T cells.
  • mice were inoculated with 3 ⁇ 10 6 HepG2 cells via the right underarm, and the day of modeling was recorded as day 0. After 7 days, it was observed that the mice were successfully modeled, and the mice were divided into 3 groups, which were given ordinary human primary T cells (control T cells), conventional GPC3-CAR-T cells, and GPC3 CAR/CNK-UT cells, and observed Clearance of tumor fluorescence by various cells in mice. All three kinds of cells were administered by tail vein injection, the dose was 2 ⁇ 10 6 /monkey, 3 times in a row (7th, 9th, 11th day), the administration date was recorded as the 7th day, and then observed every 7 days Fluorescent condition.
  • FIG. 9 shows that GPC3 CAR/CNK-UT cells have more efficient tumor clearance ability in mice than GPC3 CAR-T cells.
  • Figure 10 shows that GPC3 CAR/CNK-UT cells have a more efficient tumor clearance ability than GPC3 CAR-T.
  • mice were also studied.
  • the body weight of the experimental animals in the CNK-UT group reinfused with CNK-UT cells increased slightly compared with D0, while the body weight of the animals in the isotype control group and the negative control group of control T cells reinfused with GPC3-CAR-T cells increased with the experimental results.
  • the time extension gradually decreases.
  • the above results suggest that after the experimental animals were treated with different methods, the tumors of mice injected with control T cells and GPC3 CAR-T cells still grew, while GPC3 CAR/CNK-UT cells were more effective than GPC3 CAR-T cells. It has stronger lethality and can clear tumor cells 21 days after infusion without recurrence. Moreover, the reinfusion of GPC3 CAR/CNK-UT has no cytotoxicity, the survival period of the animals is prolonged, the living conditions of the animals are improved, and the body weight is increased.
  • FIG 11 shows that GPC3 CAR/CNK-UT can specifically recognize and kill triple-negative breast cancer cell MDA-MB453.
  • T cells and tumor cells In the co-culture experiment of T cells and tumor cells, the effect-to-target ratio was 1:5. Untransfected T cells, conventional GPC3 CAR-T cells, CNK-UT cells and GPC3 CAR/CNK-UT cells were added to MDA-MB453 cells, respectively. After 24 hours of co-cultivation, all cells were digested and detected by flow cytometry. CD45 was used to distinguish tumor cells from T cells, and the expression of NK element NKG2D and surface activation marker CD25 in CD8 and CD4 cells was analyzed.
  • MDA-MB453 is a triple-negative breast cancer cell line, and the cells do not express GPC3, so GPC3 CAR-T cannot clear MDA-MB453 (after 24 hours of co-culture, there are still a large number of CD45-negative tumor cells in the system), while CNK-UT The product can efficiently kill and eliminate tumor cells.
  • CD45(+) T cells CD8(+) and CD4(+) T cells highly express the CNK component NKG2D, and up-regulate the T cell activation marker CD25.
  • FIG. 12 shows that GPC3 CAR/CNK-UT can specifically recognize and kill acute myeloid leukemia cell line THP1.
  • THP1 is a leukemia cell line, and the cells do not express GPC3, so GPC3 CAR-T cannot clear THP1 (after 24 hours of co-culture, there are still a large number of CD3(-), CD123(+), and CLL1(+) tumors in the system cells, while GPC3 CAR/CNK-UT products can efficiently kill and eliminate tumor cells, and detect the up-regulated T cell activation markers CD25 and CD137 in CD3(+) T cells, indicating that GPC3 CAR/CNK-UT can specifically Recognize THP1 cells, activate and kill tumor cells.
  • Example 9 The ability of CNK-UT cells upgraded with NCR elements to kill and clear different tumors
  • the renal cell carcinoma (Renal Cell Carcinoma, RCC) tumor cell line 786-O was purchased from Punuo Sai (CL-0010)
  • the renal cell carcinoma (Renal Cell Carcinoma, RCC) tumor cell line ACHN was purchased from Punuo Sai (CL-0010).
  • epithelial adenoma (Lung epithelial carcinoma) tumor cell line A549 was purchased from Punuo Sai (CL-0016)
  • acute myeloid leukemia (Acute myeloid leukemia) tumor cell line UL60 was purchased from Punuo Sai (CL-0110) .
  • NCR2 CAR/CNK-UT cells Referring to Example 3-Example 5, the nucleotide sequence (SEQ ID NO.124) of the whole gene synthesis NCR2-CD28-CD3 ⁇ (SEQ ID NO.124, the amino acid sequence of its encoded protein is as SEQ ID NO.123 shown); the nucleotide sequence of the whole gene synthesis DAP10-CD3 ⁇ -T2A-NKG2D-p2A-anti-TCR scFv-AdE3 was connected to the lentiviral vector pCDH-CMV-MCS-EF1 ⁇ -Puro by molecular cloning, and prepared After plasmid and lentivirus, T cells were transfected and expanded and cultured to obtain NCR2 CAR/CNK-UT cells.
  • SEQ ID NO.124 the nucleotide sequence of the whole gene synthesis DAP10-CD3 ⁇ -T2A-NKG2D-p2A-anti-TCR scFv-AdE
  • Figure 13 shows the recognition, specific killing and activation ability of CNK-UT cells to renal cell carcinoma 786-O cells.
  • T cells and tumor cells For the co-culture experiment of T cells and tumor cells, take a 24-well plate, count 0.5 ⁇ 10 6 cells/well of the RCC tumor cell line 786-O for plating, and add untransfected T cells and NCR2 CAR/CNK-UT cells (NCR2-CD28-CD3 ⁇ ; DAP10-CD3 ⁇ -T2A-NKG2D-p2A-anti-TCR scFv-AdE3), after 24h, all cells were harvested by centrifugation, and 7-AAD, CD45 - staining of APC-Cy5.5, CD8-APC, NKG2D-PE-Cy7, NKp44-PE, CD137-Pacific Blue (purchased from Biolegend), and then detected by flow cytometry.
  • NCR2 CAR/CNK-UT cells NCR2 CAR/CNK-UT cells
  • NCR2 CAR/CNK-UT cells have a good killing function on 786-O cells.
  • Figure 14 shows the ability of CNK-UT cells to recognize, specifically kill and activate renal cell carcinoma ACHN cells.
  • T cells and tumor cells For the co-culture experiment of T cells and tumor cells, take a 24-well plate, count 0.5 ⁇ 10 6 cells/well of the RCC cell line ACHN for plating, and add untransfected T cells and NCR2 CAR/CNK-UT cells (NCR2-CD28-CD3 ⁇ ; DAP10-CD3 ⁇ -T2A-NKG2D-p2A-anti-TCR scFv-AdE3), after 24h, all the cells were harvested by centrifugation, and 7-AAD, CD45-APC- Cy5.5, CD8-APC, NKG2D-PE-Cy7, NKp44-PE, CD137-Pacific Blue (purchased from Biolegend) were stained, and then detected by flow cytometry.
  • NCR2 CAR/CNK-UT cells NCR2 CAR/CNK-UT cells
  • NCR2 CAR/CNK-UT cells have a good killing function on ACHN tumor cells.
  • Figure 15 shows the ability of CNK-UT cells to recognize, specifically kill and activate lung epithelial adenoma A549 cells.
  • T cells and tumor cells For the co-culture experiment of T cells and tumor cells, take a 24-well plate, count 0.5 ⁇ 10 6 cells/well of the lung cancer cell line A549 for plating, and add untransfected T cells and NCR2 CAR/CNK-UT cells (NCR2-CD28-CD3 ⁇ ; DAP10-CD3 ⁇ -T2A-NKG2D-p2A-anti-TCR scFv-AdE3), after 24h, all the cells were harvested by centrifugation, and 7-AAD, CD45-APC- Cy5.5, CD8-APC, NKG2D-PE-Cy7, NKp44-PE, CD137-Pacific Blue (purchased from Biolegend) were stained, and then detected by flow cytometry.
  • NCR2 CAR/CNK-UT cells NCR2-CD28-CD3 ⁇ ; DAP10-CD3 ⁇ -T2A-NKG2D-p2A-anti-TCR scFv-AdE3
  • NCR2 CAR/CNK-UT cells have a good killing function on A549 tumor cells.
  • Figure 16 shows the ability of CNK-UT cells to recognize, specifically kill and activate the AML cell line UL60.
  • T cells and tumor cells For the co-culture experiment of T cells and tumor cells, take a 24-well plate, count 0.5 ⁇ 10 6 cells/well of the lung cancer cell line UL60 for plating, and add untransfected T cells and NCR2 CAR/CNK-UT cells (NCR2-CD28-CD3 ⁇ ; DAP10-CD3 ⁇ -T2A-NKG2D-p2A-anti-TCR scFv-AdE3), after 24h, all the cells were harvested by centrifugation, and 7-AAD, CD45-APC- Cy5.5, CD8-APC, NKG2D-PE-Cy7, NKp44-PE, CD137-Pacific Blue (purchased from Biolegend) were stained, and then detected by flow cytometry.
  • NCR2 CAR/CNK-UT cells NCR2 CAR/CNK-UT cells
  • Example 10 Optimal Design of UT Elements Improves the Recognition and Specific Killing of CNK-UT Cells to Tumor Cell Line U937
  • CNK-UT (UL16) cells and CNK-UT (UL16/E3-19K) cells referring to Example 3-Example 5, the nucleotide sequence of the whole gene synthesis DAP10-CD3 ⁇ -T2A-NKG2D (its encoded protein The amino acid sequence is shown in SEQ ID NO.125); the nucleotide sequence of the whole gene synthesis anti-TCR scFv-AdE3-p2A-UL16 (the amino acid sequence of its encoded protein is shown in SEQ ID NO.126); the whole gene synthesis The nucleotide sequence of anti-TCR scFv-AdE3-p2A-UL16-p2A-E3-19K (the amino acid sequence of its encoded protein is shown in SEQ ID NO.127), connected to the lentiviral vector pCDH- CMV-MCS-EF1 ⁇ -Puro, after preparing plasmids and lentiviruses, transfected T cells and cultured to
  • Figure 17 shows the ability of CNK-UT cells to recognize, specifically kill and activate AML cell line U937 (purchased from U937, Cat. No. CL-0239).
  • T cells and tumor cells For the co-culture experiment of T cells and tumor cells, take a 24-well plate, count the lung cancer cell line U937-GFP 0.5 ⁇ 10 6 cells/well for plating, and add untransfected T cells, CNK- UT(UL16) cells (DAP10-CD3 ⁇ -T2A-NKG2D; anti-TCR scFv-AdE3-p2A-UL16) and CNK-UT(UL16/E3-19K) cells (DAP10-CD3 ⁇ -T2A-NKG2D; anti-TCR scFv -AdE3-p2A-UL16-p2A-E3-19K), after 24h, all cells were harvested by centrifugation, and then 7-AAD, CD33-APC-Cy5.5, CD8-Pacific Blue, NKG2D-APC, CD137-PE-Cy7 (purchased from Biolegend) staining, followed by flow cytometry for detection.
  • CNK-UT (UL16/E3-19K) design has a more efficient killing function on U937 tumor cells than CNK-UT (UL16) cells.
  • CD33(+) tumor cells in the control group accounted for 69.8%, and the control T cells did not up-regulate the activation marker CD137; while in the co-culture system of CNK-UT(UL16) cells, only 4.69% of CD33+, GFP+ tumor cells remained, At the same time, 23.07% of T cells up-regulated the expression of CD137; while in the co-culture system of CNK-UT (UL16/E3-19K) cells, only 0.079% of tumor cells remained, and 54.7% of T cells up-regulated the expression of CD137.
  • Example 11 Optimized design of NK elements improves CNK-UT cells against tumor cell lines
  • NCR1 CAR-T cells NCR2 CNK-UT cells and NCR1/2 CNK-UT cells: Refer to Example 3-Example 5, the nucleotide sequence of the whole gene synthesis NCR1-CD28-CD3 ⁇ (the amino acid sequence of its encoded protein As shown in SEQ ID NO.128); the nucleotide sequence of the whole gene synthesis DAP10-CD3 ⁇ -T2A-NKG2D-p2A-anti-TCR scFv-AdE3 (the amino acid sequence of its encoded protein is shown in SEQ ID NO.117)
  • the whole gene synthesizes the nucleotide sequence of NCR2-CD28-CD3 ⁇ (the amino acid sequence of its encoded protein is shown in SEQ ID NO.123); the whole gene synthesizes the nucleoside of NCR2-CD28-CD3 ⁇ -p2A-NCR1-CD28-CD3 ⁇ acid sequence (the amino acid sequence of its encoded protein is shown in SEQ ID NO.129), connected
  • Figure 18 shows the ability of CNK-UT cells to recognize, specifically kill and activate AML cell line U937.
  • NCR1 CAR-T cells NCR1-CD28-CD3 ⁇
  • NCR2 CNK-UT cells DAP10-CD3 ⁇ -T2A-NKG2D-p2A-anti-TCR scFv-AdE3; NCR2-CD28-CD3 ⁇
  • NCR1/2 CNK-UT cells UT cells DAP10-CD3 ⁇ -T2A-NKG2D-p2A-anti-TCR scFv-AdE3; NCR2-CD28-CD3 ⁇
  • the NK recognition element can significantly improve the killing function of CNK-UT cells on U937 tumor cells.
  • Figure 19 shows the ability of CNK-UT cells to recognize, specifically kill and activate the pancreatic cancer cell line PANC-1 (purchased from Proceed, Cat. No. CL-0184).
  • NCR1 CAR-T cells Preparation of NCR1 CAR-T cells, NCR2 CNK-UT cells and NCR1/2 CNK-UT cells: Refer to Example 3-Example 5, the nucleotide sequence of the whole gene synthesis NCR1-CD28-CD3 ⁇ -p2A-tEGFR (which encodes The amino acid sequence of the protein is shown in SEQ ID NO.130); the nucleotide sequence of the whole gene synthesis DAP10-CD3 ⁇ -T2A-NKG2D-p2A-anti-TCR scFv-AdE3 (the amino acid sequence of its encoded protein is shown in SEQ ID NO.
  • NCR2-CD28-CD3 ⁇ -p2A-tEGFR the amino acid sequence of its coded protein is shown in SEQ ID NO.131
  • the whole gene synthesizes NCR2-CD28-CD3 ⁇ -p2A-
  • the nucleotide sequence of NCR1-CD28-CD3 ⁇ (the amino acid sequence of its coded protein is shown in SEQ ID NO.129), is connected to the lentiviral vector pCDH-CMV-MCS-EF1 ⁇ -Puro by the mode of molecular cloning, prepares plasmid and After lentivirus, T cells were transfected and expanded to obtain NCR1 CAR-T cells, NCR2 CNK-UT cells and NCR1/2 CNK-UT cells.
  • T cells and tumor cells For the co-culture experiment of T cells and tumor cells, take a 24-well plate, count the pancreatic cancer cell line PANC-1-GFP 0.5 ⁇ 10 6 cells/well for plating, and add untransfected T cells according to the effect-to-target ratio of 1:1.
  • NCR1 CAR-T cells NCR1-CD28-CD3 ⁇ -p2A-tEGFR
  • NCR2 CNK-UT cells DAP10-CD3 ⁇ -T2A-NKG2D-p2A-anti-TCR scFv-AdE3; NCR2-CD28-CD3 ⁇ -p2A-tEGFR
  • NCR1/2 CNK-UT DAP10-CD3 ⁇ -T2A-NKG2D-p2A-anti-TCR scFv-AdE3; NCR2-CD28-CD3 ⁇ -p2A-NCR1-CD28-CD3 ⁇
  • co-cultured for 48h and all cells were harvested by centrifugation , for 7-AAD, CD33-APC-Cy5.5, CD8-Pacific Blue, NKG2D-APC, NKp44-PE or EGFR-PE, CD137-PE-Cy7 (purchased from Biolegend) staining, and then flow cytometry Technology is

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Abstract

提供了一种通用型T细胞(CNK-UT)及其应用。所述CNK-UT具有广谱的肿瘤细胞和病毒感染细胞识别和杀伤能力。

Description

通用型T细胞及其应用 技术领域
本公开涉及细胞治疗领域，更具体地，涉及一种通用型T细胞及其应用，特别是嵌合自然杀伤受体通用型T细胞(CNK-UT)及其应用。
背景技术
细胞治疗，通过基因工程技术改造免疫细胞使其能呈递肿瘤相关抗原或表达特异性识别疾病细胞的受体，在体外指数级扩增后回输至患者体内，激活体内免疫系统对肿瘤细胞进行攻击或直接特异性识别并杀伤疾病细胞的疗效。2012年CD19靶向CAR-T细胞实现了人类医学史上首次定向清除B细胞白血病患者体内肿瘤细胞，成为继骨髓干细胞移植治疗技术后又一项能真正治愈白血病的治疗技术，开启了精准医学细胞治疗的新时代。这项技术有望应用于各种血液肿瘤和实体瘤的治疗上。但目前常规CAR-T治疗实体肿瘤的临床疗效不佳，其原因：(1)CAR-T的杀伤功能高度依赖于CAR结构对肿瘤相关抗原TAA的识别，但由于实体肿瘤的异质性，肿瘤细胞表面靶点蛋白表达存在很大差异；目前单一靶向的CAR-T无法完全识别和杀伤所有肿瘤细胞，从而导致肿瘤的免疫逃逸和复发转移；(2)实体肿瘤的直接免疫抑制效应，表达PD-L1，B3H7等，直接抑制T细胞的激活；(3)实体肿瘤的免疫抑制微环境，有大量的免疫抑制细胞，如TAM、Treg、MDSC，传统CAR-T的设计很难突破肿瘤内部的免疫抑制细胞的限制；(4)传统CAR-T是个性化的细胞治疗，T细胞来源患者本身，如果患者已经接受大量的放化疗等，免疫系统功能受损，很难从患者外周血分离出足够数量的T细胞，即使分离出来改造，T细胞的增殖和杀伤功能还是很薄弱，因此很难发挥治疗效果。
免疫系统具有强大的可塑性和具有近乎无限的检测入侵病毒，细菌，异源细胞和病变细胞的能力。这种非凡的免疫监测能力主要通过体液免疫和细胞免疫实现的，其中包括两种重要分子结构：免疫球蛋白和T细胞受体(TCR)。TCR是T细胞的定义结构，是一种跨膜异源二聚体，由通过二硫键连接的α和β链或δ和γ链组成。在这些链中，互补决定区(CDR)决定了TCR将结合的抗原。在TCR中，TCRα和TCRβ亚基(或γδT细胞中的TCRγ和TCRδ)负责识别主要组织相容性复合体(MHC)/抗原配体。
人类MHCⅠ类基因区包括HLA-A、B、C位点的等位基因，编码HLA-A抗原、B抗原和C抗原等经典的Ⅰ类抗原(分子)，称为HLA-A、HLA-B和HLA-C。这些抗原分子存在于所有体细胞的表面，并与细胞内蛋白质表位肽段结合，供免疫系统进行识别。如果细胞产生突变蛋白，或有外来细菌或病毒入侵，细胞将这些突变蛋白或异源蛋白抗原表位呈递后，免疫细胞进行识别后会进行免疫攻击和杀伤，从而清除病变细胞，病菌和病毒入侵细胞。
由于MHC类分子是呈递病毒抗原，触发免疫系统针对病毒病原体的攻击的关键因素，在数百万年的进化中，病毒逐渐形成了靶向抑制MHC类分子的策略，以避免免疫监视和清除。其中非常典型的例子是人巨细胞病毒(HCMV)，与其宿主一起进化，通过诸多基因组功能蛋白实现免疫逃逸。HCMV独特的短(unique short，US)基因组区域编码至少五种糖蛋白(US2、US3、US6、US10、US11)，这些糖蛋白使用专门的机制下调MHC I类分子，从而阻碍病毒抗原呈递给细胞毒性T淋巴细胞(CTL)。
CMV US糖蛋白能够劫持内质网相关降解(ER-associated degradation，ERAD)机制来抑制MHC分子介导的病毒抗原呈递，从而逃避免疫监视系统。ER相关降解(ERAD)是一种蛋白质清除系统。内质网中错误折叠、错误组装或代谢调节的蛋白质通过特定的膜穿透机制选择性地从内质网脱位到胞质溶胶中，随后被胞质泛素蛋白酶体系统(ubiquitin proteasome system，UPS)降解。
HCMV US2和US11这两种蛋白都是ER驻留I型整合膜糖蛋白，它们共同选择该ERAD途径以促进MHC I类重链的降解，从而抑制MHC I类抗原呈递。两种蛋白中的任何一种蛋白质的表达都会导致新合成的MHC I类重链快速降解。US2和US11通过其腔结构域与重链结合并募集宿主细胞蛋白，这些蛋白通过“拉动”重链的胞质尾部从内质网膜中提取多肽。MHC I类分子易位进入细胞质后，被泛素化，并被蛋白酶体降解。
除了I类分子外，US2还导致II类途径的两种蛋白质DR-α和DM-α以及HFE(一种参与铁调节的非经典主要组织相容性复合体(MHC)I类蛋白质)的降解。
US2的腔结构域负责结合MHC I类和II类分子，跨膜结构域(TM)和细胞质结构域(CT)与ER相关降解途径的细胞成分相互作用，并有助于易位和促进I类和II类这两种蛋白的酶解。US2的胞质尾部足以与信号肽肽酶(SPP)相互作用，SPP是US2依赖性MHC I位错复合物的必要成分。此外，US2通过其TM结构域与内质网驻留RING型E3连接酶TRC8相互作用，这也有助于US2尾锚定MHC I和II分子的泛素化和蛋白酶体降解。
相比之下，US11诱导的MHC-I分子降解需要Derlin1而不是SPP。US11的ER腔域与MHC-I重链的腔域相互作用，而US11的TM域与Derlin-1结合。因此，US11的主要功能可能是将MHC-I分子传递到Derlin-1，然后诱导它们错位到胞质溶胶以进行蛋白酶体降解。此外，US11激活未折叠蛋白。通过Derlin-1，US11与作为ERAD RING E3连接酶的TMEM129相关联并招募Ube2J2在US11诱导降解之前泛素化MHC-I。
US3糖蛋白不是促进MHC蛋白的降解，而是与载有肽的MHC I类异二聚体物理结合，导致I类复合物保留在 ER中并抑制不变链与ER中II类DR-αβ二聚体的结合，导致II类复合物的错误定位和减少的肽负载。因此，在HCMV感染的早期阶段，US3能够干扰MHC I类分子的细胞内转运和成熟。US3是一种内质网驻留膜蛋白。结构域交换实验表明，US3的腔结构域足以使US3本身保持内质网，而腔内结构域和跨膜结构域对于内质网中I类MHC分子的保留都是必需的。
除了MHC I分子外，US2和US3糖蛋白通过破坏或消除II类蛋白的功能来抑制II类抗原呈递。US2与II类DR结合并导致仅II类DRαβ复合物的α链的快速有效的蛋白酶体介导的降解。US2还导致DMα链的降解，该DM是将抗原肽加载到II类DR复合物上所需的MHC II类复合物。HCMV US3与II类DRαβ异源二聚体结合，抑制不变链(Ii)的结合，导致细胞内错误定位并减少DR复合物的肽负载。
HCMV US10编码内质网膜糖蛋白，该糖蛋白也与MHC I类抗原呈递的成分相互作用。US10结合游离的I类重链并延迟它们从ER的运输。但是，US10不影响US2或US11。
腺病毒基因产物E3/19K(E19)也可以在分泌途径中保留I类分子并干扰抗原呈递。E19也是内质网驻留糖蛋白，它可以消除主要组织相容性复合体I类(MHC-I)和MHC-I相关链A和B(MICA/B)分子的细胞表面转运。E3/19K包含三个功能模块：用于与MHC-I和MICA/B分子相互作用的腔域、跨膜域和细胞质尾中的二赖氨酸基序，该基序可将高尔基体返回到内质网。研究表明，需要跨膜结构域(TMD)和ER返回信号来确保有效的ER定位、MHC-I和MICA/B分子的转运抑制以及蛋白酶体降解。
与US2、US3、US10和US11不同，HCMV L蛋白US6通过完全不同的策略影响抗原呈递。相反于与游离I类重链或完全组装的I类复合物相互作用，US6通过TAP复合物(TAP1/2)抑制胞质肽的易位。US6与TAP1的ER腔侧结合并导致构象变化，从而阻止ATP的结合。US6的ER-腔结构域中的残基89-108有助于US6与TAP的结合，并且是这种抑制的充分必要条件。这种对TAP活性的抑制不仅影响经典MHC I类等位基因的表达，还会影响胎儿细胞滋养层细胞中非经典等位基因HLA-C和HLA-G的表达。
作为HCMV US6蛋白，HSV ICP47在感染周期的早期表达，对于体外复制是可有可无的，也可以应用相同的策略来阻止I类分子组装。ICP47阻断TAP介导的肽转运并与TAP1-TAP2复合物紧密结合。ICP47阻断TAP机制的一个线索是它表现出高物种选择性。HSV1和HSV2I CP47均抑制猿、猴、猪、狗和牛TAP，对小鼠、大鼠、豚鼠或兔TAP几乎没有影响。ICP47对人TAP的亲和力比对小鼠TAP的亲和力高约100倍。ICP47抑制肽与TAP的结合，但不影响ATP结合。ICP47对TAP的亲和力比大多数肽高10-1000倍，可作为肽与TAP结合的竞争性抑制剂，并被认为直接与肽结合位点结合。
α/βT淋巴细胞通过称为αβT细胞抗原受体(TCR)CD3复合物的多聚蛋白集合识别肽-MHC配体。该结构由结合抗原的可变αβTCR二聚体和参与TCR.CD3表面转运、稳定和信号转导的三个不变二聚体(CD3γε、δε和ζζ)组成。αβT细胞受体(αβTCR)在大多数(约95％)T细胞上表达，并通过识别主要组织相容性复合体(MHC)锚定抗原在T细胞活化中发挥关键作用。因此，TCR介导的T细胞活化是同种异体造血细胞移植(allo-HCT)和同种异体CAR-T细胞治疗过程中移植物抗宿主病(GVHD)发病机制的关键步骤。
人类白细胞抗原(HLA)系统或复合物是一组由人类主要组织相容性复合物(MHC)基因复合物编码的相关蛋白质。这些细胞表面蛋白负责调节免疫系统。在治疗性移植环境中，当同种异体移植物上的供体HLA与受体不同时，就会发生“HLA不匹配”。HLA不匹配导致同种异体反应性T细胞的激活，这会在移植后的六个月内引起急性细胞排斥(ACR)。错配的供体HLA抗原也是新生供体特异性HLA抗体(dnDSA)开发的靶标，其在急性和慢性移植T细胞排斥中发挥增强作用。因此，为了产生用于安全同种异体输注和治疗目的的通用T细胞，建议通过基因破坏TCR来有效阻断移植物抗宿主病(GVHD)。此外，有必要抑制同种异体T细胞上的HLA表达，以减少受体免疫系统对同种异体T细胞TCRαβ和/或HLA I类的排斥反应。
因此，需要开发新的细胞治疗方法，以便能克服现有细胞治疗中面临的肿瘤靶标差异性，肿瘤免疫抑制环境，个性化治疗细胞来源问题，无法标准化和规模化生产，杀伤效率低等问题。
发明内容
本公开的目的是为了克服现有肿瘤细胞治疗中面临的肿瘤靶标多样性，肿瘤免疫抑制环境，个性化治疗细胞来源问题，无法标准化和规模化生产，杀伤效率低等的问题，本公开的一个目的是提供一种通用T(Universal T，UT)组件，其可以劫持ERAD机制以阻止ER中的TCR、MHC分子，阻止其运输并促进其易位到细胞质中，以通过蛋白酶体进行泛素化和降解。这种设计可以针对任何蛋白质(包括内源性蛋白质或外源性蛋白质)，以有效抑制表达和快速降解以达到治疗目的。
UT组件的概念是基于发现病毒ER驻留糖蛋白可以劫持ERAD调节机制来抑制/阻断MHC分子组装、运输或促进其泛素化和蛋白酶体降解，从而抑制MHC介导的病毒抗原呈递以逃避免疫监视。TCR的有效下调将显着抑制TCR介导的免疫攻击并减少T细胞同种异体输血过程中的GVHD。包括天然病毒内质网驻留糖蛋白可以进一步抑制MHC分子，从而阻止肽呈递给受体CD8+T细胞并抑制同种异体T细胞的免疫识别。因此，UT组件可以提高输注后同种异体T细胞的相容性和长期持久性。对于细胞治疗目的，TCRαβ和MHC分子表面表达有缺陷的通用T细胞可以进一步基因工程用于治疗目的。
本公开的另一个目的是提供通过导入NK元件，特别是优化重组的NK元件，让T细胞能象NK细胞一样高效广谱的识别所有病毒感染细胞和肿瘤细胞，通过优化转导元件，CNK-T细胞能高效激活并杀伤肿瘤细胞。因为NK靶点包括MICA、MICB和ULBP1–6等家族成员蛋白，能广泛表达在各类肿瘤细胞并覆盖肿瘤进展的不同阶 段，所以，CNK技术可有效的解决单一CAR-T的脱靶效应，消除肿瘤免疫逃逸的机率。同时，CNK在设计中导入的复合型转接器(Chimeric Adaptor)，能有效的转导和放大NK信号，跨越如PD1信号的免疫检查点的限制，高效激活T细胞，并实现对肿瘤细胞的杀伤。复合型转接器(Chimeric Adaptor)实现放大CNK-T细胞信号，能抵抗肿瘤环境中的免疫抑制，实现对T细胞的激活，实现对肿瘤细胞的杀伤；再有CNK-T细胞通过NK识别靶点，还能清除MDSC等免疫抑制细胞。病毒感染细胞后，表达特定功能蛋白，MHCI的组装或转运或直接促进MHCI分子的定向降解，从而抑制病毒抗原表位的呈递，产生免疫逃逸。
本公开通过重新设计病毒元件，靶向TCR分子，实现TCR分子内质网滞留和定向降解，同时，本公开还通过导入特定病毒元件，实现MHCI分子的表达抑制或定向降解，从而实现T细胞通用型改造。
在本公开中验证了CNK-UT具有广谱的肿瘤识别和杀伤能力。本公开进一步设计复合特定靶点CAR/CNK-UT产品，论证了CAR/CNK-UT产品比常规CAR-T对肿瘤细胞具有更强大的杀伤和激活功能；在动物实验，CAR/CNK-UT产品也具有更高效的肿瘤清除能力。UT技术实现了异体通用型的改造，T细胞来源健康供者，从而实现了CNK-UT产品的标准化和规模化生产，可以提前制备，并保证T细胞对肿瘤细胞杀伤和激活的功能。
附图说明
图1示出了四种结构的CNK-UT多功能复合体(multi-functional complex)，其中，图1A为基本CNK-UT多功能复合体；图1B为增加靶向MHC I结合蛋白分子结构域的CNK-UT多功能复合体；图1C为增加靶向MHC I的结合蛋白分子结构域和复合型转接器的CNK-UT多功能复合体；图1D为增加靶向MHC I的结合蛋白分子结构域和CAR或TCR等靶向杀伤肿瘤细胞的受体的CNK-UT多功能复合体。
图2示出了四种表达CNK-UT多功能复合体的CNK-UT元件的结构，其中，如图2A所示，单一慢病毒EF1α启动子表达载体，启动表达CNK-UT元件其中一种组合：DAP10-DAP12 ICD-T2A-NKG2D-p2A-anti-TCR-AdE3 ERAD；如图2B所示，利用单一慢病毒EF1α启动子表达载体，启动表达CNK-UT元件其中一种组合：DAP10-DAP12 ICD-T2A-NKG2D-p2A-anti-TCR-US2 ERAD；如图2C所示，利用单一慢病毒表达载体，利用EF1α和CMV启动子，分别调控DAP10-CD3ζ-T2A-NKG2D-p2A-anti-TAA scFv-DAP10和anti-TCR-AdE3 ERAD-E2A-AdE3多基因的表达，实现在单个载体上表达多个CNK-UT功能元件；如图2D所示，利用两种不同慢病毒表达载体，分别调控anti-TAA scFv-CD28/4-1BB-CD3ζ-T2A-CD3ζ-p2A-NKG2D和anti-TCR-AdE3 ERAD-T2A-AdE3的表达，共转染T细胞后，实现同一T细胞上表达CNK-UT多个功能元件。
图3示出了一种CNK-UT(DAP10-CD3ζ-T2A-NKG2D-p2A-anti-TCR scFv-AdE3)细胞基本表型的流式细胞检测结果。
图4示出了CNK-UT细胞对人结肠腺瘤细胞系HT29的识别和特异性杀伤结果，其中，图4A示出了人结肠腺瘤细胞系HT29不同NK靶点蛋白的表达情况；图4B示出了CNK-UT细胞具有对HT29高效的杀伤和激活功能。
图5示出了CNK-UT细胞对MDA-MB453的识别和特异性杀伤，其中，图5A示出了三阴乳腺癌细胞系MDA-MB453不同NK靶点蛋白的表达情况；图6B示出了CNK-UT细胞对THP1显示高效的杀伤和激活功能。
图6示出了CNK-UT细胞对THP1的识别和特异性杀伤，其中，图6A示出了急性髓系白血病细胞THP1不同NK靶点的表达情况；图B示出了CAR/CNK-UT细胞比常规CAR-T细胞显示对HepG2细胞更高效的杀伤和激活功能。
图7示出了CNK-UT细胞能升级常规CAR-T技术，实现更强大的靶向杀伤和激活能力，其中，图7A示出了肝细胞癌(HCC)HepG2的GPC3、PD-L1和不同NK靶点的表达情况；图7B示出了
图8示出了CNK-UT细胞能升级常规CAR-T技术，实现对高表达PD-L1肿瘤细胞的靶向杀伤和激活能力，其中，图8A示出了肝细胞癌(HCC)PLC的GPC3、PD-L1和不同NK靶点的表达情况；图8B示出了CNK-UT细胞比常规CAR-T细胞显示对HepG2细胞更高效的杀伤和激活功能。
图9示出了GPC3 CAR/CNK-UT细胞比GPC3 CAR-T细胞在小鼠体内具有更高效的肿瘤清除能力。
图10示出了GPC3 CAR/CNK-UT细胞比GPC3 CAR-T具有高效的肿瘤清除能力。
图11示出了GPC3 CAR/CNK-UT能特异性的识别和杀伤三阴乳腺癌细胞MDA-MB453。
图12示出了GPC3 CAR/CNK-UT能特异性的识别和杀伤急性髓系白血病细胞系THP1。
图13示出了CNK-UT细胞对肾细胞癌786-O细胞的识别，特异性杀伤和激活能力。
图14示出了CNK-UT细胞对肾细胞癌ACHN细胞的识别，特异性杀伤和激活能力。
图15示出了CNK-UT细胞对肺上皮腺瘤A549细胞的识别，特异性杀伤和激活能力。
图16示出了CNK-UT细胞对AML细胞系UL60的识别，特异性杀伤和激活能力。
图17示出了CNK-UT细胞对AML细胞系U937(购自U937，货号CL-0239)的识别，特异性杀伤和激活能力。
图18示出了CNK-UT细胞对AML细胞系U937的识别，特异性杀伤和激活能力。
图19示出了CNK-UT细胞对胰腺癌细胞系PANC-1(购自普诺赛，货号CL-0184)的识别，特异性杀伤和激活能力。
具体实施方式
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并 且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
术语“嵌合自然杀伤受体”(chimeric natural killer receptor，CNK)是指含有嵌合型NK激活受体组件和嵌合型NK信号转接组件的组合物。
术语“CNK T细胞”是指含有CNK构建体的T细胞。
术语“嵌合自然杀伤受体通用型T细胞”(chimeric natural killer receptor-Universal T cell，CNK-UT)是指表达NK激活受体组件构建体、CNK信号转接组件构建体和UT组件构建体的T细胞。
术语“CAR/CNK-UT”是嵌合抗原受体和嵌合自然杀伤受体通用型T细胞(chimeric antigen receptor&chimeric natural killer receptor-Universal T cell，CNK-UT)，是指表达靶向特定肿瘤抗原CAR结构，NK激活受体组件构建体、CNK信号转接组件构建体和UT组件构建体的T细胞。
如在本文中所使用的术语“约”指可测量的值，例如量、持续时间等，并且包括与指定值相差±20％、±10％、±5％、±1％、±0.5％或±0.1％的变化方式。
如在本文中所使用的术语“抗体”指与抗原特异性结合的免疫球蛋白分子。抗体可以是源自天然来源或重组来源的完整免疫球蛋白并且可以是完整免疫球蛋白的免疫反应部分。抗体通常是免疫球蛋白分子的四聚体。本公开中的抗体可以以多种形式存在，包括例如多克隆抗体、单克隆抗体、Fv、Fab和F(ab)，以及单链抗体和人源化抗体。
如在本文中所使用的术语“共刺激配体”包括在抗原呈递细胞(例如APC、树突状细胞、B细胞和其他免疫细胞)上的分子，其特异性结合T细胞上的关联共刺激分子，从而提供信号，该信号除了通过例如TCR/CD3复合物与载有肽的MHC分子结合提供的初级信号外，还介导T细胞响应，包括但不限于增殖、激活、分化等。共刺激配体可包括但不限于CD7、B7-1(CD80)、B7-2(CD86)、PD-L1、PD-L2、4-1BBL、OX40L、诱导型共刺激配体(ICOS-L)、细胞间粘附分子(ICAM)、CD30L、CD40、CD70、CD83、HLA-G、MICA、MICB、HVEM、淋巴毒素β受体、3/TR6、ILT3、ILT4、HVEM，与Toll配体受体结合的激动剂或抗体以及与B7-H3特异性结合的配体。共刺激配体还包括尤其是与T细胞上存在的共刺激分子特异性结合的抗体，例如但不限于CD27、CD28、4-1BB、OX40、CD30、CD40、PD-1、ICOS、淋巴细胞功能相关抗原-1(LFA-1)、CD2、CD7LIGHT、NKG2C、B7-H3和与CD83特异性结合的配体。
“共刺激分子”或“共刺激受体”指T细胞上的关联结合配偶体，其与共刺激配体特异性结合，从而介导T细胞的共刺激响应，例如但不限于增殖。共刺激分子包括但不限于MHCI类分子、BTLA、Toll配体受体。共刺激分子还包括非天然工程化蛋白。
如在本文中所使用的“共刺激信号”指与初级信号例如TCR/CD3连接结合，导致T细胞增殖和/或关键分子的上调或下调的信号。
术语“刺激”是指通过刺激性分子(例如TCR/CD3复合物)与其关联配体结合诱导的初级响应，从而介导信号转导事件，例如但不限于，通过TCR/CD3复合物进行信号转导。刺激可以介导某些分子的改变的表达，例如TGF-B的下调，和/或细胞骨架结构的重组，等等。
如在本文中所使用的术语“刺激性分子”指T细胞上与抗原呈递细胞上存在的关联刺激性配体特异性结合的分子。
如在本文中所使用的“刺激性配体”指当存在于抗原呈递细胞(例如APC、树突状细胞、B细胞等)上时可以与T细胞上的关联结合配偶体(在本文中称为“刺激性分子”)特异性结合，从而介导T细胞的初级应答，包括但不限于激活、免疫应答的启动、增殖等的配体。刺激性配体在本领域中是众所周知的，并且包括，尤其是，载有肽的MHCI类分子、抗CD3抗体、超激动剂抗CD28抗体，和超激动剂抗CD2抗体。
“载体”是包含分离的核酸并且可以用于将分离的核酸递送至细胞内部的物质组合物。许多载体是本领域已知的，包括但不限于线性多核苷酸、与离子或两性化合物相关的多核苷酸、质粒和病毒。因此，术语“载体”包括自主复制质粒或病毒。该术语也应解释为包括有助于核酸转移到细胞中的非质粒和非病毒化合物，例如聚赖氨酸化合物，脂质体等。病毒载体的实例包括但不限于慢病毒、腺病毒载体、腺相关病毒载体、逆转录病毒载体等。非病毒载体的实例包括但不限于CRISPR载体系统、Sleeping Beauty转座子系统等。如在本文中所使用的“激活”指已经被充分刺激以诱导可检测的细胞增殖的T细胞的状态。激活还可以与诱导的细胞因子产生和可检测的效应子功能相关。术语“激活的T细胞”尤其指正在进行细胞分裂的T细胞。
在一方面，本公开提供了一种多功能复合体(multi-functional complex)，其包含以下组件：
(1)NK激活受体组件，其至少包含NK细胞激活受体或其功能变体，所述NK细胞激活受体包含：(a)NK细胞激活受体胞外结构域(ED)或其功能变体，(b)NK细胞激活受体跨膜结构域(TMD)或其功能变体，以及(c)NK细胞激活受体胞内结构域(ICD)或其功能变体；任选地，所述NK细胞激活受体胞外结构域或其功能变体、所述NK细胞激活受体跨膜结构域或其功能变体和/或所述NK细胞激活受体胞内结构域或其功能变体之间包含铰链或接头；
(2)CNK信号转接组件，其至少包含(i)NK细胞信号转换器(adaptor)或其功能变体，所述NK细胞信号转换器包含：(a)NK细胞信号转换器胞外结构域(ED)或其功能变体，(b)NK细胞信号转换器跨膜结构域(TMD)或其功能变体，以及(c)NK细胞信号转换器胞内结构域(ICD)或其功能变体；任选地，所述NK细 胞信号转换器胞外结构域或其功能变体、所述NK细胞信号转换器跨膜结构域或其功能变体和/或所述NK细胞信号转换器胞内结构域或其功能变体之间包含铰链或接头；和
(3)UT组件，其至少包含(i)靶向降解TCR、MHC和/或NK细胞靶点的重组蛋白分子或其功能变体，所述靶向降解TCR、MHC和/或NK细胞靶点的重组蛋白分子包含：(a)靶向TCR、MHC和/或NK细胞靶点的结合蛋白分子结构域或其功能变体，(b)病毒内质网(ER)驻留糖蛋白的跨膜结构域或其功能变体，以及(c)病毒内质网驻留糖蛋白的胞质结构域或其功能变体；所述病毒内质网驻留糖蛋白的跨膜结构域或其功能变体和病毒内质网驻留糖蛋白的胞质结构域或其功能变体形成ERAD降解结构域；任选地，所述靶向TCR的结合蛋白分子结构域或其功能变体、所述病毒内质网驻留糖蛋白的跨膜结构域或其功能变体和/或所述病毒内质网驻留糖蛋白的胞质结构域或其功能变体之间包含铰链或接头。
任选地，所述NK激活受体组件、所述CNK信号转接组件和/或所述UT组件之间包含铰链或接头。
在一些实施方案中，所述NK激活受体组件中的所述NK细胞激活受体选自NKG2D、NKG2C、NKG2E、NKG2F、NKG2H、CD94、KIR2DL4、KIR2DS1、KIR2DS2、KIR2DS4、KIR3DS1，天然细胞毒性受体、TRAIL、DNAM-1、CD16a、2B4、NTB-A、CRACC以及NKp80；优选地，所述天然细胞毒性受体选自NKp46、NKp44和NKp30。
在一些优选的实施方案中，所述NK细胞激活受体是哺乳动物来源的NK细胞激活受体；优选地，所述哺乳动物选自人、灵长类动物、鼠、马、牛、绵羊、山羊、猫、猪、狗、美洲驼、羊驼、大象、松鼠、豚鼠。
在一些优选的实施方案中，所述NK细胞激活受体是包含不同来源NK细胞激活受体结构域的重组NK细胞激活受体。
在一些优选的实施方案中，所述NK细胞激活受体是人源的NK细胞激活受体；优选地，所述NK细胞激活受体是包含不同的人源NK细胞激活受体结构域的重组NK细胞激活受体。
在一些优选的实施方案中，所述NK细胞激活受体是鼠源的NK细胞激活受体；优选地，所述NK细胞激活受体是包含不同的鼠源NK细胞激活受体结构域的重组NK细胞激活受体。
在一些优选的实施方案中，所述NK细胞激活受体是包含人源和鼠源NK细胞激活受体结构域的重组NK细胞激活受体。
在一些优选的实施方案中，所述NK细胞激活受体的胞外结构域为人或鼠的NK细胞激活受体的胞外结构域。
在一些优选的实施方案中，所述NK细胞激活受体的跨膜结构域为人或鼠的NK细胞激活受体的跨膜结构域。
在一些优选的实施方案中，所述NK细胞激活受体的胞内结构域为人或鼠的NK细胞激活受体的胞内结构域。
在一些优选的实施方案中，所述NK细胞激活受体功能变体选自NK细胞激活受体的突变体，野生型融合蛋白，或野生型与突变型的融合蛋白。
在一些优选的实施方案中，人NKG2D的胞外域包含与SEQ ID NO.1所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKG2D的胞外域的氨基酸序列如SEQ ID NO.1所示。
在一些优选的实施方案中，人NKG2D的全长序列包含与SEQ ID NO.2所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKG2D的全长序列的氨基酸序列如SEQ ID NO.2所示。
在一些优选的实施方案中，小鼠NKG2D的胞外域包含与SEQ ID NO.3所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；小鼠NKG2D的胞外域的氨基酸序列如SEQ ID NO.3所示。
在一些优选的实施方案中，小鼠NKG2D的全长序列包含与SEQ ID NO:4所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；小鼠NKG2D的全长序的氨基酸序列如SEQ ID NO.4所示。
在一些优选的实施方案中，人鼠重组NKG2D的全长序列包含与SEQ ID NO:5所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人鼠重组NKG2D的全长序列的氨基酸序列如SEQ ID NO.5所示。
在一些优选的实施方案中，人NKG2C的全长序列包含与SEQ ID NO.6所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKG2C的全长序列的氨基酸序列如SEQ ID NO.6所示。
在一些优选的实施方案中，人NKG2E的全长序列包含与SEQ ID NO：7所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKG2E的全长序列的氨基酸序列如SEQ ID NO.7所示。
在一些优选的实施方案中，人NKG2F的全长序列包含与SEQ ID NO：8所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKG2F的全长序列的氨基酸序列如SEQ ID NO.8所示。
在一些优选的实施方案中，人CD94的全长序列包含与SEQ ID NO.9所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；的氨基酸序列如SEQ ID NO.9所示。
在一些优选的实施方案中，人KIR2DL4的全长序列包含与SEQ ID NO：10所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人KIR2DL4的全长序列的氨基酸序列如SEQ ID NO.10所示。
在一些优选的实施方案中，人KIR2DS1的全长序列包含与SEQ ID NO：11所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人KIR2DS1的全长序列的氨基酸序列如SEQ ID NO.11所示。
在一些优选的实施方案中，人KIR2DS2的全长序列包含与SEQ ID NO：12所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人KIR2DS2的全长序列的氨基酸序列如SEQ ID NO.12所示。
在一些优选的实施方案中，人KIR2DS4的全长序列包含与SEQ ID NO：13所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人KIR2DS4的全长序列的氨基酸序列如SEQ ID NO.13所示。
在一些优选的实施方案中，人KIR3DS1的全长序列包含与SEQ ID NO：14所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人KIR3DS1的全长序列的氨基酸序列如SEQ ID NO.14所示。
在一些优选的实施方案中，人NKp46的全长序列包含与SEQ ID NO.15所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKp46的全长序列的氨基酸序列如SEQ ID NO.15所示。
在一些优选的实施方案中，人NKp44的全长序列包含与SEQ ID NO.16所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKp44的全长序列的氨基酸序列如SEQ ID NO.16所示。
在一些优选的实施方案中，人NKp30的全长序列包含与SEQ ID NO.17所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKp30的全长序列的氨基酸序列如SEQ ID NO.17所示。
在一些优选的实施方案中，人DNAM1的全长序列包含与SEQ ID NO：18所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DNAM1的全长序列的氨基酸序列如SEQ ID NO.18所示。
在一些优选的实施方案中，人TRAIL的全长序列包含与SEQ ID NO：19所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人TRAIL的全长序列的氨基酸序列如SEQ ID NO.19所示。
在一些优选的实施方案中，人CD16a的全长序列包含与SEQ ID NO：20所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人CD16a的全长序列的氨基酸序列如SEQ ID NO.20所示。
在一些优选的实施方案中，人2B4的全长序列包含与SEQ ID NO：21所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人2B4的全长序列的氨基酸序列如SEQ ID NO.21所示。
在一些优选的实施方案中，人NTB-A的全长序列包含与SEQ ID NO：22所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NTB-A的全长序列的氨基酸序列如SEQ ID NO.22所示。
在一些优选的实施方案中，人CRACC的全长序列包含与SEQ ID NO：23所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人CRACC的全长序列的氨基酸序列如SEQ ID NO.23所示。
在一些优选的实施方案中，人NKp80的全长序列包含与SEQ ID NO：24所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKp80的全长序列的氨基酸序列如SEQ ID NO.24所示。
在一些实施方案中，所述CNK信号转接组件中的所述NK细胞信号转换器为DAP10或DAP12。
在一些优选的实施方案中，所述NK细胞信号转换器是哺乳动物来源的NK细胞信号转换器；优选地，所述哺乳动物选自人、灵长类动物、鼠、马、牛、绵羊、山羊、猫、猪、狗、美洲驼、羊驼、大象、松鼠、豚鼠。
在一些优选的实施方案中，所述NK细胞信号转换器是包含不同来源NK细胞信号转换器结构域的重组NK细胞信号转换器。
在一些优选的实施方案中，所述NK细胞信号转换器是人源的NK细胞信号转换器；优选地，所述NK细胞信号转换器是包含不同的人源NK细胞信号转换器结构域的重组NK细胞信号转换器。
在一些优选的实施方案中，所述NK细胞信号转换器是鼠源的NK细胞信号转换器；优选地，所述NK细胞信号转换器是包含不同的鼠源NK细胞信号转换器结构域的重组NK细胞信号转换器。
在一些优选的实施方案中，所述NK细胞信号转换器是包含人源和鼠源NK细胞信号转换器结构域的重组NK细胞信号转换器。
在一些优选的实施方案中，所述NK细胞信号转换器的胞外结构域为人或鼠的NK细胞信号转换器的胞外结构域。
在一些优选的实施方案中，所述NK细胞信号转换器的跨膜结构域为人或鼠的NK细胞信号转换器的跨膜结构域。
在一些优选的实施方案中，所述NK细胞信号转换器的胞内结构域为人或鼠的NK细胞信号转换器的胞内结构域；
在一些优选的实施方案中，所述CNK细胞信号转换器功能变体选自DAP10突变体或DAP12的突变体，或DAP10和DAP12的融合蛋白，或野生型DAP10或DAP12与突变型DAP10或DAP12的融合蛋白。
在一些优选的实施方案中，所述CNK信号转接组件还包含(ii)免疫受体活化信号传导结构域(ITAM)和/或(iii)T细胞共刺激信号传导结构域。
在一些优选的实施方案中，所述NK细胞信号转换器或其功能变体、所述免疫受体活化信号传导结构域(ITAM)和/或所述T细胞共刺激信号传导结构域之间包含铰链或接头；优选地，所述NK细胞信号转换器或其功能变体与所述免疫受体活化信号传导结构域(ITAM)域融合。
在一些优选的实施方案中，所述免疫受体活化信号传导结构域(ITAM)来自免疫受体的胞内激活信号传导结构域；优选地，所免疫受体选自TCRζ、CD2、CD3γ、CD3δ、CD3ε、CD3ζ、CD5、CD22、FcRγ、CD66d、FcαRI、FcγRI、FcγRII、FcγRIII、Dectin-1、CLEC-1、CD72、CD79A、CD79B；优选地，所述免疫受体活化信号传导结构域(ITAM)与NK细胞信号转换器或其功能变体融合；优选地，所述免疫受体是CD3ζ。
在一些优选的实施方案中，所述T细胞共刺激信号传导结构域来自共刺激分子的胞内信号结构域；优选地，所述共刺激分子选自MHC I类分子、TNF受体蛋白、免疫球蛋白样蛋白质、细胞因子受体、整联蛋白、淋巴细胞活化信号分子(SLAM蛋白)、活化NK细胞受体、BTLA、Toll配体受体、OX40、CD2、CD7、CD16、CD27、CD28、CD30、CD40、CD38、CD35、CD79A、CD79B、CDS、ICAM-1、LFA-1、(CD11a/CD18)、4-1BB(CD137)、B7-H3、CDS、ICAM-1、ICOS(CD278)、GITR、BAFFR、LIGHT、HVEM(LIGHTR)、KIRDS2、SLAMF7、NKp80(KLRF1)、NKp44、NKp30、NKp46、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、NKG2D、NKG2C、NCR、DAP10、DAP12、TNFR2、TRANCE/RANKL、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100SEMA4D)、CD69、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、CD19a、与CD83特异性结合的配体、CARD11、FcRa、FcRp、FcRy、Fyn、HVEM、ICOS、Lck、LAG3、LAT、LRP、NOTCH1、Wnt、OX40、ROR2、Ryk、SLAMF1、Slp76、pTa、TCRa、TCRp、TRIM、ZAP70、PTCH2。
在一些优选的实施方案中，人DAP10的全长序列包含与SEQ ID NO.25所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10的全长序列的氨基酸序列如SEQ ID NO.25所示。
在一些优选的实施方案中，人DAP10的全长序列包含与SEQ ID NO.26所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10的全长序列的氨基酸序列如SEQ ID NO.26所示。
在一些优选的实施方案中，人DAP10的跨膜结构域包含与SEQ ID NO.27所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10的跨膜结构域的氨基酸序列如SEQ ID NO.27所示。
在一些优选的实施方案中，人DAP12的全长序列包含与SEQ ID NO.28所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP12的全长序列的氨基酸序列如SEQ ID NO.28所示。
在一些优选的实施方案中，人DAP12的跨膜结构域包含与SEQ ID NO.29所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP12的跨膜结构域的氨基酸序列如SEQ ID NO.29所示。
在一些优选的实施方案中，人DAP10和人DAP12的跨膜结构域融合蛋白包含与SEQ ID NO.30所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10和人DAP12的跨膜结构域的氨基酸序列如SEQ ID NO.30所示。
在一些优选的实施方案中，人DAP10-DAP12融合蛋白序列包含与SEQ ID NO.31所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10-DAP12融合蛋白序列的氨基酸序列如SEQ ID NO.31所示。
在一些优选的实施方案中，人CD3zeta胞内信号传导结构域序列包含与SEQ ID NO.32所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序 列，更优选具有98％或99％以上同一性的氨基酸序列；人CD3zeta胞内信号传导结构域序列的氨基酸序列如SEQ ID NO.32所示。
在一些优选的实施方案中，人DAP10-CD3zeta序列包含与SEQ ID NO.33所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10-CD3zeta序列的氨基酸序列如SEQ ID NO.33所示。
在一些优选的实施方案中，人DAP12-CD3zeta序列包含与SEQ ID NO.34所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP12-CD3zeta序列的氨基酸序列如SEQ ID NO.34所示。
在一些优选的实施方案中，人DAP10-DAP12-CD3zeta序列包含与SEQ ID NO.35所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10-DAP12-CD3zeta序列的氨基酸序列如SEQ ID NO.35所示。
在一些优选的实施方案中，人41BB胞内信号传导结构域序列包含与SEQ ID NO.36所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人41BB胞内信号传导结构域序列的氨基酸序列如SEQ ID NO.36所示。
在一些优选的实施方案中，人DAP10-41BB序列包含与SEQ ID NO.37所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10-41BB序列的氨基酸序列如SEQ ID NO.37所示。
在一些优选的实施方案中，人DAP10-41BB-CD3zeta序列包含与SEQ ID NO.38所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10-41BB-CD3zeta序列的氨基酸序列如SEQ ID NO.38所示。
在一些优选的实施方案中，人CD28胞内信号传导结构域序列包含与SEQ ID NO.39所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人CD28胞内信号传导结构域序列的氨基酸序列如SEQ ID NO.39所示。
在一些优选的实施方案中，人DAP10-CD28序列包含与SEQ ID NO.40所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10-CD28序列的氨基酸序列如SEQ ID NO.40所示。
在一些优选的实施方案中，人DAP10-CD28-CD3zeta序列包含与SEQ ID NO.41所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10-CD28-CD3zeta序列的氨基酸序列如SEQ ID NO.41所示。
在一些优选的实施方案中，人DAP12-41BB序列包含与SEQ ID NO.42所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP12-41BB序列的氨基酸序列如SEQ ID NO.42所示。
在一些优选的实施方案中，人DAP12-41BB-CD3zeta序列包含与SEQ ID NO.43所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP12-41BB-CD3zeta序列的氨基酸序列如SEQ ID NO.43所示。
在一些优选的实施方案中，人DAP12-CD28序列包含与SEQ ID NO.44所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP12-CD28序列的氨基酸序列如SEQ ID NO.44所示。
在一些优选的实施方案中，人DAP12-CD28-CD3zeta序列包含与SEQ ID NO.45所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP12-CD28-CD3zeta序列的氨基酸序列如SEQ ID NO.45所示。
在一些实施方案中，在所述UT组件的靶向降解TCR的重组蛋白分子中，所述靶向TCR的结合蛋白分子结构域或其功能变体来自TCR抗体或其功能片段或其组合。
在一些优选的实施方案中，所述抗体选自TCRα抗体、TCRβ抗体、TCRαβ抗体、TCRγ抗体、TCRδ抗体、TCRγδ抗体、TCR Vδ2抗体、TCR Cβ1抗体；所述抗体的功能片段选自Fd、Fv、Fab、Fab’、F(ab’)2、Fv(scFv)、单链抗体(scFv)或纳米抗体(nanobody)、双链抗体、三链抗体和四链抗体；优选地，所述TCR抗体是TCR单链抗体；优选地，所述TCR单链抗体的氨基酸序列包含与SEQ ID NO.116所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述TCR单链抗体的全长序列的氨基酸序列如SEQ ID NO.116所示。
在一些优选的实施方案中，所述UT组件中的ERAD降解结构域来自HCMV糖蛋白US2、US3、US11或 US10、腺病毒E3-19K或HHV-7US21。
在一些优选的实施方案中，所述HCMV糖蛋白US2的全长序列包含与SEQ ID NO.46所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US2的全长序列的氨基酸序列如SEQ ID NO.46所示。
在一些优选的实施方案中，所述HCMV糖蛋白US2的HLA结合结构域包含与SEQ ID NO.47所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US2的HLA结合结构域的氨基酸序列如SEQ ID NO.47所示。
在一些优选的实施方案中，所述HCMV糖蛋白US2的ERAD降解结构域包含与SEQ ID NO.48所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US2的ERAD降解结构域的氨基酸序列如SEQ ID NO.48所示。
在一些优选的实施方案中，所述HCMV糖蛋白US3的全长序列包含与SEQ ID NO.49所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US3的全长序列的氨基酸序列如SEQ ID NO.49所示。
在一些优选的实施方案中，所述HCMV糖蛋白US3的HLA结合结构域包含与SEQ ID NO.50所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US3的HLA结合结构域的氨基酸序列如SEQ ID NO.50所示。
在一些优选的实施方案中，所述HCMV糖蛋白US3的ERAD降解结构域包含与SEQ ID NO.51所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US3的ERAD降解结构域的氨基酸序列如SEQ ID NO.51所示。
在一些优选的实施方案中，所述HCMV糖蛋白US11的全长序列包含与SEQ ID NO.52所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US11的全长序列的氨基酸序列如SEQ ID NO.52所示。
在一些优选的实施方案中，所述HCMV糖蛋白US11的MHC结合结构域包含与SEQ ID NO.53所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US11的MHC结合结构域的氨基酸序列如SEQ ID NO.53所示。
在一些优选的实施方案中，所述HCMV糖蛋白US11的ERAD降解结构域包含与SEQ ID NO.54所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US11的ERAD降解结构域的氨基酸序列如SEQ ID NO.54所示。
在一些优选的实施方案中，所述HCMV糖蛋白US10的全长序列包含与SEQ ID NO.55所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US10的全长序列的氨基酸序列如SEQ ID NO.55所示。
在一些优选的实施方案中，所述HCMV糖蛋白US10的HLA结合结构域包含与SEQ ID NO.56所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US10的HLA结合结构域的氨基酸序列如SEQ ID NO.56所示。
在一些优选的实施方案中，所述HCMV糖蛋白US10的ERAD降解结构域包含与SEQ ID NO.57所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US10的ERAD降解结构域的氨基酸序列如SEQ ID NO.57所示。
在一些优选的实施方案中，所述腺病毒E3-19K的全长序列包含与SEQ ID NO.58所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述腺病毒E3-19K的全长序列的氨基酸序列如SEQ ID NO.58所示。
在一些优选的实施方案中，所述腺病毒E3-19K的MHC结合结构域包含与SEQ ID NO.59所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述腺病毒E3-19K的MHC结合结构域的氨基酸序列如 SEQ ID NO.59所示。
在一些优选的实施方案中，所述腺病毒E3-19K的ERAD降解结构域包含与SEQ ID NO.60所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述腺病毒E3-19K的ERAD降解结构域的氨基酸序列如SEQ ID NO.60所示。
在一些优选的实施方案中，所述HHV-7US21的全长序列包含与SEQ ID NO.61所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HHV-7US21的全长序列的氨基酸序列如SEQ ID NO.61所示。
在一些优选的实施方案中，所述HHV-7US21的MHC结合结构域包含与SEQ ID NO.62所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HHV-7US21的MHC结合结构域的氨基酸序列如SEQ ID NO.62所示。
在一些优选的实施方案中，所述HHV-7US21的ERAD降解结构域包含与SEQ ID NO.63所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HHV-7US21的ERAD降解结构域的氨基酸序列如SEQ ID NO.63所示。
在一些实施方案中，所述UT组件还包含(ii)靶向MHC I和/或MHC II的结合蛋白分子结构域或其功能变体。
在一些优选的实施方案中，所述靶向MHC I和/或MHC II的结合蛋白分子结构域或其功能变体为靶向HLA的结合蛋白分子结构域或其功能变体。
在一些优选的实施方案中，所述靶向MHC I和/或MHC II的结合蛋白分子结构域或其功能变体来自抑制MHC分子表达或促进其降解的病毒内质网蛋白；优选地，所述病毒内质网糖蛋白选自HCMV US6、HSV ICP47、CPXV012、HPV E6/E7、EBV BNFL2a或BHV UL49.5；优选地，所述靶向MHC I和/或MHC II的结合蛋白分子结构域或其功能变体包含TAP结合结构域。
在一些优选的实施方案中，所述HCMV US6的全长序列包含与SEQ ID NO.64所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV US6的全长序列的氨基酸序列如SEQ ID NO.64所示。
在一些优选的实施方案中，所述HHV-7US6的TAP结合结构域包含与SEQ ID NO.65所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HHV-7US6的TAP结合结构域的氨基酸序列如SEQ ID NO.65所示。
在一些优选的实施方案中，所述HSV ICP47的全长序列包含与SEQ ID NO.66所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HSV ICP47的全长序列的氨基酸序列如SEQ ID NO.66所示。
在一些优选的实施方案中，所述HSV ICP47的TAP结合结构域包含与SEQ ID NO.67所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HSV ICP47的TAP结合结构域的氨基酸序列如SEQ ID NO.67所示。
在一些优选的实施方案中，所述CPXV012的全长序列包含与SEQ ID NO.68所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述CPXV012的全长序列的氨基酸序列如SEQ ID NO.68所示。
在一些优选的实施方案中，所述CPXV012的TAP结合结构域包含与SEQ ID NO.69所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述CPXV012的TAP结合结构域的氨基酸序列如SEQ ID NO.69所示。
在一些优选的实施方案中，所述EBV BNFL2a的全长序列包含与SEQ ID NO.70所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述EBV BNFL2a的全长序列的氨基酸序列如SEQ ID NO.70所示。
在一些优选的实施方案中，所述EBV BNFL2a的TAP结合结构域包含与SEQ ID NO.71所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述EBV BNFL2a的TAP结合结构域的氨基酸序列如SEQ ID NO.71所示。
在一些优选的实施方案中，所述BHV UL49.5的全长序列包含与SEQ ID NO.72所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优 选具有98％或99％以上同一性的氨基酸序列；所述BHV UL49.5的全长序列的氨基酸序列如SEQ ID NO.72所示。
在一些优选的实施方案中，所述BHV UL49.5的TAP结合结构域包含与SEQ ID NO.73所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述BHV UL49.5的TAP结合结构域的氨基酸序列如SEQ ID NO.73所示。
在一些优选的实施方案中，所述靶向MHC I和/或MHC II的结合蛋白分子结构域或其功能变体来自降解MHC和/或MHC II分子的病毒糖蛋白；优选地，病毒糖蛋白选自HCMV糖蛋白US2、US3、US11或US10、腺病毒E3-19K或HHV-7US21。
在一些优选的实施方案中，所述US2的全长序列包含与SEQ ID NO.74所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述US2的全长序列的氨基酸序列如SEQ ID NO.74所示。
在一些优选的实施方案中，所述US2的HLA结合结构域包含与SEQ ID NO.75所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述US2的HLA结合结构域的氨基酸序列如SEQ ID NO.75所示。
在一些优选的实施方案中，所述US2的ERAD降解结构域包含与SEQ ID NO.76所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述US2的ERAD降解结构域的氨基酸序列如SEQ ID NO.76所示。
在一些优选的实施方案中，所述US3的全长序列包含与SEQ ID NO.77所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述US3的全长序列的氨基酸序列如SEQ ID NO.77所示。
在一些优选的实施方案中，所述US3的HLA结合结构域包含与SEQ ID NO.78所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述US3的HLA结合结构域的氨基酸序列如SEQ ID NO.78所示。
在一些优选的实施方案中，所述US3的ERAD降解结构域包含与SEQ ID NO.79所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述US3的ERAD降解结构域的氨基酸序列如SEQ ID NO.79所示。
在一些优选的实施方案中，所述US11的全长序列包含与SEQ ID NO.80所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述US11的全长序列的氨基酸序列如SEQ ID NO.80所示。
在一些优选的实施方案中，所述US11的HLA结合结构域包含与SEQ ID NO.81所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述US11的HLA结合结构域的氨基酸序列如SEQ ID NO.81所示。
在一些优选的实施方案中，所述US11的ERAD降解结构域包含与SEQ ID NO.82所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述US11的ERAD降解结构域的氨基酸序列如SEQ ID NO.82所示。
在一些优选的实施方案中，所述靶向MHC I和/或MHC II的结合蛋白分子结构域或其功能变体还包含定向抑制或降解MICA、MICB、ULBP1、ULBP2、ULBP3、ULBP4、ULBP5或ULBP6的NK靶点蛋白的病毒蛋白；优选地，所述病毒蛋白选自HCMV UL16、UL141、UL142或腺病毒E3-19K。
在一些优选的实施方案中，所述HCMV UL16的全长序列包含与SEQ ID NO.83所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV UL16的全长序列的氨基酸序列如SEQ ID NO.83所示。
在一些优选的实施方案中，所述HCMV UL16的NK靶点蛋白结合结构域包含与SEQ ID NO.84所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV UL16的NK靶点蛋白结合结构域的氨基酸序列如SEQ ID NO.84所示。
在一些优选的实施方案中，所述HCMV UL16的ERAD降解结构域包含与SEQ ID NO.85所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV UL16的ERAD降解结构域的氨基酸序列如SEQ ID NO.85所示。
在一些优选的实施方案中，所述HCMV UL141的全长序列包含与SEQ ID NO.86所示氨基酸序列具有80％或 以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV UL141的全长序列的氨基酸序列如SEQ ID NO.86所示。
在一些优选的实施方案中，所述HCMV UL141的NK靶点蛋白结合结构域包含与SEQ ID NO.87所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV UL141的NK靶点蛋白结合结构域的氨基酸序列如SEQ ID NO.87所示。
在一些优选的实施方案中，所述HCMV UL141的ERAD降解结构域包含与SEQ ID NO.88所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV UL141的ERAD降解结构域的氨基酸序列如SEQ ID NO.88所示。
在一些优选的实施方案中，所述HCMV UL142的全长序列包含与SEQ ID NO.89所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV UL142的全长序列的氨基酸序列如SEQ ID NO.89所示。
在一些优选的实施方案中，所述HCMV UL142的MICA、ULBP3结合结构域包含与SEQ ID NO.90所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV UL142的MICA、ULBP3结合结构域的氨基酸序列如SEQ ID NO.90所示。
在一些优选的实施方案中，所述HCMV UL142的高尔基驻留结构域包含与SEQ ID NO.91所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV UL142的高尔基驻留结构域的氨基酸序列如SEQ ID NO.91所示。
在一些优选的实施方案中，所述靶向MHC I和/或MHC II的结合蛋白分子结构域或其功能变体还包含把MHC I分子从高尔基体转运到溶酶体进行降解的病毒蛋白；优选地，所述病毒蛋白选自HIV Nef、HIV Vpu、HHV-7U21、HHV-8 KK3、HHV-8 KK5、MHV-68 MK3和HTLV-1 p12。
在一些优选的实施方案中，所述HIV Nef包含与SEQ ID NO.92所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HIV Nef的氨基酸序列如SEQ ID NO.92所示。
在一些优选的实施方案中，所述HIV Vpu包含与SEQ ID NO.93所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HIV Vpu的氨基酸序列如SEQ ID NO.93所示。
在一些优选的实施方案中，所述HHV-8 KK3包含与SEQ ID NO.94所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HHV-8 KK3的氨基酸序列如SEQ ID NO.94所示。
在一些优选的实施方案中，所述HHV-8 KK5包含与SEQ ID NO.95所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HHV-8 KK5的氨基酸序列如SEQ ID NO.95所示。
在一些优选的实施方案中，所述MHV-68 MK3包含与SEQ ID NO.96所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述MHV-68 MK3的氨基酸序列如SEQ ID NO.96所示。
在一些优选的实施方案中，所述HTLV-1 p12包含与SEQ ID NO.97所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HTLV-1 p12的氨基酸序列如SEQ ID NO.97所示。
在一些优选的实施方案中，所述靶向MHC I和/或MHC II的结合蛋白分子结构域或其功能变体还包含介导MHC-多肽分子从高尔基体返回内质网并促进其降解的病毒蛋白；优选地，所述病毒蛋白包含MHC结合结构和KDEL receptor结合结构域；优选地，所述病毒蛋白是Cowpox Virus蛋白CPXV203。
在一些优选的实施方案中，所述牛痘病毒蛋白CPXV203的全长序列包含与SEQ ID NO.98所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述牛痘病毒蛋白CPXV203的全长序列的氨基酸序列如SEQ ID NO.98所示。
在一些优选的实施方案中，所述牛痘病毒蛋白CPXV203的MHC结合结构域包含与SEQ ID NO.99所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述牛痘病毒蛋白CPXV203的MHC结合结构域的氨基酸序列如SEQ ID NO.99所示。
在一些优选的实施方案中，所述牛痘病毒蛋白CPXV203的KDEL受体结合结构域包含与SEQ ID NO.100所 示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述牛痘病毒蛋白CPXV203的KDEL受体结合结构域的氨基酸序列如SEQ ID NO.100所示。
在一些优选的实施方案中，所述牛痘病毒蛋白CPXV203的全长序列包含与SEQ ID NO.101所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述牛痘病毒蛋白CPXV203的全长序列的氨基酸序列如SEQ ID NO.101所示。
在一些优选的实施方案中，所述牛痘病毒蛋白CPXV203的MHC结合结构域包含与SEQ ID NO.102所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述牛痘病毒蛋白CPXV203的MHC结合结构域的氨基酸序列如SEQ ID NO.102所示。
在一些优选的实施方案中，所述牛痘病毒蛋白CPXV203的KDEL受体结合结构域包含与SEQ ID NO.103所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述牛痘病毒蛋白CPXV203的KDEL受体结合结构域的氨基酸序列如SEQ ID NO.103所示。
在一些实施方案中，所述多功能复合体还包含组件(4)复合型转接器(Chimeric adaptor)组件和/或靶向杀伤肿瘤细胞受体组件；
所述(4)复合型转接器包含：(i)靶向肿瘤的胞外识别结构域；(ii)跨膜结构域；和(iii)胞内信号传导结构域；任选地，所述靶向肿瘤的胞外识别结构域、跨膜结构域和/或胞内信号结构域之间包含铰链或接头；
优选地，所述复合型转接器组件的靶向肿瘤的胞外识别结构域选自肿瘤抗原特异结合结构域、肿瘤微环境靶抗原结合结构域和/或靶向肿瘤微环境的趋化受体。
在一些优选的实施方案中，所述靶向肿瘤的胞外识别结构域选自能靶向识别肿瘤相关抗原的抗体或其功能片段、TCR或其组合；所述抗体的功能片段选自Fd、Fv、Fab、Fab’、F(ab’)2、Fv(scFv)、单链抗体(scFv)或纳米抗体(nanobody)、双链抗体、三链抗体和四链抗体。
在一些优选的实施方案中，所述复合型转接器组件的跨膜结构域选自NK细胞激活受体跨膜结构域、DAP10跨膜结构域、DAP12跨膜结构域、CD8跨膜结构域、CD28跨膜结构域、CD4跨膜结构域、4-1BB跨膜结构域、OX40跨膜结构域、ICOS跨膜结构域、CTLA-4跨膜结构域、PD-1跨膜结构域、LAG-3跨膜结构域、2B4跨膜结构域和BTLA跨膜结构域以及其组合；优选地，所述NK细胞激活受体选自NKG2D、NKG2C、NKG2E、NKG2F、NKG2H、CD94、KIR2DL4、KIR2DS1、KIR2DS2、KIR2DS4、KIR3DS1，天然细胞毒性受体、TRAIL、DNAM-1、CD16a、2B4、NTB-A、CRACC以及NKp80；优选地，所述天然细胞毒性受体选自NKp46、NKp44和NKp30。
在一些优选的实施方案中，所述复合型转接器组件的胞内信号传导结构域包括NK细胞激活受体的胞内信号结构域和/或共刺激信号传导结构域。
在一些优选的实施方案中，所述NK细胞激活受体选自NKG2D、NKG2C、NKG2E、NKG2F、NKG2H、CD94、KIR2DL4、KIR2DS1、KIR2DS2、KIR2DS4、KIR3DS1，天然细胞毒性受体、TRAIL、DNAM-1、CD16a、2B4、NTB-A、CRACC以及NKp80。
在一些优选的实施方案中，所述胞内信号结构域还包括共刺激信号传导结构域；优选地，所述共刺激信号传导结构域选自T细胞共刺激信号传导结构域；包括，但不限于衍生自MHC I类分子、TNF受体蛋白、免疫球蛋白样蛋白质、细胞因子受体、整联蛋白、淋巴细胞活化信号分子(SLAM蛋白)、活化NK细胞受体、BTLA、Toll配体受体、OX40、CD2、CD7、CD16、CD27、CD28、CD30、CD40、CD38、CD35、CD79A、CD79B、CDS、ICAM-1、LFA-1，(CD11a/CD18)、4-1BB(CD137)、B7-H3、CDS、ICAM-1、ICOS(CD278)、GITR、BAFFR、LIGHT、HVEM(LIGHTR)、KIRDS2、SLAMF7、NKp80(KLRF1)、NKp44、NKp30、NKp46、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、NKG2D、NKG2C、NCR、DAP10、DAP12、TNFR2、TRANCE/RANKL、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100SEMA4D)、CD69、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、CD19a和与CD83特异性结合的配体，CARD11、FcRa、FcRp、FcRy、Fyn、HVEM、ICOS、Lck、LAG3、LAT、LRP、NOTCH1、Wnt、OX40、ROR2、Ryk、SLAMF1、Slp76、pTa、TCRa、TCRp、TRIM、ZAP70、PTCH2等胞内信号结构域；更优选地，所述共刺激信号传导结构域选自NKG2D胞内信号结构域，DAP10胞内信号结构域，DAP12胞内信号结构域，NCR胞内信号结构域，CD28胞内信号结构域、4-1BB胞内信号结构域、OX40胞内信号结构域、ICOS胞内信号结构域；
所述靶向杀伤肿瘤细胞受体组件包含(i)靶向肿瘤抗原的胞外识别结构域；(ii)跨膜结构域；和(iii)胞内共刺激信号传导结构域；(iv)T细胞活化信号传导结构域(ITAM)；任选地，所述靶向肿瘤抗原的胞外识别结构域、跨膜结构域、胞内共刺激信号传导结构域和/或T细胞活化信号传导结构域(ITAM)之间包含铰链或接头；
所述靶向杀伤肿瘤细胞受体组件的跨膜结构域选自CD8跨膜结构域、T细胞受体的α和/或β链跨膜结构域、CD28跨膜结构域、CD3ε跨膜结构域、CD45跨膜结构域、CD4跨膜结构域、CD5跨膜结构域、CD8跨膜结构域、CD9跨膜结构域、CD16跨膜结构域、CD22跨膜结构域、CD33跨膜结构域、CD37跨膜结构域、CD64跨膜结构域、CD80跨膜结构域、CD86跨膜结构域、CD134跨膜结构域、CD137跨膜结构域、CD154跨膜结构域、GITR跨膜结构域以及其组合；
所述T细胞活化信号传导结构域衍生自CD3ζ、共同FcRγ(FCER1G)、FcγRIIa、FcRβ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、CD278(“ICOS”)、FcεRI CD66d、DAP10和DAP12等胞内信号结构域。
在一些优选的实施方案中，所述接头是柔性接头；优选地，所述柔性接头包含所示的氨基酸序列(Gly(x)Ser(y))n，其中n是1到10的整数，并且x和y独立地是0到10的整数，前提是x和y不都是0；更优选地，所述接头包含SEQ ID NO.105所示的氨基酸序列或SEQ ID NO.106所示的氨基酸序列。
在一些优选的实施方案中，所述接头是铰链；优选地，所述铰链是IgG1铰链或IgG4铰链。
在一些优选的实施方案中，所述IgG1铰链包含与SEQ ID NO.106所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述IgG1铰链的氨基酸序列如SEQ ID NO.106所示。
在一些优选的实施方案中，所述IgG4铰链包含与SEQ ID NO.107所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述IgG4铰链的氨基酸序列如SEQ ID NO.107所示。
在一些优选的实施方案中，所述NK激活受体组件、CNK信号转接组件和/或UT组件之间包含可切割肽；例如，T2A肽、GSG-T2A肽、E2A肽、GSG-E2A肽、F2A肽、GSG-F2A肽、P2A肽或GSG-P2A肽。
在一些优选的实施方案中，所述T2A包含与SEQ ID NO.108所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述T2A的氨基酸序列如SEQ ID NO.108所示。
在一些优选的实施方案中，GSG-T2A肽的氨基酸序列如SEQ ID NO.109所示。
在一些优选的实施方案中，所述P2A包含与SEQ ID NO.110所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述P2A的氨基酸序列如SEQ ID NO.110所示。
在一些优选的实施方案中，GSG-P2A肽的氨基酸序列如SEQ ID NO.111所示。
在一些优选的实施方案中，所述E2A包含与SEQ ID NO.112所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述E2A的氨基酸序列如SEQ ID NO.112所示。
在一些优选的实施方案中，GSG-E2A肽的氨基酸序列如SEQ ID NO.113所示。
在一些优选的实施方案中，所述F2A包含与SEQ ID NO.114所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述F2A的氨基酸序列如SEQ ID NO.114所示。
在一些优选的实施方案中，GSG-F2A肽的氨基酸序列如SEQ ID NO.115所示。
在一些优选的实施方案中，所述多功能复合体包含与SEQ ID NO.116所示的氨基酸序列有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述多功能复合体的氨基酸序列包含如SEQ ID NO.116所示的TCR抗体单链抗体。
在一些优选的实施方案中，所述多功能复合体包含与SEQ ID NO.117所示的氨基酸序列有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述多功能复合体的氨基酸序列如SEQ ID NO.117所示。
在一些优选的实施方案中，所述多功能复合体包含与SEQ ID NO.121所示的氨基酸序列有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述多功能复合体的氨基酸序列如SEQ ID NO.121所示。
在一些优选的实施方案中，所述多功能复合体包含与SEQ ID NO.123所示的氨基酸序列有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述多功能复合体的氨基酸序列如SEQ ID NO.123所示。
在一些优选的实施方案中，所述多功能复合体包含与SEQ ID NO.125所示的氨基酸序列有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述多功能复合体的氨基酸序列如SEQ ID NO.125所示。
在一些优选的实施方案中，所述多功能复合体包含与SEQ ID NO.12所示的氨基酸序列有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述多功能复合体的氨基酸序列如SEQ ID NO.126所示。
在一些优选的实施方案中，所述多功能复合体包含与SEQ ID NO.127所示的氨基酸序列有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％ 或99％以上同一性的氨基酸序列；所述多功能复合体的氨基酸序列如SEQ ID NO.127所示。
在一些优选的实施方案中，所述多功能复合体包含与SEQ ID NO.128所示的氨基酸序列有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述多功能复合体的氨基酸序列如SEQ ID NO.128所示。
在一些优选的实施方案中，所述多功能复合体包含与SEQ ID NO.129所示的氨基酸序列有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述多功能复合体的氨基酸序列如SEQ ID NO.129所示。
在一些优选的实施方案中，所述多功能复合体包含与SEQ ID NO.130所示的氨基酸序列有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述多功能复合体的氨基酸序列如SEQ ID NO.130所示。
在一些优选的实施方案中，所述多功能复合体包含与SEQ ID NO.131所示的氨基酸序列有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述多功能复合体的氨基酸序列如SEQ ID NO.131所示。
在一方面，本公开提供了一种编码所述多功能复合体的核酸分子。
在一些优选的实施方案中，所述核酸分子是DNA或RNA。
在一些优选的实施方案中，所述RNA是mRNA。
在一些优选的实施方案中，所述核酸分子包含与SEQ ID NO.118所示的核苷酸序列有80％或以上同一性的核苷酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的核苷酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述核酸分子的核苷酸序列SEQ ID NO.118所示。
在一些优选的实施方案中，所述核酸分子包含与SEQ ID NO.122所示的核苷酸序列有80％或以上同一性的核苷酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的核苷酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述核酸分子的核苷酸序列SEQ ID NO.122所示。
在一些优选的实施方案中，所述核酸分子包含与SEQ ID NO.124所示的核苷酸序列有80％或以上同一性的核苷酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的核苷酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述核酸分子的核苷酸序列SEQ ID NO.124所示。
在一方面，本公开提供了一种含有所述核酸的表达载体。
在一些优选的实施方案中，所述载体选自质粒、粘粒、病毒载体、RNA载体或线性或圆形DNA或RNA分子。
在一些优选的实施方案中，所述病毒载体选自逆转录病毒、腺病毒、细小病毒(例如，腺伴随病毒)、冠状病毒、负链RNA病毒诸如正粘病毒(例如，流感病毒)、弹状病毒(例如，狂犬病和水疱性口炎病毒)、副粘病毒(例如，麻疼和仙台)、正链RNA病毒诸如小RNA病毒和甲病毒和双链DNA病毒，所述双链DNA病毒包括腺病毒、疱疹病毒(例如，单纯疱疹病毒1和2型、愛泼斯坦-巴尔病毒、巨细胞病毒)和痘病毒(例如，牛痘病毒、鸡痘病毒和金丝雀痘病毒)、诺沃克病毒、披膜病毒、黄病毒、呼肠孤病毒、乳多泡病毒、嗜肝DNA病毒、杆状病毒和肝炎病毒。
在一些优选的实施方案中，所述逆转录病毒选自禽造白细胞组织增生-肉瘤、哺乳动物C-型、B-型病毒、D-型病毒、HTLV-BLV集合、慢病毒、泡沫病毒。
在一些优选的实施方案中，所述慢病毒载体选自HIV-1、HIV-2、SIV、FIV、BIV、EIAV、CAEV或绵羊脱髓鞘性脑白质炎慢病毒。
在一些优选的实施方案中，所述NK激活受体组件、CNK信号转接组件和/或UT组件可在同一载体同一启动子，或不同启动子下调控表达，或多种载体中进行表达。
在一些优选的实施方案中，所述载体为慢病毒载体，所述编码NK激活受体组件、CNK信号转接组件和/或UT组件的基因之间包含可切割肽编码基因；优选地，所述可切割肽为2A接头；所述2A接头选自T2A、P2A、E2A和F2A。
在一些优选的实施方案中，所述载体还包含启动子；优选地，所述启动子为EF1α启动子或CMV启动子。
在一方面，本公开提供了一种免疫细胞，其包含所述的核酸或所述的表达载体。
在一些优选的实施方案中，免疫细胞选自T细胞，NKT细胞，NK细胞，B细胞，单核细胞，巨噬细胞等
在一方面，本公开提供了一种制造免疫细胞的方法，其包括用选自下述的方法将所述的核酸或所述的表达载体引入细胞：电穿孔、声穿孔、基因枪(例如用Au-颗粒的基因枪)、脂质转染、聚合物转染、纳米颗粒或多聚复合体。
在一方面，本公开提供了一种药物组合物，其包含的多功能复合体、所述的核酸、所述的表达载体、所述的免疫细胞和/或所述方法制造的免疫细胞，以及药学上可接受的载体。
在一方面，本公开提供了一种的多功能复合体、所述的核酸、所述的表达载体、所述的免疫细胞、所述方法制造的免疫细胞和/或所述的药物组合物在制备治疗疾病的药物中的用途。
在一方面，本公开提供了一种治疗疾病的方法，其包括向受试者施用的多功能复合体、所述的核酸、所述的表达载体、所述的免疫细胞和/或所述的药物组合物。
在一些优选的实施方案中，所述疾病包括各类实体肿瘤和血液肿瘤，病毒感染性疾病，自身免疫性疾病。
在一些优选的实施方案中，所述实体肿瘤选自神经系统肿瘤，头颈部肿瘤，胸部肿瘤，消化系统肿瘤，泌尿生殖系统肿瘤，软组织和皮肤肿瘤，骨肿瘤等。
在一些优选的实施方案中，神经系统肿瘤包括弥漫性胶质瘤，弥漫性星形细胞瘤和间变性星形细胞瘤，胶质母细胞瘤，少突胶质细胞瘤，少突星形细胞瘤，儿童弥漫性胶质瘤，其他星形细胞瘤，室管膜瘤，神经元和混合性神经元-神经胶质肿瘤，髓母细胞瘤，其他胚胎性肿瘤，神经鞘瘤，脑膜瘤，孤立性纤维性肿瘤和血管周细胞瘤等。
在一些优选的实施方案中，头颈部肿瘤包括鼻腔及鼻窦恶性肿瘤，鼻咽癌，口腔癌，喉癌，涎腺肿瘤，颅内肿瘤，甲状腺癌，舌癌等。
在一些优选的实施方案中，胸部肿瘤包括肺癌，食管癌，贲门癌，乳腺癌，纵膈肿瘤等。
在一些优选的实施方案中，消化系统肿瘤包括胃癌，大肠癌及乙状结肠和直肠癌，肝癌，胰腺癌与壶腹周围癌，胆道癌，小肠恶性肿瘤等。
在一些优选的实施方案中，泌尿生殖系统肿瘤包括肾癌，前列腺癌，膀胱癌，睾丸恶性肿瘤，阴茎癌，子宫颈癌，子宫内膜癌，卵巢癌等。
在一些优选的实施方案中，软组织和皮肤肿瘤包括恶性纤维组织细胞瘤，横纹肌肉瘤，滑膜肉瘤，皮肤恶性黑色素瘤等。
在一些优选的实施方案中，骨肿瘤包括骨肉瘤，尤文氏肉瘤等。
在一些优选的实施方案中，所述结肠癌是结肠腺瘤。
在一些优选的实施方案中，所述乳腺癌是三阴乳腺癌细胞。
在一些优选的实施方案中，所述肝癌是肝细胞癌。
在一些优选的实施方案中，所述疾病是血液肿瘤选自白血病、淋巴瘤(HL)、多发性骨髓瘤(MM)、骨髓增生异常综合症(MDS)等
在一些优选的实施方案中，所述白血病是B细胞急性淋巴性白血病，T细胞急性淋巴性白血病，急性髓系白血病等。
在一些优选的实施方案中，病毒感染性疾病包括：呼吸道病毒性疾病，胃肠道病毒性疾病，肝脏病毒性疾病，皮肤和黏膜病毒性疾病，眼病毒性疾病，中枢神经系统病毒性疾病，淋巴细胞性病毒性疾病，虫传病毒性疾病，慢病毒感染疾病等。
在一些优选的实施方案中，呼吸道病毒性疾病包括鼻病毒、腺病毒、呼吸道合胞病毒、副流感病毒和冠状病毒等的感染；流行性感冒；流行性腮腺炎等。
在一些优选的实施方案中，胃肠道病毒性疾病包括脊髓灰质炎；库克萨基病毒感染；ECHO病毒感染；病毒性胃肠炎：包括轮状病毒性胃肠炎、诺瓦克病毒性胃肠炎、腺病毒性胃肠炎、星状病毒性胃肠炎、冠状病毒性胃肠炎和杯状病毒性胃肠炎等。
在一些优选的实施方案中，肝脏病毒性疾病包括甲型病毒性肝炎、乙型病毒性肝炎、丙型病毒性肝炎、丁型病毒性肝炎、戊型病毒性肝炎、EB病毒性肝炎和巨细胞病毒性肝炎等。
在一些优选的实施方案中，皮肤和黏膜病毒性疾病包括麻疹、风疹、幼儿急疹、水痘及带状疱疹、天花、单纯疱疹病毒感染、狂犬病和口蹄疫等。
在一些优选的实施方案中，眼病毒性疾病包括流行性角膜结膜炎、滤泡性结膜炎和疱疹性角膜结膜炎等。
在一些优选的实施方案中，中枢神经系统病毒性疾病包括流行性乙型脑炎、西方马脑炎、东方马脑炎、圣路易脑炎、委内瑞拉马脑炎、墨累山谷脑炎、加利福尼亚脑炎、森林脑炎和淋巴细胞脉络丛脑膜炎等。
在一些优选的实施方案中，淋巴细胞性病毒性疾病包括传染性单核细胞增多症、巨细胞病毒感染和获得性免疫缺陷综合征等。
在一些优选的实施方案中，虫传病毒性疾病包括病毒性出血热：包括流行性出血热、黄热病、克里米亚－刚果出血热、裂谷热、阿根廷出血热、玻利维亚出血热、拉萨热、鄂木斯克出血热、马尔堡病和埃波拉出血热等；登革热和登革出血热；西尼罗热；科罗拉多蜱传热；白蛉热等
优选地，慢病毒感染疾病包括亚急性硬化性全脑炎、库鲁病、进行性多灶性白质脑病和亚急性海绵样脑病(皮质纹状体脊髓变性)等。
在一些优选的实施方案中，自身免疫性疾病包括器官特异性自身免疫病和系统性自身免疫病
优选地，器官特异性自身免疫病包括慢性淋巴细胞性甲状腺炎、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌无力、溃疡性结肠炎、恶性贫血伴慢性萎缩性胃炎、肺出血肾炎综合征、寻常天疱疮、类天疱疮、原发性胆汁性肝硬化、多发性脑脊髓硬化症、急性特发性多神经炎等。
在一些优选的实施方案中，系统性自身免疫病包括系统性红斑狼疮、类风湿关节炎、系统性血管炎、硬皮病、天疱疮、皮肌炎、混合性结缔组织病、自身免疫性溶血性贫血、甲状腺自身免疫病、溃疡性结肠炎等。
在一方面，本公开提供了一种刺激受试者中的免疫应答的方法，所述方法包括给予受试者有效量的多功能复合体、所述的核酸、所述的表达载体、所述的免疫细胞、所述方法制造的免疫细胞和/或所述的药物组合物。
本公开还提供了细胞、细胞群以及包含所述细胞和群的组合物(包括药物和治疗组合物)，例如通过所提供的方法产生的细胞和群，以及方法，例如用于施用所述细胞和组合物至受试者如患者的治疗方法。
还提供了包括用于施用的细胞的组合物，包括药物组合物和制剂，例如包括用于以给定剂量或其分数施用的 数量的细胞的单位剂量形式组合物。药物组合物和制剂通常包含一种或多种任选的药学上可接受的载体或赋形剂。在一些实施方式中，组合物包含至少一种另外的治疗剂。
术语“药物制剂”指形式为允许其中所含活性成分的生物活性有效，并且不包含对将要施用该制剂的受试者有不可接受的毒性的另外组分的制备物。
“药学上可接受的载体”指药物制剂中除活性成分之外的成分，其对受试者无毒。药学上可接受的载体包括但不限于缓冲剂、赋形剂、稳定剂或防腐剂。
在一些方面，载体的选择部分地由特定细胞和/或施用方法来确定。因此，存在多种合适的制剂。例如，药物组合物可包含防腐剂。合适的防腐剂可包括例如对羟基苯甲酸甲酯、对羟基苯甲酸丙酯、苯甲酸钠和苯扎氯铵。在一些方面，使用两种或更多种防腐剂的混合物。防腐剂或其混合物通常以组合物总重量的约0.0001重量％至约2重量％的量存在。药学上可接受的载体通常在使用的剂量和浓度下对受体无毒，并包括但不限于：缓冲剂，例如磷酸盐、柠檬酸盐和其他有机酸；抗氧化剂，包括抗坏血酸和蛋氨酸；防腐剂(例如十八烷基二甲基苄基氯化铵；氯化六甲双铵(hexamethonium chloride)；苯扎氯铵；苄索氯铵；苯酚、丁醇或苄醇；对羟基苯甲酸烷基酯，例如对羟基苯甲酸甲酯或对羟基苯甲酸丙酯；邻苯二酚；间苯二酚；环己醇；3-戊醇和间甲酚)；低分子量(少于约10个残基)多肽；蛋白质，例如血清白蛋白、明胶或免疫球蛋白；亲水性聚合物，例如聚乙烯吡咯烷酮；氨基酸，例如甘氨酸、谷氨酰胺、天冬酰胺、组氨酸、精氨酸或赖氨酸；单糖、二糖和其他碳水化合物，包括葡萄糖、甘露糖或糊精；螯合剂，如EDTA；糖，例如蔗糖、甘露醇、海藻糖或山梨糖醇；盐形成抗衡离子，例如钠；金属络合物(例如锌蛋白络合物)；和/或非离子表面活性剂，例如聚乙二醇(PEG)。
在一些方面，缓冲剂包括在组合物中。合适的缓冲剂包括例如柠檬酸、柠檬酸钠、磷酸、磷酸钾和各种其他酸和盐。在一些方面，使用两种或更多种缓冲剂的混合物。缓冲剂或其混合物通常以组合物总重量的约0.001重量％至约4重量％的量存在。制备可施用的药物组合物的方法是已知的。
制剂可以包括水溶液。制剂或组合物还可包含多于一种可用于正在用细胞治疗的特定适应症、疾病或病症的活性成分，优选具有与细胞互补的活性的那些，其中各自的活性不会彼此不利地影响。这样的活性成分合适地以对于预期目的有效的量组合存在。因此，在一些实施方式中，药物组合物还包含其他药物活性剂或药物，例如化学治疗剂例如天冬酰胺酶、白消安、卡铂、顺铂、柔红霉素、阿霉素、氟尿嘧啶、吉西他滨、羟基脲、甲氨蝶呤、紫杉醇、利妥昔单抗、长春碱和/或长春新碱。
在一些实施方式中，药物组合物包含有效治疗或预防疾病或病症的量的细胞，例如治疗有效量或预防有效量。在一些实施方式中，通过定期评估所治疗的受试者来监测治疗或预防功效。可以通过单次推注施用细胞、多次推注施用细胞，或连续输注施用细胞来递送期望剂量。
细胞和组合物可以使用标准施用技术、制剂和/或装置来施用。细胞的施用可以是自体的或异源的。例如免疫应答细胞或祖细胞可以从一个受试者获得，并施用于相同受试者或不同的、相容的受试者。外周血衍生的免疫应答细胞或其后代(例如体内、离体或体外衍生的)可以通过局部注射施用，包括导管施用、全身注射、局部注射、静脉注射或肠胃外施用。当施用治疗组合物(例如包含遗传修饰的免疫应答细胞的药物组合物)时，通常将其配制为单位剂量可注射形式(溶液、悬浮液、乳剂)。
制剂包括用于口服、静脉内、腹膜内、皮下、肺、透皮、肌内、鼻内、颊、舌下或栓剂施用的那些。在一些实施方式中，细胞群是肠胃外施用的。如在本文中所使用的术语“肠胃外”包括静脉内、肌内、皮下、直肠、阴道和腹膜内施用。在一些实施方式中，细胞通过静脉内，腹膜内或皮下注射施用使用外周全身递送而施用至受试者。
在一些实施方式中，组合物作为无菌液体制备物提供，例如等渗水溶液、悬浮液、乳剂、分散液或粘性组合物，其在某些方面可以缓冲至选定的pH。液体制备物通常比凝胶、其他粘性组合物和固体组合物更容易制备。另外，液体组合物在某种程度上更方便施用，尤其是通过注射。另一方面，粘性组合物可以在适当的粘度范围内配制以提供与特定组织更长的接触时间。液体或粘性组合物可包含载体，其可以是溶剂或分散介质，包含例如水、盐水、磷酸盐缓冲盐水、多元醇(例如甘油、丙二醇、液体聚乙二醇)及其合适的混合物。
无菌可注射溶液可以通过将细胞引入溶剂中来制备，例如与合适的载体、稀释剂或赋形剂如无菌水、生理盐水、葡萄糖、右旋糖等混合。组合物可以包含辅助物质，例如润湿剂、分散剂或乳化剂(例如甲基纤维素)、pH缓冲剂、胶凝或增粘添加剂、防腐剂、矫味剂和/或颜色，这取决于施用途径和期望制备物。在某些方面，可以参考标准文本以制备合适的制备物。
可以添加增强组合物的稳定性和无菌性的各种添加剂，包括抗微生物防腐剂、抗氧化剂、螯合剂和缓冲剂。微生物作用的预防可以通过各种抗细菌和抗真菌剂来确保，例如对羟基苯甲酸酯、氯丁醇、苯酚和山梨酸。可注射药物形式的延长吸收可通过使用延迟吸收的试剂例如单硬脂酸铝和明胶来实现。
用于体内施用的制剂通常是无菌的。无菌性可以通过例如经过无菌滤膜过滤而容易地实现。
还提供了施用细胞、群体和组合物以治疗或预防疾病，病症和紊乱(包括癌症)的方法，以及这样的细胞、群体和组合物治疗或预防疾病，病症和紊乱(包括癌症)的用途。在一些实施方式中，将细胞、群体和组合物施用于患有特定疾病或病症的待治疗受试者或患者，例如通过过继细胞疗法(例如过继T细胞疗法)。在一些实施方式中，将通过提供的方法制备的细胞和组合物(例如工程化组合物和孵育和/或其他加工步骤后的生产末端产物)施用于受试者，例如患有疾病或病症或处于疾病或病症风险的受试者。在一些方面，方法由此治疗例如疾病或病症的一种或多种症状，例如通过减轻表达被工程化T细胞识别的抗原的癌症中的肿瘤负担。
用于过继细胞疗法的细胞施用方法是已知的，并且可以与所提供的方法和组合物结合使用。例如，过继性T细胞疗法的方法在例如Gruenberg等的美国专利申请公开号2003/0170238；Rosenberg的美国专利号4,690,915；Rosenberg(2011)Nat Rev Clin Oncol.8(10):577-85)中描述。参见例如Themeli等(2013)Nat Biotechnol.31(10):928-933；Tsukahara等(2013)Biochem Biophys Res Commun 438(1):84-9；Davila等(2013)PLoS ONE 8(4):e61338。
如在本文中所使用的“受试者”是哺乳动物，例如人或其他动物，并且通常是人。在一些实施方式中，向其施用细胞、细胞群或组合物的受试者例如患者是哺乳动物，通常是灵长类动物例如人。在一些实施方式中，灵长类动物是猴子或猿。受试者可以是男性或女性，并且可以是任何合适的年龄，包括婴儿、少年、青少年、成人和老年受试者。在一些实施方式中，受试者是非灵长类哺乳动物，例如啮齿动物。
如在本文中所使用的“治疗”(及其语法变化，例如“治疗”或“治疗”)指完全或部分改善或减轻疾病或病症或紊乱，或与之相关的症状、不良作用或结果或表型。期望的治疗效果包括但不限于预防疾病的发生或复发、症状的减轻、疾病的任何直接或间接病理后果的减轻、预防转移、降低疾病进展速度、缓解或减轻疾病状态以及缓解或改善预后。这些术语并不暗示完全治愈疾病或完全消除任何症状或对所有症状或后果的影响。
如在本文中所使用的“延缓疾病的发展”是指推迟、阻碍、减缓、阻滞、稳定、抑制和/或延迟疾病(例如癌症)的发展。该延缓可以具有不同的时间长度，这取决于疾病的历史和/或被治疗的个体。对本领域技术人员显而易见的是，足够或明显的延迟实际上可以包括预防，因为个体不发生该疾病。例如，晚期癌症例如转移的发展可能被延迟。
如在本文中所使用的“预防”包括针对可易患疾病但尚未被诊断患有该疾病的受试者中该疾病的发生或复发提供预防。在一些实施方式中，提供的细胞和组合物用于延迟疾病的发展或减慢疾病的进展。
如在本文中所使用的“抑制”功能或活性是指当与相同条件(除感兴趣的条件或参数)相比时，或者与另外的条件相比时，降低功能或活性。例如，抑制肿瘤生长的细胞与在该细胞不存在下的肿瘤的生长速率相比降低肿瘤的生长速率。
在施用的上下文中，试剂(例如嵌合抗原受体、多核苷酸、载体、药物制剂、细胞或组合物)的“有效量”指在必要的剂量/量和时间段下，有效达到期望结果例如治疗或预防结果的量。
试剂(例如药物制剂或细胞)的“治疗有效量”指在必要的剂量和时间段下，有效达到期望治疗效果(例如用于治疗疾病、病症、或紊乱，和/或治疗的药代动力学或药效学作用)的量。治疗有效量可以根据例如疾病状态、受试者的年龄、性别和体重以及所施用的细胞群等因素而变化。在一些实施方式中，提供的方法包括以有效量例如治疗有效量施用细胞和/或组合物。
“预防有效量”指在必要的剂量和时间段下，有效达到期望预防结果的量。通常但不是必须的，因为在疾病之前或早期在受试者中使用预防剂量，所以预防有效量将小于治疗有效量。在较低肿瘤负荷的情况下，在某些方面中预防有效量将高于治疗有效量。
在某些实施方式中，将细胞或细胞的个体群体或亚型以约一百万至约一千亿个细胞的范围施用于受试者，例如100万至约500亿个细胞(例如约500万个细胞、约2500万个细胞、约5亿个细胞、约10亿个细胞、约50亿个细胞、约200亿个细胞、约300亿个细胞、约400亿个细胞、或任意上述两个值定义的范围)，例如约1000万到约1000亿个细胞(例如约2000万个细胞、约3000万个细胞、约4000万个细胞、约6000万个细胞、约7000万个细胞、约8000万个细胞、约9000万个细胞、约100亿个细胞、约250亿个细胞、约500亿个细胞、约750亿个细胞、约900亿个细胞，或任意上述两个值定义的范围)，和在某些情况下，约1亿个细胞至约500亿个细胞(例如，约1.2亿个细胞、约2.5亿个细胞、约3.5亿个细胞、约4.5亿个细胞、约6.5亿个细胞、约8亿个细胞、约9亿个细胞、约30亿个细胞、约300亿个细胞、约450亿个细胞)或这些范围之间的任何值。
在一些实施方式中，总细胞的剂量和/或细胞的个体亚群的剂量在处于或大约10 4个细胞/千克(kg)体重至处于或大约10 9个细胞/千克(kg)体重之间的范围内，例如在10 5和10 6个细胞/kg体重之间，例如，至少或至少大约或处于或大约1×10 5个细胞/kg、1.5×10 5个细胞/kg，2×10 5个细胞/kg或1×10 6个细胞/kg体重。例如，在一些实施方式中，细胞以在处于或大约10 4和处于或约10 9个T细胞/千克(kg)体重之间或其一定误差范围内施用，例如在10 5和10 6个T细胞/kg体重之间，例如至少或至少大约或处于或大约1×10 5个T细胞/kg、1.5×10 5个T细胞/kg，2×10 5个T细胞/kg、或1×10 6个T细胞/kg体重。
细胞可以通过任何合适的方式来施用，例如通过推注，通过注射，例如静脉内或皮下注射、眼内注射、眼周注射、视网膜下注射、玻璃体内注射、经中隔注射、巩膜下注射、脉络膜内注射、前房内注射、会阴下注射、结膜下注射、眼球筋膜囊下(sub-Tenon)注射、球后注射、球周注射，或后近巩膜(posterior juxtascleral)递送。在一些实施方式中，它们通过肠胃外、肺内和鼻内施用，并且如果局部治疗是期望的，则通过病灶内施用。肠胃外输注包括肌内、静脉内、动脉内、腹膜内或皮下给药。在一些实施方式中，给定剂量通过单次推注施用细胞来施用。在一些实施方式中，其通过多次推注施用细胞来施用，例如，在不超过3天的期间，或通过连续输注施用细胞来施用。
在一些实施方式中，提供了重复剂量方法，其中给予第一剂量的细胞，然后给予一个或多个第二连续剂量。当以过继治疗方法施用于受试者时，通常设计细胞的多剂量的时机和大小以增加表达抗原的T细胞(例如表达CAR的T细胞)的功效和/或活性和/或功能。在一些实施方式中，重复给药减少了当抑制性免疫分子例如PD-1和/或PD-L1在表达抗原的例如表达CAR的T细胞上被上调时可发生的下调或抑制活性。方法包括施用第一剂量，通常随后是一个或多个连续剂量，并且在不同剂量之间具有特定的时间范围。
在过继细胞疗法的背景下，给定“剂量”的施用包括作为单一组合物和/或单次不间断施用(例如，作为单次注射或连续输注)的给定量或数量的细胞的施用，并且还包括在指定的时间段(不超过3天)中，以多个个体组合物或输注提供的给定量或数量的细胞数量作为分次剂量的施用。因此，在某些情况下，第一或连续剂量是在单个时间点给予或启动的指定数量细胞的单次或连续施用。然而，在某些情况下，第一或连续剂量在不超过三天的时间段内以多次注射或输注施用，例如三天或两天、每天一次，或在一天时间内多次输注。
在下面对本公开的实施例的详细描述中，参考了附图。在附图中相似的附图标记指示相似的元素，并且在附图中以说明的方式示出了可以实践本公开的特定实施方式。对这些实施方式进行了足够详细的描述，以使本领域技术人员能够实施本公开。在其他情况下，未详细示出公知的过程、结构和技术，以免混淆对本说明书的理解。因此，以下详细描述不应被理解为限制性的，并且本公开的技术方案仅由所附权利要求书限定。
实施例
实施例1：CNK-UT多功能复合体的设计
本实施例设计了四种结构的CNK-UT多功能复合体(multi-functional complex)。其中，第一种结构是基本CNK-UT多功能复合体，其它三种结构是在第一种结构的基础上再进一步增加其它组件(module)而形成的具有更多功能的CNK-UT多功能复合体。
1.1基本CNK-UT多功能复合体
在一些实施方式中，使用基本CNK-UT多功能复合体，其示意图如图1A所示，其包含三种组件：(1)NK激活受体组件；(2)CNK信号转接组件；和(3)UT组件。任选地，所述NK激活受体组件、所述CNK信号转接组件和/或所述UT组件之间包含铰链或接头。
其中：
(1)NK激活受体组件，其至少包含NK细胞激活受体或其功能变体，所述NK细胞激活受体包含：(a)NK细胞激活受体胞外结构域(ED)或其功能变体，(b)NK细胞激活受体跨膜结构域(TMD)或其功能变体，以及(c)NK细胞激活受体胞内结构域(ICD)或其功能变体；任选地，所述NK细胞激活受体胞外结构域或其功能变体、所述NK细胞激活受体跨膜结构域或其功能变体和/或所述NK细胞激活受体胞内结构域或其功能变体之间包含铰链或接头；
所述NK细胞激活受体选自NKG2D、NKG2C、NKG2E、NKG2F、NKG2H、CD94、KIR2DL4、KIR2DS1、KIR2DS2、KIR2DS4、KIR3DS1，天然细胞毒性受体(Natural Cytotoxicity Receptors，NCR)、TRAIL、信号淋巴激活分子(signaling lymphocytic activation molecule，SLAM)家族分子2B4(又名CD244)、DNAX附件分子1(DNAM-1，又名CD226)、CD16a、2B4、NTB-A、CRACC(CS1)以及NKp80；其中，所述天然细胞毒性受体包括NKp46(又名NCR1或CD335)、NKp44(又名NCR2或CD336)和NKp30(又名NCR3或CD337)
在一些优选的实施方案中，CNK多功能复合体所包含选自SEQ ID NO.1～24所示的氨基酸序列。
(2)CNK信号转接组件，其至少包含(i)NK细胞信号转换器(adaptor)或其功能变体，所述NK细胞信号转换器包含：(a)NK细胞信号转换器胞外结构域(ED)或其功能变体，(b)NK细胞信号转换器跨膜结构域(TMD)或其功能变体，以及(c)NK细胞信号转换器胞内结构域(ICD)或其功能变体；任选地，所述NK细胞信号转换器胞外结构域或其功能变体、所述NK细胞信号转换器跨膜结构域或其功能变体和/或所述NK细胞信号转换器胞内结构域或其功能变体之间包含铰链或接头
所述CNK信号转接组件中的所述NK细胞信号转换器为DAP10或DAP12。
在一些优选的实施方案中，所述CNK细胞信号转换器功能变体选自DAP10或DAP12的突变体，或DAP10和DAP12的融合蛋白，或野生型DAP10或DAP12与突变型DAP10或DAP12的融合蛋白。
所述CNK细胞信号转换器功能变体选自DAP10突变体或DAP12的突变体，或DAP10和DAP12的融合蛋白，或野生型DAP10或DAP12与突变型DAP10或DAP12的融合蛋白。
在一些优选的实施方案中，所述CNK信号转接组件还包含(ii)免疫受体活化信号传导结构域(ITAM)和/或(iii)T细胞共刺激信号传导结构域。
在一些优选的实施方案中，所述NK细胞信号转换器或其功能变体、所述免疫受体活化信号传导结构域(ITAM)和/或所述T细胞共刺激信号传导结构域之间包含铰链或接头；优选地，所述NK细胞信号转换器或其功能变体与所述免疫受体活化信号传导结构域(ITAM)域融合。
在一些优选的实施方案中，所述免疫受体活化信号传导结构域(ITAM)来自免疫受体的胞内激活信号传导结构域；优选地，所免疫受体选自TCRζ、CD2、CD3γ、CD3δ、CD3ε、CD3ζ、CD5、CD22、FcRγ、CD66d、FcαRI、FcγRI、FcγRII、FcγRIII、Dectin-1、CLEC-1、CD72、CD79A、CD79B；优选地，所述免疫受体活化信号传导结构域(ITAM)与NK细胞信号转换器或其功能变体融合；优选地，所述免疫受体是CD3ζ；
在一些优选的实施方案中，所述T细胞共刺激信号传导结构域来自共刺激分子的胞内信号结构域。
在一些优选的实施方案中，CNK信号转接组件包含选自SEQ ID NO.25～45所示的氨基酸序列。
(3)UT组件，其至少包含(i)靶向降解TCR、MHC和/或NK细胞靶点的重组蛋白分子或其功能变体，所述靶向降解TCR、MHC和/或NK细胞靶点的重组蛋白分子包含：(a)靶向TCR、MHC和/或NK细胞靶点的结合蛋白分子结构域或其功能变体，(b)病毒内质网(ER)驻留糖蛋白的跨膜结构域或其功能变体，以及(c)病毒内质网驻留糖蛋白的胞质结构域或其功能变体；所述病毒内质网驻留糖蛋白的跨膜结构域或其功能变体和病毒内质 网驻留糖蛋白的胞质结构域或其功能变体形成ERAD降解结构域；任选地，所述靶向TCR的结合蛋白分子结构域或其功能变体、所述病毒内质网驻留糖蛋白的跨膜结构域或其功能变体和/或所述病毒内质网驻留糖蛋白的胞质结构域或其功能变体之间包含铰链或接头。
在所述UT组件的靶向降解TCR的重组蛋白分子中，所述靶向TCR的结合蛋白分子结构域或其功能变体来自TCR抗体或其功能片段或其组合。
在一些优选的实施方案中，所述抗体选自TCRα抗体、TCRβ抗体、TCRαβ抗体、TCRγ抗体、TCRδ抗体、TCRγδ抗体、TCR Vδ2抗体、TCR Cβ1抗体；所述抗体的功能片段选自Fd、Fv、Fab、Fab’、F(ab’)2、Fv(scFv)、单链抗体(scFv)或纳米抗体(nanobody)、双链抗体、三链抗体和四链抗体；优选地，所述TCR抗体是TCR单链抗体。在一些优选的实施方案中，所述靶向TCR的结合蛋白分子为TCRαβ抗体。在一些优选的实施方案中，所述TCR单链抗体的氨基酸序列包含与SEQ ID NO.116所示的氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述TCR单链抗体的全长序列的氨基酸序列如SEQ ID NO.116所示。
在一些优选的实施方案中，所述UT组件中的ERAD降解结构域来自HCMV糖蛋白US2、US3、US11或US10、腺病毒E3-19K或HHV-7US21。
在一些优选的实施方案中，所述UT组件中的ERAD降解结构域包含选自SEQ ID NO.46～63所示氨基酸序列。
可以通过基因合成技术合成表达所述基本CNK-UT多功能复合体的多种组件的基因元件片段。采用多顺反子表达方案，利用自剪切多肽2A(self-cleaving 2A peptide，2A:T2A、p2A、E2A、F2A)等元件连接不同组件的基因元件片段，获得基本CNK-UT多功能复合体基因片段；通过分子克隆技术将合成的基本CNK-UT多功能复合体基因片段克隆到慢病毒载体，转染T细胞，实现多种不同元件的同时表达，从而实现T细胞对NK靶点的特异性识别和杀伤，同时也借助UT元件有效抑制和降解TCR。或者，可以将不同的功能元件放在同一个慢病毒载体的不同启动子下，转染T细胞，实现多种CNK-UT元件的同时表达；或者，将不同的功能元件放在两种慢病毒载体上，同时转染T细胞，也可以实现多种CNK-UT元件的同时表达。
1.2增加靶向MHC结合蛋白分子结构域的CNK-UT多功能复合体
在一些实施方式中，使用同时降解或抑制TCR和MHC I和/或MHC II的CNK-UT多功能复合体，其示意图如图1B所示，其也包含三种组件：(1)NK激活受体组件；(2)CNK信号转接组件；和(3)UT组件：包含靶向降解TCR的重组蛋白分子结构域或其功能变体和靶向MHC I和/或MHC II的结合蛋白分子结构域或其功能变体，其与基本CNK-UT多功能复合体相比，不同之处在于(3)UT组件增加了靶向MHC I的结合蛋白分子结构域或其功能变体。任选地，所述NK激活受体组件、CNK信号转接组件、UT组件之间通过铰链或接头连接。
在一些实施方案中，所述靶向MHC I和/或MHC II的结合蛋白分子结构域或其功能变体为靶向HLA的结合蛋白分子结构域或其功能变体。
在一些实施方案中，所述靶向MHC I的结合蛋白分子结构域可以来自抑制MHC I分子的病毒内质网蛋白。在一些实施方案中，所述抑制MHC I分子的病毒内质网糖蛋白选自人巨细胞病毒(human cytomegalovirus，HCMV)US6、单纯疱疹病毒(herpes simplex virus，HSV)ICP47、牛痘病毒(cowpox virus，CPXV)CPXV12、牛疱疹病毒(bovine herpesvirus，BHV)UL49.5、或爱泼斯坦巴尔病毒(Epstein Barr virus，EBV)BNFL2a等。上述病毒内质网蛋白通过与抗原处理相关转运体(transporter associated with antigen processing，TAP)结合，从而阻止TAP介导的多肽转运到内质网，并抑制MHC分子的组装，实现对MHC I的表达抑制。在一些优选的实施方案中，所述靶向MHC I和/或MHC II的结合蛋白分子结构域或其功能变体包含TAP结合结构域。在一些优选的实施方案中，所述TAP结合结构域包含选自SEQ ID NO.64～73所示的氨基酸序列。
在一些实施方案中，所述靶向MHC I的结合蛋白分子结构域还可以来自降解MHC I分子的病毒糖蛋白。在一些实施方案中，所述病毒糖蛋白选自HCMV糖蛋白US2、US3、US11、US10或腺病毒E19等。在一些优选的实施方案中，所述靶向MHC I和/或MHC II的结合蛋白分子结构域或其功能变体包含MHC结合结构域。在一些优选的实施方案中，所述MHC结合结构域包含选自SEQ ID NO.74～82所示的氨基酸序列。
在一些实施方案中，所述靶向MHC I的结合蛋白分子结构域还可以来自定向抑制或降解MICA、MICB、ULBP1-6等NK靶点蛋白的病毒蛋白。在一些实施方案中，所述病毒蛋白选自HCMV UL16、UL141、UL142或腺病毒E3-19K9等。在一些优选的实施方案中，所述病毒蛋白的氨基酸序列如SEQ ID NO.83～91所示的氨基酸序列。
在一些实施方案中，所述靶向MHC I的结合蛋白分子结构域还可以来自把MHC I分子从高尔基体转运到溶酶体进行降解的病毒蛋白。在一些实施方案中，所述病毒蛋白选自HIV Nef、HIV Vpu、HHV-7 U21、HHV-8 KK3、HHV-8 KK5、MHV-68 MK3和HTLV-1 p12等。在一些优选的实施方案中，所述病毒蛋白包含选自SEQ ID NO.92～97所示的氨基酸序列。
在一些实施方案中，所述靶向MHC I和/或MHC II的结合蛋白分子结构域或其功能变体还包含介导MHC-多肽分子从高尔基体返回内质网并促进其降解的病毒蛋白；优选地，所述病毒蛋白包含MHC结合结构和KDEL receptor结合结构域。在一些实施方案中，所述病毒蛋白是Cowpox Virus蛋白CPXV203。在一些优选的实施方案中，所述病毒蛋白包含选自SEQ ID NO.98～103所示的氨基酸序列。
采用与获得基本CNK-UT多功能复合体基因片段相同的方式，获得增加了靶向MHC I结合蛋白分子结构域的 CNK-UT多功能复合体基因片段；通过分子克隆技术将合成的CNK-UT多功能复合体基因片段克隆到慢病毒载体，转染T细胞，实现多种不同元件的同时表达，从而实现T细胞对NK靶点的特异性识别和杀伤，也借助UT元件有效抑制和降解TCR和MHC I的表达。
1.3增加靶向MHC I的结合蛋白分子结构域和复合型转接器的CNK-UT多功能复合体
在一些实施方式中，使用同时降解TCR和MHC I并实现CNK-T细胞对肿瘤抗原特异性的识别和激活的多功能复合体，其示意图如图1C所示，其包含四种组件：(1)NK激活受体组件；(2)CNK信号转接组件；(3)UT组件；和(4)复合型转接器(Chimeric adaptor)组件，其与增加靶向MHC I结合蛋白分子结构域的CNK-UT多功能复合体相比，不同之处在于增加了(4)复合型转接器组件。任选地，所述NK激活受体组件、CNK信号转接组件、UT组件和/或复合型转接器组件之间通过铰链或接头连接。
所述(4)复合型转接器包含：(i)靶向肿瘤的胞外识别结构域；(ii)跨膜结构域；和(iii)胞内信号传导结构域；任选地，所述靶向肿瘤的胞外识别结构域、跨膜结构域和/或胞内信号结构域之间包含铰链或接头；
在一些实施方式中，所述复合型转接器组件的靶向肿瘤的胞外识别结构域选自肿瘤抗原特异结合结构域、肿瘤微环境靶抗原结合结构域和/或靶向肿瘤微环境的趋化受体；
在一些实施方式中，所述靶向肿瘤的胞外识别结构域选自能靶向识别肿瘤相关抗原的抗体或其功能片段、TCR或其组合；所述抗体的功能片段选自Fd、Fv、Fab、Fab’、F(ab’)2、Fv(scFv)、单链抗体(scFv)或纳米抗体(nanobody)、双链抗体、三链抗体和四链抗体；
在一些实施方式中，所述复合型转接器组件的跨膜结构域选自NK细胞激活受体跨膜结构域、DAP10跨膜结构域、DAP12跨膜结构域、CD8跨膜结构域、CD28跨膜结构域、CD4跨膜结构域、4-1BB跨膜结构域、OX40跨膜结构域、ICOS跨膜结构域、CTLA-4跨膜结构域、PD-1跨膜结构域、LAG-3跨膜结构域、2B4跨膜结构域和BTLA跨膜结构域以及其组合；优选地，所述NK细胞激活受体选自NKG2D、NKG2C、NKG2E、NKG2F、NKG2H、CD94、KIR2DL4、KIR2DS1、KIR2DS2、KIR2DS4、KIR3DS1，天然细胞毒性受体、TRAIL、DNAM-1、CD16a、2B4、NTB-A、CRACC以及NKp80；优选地，所述天然细胞毒性受体选自NKp46、NKp44和NKp30；
在一些实施方式中，所述复合型转接器组件的胞内信号传导结构域包括NK细胞激活受体的胞内信号结构域和/或共刺激信号传导结构域。
在一些实施方式中，所述NK细胞激活受体选自NKG2D、NKG2C、NKG2E、NKG2F、NKG2H、CD94、KIR2DL4、KIR2DS1、KIR2DS2、KIR2DS4、KIR3DS1，天然细胞毒性受体、TRAIL、DNAM-1、CD16a、2B4、NTB-A、CRACC以及NKp80；
在一些实施方式中，所述胞内信号结构域还包括共刺激信号传导结构域；优选地，所述共刺激信号传导结构域选自T细胞共刺激信号传导结构域；包括，但不限于衍生自MHC I类分子、TNF受体蛋白、免疫球蛋白样蛋白质、细胞因子受体、整联蛋白、淋巴细胞活化信号分子(SLAM蛋白)、活化NK细胞受体、BTLA、Toll配体受体、OX40、CD2、CD7、CD16、CD27、CD28、CD30、CD40、CD38、CD35、CD79A、CD79B、CDS、ICAM-1、LFA-1，(CD11a/CD18)、4-1BB(CD137)、B7-H3、CDS、ICAM-1、ICOS(CD278)、GITR、BAFFR、LIGHT、HVEM(LIGHTR)、KIRDS2、SLAMF7、NKp80(KLRF1)、NKp44、NKp30、NKp46、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、NKG2D、NKG2C、NCR、DAP10、DAP12、TNFR2、TRANCE/RANKL、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100SEMA4D)、CD69、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、CD19a和与CD83特异性结合的配体，CARD11、FcRa、FcRp、FcRy、Fyn、HVEM、ICOS、Lck、LAG3、LAT、LRP、NOTCH1、Wnt、OX40、ROR2、Ryk、SLAMF1、Slp76、pTa、TCRa、TCRp、TRIM、ZAP70、PTCH2等胞内信号结构域；更优选地，所述共刺激信号传导结构域选自NKG2D胞内信号结构域，DAP10胞内信号结构域，DAP12胞内信号结构域，NCR胞内信号结构域，CD28胞内信号结构域、4-1BB胞内信号结构域、OX40胞内信号结构域、ICOS胞内信号结构域。
采用与获得基本CNK-UT多功能复合体基因片段相同的方式，获得增加靶向MHC I的结合蛋白分子结构域和复合型转接器的CNK-UT多功能复合体基因片段；通过分子克隆技术将合成的CNK-UT多功能复合体基因片段克隆到慢病毒载体，转染T细胞，实现四种不同元件的同时表达，从而实现T细胞对NK靶点的特异性识别和杀伤，也借助UT元件有效抑制和降解TCR和MHC I的表达；并通过复合型的转接器实现CNK-T细胞对肿瘤抗原特异性的识别和激活。
1.4增加靶向MHC I的结合蛋白分子结构域和CAR或TCR等靶向杀伤肿瘤细胞的受体的CNK-UT多功能复合体
在一些实施方式中，使用同时降解TCR和MHC I并实现对肿瘤细胞的特异性识别和杀伤效应的多功能复合体，其示意图如图1D所示，其包含四种组件：(1)NK激活受体组件；(2)CNK信号转接组件；(3)UT组件；和(4)靶向杀伤肿瘤细胞受体组件，其与增加靶向MHC I结合蛋白分子结构域的CNK-UT多功能复合体相比，不同之处在于增加了(4)靶向杀伤肿瘤细胞受体组件。任选地，所述NK激活受体组件、CNK信号转接组件、 UT组件和/或靶向杀伤肿瘤细胞受体组件之间通过铰链或接头连接。
所述(4)靶向杀伤肿瘤细胞受体组件包含(i)靶向肿瘤抗原的胞外识别结构域；(ii)跨膜结构域；和(iii)胞内共刺激信号传导结构域；(iv)T细胞活化信号传导结构域(ITAM)；任选地，所述靶向肿瘤抗原的胞外识别结构域、跨膜结构域、胞内共刺激信号传导结构域和/或T细胞活化信号传导结构域(ITAM)之间包含铰链或接头。
所述靶向杀伤肿瘤细胞受体组件的跨膜结构域选自CD8跨膜结构域、T细胞受体的α和/或β链跨膜结构域、CD28跨膜结构域、CD3ε跨膜结构域、CD45跨膜结构域、CD4跨膜结构域、CD5跨膜结构域、CD8跨膜结构域、CD9跨膜结构域、CD16跨膜结构域、CD22跨膜结构域、CD33跨膜结构域、CD37跨膜结构域、CD64跨膜结构域、CD80跨膜结构域、CD86跨膜结构域、CD134跨膜结构域、CD137跨膜结构域、CD154跨膜结构域、GITR跨膜结构域以及其组合
所述T细胞活化信号传导结构域衍生自CD3ζ、共同FcRγ(FCER1G)、FcγRIIa、FcRβ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、CD278(“ICOS”)、FcεRI CD66d、DAP10和DAP12等胞内信号结构域
在一些优选的实施方式中，所述(4)靶向杀伤肿瘤细胞受体组件为CAR或TCR靶向杀伤肿瘤细胞的受体。
采用与获得基本CNK-UT多功能复合体基因片段相同的方式，获得增加靶向MHC I的结合蛋白分子结构域和CAR或TCR等靶向杀伤肿瘤细胞的受体的CNK-UT多功能复合体基因片段；通过分子克隆技术将合成的CNK-UT多功能复合体基因片段克隆到慢病毒载体，转染T细胞，实现四种不同元件的同时表达，从而实现T细胞对NK靶点的特异性识别和杀伤，也借助UT元件有效抑制和降解TCR和MHC I的表达；并通过CAR或TCR的结构实现对肿瘤细胞的特异性识别和杀伤效应，协同CNK元件，更高效的对肿瘤细胞进行清除。
实施例2：表达CNK-UT多功能复合体的CNK-UT元件的设计
本实施例设计了四种表达CNK-UT多功能复合体的CNK-UT元件。
2.1 CNK-UT元件设计结构1
在一些实施方式中，如图2A所示，利用单一慢病毒EF1α启动子表达载体，利用自剪切多肽2A等元件来连接CNK-UT元件其中一种组合：DAP10-DAP12 ICD-T2A-NKG2D-p2A-anti-TCR-AdE3 ERAD，实现在单个载体上表达多个功能元件。
2.2 CNK-UT元件设计结构2
在一些实施方式中，如图2B所示，利用单一慢病毒EF1α启动子表达载体，利用自剪切多肽2A等元件来连接CNK-UT元件其中一种组合：DAP10-DAP12 ICD-T2A-NKG2D-p2A-anti-TCR-US2 ERAD，实现在单个载体上表达多个功能元件。
2.3 CNK-UT元件设计结构3
在一些实施方式中，如图2C所示，利用单一慢病毒表达载体，采用双启动子和多顺反子表达方案，利用EF1α和CMV启动子，分别调控DAP10-CD3ζ-T2A-NKG2D-p2A-anti-TAA scFv-DAP10和anti-TCR-AdE3 ERAD-E2A-AdE3多基因的表达，实现在单个载体上表达多个功能元件。
2.4CNK-UT元件设计结构4
在一些实施方式中，如图2D所示，利用两种不同慢病毒表达载体，分别调控anti-TAA scFv-CD28/4-1BB-CD3ζ-T2A-DAP10-CD3ζ-T2A-NKG2D和anti-TCR-AdE3 ERAD-T2A-AdE3的表达，制备出不同慢病毒后共转染T细胞后，实现同一T细胞上表达多个功能元件。
实施例3：靶向NKG2DL的特异性嵌合抗原受体的表达质粒的制备
全基因合成CNK-UT的核苷酸序列(其核苷酸序列如SEQ ID NO.118所示，其编码蛋白如SEQ ID NO.117所示，其中嵌合抗原受体中的功能片段按以下顺序连接DAP10-CD3ζ-T2A-NKG2D-p2A-anti-TCR scFv-AdE3)，通过分子克隆的方式连接到慢病毒载体pCDH-CMV-MCS-EF1α-Puro(购自优宝生物)或pLVX-EF1α-AcGFP1-C1 Vector(Takara)，构建CNK-UT慢病毒目标载体，并通过测序确定正确后，用质粒转化DH5α感受态细胞(Thermo Fisher)，挑取单克隆菌落，大规模培养后，利用质粒抽提试剂盒PureLink TM HiPure Plasmid Maxiprep Kit(Thermo Fisher)进行质粒生产和纯化，获得CNK-UT慢病毒质粒。
实施例4：CNK-UT的病毒制备
将实施例3获得的CNK-UT慢病毒质粒，以及包装质粒psPAX2和pMD2.G(购自Addgene，货号:12259&12260)，按照1.64pmol：1.3pmol：0.72pmol的比例用聚乙烯亚胺转染试剂(408727，Sigma)按DNA:μg PEI＝1:3的比例进行转染，共转染293T细胞(为ATCC产品，产品编号为CRL3216TM)；具体的包装质粒的制备方法参见PureLink TM HiPure Plasmid Maxiprep Kit(K210006，Thermo)说明书；具体的转染操作过程参见Sigma转染说明书。转染后16小时更换为完全培养基(购买自Life Technologies公司，产品货号：11995-065)，培养24小时、48小时和72小时后，分别收集慢病毒上清液，合并，于-80℃，3000rpm离心10～15分钟后经0.45μm滤膜过滤，最后于4℃，25000rpm超离心2～3小时，浓缩病毒，收集慢病毒浓缩液，将收集的慢病毒浓缩液转入-80℃保存。
实施例5：CNK-UT细胞的制备
取健康供者的新鲜外周血，1:1加入PBS+EDTA，通过Ficoll密度梯度离心分离新鲜的外周血单核细胞；再利用CD3分选磁珠，MS分离柱和MiniMACS TM分离装置(Miltenyi Biotec)进行CD3 +T细胞的阳性分选和洗脱，分离出的CD3 +T细胞再通过偶联抗-CD3抗体和抗-CD28抗体的磁珠(Human T-Activator CD3/CD28，Invitrogen，货号：11161D)进行刺激培养，具体步骤如下：将外周血单核细胞稀释至浓度为1×10 6个单个细胞/ml，铺在24孔板中，再以1:1的比例将磁珠加入细胞并混匀，重悬于培养液(OpTmizerTM T-Cell Expansion SFM，A1048503，Life Technologies)，并加入IL2 50U/ml和IL15 5ng/ml，在37℃、5％CO 2培养箱中培养1天后进行装载有CNK-UT元件的实施例4制备的慢病毒转染。具体步骤如下：取实施例4制备的慢病毒浓缩液，按照MOI＝3-5的值将病毒液加入细胞，并加入Polybrene 10μg/ml，放入平角离心机后，低速(500g-1000g/min)离心30～60分钟，然后放入培养箱中37℃培养，感染后48小时后获得表达CNK-UT的细胞(CNK-UT细胞)，可进行流式检测细胞表型，继续培养8天后可进行下一步的表型检测和细胞功能实验。
实施例6：CNK-UT细胞的表型检测
图3是一种CNK-UT(DAP10-CD3ζ-T2A-NKG2D-p2A-anti-TCR scFv-AdE3)细胞基本表型的流式细胞检测结果。在制备例2中培养10天的细胞，分别取5×10 5未转染的T细胞和转染CNK-UT病毒载体的T细胞，经过抗体CD8-Pacific Blue、CD4-APC、NKG2D-PE-Cy7、TCR-αβ-APC-Cy7(所有抗体均购自Biolegend)染色，再通过使用流式细胞仪(BD FACSCanto II)进行细胞表型鉴定。结果显示，未转染的T细胞，CD8表达NKG2D且表达TCR-αβ，CD4不表达NKG2D，高表达TCR-αβ；而经过转染改造的CNK-UT，CD8和CD4 T细胞都高表达NKG2D，不表达TCR-αβ。
实施例7：CNK-UT细胞的广谱肿瘤杀伤功能检测
本实施例研究实施例5制备的CNK-UT细胞的广谱肿瘤杀伤功能。其中，人结肠腺瘤细胞系HT29购自Procell(CL-0118)；三阴乳腺癌细胞MDA-MB453购自Procell(CL-0152)；急性髓系白血病细胞系THP1购自Procell(CL-0233)；肝细胞癌(HCC)HepG2购自Procell(CL-0103)；肝细胞癌(HCC)PLC购自Procell(CL-0415)。
7.1 CNK-UT细胞对人结肠腺瘤细胞系HT29的识别和特异性杀伤
本实验中使用的CNK-UT细胞是实施例5制备的细胞。
图4示出了CNK-UT细胞对人结肠腺瘤细胞系HT29的识别和特异性杀伤。其中：
A：人结肠腺瘤细胞系HT29不同NK靶点蛋白的表达情况
人结肠腺瘤细胞系HT29 0.5×10 6细胞经过胰酶消化下来，分别染MICA-APC、MICB-APC、ULBP1-APC、ULBP2-APC、ULBP3-APC，并用IgG同型(IgG isotype)抗体作为对照(购自R&D Systems)，再通过使用流式细胞仪(BD FACSCanto II)进行细胞表型鉴定。结果显示：HT29细胞高表达ULBP2，弱表达MICA、MICB、ULBP1和ULBP3。
B：CNK-UT细胞具有对HT29高效的杀伤和激活功能
T细胞和肿瘤细胞的共培养实验，取24孔板，计数人结肠腺瘤细胞系HT29 0.5×10 6细胞/孔进行铺板，待肿瘤细胞生长密度达80％以上，按效靶比1:5，分别加入未转染T细胞和CNK-UT细胞(DAP10-CD3ζ-T2A-NKG2D-p2A-anti-TCR scFv-AdE3)，24h后，所有细胞消化收获后，进行CD45-PerCP-Cy5.5、CD8-Pacific Blue、CD4-APC、NKG2D-PE-Cy7、CD25-APC-Cy7(购自Biolegend)的染色，而后进行流式细胞仪技术进行检测。分析结果时，通过CD45区别CD45(-)肿瘤细胞和CD45(+)T细胞，并进一步分析CD8，CD4 T细胞表面的NKG2D和激活标志物CD25的表达。实验数据表明，59.0％的HT-29细胞上调MICA的表达，40.1％的细胞上调MICB的表达，56.1％的细胞上调ULBP1的表达，84.4％的细胞上调ULBP2的表达，23.7％的细胞上调ULBP3的表达(图4A)，CNK-UT细胞对HT29细胞具有良好的杀伤功能，对照组的共培养系统中69.5％的细胞是肿瘤细胞，而T细胞不表达CD25；而CNK-UT细胞的共培养体系中，从FSC/SSC图中可发现，绝大多数肿瘤细胞已经被清除，T细胞的比例显著增加占84.1％，而且36.345％的CD8+T细胞上调CD25表达，19.08％的CD4+T细胞上调CD25的表达(图4B)。
7.2 CNK-UT细胞对MDA-MB453的识别和特异性杀伤
本实验中使用的CNK-UT细胞是实施例5制备的细胞。
图5示出了CNK-UT细胞对MDA-MB453的识别和特异性杀伤。
A：三阴乳腺癌细胞系MDA-MB453不同NK靶点蛋白的表达情况
MDA-MB453 0.5×10 6细胞经过胰酶消化下来，分别染MICA-APC、MICB-APC、ULBP1-APC、ULBP2-APC、ULBP3-APC，IgG同型抗体作为对照(购自R&D Systems)，再通过流式细胞仪(BD FACSCanto II)进行细胞表型鉴定。结果显示：MDA-MB453细胞高表达MICA、ULBP2，弱表达MICB、ULBP1和ULBP3。
B：CNK-UT细胞对MDA-MB453显示高效的杀伤和激活功能
T细胞和肿瘤细胞的共培养实验，取24孔板，计数乳腺癌细胞系MDA-MB453 0.5×10 6细胞/孔进行铺板，待肿瘤细胞生长密度达80％以上，按效靶比1:5，分别加入未转染T细胞和CNK-UT细胞(DAP10-CD3ζ-T2A-NKG2D-p2A-anti-TCR scFv-AdE3)，24h后，所有细胞消化收获后，进行CD45-PerCP-Cy5.5、CD8-Pacific Blue、 CD4-APC、NKG2D-PE-Cy7、CD25-APC-Cy7(购自Biolegend)的染色，而后进行流式细胞仪技术进行检测。实验数据说明，MDA-MB453细胞83.7％上调MICA的表达，33.6％的细胞上调MICB的表达，31.2％的细胞上调ULBP1的表达，95.0％的细胞上调ULBP2的表达，6.23％的细胞上调ULBP3的表达(图5A)；CNK-UT细胞对MDA-MB453细胞具有良好的杀伤功能，对照组的共培养系统中48.9％的细胞是肿瘤细胞，而T细胞不表达CD25；而CNK-UT细胞的共培养体系中，只剩下6.74％是肿瘤细胞，而且14.8％的CD8+T细胞上调CD25表达，28.1％的CD4+T细胞上调CD25的表达(图5B)。实验结果表明，CNK-UT细胞对MDA-MB453细胞具有良好的杀伤功能，并实现特异性的激活。
7.3 CNK-UT细胞对THP1的识别和特异性杀伤
本实验中使用的CNK-UT细胞是实施例5制备的细胞。
图6示出了CNK-UT细胞对THP1的识别和特异性杀伤
A：急性髓系白血病细胞THP1不同NK靶点的表达情况
0.5×10 6THP1细胞经离心下来，分别染MICA-APC、MICB-APC、ULBP1-APC、ULBP2-APC、ULBP3-APC，IgG同型抗体作为对照(购自R&D Systems)，再通过流式细胞仪(BD FACSCanto II)进行细胞表型鉴定。结果显示：THP1细胞高表达ULBP1、ULBP2、ULBP3，弱表达MICA、MICB。
B：CNK-UT细胞对THP1显示高效的杀伤和激活功能
T细胞和肿瘤细胞的共培养实验，取24孔板，计数AML细胞系THP1 0.5×10 6细胞/孔进行铺板，按效靶比1:5，分别加入未转染T细胞和CNK-UT细胞(DAP10-CD3ζ-T2A-NKG2D-p2A-anti-TCR scFv-AdE3)，24h后，所有细胞离心收获后，进行CD45-PerCP-Cy5.5、CD8-Pacific Blue、CD4-APC、CD137-PE-Cy7、CD25-APC-Cy7(购自Biolegend)的染色，而后进行流式细胞仪技术进行检测。实验数据表明，THP1细胞27.9％上调MICB的表达，99.0％的细胞上调ULBP1的表达，95.8％的细胞上调ULBP2的表达，99.1％的细胞上调ULBP3的表达(图6A)；CNK-UT细胞对THP1细胞具有良好的杀伤功能，对照组的共培养系统中92.6％细胞是肿瘤细胞，而T细胞不上调CD25；；而CNK-UT细胞的共培养体系中，只剩下12.3％的肿瘤细胞，而且49.1％CD8+T细胞上调CD25表达，33.59％CD8+T细胞上调CD137的表达；33.9％CD4+T细胞上调CD25的表达，11.28％CD4+细胞上调CD137的表达。实验结果证实，CNK-UT细胞对THP1细胞具有良好的杀伤功能，清除共培养体系中的THP1，并实现特异性的激活，上调T细胞激活标志物CD25和CD137的表达。
7.4 CNK-UT可协同常规CAR-T技术增强对肿瘤细胞的识别和特异性杀伤
本实验中使用的CNK-UT细胞是实施例5制备的细胞。
图7示出了CNK-UT细胞能升级常规CAR-T技术，实现更强大的靶向杀伤和激活能力。
A：肝细胞癌(HCC)HepG2的GPC3、PD-L1和不同NK靶点的表达情况
0.5×10 6HepG2细胞经过胰酶消化下来，分别染GPC3-APC、PD-L1-APC、MICA-APC、MICB-APC、ULBP1-APC、ULBP2-APC、ULBP3-APC，IIgG同型抗体作为对照(购自R&D Systems)，再通过流式细胞仪(BD FACSCanto II)进行细胞表型鉴定。结果显示：HepG2细胞高表达GPC3，弱表达PD-L1、MICA、MICB、ULBP1和ULBP2。
B：CAR/CNK-UT细胞比常规CAR-T细胞显示对HepG2细胞更高效的杀伤和激活功能
制备GPC3 CAR-T细胞：参考实施例3-实施例5，全基因合成anti-GPC3 41BB-CD3ζ核苷酸序列(SEQ ID NO.120，其编码蛋白的氨基酸序列如SEQ ID NO.119所示)，通过分子克隆的方式连接到慢病毒载体pCDH-CMV-MCS-EF1α-Puro，制备质粒和慢病毒后，转染T细胞，获得常规GPC3 CAR-T细胞。
制备GPC3 CAR/CNK-UT细胞：参考实施例3-实施例5，全基因合成anti-GPC3 41BB-CD3ζ-T2A-DAP10-CD3ζ-T2A-NKG2D-p2A-anti-TCR scFv-AdE3核苷酸序列(SEQ ID NO.122，其编码蛋白的氨基酸序列如SEQ ID NO.121所示)，通过分子克隆的方式连接到慢病毒载体pCDH-CMV-MCS-EF1α-Puro，制备质粒和慢病毒后，转染T细胞并进行扩增培养，获得GPC3 CAR/CNK-UT细胞。
T细胞和肿瘤细胞的共培养实验，取24孔板，计数0.5×10 6细胞/孔进行铺板，待肿瘤细胞生长密度达80％以上，按效靶比1:5，分别加入常规GPC3 CAR-T(anti-GPC3-41BB-CD3ζ)细胞，CNK-UT细胞(DAP10-CD3ζ-T2A-NKG2D-p2A-anti-TCR scFv-AdE3)和GPC3 CAR/CNK-UT细胞(anti-GPC3 41BB-CD3ζ-T2A-DAP10-CD3ζ-T2A-NKG2D-p2A-anti-TCR scFv-AdE3)，24h后，所有细胞消化收获后，进行CD45-PerCP-Cy5.5、CD8-Pacific Blue、CD4-APC、NKG2D-PE、CD137-PE-Cy7(购自Biolegend)的染色，所有细胞消化后进行流式细胞仪技术进行检测，通过CD45区别肿瘤细胞和T细胞，再分析CD8和CD4细胞表面激活标志物CD137的表达。实验数据表明，HepG2细胞95.0％高表达GPC3，69.1％细胞上调PD-L1表达，18.2％的细胞表达MICA，25.6％细胞上调MICB，16.0％细胞上调ULBP1，5.23％的细胞上调ULBP2的表达，在E:T＝1:5共培养24h后，对照组T细胞共培养系统中39.1％细胞是肿瘤细胞，CD4+、CD8+T细胞不上调CD25；GPC3 CAR-T共培养体系中，只剩下11.1％的肿瘤细胞，20.1％CD8+T细胞上调CD137表达；10.3％CD4+T细胞上调CD137的表达；在CNK-UT共培养系统中，只剩下8.01％的肿瘤细胞，57.9％CD8+T细胞上调CD137表达；33.1％CD4+T细胞上调CD137的表达；GPC3 CAR/CNK-UT共培养系统中，只剩下4.86％的肿瘤细胞，50.3％CD8+T细胞上调CD137表达；35.42％CD4+T细胞上调CD137的表达。因此，CNK-UT细胞对比常规CAR-T细胞对HepG2细胞具有良好的杀伤功能，并实现特异性的激活，上调CD137的表达；集成GPC3 41BB-Z CAR和CNK-UT元件的GPC3 CAR/CNK-UT细胞具有更加高效的肿瘤杀伤和激活功能。
7.5 CNK-UT细胞对高表达PD-L1肿瘤细胞的靶向杀伤和激活能力
本实验中使用的CNK-UT细胞是实施例5制备的细胞。
图8示出了CNK-UT细胞能升级常规CAR-T技术，实现对高表达PD-L1肿瘤细胞的靶向杀伤和激活能力。
A：肝细胞癌(HCC)PLC的GPC3、PD-L1和不同NK靶点的表达情况
0.5×10 6PLC细胞经过胰酶消化下来，分别染GPC3-APC、PD-L1-APC、MICA-APC、MICB-APC、ULBP1-APC、ULBP2-APC、ULBP3-APC，IgG同型抗体作为对照(购自R&D Systems)，再通过流式细胞仪(BD FACSCanto II)进行细胞表型鉴定。结果显示：PLC细胞高表达PD-L1和ULBP1，弱表达GPC3、MICA、MICB和ULBP2。
B：CNK-UT细胞比常规CAR-T细胞显示对HepG2细胞更高效的杀伤和激活功能
T细胞和肿瘤细胞的共培养实验，效靶比1:5，未转染T细胞，常规GPC3 CAR-T细胞，CNK-UT细胞和GPC3 CAR/CNK-UT细胞分别加入PLC细胞中，共培养24h后，所有细胞消化后进行流式细胞仪技术进行检测，通过CD45区别肿瘤细胞和T细胞，再分析CD8/CD4细胞表面激活标志物CD25和CD137的表达。实验数据表明，PLC细胞35.9％高表达GPC3，97.8％细胞上调PD-L1表达，56.9％的细胞表达MICA，40.9％细胞上调MICB，68.7％细胞上调ULBP1，25.7％的细胞上调ULBP2的表达；在E:T＝1:5共培养24h后，对照组T细胞共培养系统中30.3％细胞是肿瘤细胞，未转染的CD4+，CD8+T细胞都不上调CD137的表达；而GPC3 CAR-T共培养体系中，高达47.0％都是肿瘤细胞，只有6.89％CD8+T细胞上调CD137表达；CD4+T细胞基本不上调CD137的表达；在CNK-UT共培养系统中，只剩下12.2％的肿瘤细胞，72.0％CD8+T细胞上调CD137表达；42.2％CD4+T细胞上调CD137的表达；GPC3 CAR/CNK-UT共培养系统中，只剩下8.94％的肿瘤细胞，73.9％CD8+T细胞上调CD137表达；36.2％CD4+T细胞上调CD137的表达。实验结果显示，由于PLC高表达PD-L1，弱表达GPC3，所以常规GPC3 CAR-T对PLC的杀伤功能很弱，但CNK-UT细胞对PLC细胞具有良好的杀伤功能，并实现特异性的激活，上调CD137的表达；集成GPC3 CAR和CNK-UT元件的GPC3 CAR/CNK-UT细胞具有更加高效的肿瘤杀伤和激活功能。
实施例8：CNK-UT细胞的肿瘤模型功能检测
本实施例采用免疫缺陷小鼠(NSG小鼠)(购自江苏集萃药康生物科技股份有限公司，货号T001475)荷瘤模型进行体内药效学研究。
8.1 CNK-UT细胞对肝癌细胞系HepG2动物模型的治疗效果
实验动物分为以下三组：
阴性对照组：给予对照T细胞，同种供者来源T细胞，经磁珠刺激扩增好的未转染T细胞；
同型对照组：给予常规GPC3 CAR-T细胞：制备GPC3 CAR-T细胞：参考实施例3-实施例5，全基因合成anti-GPC3 41BB-CD3ζ核苷酸序列，通过分子克隆的方式连接到慢病毒载体pCDH-CMV-MCS-EF1α-Puro，制备质粒和慢病毒后，转染T细胞，获得常规GPC3 CAR-T细胞。
CNK-UT组：给予GPC3 CAR/CNK-U细胞
制备GPC3 CAR/CNK-UT细胞：参考实施例3-实施例5，全基因合成anti-GPC3 41BB-CD3ζ-T2A-DAP10-CD3ζ-T2A-NKG2D-p2A-anti-TCR scFv-AdE3核苷酸序列，通过分子克隆的方式连接到慢病毒载体pCDH-CMV-MCS-EF1α-Puro，制备质粒和慢病毒后，转染T细胞并进行扩增培养，获得GPC3 CAR/CNK-UT细胞。
将实验动物分组后，分别给予对照T细胞、常规GPC3 CAR-T细胞和GPC3 CAR/CNK-UT，之后称量动物体重。15只NSG小鼠经右侧腋下接种HepG2细胞3×10 6/只，造模当天记为第0天。7天后观察见小鼠造模成功，将小鼠分为3组，分别给予普通人原代T细胞(对照T细胞)、常规GPC3-CAR-T细胞、GPC3 CAR/CNK-UT细胞，并观察各种细胞对小鼠体内肿瘤荧光的清除作用。三种细胞均以尾静脉注射的方式给药，剂量为2×10 6/只，连续3次(第7，9，11天)，给药日期记为第7天，之后每7天观察一次荧光情况。
图9示出了GPC3 CAR/CNK-UT细胞比GPC3 CAR-T细胞在小鼠体内具有更高效的肿瘤清除能力。
采用肝癌细胞系HepG2-Ffluc建模后给予2×10 6未转染T细胞(左)、GPC3 CAR-T(中)与GPC3 CAR/CNK-UT(右)，并每周进行IVIS动物荧光成像系统监测肿瘤生长情况。实验结果显示：对照T细胞组荧光持续增强，GPC3 CAR-T荧光缓慢增强，而CNK-T001组肿瘤荧光不断减弱直至消失，证实了GPC3 CAR/CNK-UT细胞在小鼠模型中比常规CART细胞有更强的抗肿瘤作用。
图10示出了GPC3 CAR/CNK-UT细胞比GPC3 CAR-T具有高效的肿瘤清除能力。
将图10中的荧光成像进行定量后绘制肿瘤荧光曲线，可见对照T细胞(蓝色)、GPC3 CAR-T(红色)均无法完全抑制肿瘤生长，而(绿色)可以显著抑制肿瘤生长，肿瘤荧光不断减弱直至消失，证实了GPC3 CAR/CNK-UT细胞在小鼠模型中的抗肿瘤作用。
以上实验结果显示，阴性对照组小鼠肿瘤逐渐增大，在28-42天之间出现第一只小鼠死亡，42天之后相继全部死亡；给予同型对照药GPC3 CAR/CAR-T细胞的同型对照组在第14天可见抑制肿瘤生长，但无法完全消除肿瘤，在第21天观察到肿瘤增大，并在28天后与阴性对照组几乎没有差别，全组小鼠在42天至56天之间相继死亡，这说明单靶点GPC3 CAR-T仅能部分抑制肿瘤，但不能完全清除，更不能预防复发；在第14天，给予GPC3 CAR/CNK-UT细胞的CNK-UT组明显降低小鼠体内的肿瘤荧光信号，至21天时信号完全消失，直到观察截止时间(第56天)，小鼠全部存活，且未见肿瘤复发。说明GPC3 CAR/CNK-UT细胞在体内具有良好抗肿瘤活性，其 活性明显优于GPC3 CAR-T，清除肿瘤后，其抗肿瘤效果可至少维持56天。
此外，还研究了不同细胞回输后小鼠体重变化。其中，回输CNK-UT细胞的CNK-UT组实验动物体重与D0相比有少量升高，而回输GPC3-CAR-T的同型对照组和对照T细胞的阴性对照组动物，体重随实验时间延长逐渐降低。
综合以上实验结果可知，以上结果提示，实验动物经不同方法治疗后，注射对照T细胞和GPC3 CAR-T细胞的小鼠肿瘤仍然生长，而GPC3 CAR/CNK-UT细胞比GPC3 CAR-T细胞的具有更强的杀伤力，能够在输注后的21天清除肿瘤细胞，且无复发。并且，GPC3 CAR/CNK-UT回输无细胞毒性，动物的生存期延长，动物生活状况有所改善，体重增加。
8.2 CNK-UT细胞对三阴乳腺癌细胞MDA-MB453的靶向杀伤和激活能力
图11示出了GPC3 CAR/CNK-UT能特异性的识别和杀伤三阴乳腺癌细胞MDA-MB453。
T细胞和肿瘤细胞的共培养实验，效靶比1:5，未转染T细胞、常规GPC3 CAR-T细胞、CNK-UT细胞和GPC3 CAR/CNK-UT细胞分别加入MDA-MB453细胞中，共培养24h后，所有细胞消化后进行流式细胞仪技术进行检测，通过CD45区别肿瘤细胞和T细胞，再分析CD8和CD4细胞NK元件NKG2D和表面激活标志物CD25的表达。
MDA-MB453是三阴乳腺癌细胞系，细胞不表达GPC3，因此GPC3 CAR-T无法对MDA-MB453进行清除(共培养24h后，体系中还存在大量CD45阴性的肿瘤细胞)，而CNK-UT产品却可以高效的杀伤和清除肿瘤细胞，检测CD45(+)T细胞中，CD8(+)、CD4(+)T细胞都高表达CNK元件NKG2D，并上调T细胞的激活标志物CD25。
8.3 CNK-UT细胞对急性髓系白血病细胞系THP1的靶向杀伤和激活能力
图12示出了GPC3 CAR/CNK-UT能特异性的识别和杀伤急性髓系白血病细胞系THP1。
T细胞和肿瘤细胞的共培养实验，效靶比1:5，未转染T细胞，GPC3 CAR-T，CNK-UT和GPC3 CAR/CNK-UT分别加入THP1细胞中，24h后，所有细胞消化后进行流式细胞仪技术进行检测，通过CD3区别肿瘤细胞和T细胞，再分析CD3(-)肿瘤细胞表面标志物CD123、CLL1的表达；和CD3(+)细胞表面激活标志物CD25、CD137的表达.
结果显示THP1是白血病细胞系，细胞不表达GPC3，因此GPC3 CAR-T无法对THP1进行清除(共培养24h后，体系中还存在大量CD3(-)、CD123(+)、CLL1(+)的肿瘤细胞，而GPC3 CAR/CNK-UT产品却可以高效的杀伤和清除肿瘤细胞，检测CD3(+)T细胞中上调T细胞的激活标志物CD25和CD137，说明GPC3 CAR/CNK-UT能特异性的识别THP1细胞，并激活和杀伤清除肿瘤细胞。
实施例9：NCR元件升级的CNK-UT细胞对不同肿瘤的杀伤和清除能力
本实施例研究基于实施例5制备的CNK-UT细胞在通过NCR元件升级后对不同肿瘤的杀伤和清除能力。其中，肾细胞癌(Renal Cell Carcinoma，RCC)肿瘤细胞系786-O购自普诺赛(CL-0010)；肾细胞癌(Renal Cell Carcinoma，RCC)肿瘤细胞系ACHN购自普诺赛(CL-0021)；上皮腺瘤(Lung epithelial carcinoma)肿瘤细胞系A549购自普诺赛(CL-0016)；急性髓系白血病(Acute myeloid leukemia)肿瘤细胞系UL60购自普诺赛(CL-0110)。
9.1 CNK-UT细胞对RCC肿瘤细胞系786-O的识别和特异性杀伤
制备NCR2 CAR/CNK-UT细胞：参考实施例3-实施例5，全基因合成NCR2-CD28-CD3ζ的核苷酸序列(SEQ ID NO.124，其编码蛋白的氨基酸序列如SEQ ID NO.123所示)；全基因合成DAP10-CD3ζ-T2A-NKG2D-p2A-anti-TCR scFv-AdE3的核苷酸序列，通过分子克隆的方式连接到慢病毒载体pCDH-CMV-MCS-EF1α-Puro，制备质粒和慢病毒后，转染T细胞并进行扩增培养，获得NCR2 CAR/CNK-UT细胞。
图13示出了CNK-UT细胞对肾细胞癌786-O细胞的识别，特异性杀伤和激活能力。
T细胞和肿瘤细胞的共培养实验，取24孔板，计数RCC肿瘤细胞系786-O 0.5×10 6细胞/孔进行铺板，分别按效靶比1:1和2:1，加入未转染T细胞和NCR2 CAR/CNK-UT细胞(NCR2-CD28-CD3ζ；DAP10-CD3ζ-T2A-NKG2D-p2A-anti-TCR scFv-AdE3)，24h后，所有细胞离心收获后，进行7-AAD，CD45-APC-Cy5.5、CD8-APC、NKG2D-PE-Cy7、NKp44-PE、CD137-Pacific Blue(购自Biolegend)的染色，而后进行流式细胞仪技术进行检测。实验数据表明；NCR2 CAR/CNK-UT细胞对786-O细胞具有良好的杀伤功能，在E:T＝1:1的培养体系中，经过48h共培养，对照组肿瘤细胞占59.9％，同时对照T细胞不表达Nkp44，不上调激活标志物CD137；而NCR2CAR/CNK-UT细胞的共培养体系中，只剩下2.29％的肿瘤细胞，同时12.8％CD8+T细胞上调CD137表达；在E:T＝2:1的培养体系中，经过48h共培养，对照组肿瘤细胞占32.9％，同时对照T细胞不表达Nkp44，不上调激活标志物CD137；而NCR2CAR/CNK-UT细胞的共培养体系中，只剩下0.83％的肿瘤细胞，而且13.3％CD8+T细胞上调CD137表达。可见，NCR元件升级可以进一步增加CNK-UT细胞对不同肿瘤的杀伤和清除能力。
9.2 CNK-UT细胞对肾细胞癌(Renal Cell Carcinoma，RCC)肿瘤细胞系ACHN的识别和特异性杀伤
图14示出了CNK-UT细胞对肾细胞癌ACHN细胞的识别，特异性杀伤和激活能力。
T细胞和肿瘤细胞的共培养实验，取24孔板，计数RCC细胞系ACHN 0.5×10 6细胞/孔进行铺板，分别按效靶比1:1和2:1，加入未转染T细胞和NCR2 CAR/CNK-UT细胞(NCR2-CD28-CD3ζ；DAP10-CD3ζ-T2A-NKG2D-p2A-anti-TCR scFv-AdE3)，24h后，所有细胞离心收获后，进行7-AAD，CD45-APC-Cy5.5、CD8-APC、NKG2D- PE-Cy7、NKp44-PE、CD137-Pacific Blue(购自Biolegend)的染色，而后进行流式细胞仪技术进行检测。实验数据表明；NCR2 CAR/CNK-UT细胞对ACHN肿瘤细胞具有良好的杀伤功能，在E:T＝1:1的培养体系中，经过48h共培养，对照组肿瘤细胞占48.1％，同时对照T细胞不表达Nkp44，不上调激活标志物CD137；而NCR2 CAR/CNK-UT细胞的共培养体系中，只剩下17.9％的肿瘤细胞，同时32.1％CD8+T细胞上调CD137表达；在E:T＝2:1的培养体系中，经过48h共培养，对照组肿瘤细胞占30.8％，同时对照T细胞不表达Nkp44，不上调激活标志物CD137；而NCR2 CAR/CNK-UT细胞的共培养体系中，只剩下6.57％的肿瘤细胞，而且30.4％CD8+T细胞上调CD137表达。
9.3 CNK-UT细胞对肺上皮腺瘤(Lung epithelial carcinoma)肿瘤细胞系A549的识别和特异性杀伤
图15示出了CNK-UT细胞对肺上皮腺瘤A549细胞的识别，特异性杀伤和激活能力。
T细胞和肿瘤细胞的共培养实验，取24孔板，计数肺癌细胞系A549 0.5×10 6细胞/孔进行铺板，分别按效靶比1:1和2:1，加入未转染T细胞和NCR2 CAR/CNK-UT细胞(NCR2-CD28-CD3ζ；DAP10-CD3ζ-T2A-NKG2D-p2A-anti-TCR scFv-AdE3)，24h后，所有细胞离心收获后，进行7-AAD，CD45-APC-Cy5.5、CD8-APC、NKG2D-PE-Cy7、NKp44-PE、CD137-Pacific Blue(购自Biolegend)的染色，而后进行流式细胞仪技术进行检测。实验数据表明；NCR2 CAR/CNK-UT细胞对A549肿瘤细胞具有良好的杀伤功能，在E:T＝1:1的培养体系中，经过48h共培养，对照组肿瘤细胞占49.4％，同时对照T细胞不表达Nkp44，不上调激活标志物CD137；而NCR2 CAR/CNK-UT细胞的共培养体系中，只剩下35.4％的肿瘤细胞，同时27.4％CD8+T细胞上调CD137表达；在E:T＝2:1的培养体系中，经过48h共培养，对照组肿瘤细胞占43.7％，同时对照T细胞不表达Nkp44，不上调激活标志物CD137；而NCR2 CAR/CNK-UT细胞的共培养体系中，只剩下5.27％的肿瘤细胞，同时34.1％CD8+T细胞上调CD137表达。
9.4 CNK-UT细胞对急性髓系白血病(Acute myeloid leukemia)肿瘤细胞系UL60的识别和特异性杀伤
图16示出了CNK-UT细胞对AML细胞系UL60的识别，特异性杀伤和激活能力。
T细胞和肿瘤细胞的共培养实验，取24孔板，计数肺癌细胞系UL60 0.5×10 6细胞/孔进行铺板，分别按效靶比1:1和2:1，加入未转染T细胞和NCR2 CAR/CNK-UT细胞(NCR2-CD28-CD3ζ；DAP10-CD3ζ-T2A-NKG2D-p2A-anti-TCR scFv-AdE3)，24h后，所有细胞离心收获后，进行7-AAD，CD45-APC-Cy5.5、CD8-APC、NKG2D-PE-Cy7、NKp44-PE、CD137-Pacific Blue(购自Biolegend)的染色，而后进行流式细胞仪技术进行检测。实验数据表明；NCR2 CAR/CNK-UT细胞对UL60肿瘤细胞具有良好的杀伤功能，在E:T＝1:1的培养体系中，经过48h共培养，对照组CD3(-)肿瘤细胞占57.3％，对照T细胞不表达Nkp44，不上调激活标志物CD137；而NCR2 CAR/CNK-UT细胞的共培养体系中，只剩下7.41％的肿瘤细胞，同时10.6％CD8+T细胞上调CD137表达；在E:T＝2:1的培养体系中，经过48h共培养，对照组肿瘤细胞占30.1％，同时对照T细胞不表达Nkp44，不上调激活标志物CD137；而NCR2 CAR/CNK-UT细胞的共培养体系中，只剩下3.7％的肿瘤细胞，同时8.11％CD8+T细胞上调CD137表达。
实施例10：优化设计UT元件提高CNK-UT细胞对肿瘤细胞系U937的识别和特异性杀伤
制备CNK-UT(UL16)细胞和CNK-UT(UL16/E3-19K)细胞：参考实施例3-实施例5，全基因合成DAP10-CD3ζ-T2A-NKG2D的核苷酸序列(其编码蛋白的氨基酸序列如SEQ ID NO.125所示)；全基因合成anti-TCR scFv-AdE3-p2A-UL16的核苷酸序列(其编码蛋白的氨基酸序列如SEQ ID NO.126所示)；全基因合成anti-TCR scFv-AdE3-p2A-UL16-p2A-E3-19K的核苷酸序列(其编码蛋白的氨基酸序列如SEQ ID NO.127所示)，通过分子克隆的方式连接到慢病毒载体pCDH-CMV-MCS-EF1α-Puro，制备质粒和慢病毒后，转染T细胞并进行扩增培养，获得CNK-UT(UL16)细胞和CNK-UT(UL16/E3-19K)细胞。
图17示出了CNK-UT细胞对AML细胞系U937(购自U937，货号CL-0239)的识别，特异性杀伤和激活能力。
T细胞和肿瘤细胞的共培养实验，取24孔板，计数肺癌细胞系U937-GFP 0.5×10 6细胞/孔进行铺板，分别按效靶比1:1，加入未转染T细胞，CNK-UT(UL16)细胞(DAP10-CD3ζ-T2A-NKG2D；anti-TCR scFv-AdE3-p2A-UL16)和CNK-UT(UL16/E3-19K)细胞(DAP10-CD3ζ-T2A-NKG2D；anti-TCR scFv-AdE3-p2A-UL16-p2A-E3-19K)，24h后，所有细胞离心收获后，进行7-AAD，CD33-APC-Cy5.5、CD8-Pacific Blue、NKG2D-APC、CD137-PE-Cy7(购自Biolegend)的染色，而后进行流式细胞仪技术进行检测。实验数据表明；CNK-UT(UL16/E3-19K)设计比CNK-UT(UL16)细胞对U937肿瘤细胞有更高效的杀伤功能，在E:T＝1:1的培养体系中，经过48h共培养，对照组CD33(+)肿瘤细胞占69.8％，对照T细胞不上调激活标志物CD137；而CNK-UT(UL16)细胞的共培养体系中，只剩下4.69％的CD33+，GFP+肿瘤细胞，同时23.07％T细胞上调CD137表达；而CNK-UT(UL16/E3-19K)细胞的共培养体系中，只剩下0.079％的肿瘤细胞，同时54.7％T细胞上调CD137表达。
实施例11：优化设计NK元件提高CNK-UT细胞对肿瘤细胞系
11.1优化设计NK元件提高CNK-UT细胞对肿瘤细胞系U937的识别和特异性杀伤
制备NCR1 CAR-T细胞、NCR2 CNK-UT细胞和NCR1/2 CNK-UT细胞：参考实施例3-实施例5，全基因合成NCR1-CD28-CD3ζ的核苷酸序列(其编码蛋白的氨基酸序列如SEQ ID NO.128所示)；全基因合成DAP10-CD3ζ-T2A-NKG2D-p2A-anti-TCR scFv-AdE3的核苷酸序列(其编码蛋白的氨基酸序列如SEQ ID NO.117所示)；全基因 合成NCR2-CD28-CD3ζ的核苷酸序列(其编码蛋白的氨基酸序列如SEQ ID NO.123所示)；全基因合成NCR2-CD28-CD3ζ-p2A-NCR1-CD28-CD3ζ的核苷酸序列(其编码蛋白的氨基酸序列如SEQ ID NO.129所示)，通过分子克隆的方式连接到慢病毒载体pCDH-CMV-MCS-EF1α-Puro，制备质粒和慢病毒后，转染T细胞并进行扩增培养，获得NCR1 CAR-T细胞、NCR2 CNK-UT细胞和NCR1/2 CNK-UT细胞。
图18示出了CNK-UT细胞对AML细胞系U937的识别，特异性杀伤和激活能力。
T细胞和肿瘤细胞的共培养实验，取24孔板，计数肺癌细胞系U937-GFP 0.5×10 6细胞/孔进行铺板，分别按效靶比1:1和2:1，加入未转染T细胞，NCR1 CAR-T细胞(NCR1-CD28-CD3ζ)，NCR2 CNK-UT细胞(DAP10-CD3ζ-T2A-NKG2D-p2A-anti-TCR scFv-AdE3；NCR2-CD28-CD3ζ)和NCR1/2 CNK-UT细胞(DAP10-CD3ζ-T2A-NKG2D-p2A-anti-TCR scFv-AdE3；NCR2-CD28-CD3ζ-p2A-NCR1-CD28-CD3ζ)，48h后在荧光显微镜下观察并拍照，实验数据表明；增加NK识别元件，可显著提高CNK-UT细胞对U937肿瘤细胞的杀伤功能。
11.2优化设计NK元件提高CNK-UT细胞对肿瘤细胞系U937的识别和特异性杀伤
图19示出了CNK-UT细胞对胰腺癌细胞系PANC-1(购自普诺赛，货号CL-0184)的识别，特异性杀伤和激活能力。
制备NCR1 CAR-T细胞、NCR2 CNK-UT细胞和NCR1/2 CNK-UT细胞：参考实施例3-实施例5，全基因合成NCR1-CD28-CD3ζ-p2A-tEGFR的核苷酸序列(其编码蛋白的氨基酸序列如SEQ ID NO.130所示)；全基因合成DAP10-CD3ζ-T2A-NKG2D-p2A-anti-TCR scFv-AdE3的核苷酸序列(其编码蛋白的氨基酸序列如SEQ ID NO.117所示)；全基因合成NCR2-CD28-CD3ζ-p2A-tEGFR的核苷酸序列(其编码蛋白的氨基酸序列如SEQ ID NO.131所示)；全基因合成NCR2-CD28-CD3ζ-p2A-NCR1-CD28-CD3ζ的核苷酸序列(其编码蛋白的氨基酸序列如SEQ ID NO.129所示)，通过分子克隆的方式连接到慢病毒载体pCDH-CMV-MCS-EF1α-Puro，制备质粒和慢病毒后，转染T细胞并进行扩增培养，获得NCR1 CAR-T细胞、NCR2 CNK-UT细胞和NCR1/2 CNK-UT细胞。
T细胞和肿瘤细胞的共培养实验，取24孔板，计数胰腺癌细胞系PANC-1-GFP 0.5×10 6细胞/孔进行铺板，分别按效靶比1:1，加入未转染T细胞，NCR1 CAR-T细胞(NCR1-CD28-CD3ζ-p2A-tEGFR)，NCR2 CNK-UT细胞(DAP10-CD3ζ-T2A-NKG2D-p2A-anti-TCR scFv-AdE3；NCR2-CD28-CD3ζ-p2A-tEGFR)和NCR1/2 CNK-UT(DAP10-CD3ζ-T2A-NKG2D-p2A-anti-TCR scFv-AdE3；NCR2-CD28-CD3ζ-p2A-NCR1-CD28-CD3ζ)，共培养48h，所有细胞离心收获后，进行7-AAD，CD33-APC-Cy5.5、CD8-Pacific Blue、NKG2D-APC、NKp44-PE or EGFR-PE，CD137-PE-Cy7(购自Biolegend)的染色，而后进行流式细胞仪技术进行检测。实验数据表明；联合NCR1，NCR2 CAR设计的CNK-UT比NCR2 CAR/CNK-UT对胰腺癌PANC-1肿瘤细胞有更高效的杀伤功能和激活能力。在E:T＝1:1的培养体系中，经过48h共培养，对照组GFP(+)，CD45(-)肿瘤细胞占65.7％，流式计数为6653，对照T细胞不上调激活标志物CD137；NCR1 CAR-T细胞的共培养体系中，GFP(+)，CD45(-)肿瘤细胞只占28.3％，流式计数为2567，同时16.65％T细胞上调CD137表达；而NCR2 CNK-UT细胞的共培养体系中，只剩下15.6％的肿瘤细胞，流式计数为1368，同时20.89％T细胞上调CD137表达。而NCR1/2 CNK-UT细胞的共培养体系中，只剩下7.71％的肿瘤细胞，流式计数为662，同时21.9％T细胞上调CD137表达。

Claims (14)

  1. 一种多功能复合体(multi-functional complex)，其包含以下组件：
    (1)NK激活受体组件，其至少包含NK细胞激活受体或其功能变体，所述NK细胞激活受体包含：(a)NK细胞激活受体胞外结构域(ED)或其功能变体，(b)NK细胞激活受体跨膜结构域(TMD)或其功能变体，以及(c)NK细胞激活受体胞内结构域(ICD)或其功能变体；任选地，所述NK细胞激活受体胞外结构域或其功能变体、所述NK细胞激活受体跨膜结构域或其功能变体和/或所述NK细胞激活受体胞内结构域或其功能变体之间包含铰链或接头；
    (2)CNK信号转接组件，其至少包含(i)NK细胞信号转换器(adaptor)或其功能变体，所述NK细胞信号转换器包含：(a)NK细胞信号转换器胞外结构域(ED)或其功能变体，(b)NK细胞信号转换器跨膜结构域(TMD)或其功能变体，以及(c)NK细胞信号转换器胞内结构域(ICD)或其功能变体；任选地，所述NK细胞信号转换器胞外结构域或其功能变体、所述NK细胞信号转换器跨膜结构域或其功能变体和/或所述NK细胞信号转换器胞内结构域或其功能变体之间包含铰链或接头；和
    (3)UT组件，其至少包含(i)靶向降解TCR、MHC和/或NK细胞靶点的重组蛋白分子或其功能变体，所述靶向降解TCR、MHC和/或NK细胞靶点的重组蛋白分子包含：(a)靶向TCR、MHC和/或NK细胞靶点的结合蛋白分子结构域或其功能变体，(b)病毒内质网(ER)驻留糖蛋白的跨膜结构域或其功能变体，以及(c)病毒内质网驻留糖蛋白的胞质结构域或其功能变体；所述病毒内质网驻留糖蛋白的跨膜结构域或其功能变体和病毒内质网驻留糖蛋白的胞质结构域或其功能变体形成ERAD降解结构域；任选地，所述靶向TCR的结合蛋白分子结构域或其功能变体、所述病毒内质网驻留糖蛋白的跨膜结构域或其功能变体和/或所述病毒内质网驻留糖蛋白的胞质结构域或其功能变体之间包含铰链或接头；
    任选地，所述NK激活受体组件、所述CNK信号转接组件和/或所述UT组件之间包含铰链或接头。
  2. 根据权利要求1所述的多功能复合体，其中，所述NK激活受体组件中的所述NK细胞激活受体选自NKG2D、NKG2C、NKG2E、NKG2F、NKG2H、CD94、KIR2DL4、KIR2DS1、KIR2DS2、KIR2DS4、KIR3DS1，天然细胞毒性受体、TRAIL、DNAM-1、CD16a、2B4、NTB-A、CRACC以及NKp80；优选地，所述天然细胞毒性受体选自NKp46、NKp44和NKp30；
    优选地，所述NK细胞激活受体是哺乳动物来源的NK细胞激活受体；优选地，所述哺乳动物选自人、灵长类动物、鼠、马、牛、绵羊、山羊、猫、猪、狗、美洲驼、羊驼、大象、松鼠、豚鼠；
    优选地，所述NK细胞激活受体是包含不同来源NK细胞激活受体结构域的重组NK细胞激活受体；
    优选地，所述NK细胞激活受体是人源的NK细胞激活受体；优选地，所述NK细胞激活受体是包含不同的人源NK细胞激活受体结构域的重组NK细胞激活受体；
    优选地，所述NK细胞激活受体是鼠源的NK细胞激活受体；优选地，所述NK细胞激活受体是包含不同的鼠源NK细胞激活受体结构域的重组NK细胞激活受体；
    优选地，所述NK细胞激活受体是包含人源和鼠源NK细胞激活受体结构域的重组NK细胞激活受体；
    优选地，所述NK细胞激活受体的胞外结构域为人或鼠的NK细胞激活受体的胞外结构域；
    优选地，所述NK细胞激活受体的跨膜结构域为人或鼠的NK细胞激活受体的跨膜结构域；
    优选地，所述NK细胞激活受体的胞内结构域为人或鼠的NK细胞激活受体的胞内结构域；
    优选地，所述NK细胞激活受体功能变体选自NK细胞激活受体的突变体，野生型融合蛋白，或野生型与突变型的融合蛋白；
    优选地，人NKG2D的胞外域包含与SEQ ID NO.1所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKG2D的胞外域的氨基酸序列如SEQ ID NO.1所示；
    优选地，人NKG2D的全长序列包含与SEQ ID NO.2所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKG2D的全长序列的氨基酸序列如SEQ ID NO.2所示；
    优选地，小鼠NKG2D的胞外域包含与SEQ ID NO.3所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；小鼠NKG2D的胞外域的氨基酸序列如SEQ ID NO.3所示；
    优选地，小鼠NKG2D的全长序列包含与SEQ ID NO:4所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；小鼠NKG2D的全长序的氨基酸序列如SEQ ID NO.4所示；
    优选地，人鼠重组NKG2D的全长序列包含与SEQ ID NO:5所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人鼠重组NKG2D的全长序列的氨基酸序列如SEQ ID NO.5所示；
    优选地，人NKG2C的全长序列包含与SEQ ID NO.6所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一 性的氨基酸序列；人NKG2C的全长序列的氨基酸序列如SEQ ID NO.6所示；
    优选地，人NKG2E的全长序列包含与SEQ ID NO：7所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKG2E的全长序列的氨基酸序列如SEQ ID NO.7所示；
    优选地，人NKG2F的全长序列包含与SEQ ID NO：8所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKG2F的全长序列的氨基酸序列如SEQ ID NO.8所示；
    优选地，人CD94的全长序列包含与SEQ ID NO.9所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；的氨基酸序列如SEQ ID NO.9所示；
    优选地，人KIR2DL4的全长序列包含与SEQ ID NO：10所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人KIR2DL4的全长序列的氨基酸序列如SEQ ID NO.10所示；
    优选地，人KIR2DS1的全长序列包含与SEQ ID NO：11所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人KIR2DS1的全长序列的氨基酸序列如SEQ ID NO.11所示；
    优选地，人KIR2DS2的全长序列包含与SEQ ID NO：12所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人KIR2DS2的全长序列的氨基酸序列如SEQ ID NO.12所示；
    优选地，人KIR2DS4的全长序列包含与SEQ ID NO：13所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人KIR2DS4的全长序列的氨基酸序列如SEQ ID NO.13所示；
    优选地，人KIR3DS1的全长序列包含与SEQ ID NO：14所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人KIR3DS1的全长序列的氨基酸序列如SEQ ID NO.14所示；
    优选地，人NKp46的全长序列包含与SEQ ID NO.15所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKp46的全长序列的氨基酸序列如SEQ ID NO.15所示；
    优选地，人NKp44的全长序列包含与SEQ ID NO.16所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKp44的全长序列的氨基酸序列如SEQ ID NO.16所示；
    优选地，人NKp30的全长序列包含与SEQ ID NO.17所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKp30的全长序列的氨基酸序列如SEQ ID NO.17所示；
    优选地，人DNAM1的全长序列包含与SEQ ID NO：18所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DNAM1的全长序列的氨基酸序列如SEQ ID NO.18所示；
    优选地，人TRAIL的全长序列包含与SEQ ID NO：19所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人TRAIL的全长序列的氨基酸序列如SEQ ID NO.19所示；
    优选地，人CD16a的全长序列包含与SEQ ID NO：20所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人CD16a的全长序列的氨基酸序列如SEQ ID NO.20所示；
    优选地，人2B4的全长序列包含与SEQ ID NO：21所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人2B4的全长序列的氨基酸序列如SEQ ID NO.21所示；
    优选地，人NTB-A的全长序列包含与SEQ ID NO：22所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NTB-A的全长序列的氨基酸序列如SEQ ID NO.22所示；
    优选地，人CRACC的全长序列包含与SEQ ID NO：23所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人CRACC的全长序列的氨基酸序列如SEQ ID NO.23所示；
    优选地，人NKp80的全长序列包含与SEQ ID NO：24所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKp80的全长序列的氨基酸序列如SEQ ID NO.24所示。
  3. 权利要求1或2所述的多功能复合物，其中，所述CNK信号转接组件中的所述NK细胞信号转换器为 DAP10或DAP12；
    优选地，所述NK细胞信号转换器是哺乳动物来源的NK细胞信号转换器；优选地，所述哺乳动物选自人、灵长类动物、鼠、马、牛、绵羊、山羊、猫、猪、狗、美洲驼、羊驼、大象、松鼠、豚鼠；
    优选地，所述NK细胞信号转换器是包含不同来源NK细胞信号转换器结构域的重组NK细胞信号转换器；
    优选地，所述NK细胞信号转换器是人源的NK细胞信号转换器；优选地，所述NK细胞信号转换器是包含不同的人源NK细胞信号转换器结构域的重组NK细胞信号转换器；
    优选地，所述NK细胞信号转换器是鼠源的NK细胞信号转换器；优选地，所述NK细胞信号转换器是包含不同的鼠源NK细胞信号转换器结构域的重组NK细胞信号转换器；
    优选地，所述NK细胞信号转换器是包含人源和鼠源NK细胞信号转换器结构域的重组NK细胞信号转换器；
    优选地，所述NK细胞信号转换器的胞外结构域为人或鼠的NK细胞信号转换器的胞外结构域；
    优选地，所述NK细胞信号转换器的跨膜结构域为人或鼠的NK细胞信号转换器的跨膜结构域；
    优选地，所述NK细胞信号转换器的胞内结构域为人或鼠的NK细胞信号转换器的胞内结构域；
    所述CNK细胞信号转换器功能变体选自DAP10突变体或DAP12的突变体，或DAP10和DAP12的融合蛋白，或野生型DAP10或DAP12与突变型DAP10或DAP12的融合蛋白；
    优选地，所述CNK信号转接组件还包含(ii)免疫受体活化信号传导结构域(ITAM)和/或(iii)T细胞共刺激信号传导结构域；
    优选地，所述NK细胞信号转换器或其功能变体、所述免疫受体活化信号传导结构域(ITAM)和/或所述T细胞共刺激信号传导结构域之间包含铰链或接头；优选地，所述NK细胞信号转换器或其功能变体与所述免疫受体活化信号传导结构域(ITAM)域融合；
    优选地，所述免疫受体活化信号传导结构域(ITAM)来自免疫受体的胞内激活信号传导结构域；优选地，所免疫受体选自TCRζ、CD2、CD3γ、CD3δ、CD3ε、CD3ζ、CD5、CD22、FcRγ、CD66d、FcαRI、FcγRI、FcγRII、FcγRIII、Dectin-1、CLEC-1、CD72、CD79A、CD79B；优选地，所述免疫受体活化信号传导结构域(ITAM)与NK细胞信号转换器或其功能变体融合；优选地，所述免疫受体是CD3ζ；
    优选地，所述T细胞共刺激信号传导结构域来自共刺激分子的胞内信号结构域；优选地，所述共刺激分子选自MHC I类分子、TNF受体蛋白、免疫球蛋白样蛋白质、细胞因子受体、整联蛋白、淋巴细胞活化信号分子(SLAM蛋白)、活化NK细胞受体、BTLA、Toll配体受体、OX40、CD2、CD7、CD16、CD27、CD28、CD30、CD40、CD38、CD35、CD79A、CD79B、CDS、ICAM-1、LFA-1、(CD11a/CD18)、4-1BB(CD137)、B7-H3、CDS、ICAM-1、ICOS(CD278)、GITR、BAFFR、LIGHT、HVEM(LIGHTR)、KIRDS2、SLAMF7、NKp80(KLRF1)、NKp44、NKp30、NKp46、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、NKG2D、NKG2C、NCR、DAP10、DAP12、TNFR2、TRANCE/RANKL、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100 SEMA4D)、CD69、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、CD19a、与CD83特异性结合的配体、CARD11、FcRa、FcRp、FcRy、Fyn、HVEM、ICOS、Lck、LAG3、LAT、LRP、NOTCH1、Wnt、OX40、ROR2、Ryk、SLAMF1、Slp76、pTa、TCRa、TCRp、TRIM、ZAP70、PTCH2；
    优选地，人DAP10的全长序列包含与SEQ ID NO.25所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10的全长序列的氨基酸序列如SEQ ID NO.25所示；
    优选地，人DAP10的全长序列包含与SEQ ID NO.26所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10的全长序列的氨基酸序列如SEQ ID NO.26所示；
    优选地，人DAP10的跨膜结构域包含与SEQ ID NO.27所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10的跨膜结构域的氨基酸序列如SEQ ID NO.27所示；
    优选地，人DAP12的全长序列包含与SEQ ID NO.28所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP12的全长序列的氨基酸序列如SEQ ID NO.28所示；
    优选地，人DAP12的跨膜结构域包含与SEQ ID NO.29所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP12的跨膜结构域的氨基酸序列如SEQ ID NO.29所示；
    优选地，人DAP10和人DAP12的跨膜结构域融合蛋白包含与SEQ ID NO.30所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10和人DAP12的跨膜结构域的氨基酸序列如SEQ ID NO.30所示；
    优选地，人DAP10-DAP12融合蛋白序列包含与SEQ ID NO.31所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10-DAP12融合蛋白序列的氨基酸序列如SEQ ID NO.31所示；
    优选地，人CD3zeta胞内信号传导结构域序列包含与SEQ ID NO.32所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人CD3zeta胞内信号传导结构域序列的氨基酸序列如SEQ ID NO.32所示；
    优选地，人DAP10-CD3zeta序列包含与SEQ ID NO.33所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10-CD3zeta序列的氨基酸序列如SEQ ID NO.33所示；
    优选地，人DAP12-CD3zeta序列包含与SEQ ID NO.34所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP12-CD3zeta序列的氨基酸序列如SEQ ID NO.34所示；
    优选地，人DAP10-DAP12-CD3zeta序列包含与SEQ ID NO.35所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10-DAP12-CD3zeta序列的氨基酸序列如SEQ ID NO.35所示；
    优选地，人41BB胞内信号传导结构域序列包含与SEQ ID NO.36所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人41BB胞内信号传导结构域序列的氨基酸序列如SEQ ID NO.36所示；
    优选地，人DAP10-41BB序列包含与SEQ ID NO.37所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10-41BB序列的氨基酸序列如SEQ ID NO.37所示；
    优选地，人DAP10-41BB-CD3zeta序列包含与SEQ ID NO.38所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10-41BB-CD3zeta序列的氨基酸序列如SEQ ID NO.38所示；
    优选地，人CD28胞内信号传导结构域序列包含与SEQ ID NO.39所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人CD28胞内信号传导结构域序列的氨基酸序列如SEQ ID NO.39所示；
    优选地，人DAP10-CD28序列包含与SEQ ID NO.40所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10-CD28序列的氨基酸序列如SEQ ID NO.40所示；
    优选地，人DAP10-CD28-CD3zeta序列包含与SEQ ID NO.41所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10-CD28-CD3zeta序列的氨基酸序列如SEQ ID NO.41所示；
    优选地，人DAP12-41BB序列包含与SEQ ID NO.42所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP12-41BB序列的氨基酸序列如SEQ ID NO.42所示；
    优选地，人DAP12-41BB-CD3zeta序列包含与SEQ ID NO.43所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP12-41BB-CD3zeta序列的氨基酸序列如SEQ ID NO.43所示；
    优选地，人DAP12-CD28序列包含与SEQ ID NO.44所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP12-CD28序列的氨基酸序列如SEQ ID NO.44所示；
    优选地，人DAP12-CD28-CD3zeta序列包含与SEQ ID NO.45所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP12-CD28-CD3zeta序列的氨基酸序列如SEQ ID NO.45所示。
  4. 根据权利要求1-3任一项所述的多功能复合体，其中，在所述UT组件的靶向降解TCR的重组蛋白分子中，所述靶向TCR的结合蛋白分子结构域或其功能变体来自TCR抗体或其功能片段或其组合；
    优选地，所述抗体选自TCRα抗体、TCRβ抗体、TCRαβ抗体、TCRγ抗体、TCRδ抗体、TCRγδ抗体、TCR Vδ2抗体、TCR Cβ1抗体；所述抗体的功能片段选自Fd、Fv、Fab、Fab’、F(ab’)2、Fv(scFv)、单链抗体(scFv)或纳米抗体(nanobody)、双链抗体、三链抗体和四链抗体；优选地，所述TCR抗体是TCR单链抗体；优选地，所述TCR单链抗体的氨基酸序列包含与SEQ ID NO.116所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述TCR单链抗体的全长序列的氨基酸序列如SEQ ID NO.116所示；
    优选地，所述UT组件中的ERAD降解结构域来自HCMV糖蛋白US2、US3、US11或US10、腺病毒E3-19K或HHV-7 US21；
    优选地，所述HCMV糖蛋白US2的全长序列包含与SEQ ID NO.46所示氨基酸序列具有80％或以上同一性的 氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US2的全长序列的氨基酸序列如SEQ ID NO.46所示；
    优选地，所述HCMV糖蛋白US2的HLA结合结构域包含与SEQ ID NO.47所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US2的HLA结合结构域的氨基酸序列如SEQ ID NO.47所示；
    优选地，所述HCMV糖蛋白US2的ERAD降解结构域包含与SEQ ID NO.48所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US2的ERAD降解结构域的氨基酸序列如SEQ ID NO.48所示；
    优选地，所述HCMV糖蛋白US3的全长序列包含与SEQ ID NO.49所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US3的全长序列的氨基酸序列如SEQ ID NO.49所示；
    优选地，所述HCMV糖蛋白US3的HLA结合结构域包含与SEQ ID NO.50所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US3的HLA结合结构域的氨基酸序列如SEQ ID NO.50所示；
    优选地，所述HCMV糖蛋白US3的ERAD降解结构域包含与SEQ ID NO.51所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US3的ERAD降解结构域的氨基酸序列如SEQ ID NO.51所示；
    优选地，所述HCMV糖蛋白US11的全长序列包含与SEQ ID NO.52所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US11的全长序列的氨基酸序列如SEQ ID NO.52所示；
    优选地，所述HCMV糖蛋白US11的MHC结合结构域包含与SEQ ID NO.53所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US11的MHC结合结构域的氨基酸序列如SEQ ID NO.53所示；
    优选地，所述HCMV糖蛋白US11的ERAD降解结构域包含与SEQ ID NO.54所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US11的ERAD降解结构域的氨基酸序列如SEQ ID NO.54所示；
    优选地，所述HCMV糖蛋白US10的全长序列包含与SEQ ID NO.55所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US10的全长序列的氨基酸序列如SEQ ID NO.55所示；
    优选地，所述HCMV糖蛋白US10的HLA结合结构域包含与SEQ ID NO.56所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US10的HLA结合结构域的氨基酸序列如SEQ ID NO.56所示；
    优选地，所述HCMV糖蛋白US10的ERAD降解结构域包含与SEQ ID NO.57所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US10的ERAD降解结构域的氨基酸序列如SEQ ID NO.57所示；
    优选地，所述腺病毒E3-19K的全长序列包含与SEQ ID NO.58所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述腺病毒E3-19K的全长序列的氨基酸序列如SEQ ID NO.58所示；
    优选地，所述腺病毒E3-19K的MHC结合结构域包含与SEQ ID NO.59所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述腺病毒E3-19K的MHC结合结构域的氨基酸序列如SEQ ID NO.59所示；
    优选地，所述腺病毒E3-19K的ERAD降解结构域包含与SEQ ID NO.60所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述腺病毒E3-19K的ERAD降解结构域的氨基酸序列如SEQ ID NO.60所示；
    优选地，所述HHV-7 US21的全长序列包含与SEQ ID NO.61所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％ 以上同一性的氨基酸序列；所述HHV-7 US21的全长序列的氨基酸序列如SEQ ID NO.61所示；
    优选地，所述HHV-7 US21的MHC结合结构域包含与SEQ ID NO.62所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HHV-7 US21的MHC结合结构域的氨基酸序列如SEQ ID NO.62所示；
    优选地，所述HHV-7 US21的ERAD降解结构域包含与SEQ ID NO.63所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HHV-7 US21的ERAD降解结构域的氨基酸序列如SEQ ID NO.63所示。
  5. 根据权利要求1-4任一项所述的多功能复合体，其中，所述UT组件还包含(ii)靶向MHC I和/或MHC II的结合蛋白分子结构域或其功能变体；
    优选地，所述靶向MHC I和/或MHC II的结合蛋白分子结构域或其功能变体为靶向HLA的结合蛋白分子结构域或其功能变体；
    优选地，所述靶向MHC I和/或MHC II的结合蛋白分子结构域或其功能变体来自抑制MHC分子表达或促进其降解的病毒内质网蛋白；优选地，所述病毒内质网糖蛋白选自HCMV US6、HSV ICP47、CPXV012、HPV E6/E7、EBV BNFL2a或BHV UL49.5；优选地，所述靶向MHC I和/或MHC II的结合蛋白分子结构域或其功能变体包含TAP结合结构域；
    优选地，所述HCMV US6的全长序列包含与SEQ ID NO.64所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV US6的全长序列的氨基酸序列如SEQ ID NO.64所示；
    优选地，所述HHV-7 US6的TAP结合结构域包含与SEQ ID NO.65所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HHV-7 US6的TAP结合结构域的氨基酸序列如SEQ ID NO.65所示；
    优选地，所述HSV ICP47的全长序列包含与SEQ ID NO.66所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HSV ICP47的全长序列的氨基酸序列如SEQ ID NO.66所示；
    优选地，所述HSV ICP47的TAP结合结构域包含与SEQ ID NO.67所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HSV ICP47的TAP结合结构域的氨基酸序列如SEQ ID NO.67所示；
    优选地，所述CPXV012的全长序列包含与SEQ ID NO.68所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述CPXV012的全长序列的氨基酸序列如SEQ ID NO.68所示；
    优选地，所述CPXV012的TAP结合结构域包含与SEQ ID NO.69所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述CPXV012的TAP结合结构域的氨基酸序列如SEQ ID NO.69所示；
    优选地，所述EBV BNFL2a的全长序列包含与SEQ ID NO.70所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述EBV BNFL2a的全长序列的氨基酸序列如SEQ ID NO.70所示；
    优选地，所述EBV BNFL2a的TAP结合结构域包含与SEQ ID NO.71所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述EBV BNFL2a的TAP结合结构域的氨基酸序列如SEQ ID NO.71所示；
    优选地，所述BHV UL49.5的全长序列包含与SEQ ID NO.72所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述BHV UL49.5的全长序列的氨基酸序列如SEQ ID NO.72所示；
    优选地，所述BHV UL49.5的TAP结合结构域包含与SEQ ID NO.73所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述BHV UL49.5的TAP结合结构域的氨基酸序列如SEQ ID NO.73所示；
    优选地，所述靶向MHC I和/或MHC II的结合蛋白分子结构域或其功能变体来自降解MHC和/或MHC II分子的病毒糖蛋白；优选地，病毒糖蛋白选自HCMV糖蛋白US2、US3、US11或US10、腺病毒E3-19K或HHV-7 US21；
    优选地，所述US2的全长序列包含与SEQ ID NO.74所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述US2的全长序列的氨基酸序列如SEQ ID NO.74所示；
    优选地，所述US2的HLA结合结构域包含与SEQ ID NO.75所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述US2的HLA结合结构域的氨基酸序列如SEQ ID NO.75所示；
    优选地，所述US2的ERAD降解结构域包含与SEQ ID NO.76所示氨基酸序列具有80％或以上同一性的氨基 酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述US2的ERAD降解结构域的氨基酸序列如SEQ ID NO.76所示；
    优选地，所述US3的全长序列包含与SEQ ID NO.77所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述US3的全长序列的氨基酸序列如SEQ ID NO.77所示；
    优选地，所述US3的HLA结合结构域包含与SEQ ID NO.78所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述US3的HLA结合结构域的氨基酸序列如SEQ ID NO.78所示；
    优选地，所述US3的ERAD降解结构域包含与SEQ ID NO.79所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述US3的ERAD降解结构域的氨基酸序列如SEQ ID NO.79所示；
    优选地，所述US11的全长序列包含与SEQ ID NO.80所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述US11的全长序列的氨基酸序列如SEQ ID NO.80所示；
    优选地，所述US11的HLA结合结构域包含与SEQ ID NO.81所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述US11的HLA结合结构域的氨基酸序列如SEQ ID NO.81所示；
    优选地，所述US11的ERAD降解结构域包含与SEQ ID NO.82所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述US11的ERAD降解结构域的氨基酸序列如SEQ ID NO.82所示；
    优选地，所述靶向MHC I和/或MHC II的结合蛋白分子结构域或其功能变体还包含定向抑制或降解MICA、MICB、ULBP1、ULBP2、ULBP3、ULBP4、ULBP5或ULBP6的NK靶点蛋白的病毒蛋白；优选地，所述病毒蛋白选自HCMV UL16、UL141、UL142或腺病毒E3-19K；
    优选地，所述HCMV UL16的全长序列包含与SEQ ID NO.83所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV UL16的全长序列的氨基酸序列如SEQ ID NO.83所示；
    优选地，所述HCMV UL16的NK靶点蛋白结合结构域包含与SEQ ID NO.84所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV UL16的NK靶点蛋白结合结构域的氨基酸序列如SEQ ID NO.84所示；
    优选地，所述HCMV UL16的ERAD降解结构域包含与SEQ ID NO.85所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV UL16的ERAD降解结构域的氨基酸序列如SEQ ID NO.85所示；
    优选地，所述HCMV UL141的全长序列包含与SEQ ID NO.86所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV UL141的全长序列的氨基酸序列如SEQ ID NO.86所示；
    优选地，所述HCMV UL141的NK靶点蛋白结合结构域包含与SEQ ID NO.87所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV UL141的NK靶点蛋白结合结构域的氨基酸序列如SEQ ID NO.87所示；
    优选地，所述HCMV UL141的ERAD降解结构域包含与SEQ ID NO.88所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV UL141的ERAD降解结构域的氨基酸序列如SEQ ID NO.88所示；
    优选地，所述HCMV UL142的全长序列包含与SEQ ID NO.89所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV UL142的全长序列的氨基酸序列如SEQ ID NO.89所示；
    优选地，所述HCMV UL142的MICA、ULBP3结合结构域包含与SEQ ID NO.90所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV UL142的MICA、ULBP3结合结构域的氨基酸序列如SEQ ID NO.90所示；
    优选地，所述HCMV UL142的高尔基驻留结构域包含与SEQ ID NO.91所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV UL142的高尔基驻留结构域的氨基酸序列如SEQ ID NO.91所示；
    优选地，所述靶向MHC I和/或MHC II的结合蛋白分子结构域或其功能变体还包含把MHC I分子从高尔基体转运到溶酶体进行降解的病毒蛋白；优选地，所述病毒蛋白选自HIV Nef、HIV Vpu、HHV-7 U21、HHV-8 KK3、HHV-8 KK5、MHV-68 MK3和HTLV-1 p12；
    优选地，所述HIV Nef包含与SEQ ID NO.92所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HIV Nef的氨基酸序列如SEQ ID NO.92所示；
    优选地，所述HIV Vpu包含与SEQ ID NO.93所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HIV Vpu的氨基酸序列如SEQ ID NO.93所示；
    优选地，所述HHV-8 KK3包含与SEQ ID NO.94所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HHV-8 KK3的氨基酸序列如SEQ ID NO.94所示；
    优选地，所述HHV-8 KK5包含与SEQ ID NO.95所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HHV-8 KK5的氨基酸序列如SEQ ID NO.95所示；
    优选地，所述MHV-68 MK3包含与SEQ ID NO.96所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述MHV-68 MK3的氨基酸序列如SEQ ID NO.96所示；
    优选地，所述HTLV-1 p12包含与SEQ ID NO.97所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HTLV-1 p12的氨基酸序列如SEQ ID NO.97所示；
    优选地，所述靶向MHC I和/或MHC II的结合蛋白分子结构域或其功能变体还包含介导MHC-多肽分子从高尔基体返回内质网并促进其降解的病毒蛋白；优选地，所述病毒蛋白包含MHC结合结构和KDEL receptor结合结构域；优选地，所述病毒蛋白是Cowpox Virus蛋白CPXV203
    所述UT组件还包含病毒功能蛋白的MHC结合结构和KDEL receptor结合结构域，介导MHC-多肽分子从高尔基体返回内质网，并促进其降解，如牛痘病毒蛋白CPXV203；
    优选地，所述牛痘病毒蛋白CPXV203的全长序列包含与SEQ ID NO.98所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述牛痘病毒蛋白CPXV203的全长序列的氨基酸序列如SEQ ID NO.98所示；
    优选地，所述牛痘病毒蛋白CPXV203的MHC结合结构域包含与SEQ ID NO.99所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述牛痘病毒蛋白CPXV203的MHC结合结构域的氨基酸序列如SEQ ID NO.99所示；
    优选地，所述牛痘病毒蛋白CPXV203的KDEL受体结合结构域包含与SEQ ID NO.100所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述牛痘病毒蛋白CPXV203的KDEL受体结合结构域的氨基酸序列如SEQ ID NO.100所示；
    优选地，所述牛痘病毒蛋白CPXV203的全长序列包含与SEQ ID NO.101所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述牛痘病毒蛋白CPXV203的全长序列的氨基酸序列如SEQ ID NO.101所示；
    优选地，所述牛痘病毒蛋白CPXV203的MHC结合结构域包含与SEQ ID NO.102所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述牛痘病毒蛋白CPXV203的MHC结合结构域的氨基酸序列如SEQ ID NO.102所示；
    优选地，所述牛痘病毒蛋白CPXV203的KDEL受体结合结构域包含与SEQ ID NO.103所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述牛痘病毒蛋白CPXV203的KDEL受体结合结构域的氨基酸序列如SEQ ID NO.103所示。
  6. 根据权利要求1-5任一项所述的多功能复合体，其中，所述多功能复合体还包含组件(4)复合型转接器(Chimeric adaptor)组件和/或靶向杀伤肿瘤细胞受体组件；
    所述(4)复合型转接器包含：(i)靶向肿瘤的胞外识别结构域；(ii)跨膜结构域；和(iii)胞内信号传导结构域；任选地，所述靶向肿瘤的胞外识别结构域、跨膜结构域和/或胞内信号结构域之间包含铰链或接头；
    优选地，所述复合型转接器组件的靶向肿瘤的胞外识别结构域选自肿瘤抗原特异结合结构域、肿瘤微环境靶抗原结合结构域和/或靶向肿瘤微环境的趋化受体；
    优选地，所述靶向肿瘤的胞外识别结构域选自能靶向识别肿瘤相关抗原的抗体或其功能片段、TCR或其组合；所述抗体的功能片段选自Fd、Fv、Fab、Fab’、F(ab’)2、Fv(scFv)、单链抗体(scFv)或纳米抗体(nanobody)、双链抗体、三链抗体和四链抗体；
    优选地，所述复合型转接器组件的跨膜结构域选自NK细胞激活受体跨膜结构域、DAP10跨膜结构域、DAP12跨膜结构域、CD8跨膜结构域、CD28跨膜结构域、CD4跨膜结构域、4-1BB跨膜结构域、OX40跨膜结构域、ICOS跨膜结构域、CTLA-4跨膜结构域、PD-1跨膜结构域、LAG-3跨膜结构域、2B4跨膜结构域和BTLA跨膜结构域以及其组合；优选地，所述NK细胞激活受体选自NKG2D、NKG2C、NKG2E、NKG2F、NKG2H、CD94、KIR2DL4、KIR2DS1、KIR2DS2、KIR2DS4、KIR3DS1，天然细胞毒性受体、TRAIL、DNAM-1、CD16a、2B4、NTB-A、CRACC以及NKp80；优选地，所述天然细胞毒性受体选自NKp46、NKp44和NKp30；
    优选地，所述复合型转接器组件的胞内信号传导结构域包括NK细胞激活受体的胞内信号结构域和/或共刺激信号传导结构域；
    优选地，所述NK细胞激活受体选自NKG2D、NKG2C、NKG2E、NKG2F、NKG2H、CD94、KIR2DL4、KIR2DS1、KIR2DS2、KIR2DS4、KIR3DS1，天然细胞毒性受体、TRAIL、DNAM-1、CD16a、2B4、NTB-A、CRACC以及NKp80；
    优选地，所述胞内信号结构域还包括共刺激信号传导结构域；优选地，所述共刺激信号传导结构域选自T细胞共刺激信号传导结构域；包括，但不限于衍生自MHC I类分子、TNF受体蛋白、免疫球蛋白样蛋白质、细胞因子受体、整联蛋白、淋巴细胞活化信号分子(SLAM蛋白)、活化NK细胞受体、BTLA、Toll配体受体、OX40、CD2、CD7、CD16、CD27、CD28、CD30、CD40、CD38、CD35、CD79A、CD79B、CDS、ICAM-1、LFA-1，(CD11a/CD18)、4-1BB(CD137)、B7-H3、CDS、ICAM-1、ICOS(CD278)、GITR、BAFFR、LIGHT、HVEM(LIGHTR)、KIRDS2、SLAMF7、NKp80(KLRF1)、NKp44、NKp30、NKp46、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、NKG2D、NKG2C、NCR、DAP10、DAP12、TNFR2、TRANCE/RANKL、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100 SEMA4D)、CD69、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、CD19a和与CD83特异性结合的配体，CARD11、FcRa、FcRp、FcRy、Fyn、HVEM、ICOS、Lck、LAG3、LAT、LRP、NOTCH1、Wnt、OX40、ROR2、Ryk、SLAMF1、Slp76、pTa、TCRa、TCRp、TRIM、ZAP70、PTCH2等胞内信号结构域；更优选地，所述共刺激信号传导结构域选自NKG2D胞内信号结构域，DAP10胞内信号结构域，DAP12胞内信号结构域，NCR胞内信号结构域，CD28胞内信号结构域、4-1BB胞内信号结构域、OX40胞内信号结构域、ICOS胞内信号结构域；
    所述靶向杀伤肿瘤细胞受体组件包含(i)靶向肿瘤抗原的胞外识别结构域；(ii)跨膜结构域；和(iii)胞内共刺激信号传导结构域；(iv)T细胞活化信号传导结构域(ITAM)；任选地，所述靶向肿瘤抗原的胞外识别结构域、跨膜结构域、胞内共刺激信号传导结构域和/或T细胞活化信号传导结构域(ITAM)之间包含铰链或接头；
    所述靶向杀伤肿瘤细胞受体组件的跨膜结构域选自CD8跨膜结构域、T细胞受体的α和/或β链跨膜结构域、CD28跨膜结构域、CD3ε跨膜结构域、CD45跨膜结构域、CD4跨膜结构域、CD5跨膜结构域、CD8跨膜结构域、CD9跨膜结构域、CD16跨膜结构域、CD22跨膜结构域、CD33跨膜结构域、CD37跨膜结构域、CD64跨膜结构域、CD80跨膜结构域、CD86跨膜结构域、CD134跨膜结构域、CD137跨膜结构域、CD154跨膜结构域、GITR跨膜结构域以及其组合
    所述T细胞活化信号传导结构域衍生自CD3ζ、共同FcRγ(FCER1G)、FcγRIIa、FcRβ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、CD278(“ICOS”)、FcεRI CD66d、DAP10和DAP12等胞内信号结构域；
    优选地，所述接头是柔性接头；优选地，所述柔性接头包含所示的氨基酸序列(Gly(x)Ser(y))n，其中n是1到10的整数，并且x和y独立地是0到10的整数，前提是x和y不都是0；更优选地，所述接头是SEQ ID NO.104所示的氨基酸序列(Gly4Ser)2或SEQ ID NO.105所示的氨基酸序列(Gly3Ser)2；
    优选地，所述接头是铰链；优选地，所述铰链是IgG1铰链或IgG4铰链；
    优选地，所述IgG1铰链包含与SEQ ID NO.106所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述IgG1铰链的氨基酸序列如SEQ ID NO.106所示；
    优选地，所述IgG4铰链包含与SEQ ID NO.107所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述IgG4铰链的氨基酸序列如SEQ ID NO.107所示；
    优选地，所述NK激活受体组件、CNK信号转接组件和/或UT组件之间包含可切割肽；所述可切割肽为T2A肽、GSG-T2A肽、E2A肽、GSG-E2A肽、F2A肽、GSG-F2A肽、P2A肽或GSG-P2A肽；
    优选地，所述T2A包含与SEQ ID NO.108所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述T2A的氨基酸序列如SEQ ID NO.108所示；
    优选地，GSG-T2A肽的氨基酸序列如SEQ ID NO.109所示。
    优选地，所述P2A包含与SEQ ID NO.110所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述P2A的氨基酸序列如SEQ ID NO.110所示；
    优选地，GSG-P2A肽的氨基酸序列如SEQ ID NO.111所示；
    优选地，所述E2A包含与SEQ ID NO.112所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述E2A的氨基酸序列如SEQ ID NO.112所示；
    优选地，GSG-E2A肽的氨基酸序列如SEQ ID NO.113所示；
    优选地，所述F2A包含与SEQ ID NO.114所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述F2A的氨基酸序列如SEQ ID NO.114所示；
    优选地，GSG-F2A肽的氨基酸序列如SEQ ID NO.115所示。
    优选地，所述多功能复合体包含与SEQ ID NO.117所示的氨基酸序列有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述多功能复合体的氨基酸序列如SEQ ID NO.117所示；
    优选地，所述多功能复合体包含与SEQ ID NO.121所示的氨基酸序列有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述多功能复合体的氨基酸序列如SEQ ID NO.121所示；
    优选地，所述多功能复合体包含与SEQ ID NO.123所示的氨基酸序列有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述多功能复合体的氨基酸序列如SEQ ID NO.123所示；
    优选地，所述多功能复合体包含与SEQ ID NO.125所示的氨基酸序列有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述多功能复合体的氨基酸序列如SEQ ID NO.125所示；
    优选地，所述多功能复合体包含与SEQ ID NO.12所示的氨基酸序列有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述多功能复合体的氨基酸序列如SEQ ID NO.126所示；
    优选地，所述多功能复合体包含与SEQ ID NO.127所示的氨基酸序列有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述多功能复合体的氨基酸序列如SEQ ID NO.127所示；
    优选地，所述多功能复合体包含与SEQ ID NO.128所示的氨基酸序列有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述多功能复合体的氨基酸序列如SEQ ID NO.128所示；
    优选地，所述多功能复合体包含与SEQ ID NO.129所示的氨基酸序列有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述多功能复合体的氨基酸序列如SEQ ID NO.129所示；
    优选地，所述多功能复合体包含与SEQ ID NO.130所示的氨基酸序列有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述多功能复合体的氨基酸序列如SEQ ID NO.130所示；
    优选地，所述多功能复合体包含与SEQ ID NO.131所示的氨基酸序列有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述多功能复合体的氨基酸序列如SEQ ID NO.131所示。
  7. 编码权利要求1-6任一项所述多功能复合体的核酸分子；
    优选地，所述核酸分子是DNA或RNA；
    优选地，所述RNA是mRNA；
    优选地，所述核酸分子包含与SEQ ID NO.118所示的核苷酸序列有80％或以上同一性的核苷酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的核苷酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述核酸分子的核苷酸序列SEQ ID NO.118所示；
    优选地，所述核酸分子包含与SEQ ID NO.122所示的核苷酸序列有80％或以上同一性的核苷酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的核苷酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述核酸分子的核苷酸序列SEQ ID NO.122所示；
    优选地，所述核酸分子包含与SEQ ID NO.124所示的核苷酸序列有80％或以上同一性的核苷酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的核苷酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述核酸分子的核苷酸序列SEQ ID NO.124所示。
  8. 含有权利要求7所述核酸的表达载体；
    优选地，所述载体选自质粒、粘粒、病毒载体、RNA载体或线性或圆形DNA或RNA分子
    优选地，所述病毒载体选自逆转录病毒、腺病毒、细小病毒(例如，腺伴随病毒)、冠状病毒、负链RNA病毒诸如正粘病毒(例如，流感病毒)、弹状病毒(例如，狂犬病和水疱性口炎病毒)、副粘病毒(例如，麻疼和仙台)、正链RNA病毒诸如小RNA病毒和甲病毒和双链DNA病毒，所述双链DNA病毒包括腺病毒、疱疹病毒(例如，单纯疱疹病毒1和2型、愛泼斯坦-巴尔病毒、巨细胞病毒)和痘病毒(例如，牛痘病毒、鸡痘病毒和金丝雀痘病毒)、诺沃克病毒、披膜病毒、黄病毒、呼肠孤病毒、乳多泡病毒、嗜肝DNA病毒、杆状病毒和肝炎病毒；
    优选地，所述逆转录病毒选自禽造白细胞组织增生-肉瘤、哺乳动物C-型、B-型病毒、D-型病毒、HTLV-BLV集合、慢病毒、泡沫病毒；
    优选地，所述慢病毒载体选自HIV-1、HIV-2、SIV、FIV、BIV、EIAV、CAEV或绵羊脱髓鞘性脑白质炎慢病毒；
    优选地，所述NK激活受体组件、CNK信号转接组件和/或UT组件可在同一载体同一启动子，或不同启动子下调控表达，或多种载体中进行表达；
    优选地，所述载体为慢病毒载体，所述编码NK激活受体组件、CNK信号转接组件和/或UT组件的基因之间包含可切割肽编码基因；优选地，所述可切割肽为2A接头；所述2A接头选自T2A、P2A、E2A和F2A；
    优选地，所述载体还包含启动子；优选地，所述启动子为EF1α启动子或CMV启动子。
  9. 免疫细胞，其包含权利要求8所述的核酸或权利要求8所述的表达载体。
    优选地，免疫细胞选自T细胞，NKT细胞，NK细胞，B细胞，单核细胞，巨噬细胞等；
  10. 制造免疫细胞的方法，其包括用选自下述的方法将权利要求8所述的核酸或权利要求8所述的表达载体引入细胞：电穿孔、声穿孔、基因枪(例如用Au-颗粒的基因枪)、脂质转染、聚合物转染、纳米颗粒或多聚复合体。
  11. 一种药物组合物，其包含权利要求1-6任一项的多功能复合体、权利要求7所述的核酸、权利要求8所述的表达载体、权利要求9所述的免疫细胞和/或权利要求10所述方法制造的免疫细胞，以及药学上可接受的载体。
  12. 权利要求1-6任一项的多功能复合体、权利要求7所述的核酸、权利要求8所述的表达载体、权利要求9所述的免疫细胞、权利要求10所述方法制造的免疫细胞和/或权利要求11所述的药物组合物在制备治疗疾病的药物中的用途。
  13. 一种治疗疾病的方法，其包括向受试者施用权利要求1-6任一项的多功能复合体、权利要求7所述的核酸、权利要求8所述的表达载体、权利要求9所述的免疫细胞和/或权利要求10所述的药物组合物；
    优选地，所述疾病包括各类实体肿瘤和血液肿瘤、病毒感染性疾病、自身免疫性疾病；
    优选地，所述实体肿瘤选自神经系统肿瘤、头颈部肿瘤、胸部肿瘤、消化系统肿瘤、泌尿生殖系统肿瘤、软组织和皮肤肿瘤、骨肿瘤等；
    优选地，神经系统肿瘤包括弥漫性胶质瘤、弥漫性星形细胞瘤和间变性星形细胞瘤、胶质母细胞瘤、少突胶质细胞瘤、少突星形细胞瘤、儿童弥漫性胶质瘤、其他星形细胞瘤、室管膜瘤、神经元和混合性神经元-神经胶质肿瘤、髓母细胞瘤、其他胚胎性肿瘤、神经鞘瘤、脑膜瘤、孤立性纤维性肿瘤和血管周细胞瘤等；
    优选地，头颈部肿瘤包括鼻腔及鼻窦恶性肿瘤、鼻咽癌、口腔癌、喉癌、涎腺肿瘤、颅内肿瘤、甲状腺癌、舌癌等；
    优选地，胸部肿瘤包括肺癌，食管癌，贲门癌，乳腺癌，纵膈肿瘤等；
    优选地，消化系统肿瘤包括胃癌，大肠癌及乙状结肠和直肠癌，肝癌，胰腺癌与壶腹周围癌，胆道癌，小肠恶性肿瘤等；
    优选地，泌尿生殖系统肿瘤包括肾癌、前列腺癌、膀胱癌、睾丸恶性肿瘤、阴茎癌、子宫颈癌、子宫内膜癌、卵巢癌等；
    优选地，软组织和皮肤肿瘤包括恶性纤维组织细胞瘤、横纹肌肉瘤、滑膜肉瘤、皮肤恶性黑色素瘤等；
    优选地，骨肿瘤包括骨肉瘤、尤文氏肉瘤等；
    优选地，所述结肠癌是结肠腺瘤；
    优选地，所述乳腺癌是三阴乳腺癌细胞；
    优选地，所述肝癌是肝细胞癌；
    优选地，所述疾病是血液肿瘤选自白血病、淋巴瘤(HL)、多发性骨髓瘤(MM)、骨髓增生异常综合症(MDS)等；
    优选地，所述白血病是B细胞急性淋巴性白血病、T细胞急性淋巴性白血病、急性髓系白血病等；
    优选地，病毒感染性疾病包括：呼吸道病毒性疾病、胃肠道病毒性疾病、肝脏病毒性疾病、皮肤和黏膜病毒性疾病、眼病毒性疾病、中枢神经系统病毒性疾病、淋巴细胞性病毒性疾病、虫传病毒性疾病、慢病毒感染疾病等；
    优选地，呼吸道病毒性疾病包括鼻病毒、腺病毒、呼吸道合胞病毒、副流感病毒和冠状病毒等的感染；流行性感冒；流行性腮腺炎等；
    优选地，胃肠道病毒性疾病包括脊髓灰质炎；库克萨基病毒感染；ECHO病毒感染；病毒性胃肠炎：包括轮状病毒性胃肠炎、诺瓦克病毒性胃肠炎、腺病毒性胃肠炎、星状病毒性胃肠炎、冠状病毒性胃肠炎和杯状病毒性胃肠炎等；
    优选地，肝脏病毒性疾病包括甲型病毒性肝炎、乙型病毒性肝炎、丙型病毒性肝炎、丁型病毒性肝炎、戊型 病毒性肝炎、EB病毒性肝炎和巨细胞病毒性肝炎等；
    优选地，皮肤和黏膜病毒性疾病包括麻疹、风疹、幼儿急疹、水痘及带状疱疹、天花、单纯疱疹病毒感染、狂犬病和口蹄疫等；
    优选地，眼病毒性疾病包括流行性角膜结膜炎、滤泡性结膜炎和疱疹性角膜结膜炎等；
    优选地，中枢神经系统病毒性疾病包括流行性乙型脑炎、西方马脑炎、东方马脑炎、圣路易脑炎、委内瑞拉马脑炎、墨累山谷脑炎、加利福尼亚脑炎、森林脑炎和淋巴细胞脉络丛脑膜炎等；
    优选地，淋巴细胞性病毒性疾病包括传染性单核细胞增多症、巨细胞病毒感染和获得性免疫缺陷综合征等；
    优选地，虫传病毒性疾病包括病毒性出血热：包括流行性出血热、黄热病、克里米亚－刚果出血热、裂谷热、阿根廷出血热、玻利维亚出血热、拉萨热、鄂木斯克出血热、马尔堡病和埃波拉出血热等；登革热和登革出血热；西尼罗热；科罗拉多蜱传热；白蛉热等；
    优选地，慢病毒感染疾病包括亚急性硬化性全脑炎、库鲁病、进行性多灶性白质脑病和亚急性海绵样脑病(皮质纹状体脊髓变性)等；
    优选地，自身免疫性疾病包括器官特异性自身免疫病和系统性自身免疫病；
    优选地，器官特异性自身免疫病包括慢性淋巴细胞性甲状腺炎、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌无力、溃疡性结肠炎、恶性贫血伴慢性萎缩性胃炎、肺出血肾炎综合征、寻常天疱疮、类天疱疮、原发性胆汁性肝硬化、多发性脑脊髓硬化症、急性特发性多神经炎等；
    优选地，系统性自身免疫病包括系统性红斑狼疮、类风湿关节炎、系统性血管炎、硬皮病、天疱疮、皮肌炎、混合性结缔组织病、自身免疫性溶血性贫血、甲状腺自身免疫病、溃疡性结肠炎等。
  14. 一种刺激受试者中的免疫应答的方法，所述方法包括给予受试者有效量的权利要求1-6任一项的多功能复合体、权利要求7所述的核酸、权利要求8所述的表达载体、权利要求9所述的免疫细胞、权利要求10所述方法制造的免疫细胞和/或权利要求11所述的药物组合物。
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