NKG2D-NKp46 cellular adaptor molecule and its uses

Abstract

Provided are NK cell adaptor molecules that target NKG2D ligands and NKp46, and uses thereof. The cellular adaptor molecule comprises at least a first binding domain comprising the extracellular domain of NKG2D capable of specifically binding to an NKG2D ligand, and a second binding domain comprising an antigen-binding fragment of NKp46. The cellular adaptor molecule can enhance the killing effect of natural killer cells against target cells with high expression of NKG2D ligands, and can be used as a therapeutic agent for diseases associated with expression of NKG2D ligands.

Description

[Technical Field]

[0001] The present invention relates to the field of biomedicine, and more particularly to a cellular adaptor molecule composed of the extracellular domain of NKG2D, a connecting segment, and an antigen-binding fragment of NKp46, which can specifically bind to an NKG2D ligand, and its applications. [Background technology]

[0002] NKG2D is a cytotoxic factor that binds to NK cells and CD8 + NKG2D is expressed on the surface of T cells, activated macrophages, and tumor-infiltrating gamma delta T cells. It usually exists as a self-assembled homodimer and, depending on the adaptor proteins DAP10 and DAP12, directly activates NK cells to exert its killing effect or acts as a costimulatory signal to promote T cell activation. Its ligands include MICA, MICB, and ULBP1-6. Generally, NKG2D is not expressed or is expressed at low levels on normal cells. However, upon stressful conditions such as abnormal transformation, viral infection, or DNA damage, NKG2D ligand expression is significantly upregulated and can be identified by the immune system as a marker of abnormal cells. The NKG2D-NKG2D ligand signaling chain is an important mechanism for the immune system to exert its immune surveillance function. Immunotherapies developed for this purpose have shown improved safety and efficacy, gradually becoming the forefront of current immunotherapy strategies. Currently, more than 10 CAR-T and CAR-NK molecules designed using NKG2D-NKG2DL as their mechanism of action are in clinical trials, with no treatment-related serious adverse events observed. Cell adaptor molecules based on the extracellular domain of NKG2D are also gaining increasing attention in the field of anti-tumor therapy. For example, the T cell bispecific antibody NKG2D-CD3 can target tumor cells and immune inhibitory cells in vivo, thereby inhibiting tumor growth and prolonging the survival of model mice. The NK cell bispecific antibody NKG2D-CD16 can effectively kill tumor cells, such as those of acute myeloid leukemia, lymphoma, and soft tissue sarcoma. NKG2D-Fc significantly improves the tumor immune microenvironment and inhibits tumor development. Many more cellular adaptor molecules are currently being developed in the art. Summary of the Invention [Problem to be solved by the invention]

[0003] An object of the present invention is to provide a cellular adaptor molecule comprising the extracellular domain of NKG2D and an antigen-binding fragment of NKp46, and uses thereof. [Means for solving the problem]

[0004] A first aspect of the present invention provides a cellular adaptor molecule, the cellular adaptor molecule comprising: (a) a first binding domain that specifically binds to an NKG2D ligand; and (b) a second binding domain that specifically binds to NKp46.

[0005] In another preferred embodiment, the first binding domain is derived from the extracellular domain of NKG2D.

[0006] In another preferred embodiment, the amino acid sequence of the first binding domain is as set forth in SEQ ID NO:1 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or identity to the amino acid sequence set forth in SEQ ID NO:1 and is capable of binding to an NKG2D ligand.

[0007] In another preferred embodiment, the second binding domain comprises an antigen-binding fragment specific for anti-NKp46.

[0008] In another preferred embodiment, the antigen-binding fragment of anti-NKp46 comprises a structure selected from the group consisting of Fab, Fab', F(ab')2, Fd, Fv, dAb, Fc, a complementarity-determining region fragment, a single-chain antibody, a single-domain antibody, or a combination thereof.

[0009] In another preferred embodiment, the antigen-binding fragment of anti-NKp46 comprises a structure selected from the group consisting of a humanized antibody, a chimeric antibody, or a combination thereof.

[0010] In another preferred embodiment, the antigen-binding fragment of NKp46 comprises a Fab fragment, a single-chain antibody (scFv), a single-domain antibody, or a combination thereof.

[0011] In another preferred embodiment, the anti-NKp46 Fab fragment and / or single chain antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is 1) HCDR1 as shown in the amino acid sequence SEQ ID NO:3; 2) HCDR2 as shown in the amino acid sequence SEQ ID NO:4; 3) comprising a heavy chain complementarity determining region (HCDR) of HCDR3 as set forth in the amino acid sequence SEQ ID NO:5; In addition, the light chain variable region is 1) LCDR1 as shown in the amino acid sequence SEQ ID NO:6; 2) LCDR2 whose amino acid sequence is YTS; 3) comprising the light chain complementarity determining region (LCDR) of LCDR3 as set forth in the amino acid sequence SEQ ID NO:7; The CDRs are based on the IMGT numbering and definition scheme.

[0012] In another preferred embodiment, the heavy chain variable region amino acid sequence of the anti-NKp46 Fab fragment or scFv is as set forth in SEQ ID NO:8 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or identity to the amino acid sequence set forth in SEQ ID NO:8.

[0013] In another preferred embodiment, the anti-NKp46 Fab fragment or scFv light chain variable region amino acid sequence is as set forth in SEQ ID NO:9 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or identity to the amino acid sequence set forth in SEQ ID NO:9.

[0014] In another preferred embodiment, the NKp46 Fab fragment amino acid sequence is as set forth in SEQ ID NO:2 or is an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or identity to the amino acid sequence set forth in SEQ ID NO:2 (sequence taken from Gauthier, Morel et al. 2019).

[0015] In another preferred embodiment, the anti-NKp46 scFv fragment amino acid sequence is as set forth in SEQ ID NO:10 or is an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or identity to the amino acid sequence set forth in SEQ ID NO:10 (sequence taken from reference Gauthier, Morel et al. 2019).

[0016] In another preferred embodiment, the heavy chain complementarity determining regions of the anti-NKp46 single domain antibody comprise: 1) HCDR1 as shown in the amino acid sequence SEQ ID NO:3; 2) HCDR2 as shown in the amino acid sequence SEQ ID NO:4; 3) Contains an HCDR3 as shown in the amino acid sequence SEQ ID NO:5.

[0017] In another preferred embodiment, the anti-NKp46 single domain antibody amino acid sequence is as set forth in SEQ ID NO:11 or is an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or identity to the amino acid sequence set forth in SEQ ID NO:11.

[0018] In another preferred embodiment, the second binding domain further comprises an antigen-binding fragment derived from an anti-CD16 antibody and / or a polypeptide of an IL15 protein.

[0019] In another preferred embodiment, the antigen-binding fragment of anti-CD16 comprises a structure selected from the group consisting of Fab, Fab', F(ab')2, Fd, Fv, dAb, Fc, a complementarity-determining region fragment, a single-chain antibody, a single-domain antibody, or a combination thereof.

[0020] In another preferred embodiment, the antigen-binding fragment of anti-CD16 comprises a structure selected from the group consisting of a humanized antibody, a chimeric antibody, or a combination thereof.

[0021] In another preferred embodiment, the anti-CD16 single domain antibody variable region comprises: 1) HCDR1 as shown in the amino acid sequence SEQ ID NO:12; 2) HCDR2 as shown in the amino acid sequence SEQ ID NO:13; 3) Contains an HCDR3 as shown in the amino acid sequence SEQ ID NO:14.

[0022] In another preferred embodiment, the amino acid sequence of the anti-CD16 single domain antibody is as set forth in SEQ ID NO:15 or is an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity to the amino acid sequence set forth in SEQ ID NO:15.

[0023] In another preferred embodiment, the amino acid sequence of the anti-CD16 Fc fragment is as set forth in SEQ ID NO:16 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or identity to the amino acid sequence set forth in SEQ ID NO:16.

[0024] In another preferred embodiment, the amino acid sequence of the IL15 protein is as set forth in SEQ ID NO:17 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or identity to the amino acid sequence set forth in SEQ ID NO:17.

[0025] In another preferred example, the cell adaptor molecule has, from the N-terminus to the C-terminus, a structure selected from the following formula (I) or (II): S-D1-L1-D2-T(I), or S-D2-L1-D1-T(II) In the formula: each "-" is independently a connecting peptide or a peptide bond; S is none or a signal peptide sequence, D1 is the first binding domain, L1 is none, a first connecting peptide, or a single-chain Fc (scFc) fragment; D2 is the second binding domain, T is either no or labeled protein.

[0026] In another preferred example, S is a signal peptide derived from mammalian CD8α.

[0027] In another preferred embodiment, the first connecting peptide is a flexible connecting peptide or a rigid connecting peptide.

[0028] In another preferred embodiment, the first connecting peptide is a glycine-serine connecting peptide.

[0029] In another preferred embodiment, the first connecting peptide is represented by the formula (G4S)n, where n is 1, 2, 3, 4, 5 or 6, and preferably n is 3.

[0030] In another preferred embodiment, the single-chain Fc fragment comprises two Fc monomers connected by a linker.

[0031] In another preferred embodiment, the single chain Fc fragment amino acid sequence is as set forth in SEQ ID NO:18.

[0032] In another preferred example, the second binding domain has, from the N-terminus to the C-terminus, a structure selected from the following formula (III) or (IV): D3-L2-D4-I(III), or D4-L2-D3-I(IV), In the formula: each "-" is independently a connecting peptide or a peptide bond; D3 is an antigen-binding fragment of NKp46, L2 is none or a second connecting peptide, D4 is none or a CD16 antigen-binding fragment, I is none or IL15 protein.

[0033] In another preferred embodiment, the second connecting peptide is a glycine-serine connecting peptide.

[0034] In another preferred embodiment, the second connecting peptide is represented by the formula (G4S)n, where n is 1, 2, 3, 4, 5 or 6, and preferably n is 3.

[0035] In another preferred embodiment, the labeled protein T is selected from a His tag and a FLAG tag.

[0036] A second aspect of the present invention provides a recombinant protein, said recombinant protein comprising a cellular adaptor molecule according to the first aspect of the present invention.

[0037] In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.

[0038] In another preferred embodiment, the recombinant protein specifically binds to an NKG2D ligand and an NK cell surface NKp46 antigen.

[0039] In another preferred embodiment, the recombinant protein further specifically binds to CD16.

[0040] A third aspect of the present invention provides a polynucleotide, the polynucleotide comprising: (1) a cellular adaptor molecule according to the first aspect of the present invention; or (2) a recombinant protein according to the second aspect of the present invention.

[0041] A fourth aspect of the present invention provides a vector, said vector comprising a polynucleotide according to the third aspect of the invention.

[0042] In another preferred embodiment, the vector includes, but is not limited to, a bacterial plasmid, a phage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a retrovirus, or other vector.

[0043] In another preferred embodiment, the vector includes, but is not limited to, pCDH, pTOMO, pGEM, pELNS, pMSGV, or a combination thereof.

[0044] A fifth aspect of the present invention provides a modified host cell, said host cell comprising a vector according to the fourth aspect of the invention or having integrated into its genome a polynucleotide according to the third aspect of the invention.

[0045] In another preferred embodiment, the host cells are cells in vivo and cells cultured ex vivo that can be transplanted into the body.

[0046] In another preferred embodiment, the immune cells are selected from NK cells.

[0047] In another preferred embodiment, the immune cells are derived from a human or non-human mammal (eg, a mouse).

[0048] A sixth aspect of the present invention provides an antibody conjugate, the antibody conjugate comprising: (a) an antibody moiety selected from the group consisting of the cellular adaptor molecules according to the first aspect of the invention; and (b) a coupling moiety coupled to said antibody moiety selected from the group consisting of a detectable marker, a drug, or a combination thereof.

[0049] In another preferred embodiment, the detectable marker comprises a radionuclide.

[0050] In another preferred embodiment, the drug comprises a toxin, a cytokine, or an enzyme.

[0051] In another preferred example, the conjugate is selected from a fluorescent or luminescent marker, a radioactive marker, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product, a radionuclide, a biotoxin, a cytokine (e.g., IL-2, etc.), an antibody, an antibody Fc fragment, an antibody scFv fragment, a gold nanoparticle / nanorod, a viral particle, a liposome, a nanomagnetic particle, a prodrug-activating enzyme (e.g., DT-diaphorase (DTD) or biphenylhydrolase-like protein (BPHL)), a chemotherapeutic agent (e.g., cisplatin), or any form of nanoparticle, etc.

[0052] In another preferred embodiment, the antibody moiety and the coupling moiety are coupled via a chemical bond or linker.

[0053] In another preferred embodiment, the immunoconjugate comprises a multivalent (eg bivalent) cellular adaptor molecule according to the first aspect of the invention.

[0054] In another preferred embodiment, said multivalency refers to the inclusion of multiple repeats of the cellular adaptor molecule according to the first aspect of the present invention in the amino acid sequence of said immunoconjugate.

[0055] A seventh aspect of the present invention provides a pharmaceutical composition, said pharmaceutical composition comprising: (a) an active ingredient selected from the group consisting of a cellular adaptor molecule according to the first aspect of the invention, a recombinant protein according to the second aspect of the invention, a host cell according to the fifth aspect of the invention, an antibody conjugate according to the sixth aspect of the invention, or a combination thereof; and (b) one or more pharmaceutically acceptable carriers, diluents, fillers, binders, excipients, or combinations thereof.

[0056] In another preferred embodiment, the pharmaceutical composition is a liquid formulation.

[0057] In another preferred embodiment, the pharmaceutical composition is an injection.

[0058] In another preferred example, the pharmaceutical composition comprises 0.01 to 99.99% of the cellular adaptor molecule according to the first aspect of the present invention, the recombinant protein according to the second aspect of the present invention, the host cell according to the fifth aspect of the present invention, the antibody conjugate according to the sixth aspect of the present invention, or a combination thereof, and 0.01 to 99.99% of the vector, wherein the percentages are mass percentages of the pharmaceutical composition.

[0059] In another preferred embodiment, the pharmaceutical composition is used to prevent and / or treat diseases in which NKG2D ligand expression is upregulated.

[0060] An eighth aspect of the present invention provides the use of a cellular adaptor molecule according to the first aspect of the invention, or a recombinant protein according to the second aspect of the invention, or a host cell according to the fifth aspect of the invention, or an antibody conjugate according to the sixth aspect of the invention, and / or a pharmaceutical composition according to the seventh aspect of the invention, in the preparation of a medicament for treating a disease associated with upregulation of expression of an NKG2D ligand.

[0061] In another preferred embodiment, the drug is used to prevent and / or treat a disease.

[0062] In another preferred example, the upregulation of expression refers to the ratio of the expression level of NKG2D ligand (F1) to the expression level of normal cell tissue (F0) (i.e., F1 / F0) being ≧1.5, preferably ≧2, and more preferably ≧2.5.

[0063] In another preferred example, the NKG2D ligand includes (but is not limited to) MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

[0064] In another preferred embodiment, the disease includes a disease associated with tumors, autoimmune diseases, transplant rejection, inflammation, aging, and accumulation of senescent cells.

[0065] In another preferred embodiment, the tumor includes a hematological tumor and a solid tumor.

[0066] In another preferred embodiment, the hematological tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.

[0067] In another preferred embodiment, the solid tumor is selected from the group consisting of lung cancer, ovarian cancer, colorectal cancer, liver cancer, gallbladder cancer, bile duct cancer, gastric cancer, pancreatic cancer, kidney cancer, prostate cancer, breast cancer, bladder cancer, nasopharyngeal cancer, non-small cell lung cancer, glioblastoma, neuroblastoma, melanoma, or a combination thereof.

[0068] In another preferred example, the disease associated with the accumulation of senescent cells is selected from the group consisting of organ degenerative diseases caused by cellular senescence, such as muscular dystrophy, fatty liver, heart failure, atherosclerosis, diabetes, myocardial hypertrophy, osteoporosis, tissue / organ fibrosis, Alzheimer's disease, Parkinson's syndrome, arthritis, and chronic obstructive pulmonary disease, or a combination thereof.

[0069] In another preferred embodiment, the drug is used to inhibit cells in which NKG2D ligand expression is upregulated, preferably including human liver cancer cell line MHCC97H, human liver cancer cell line SMMC7721, human pancreatic cancer cell line ASPC1, or a combination thereof.

[0070] In another preferred embodiment, the senescent cells are selected from the group consisting of lung cells, adipocytes, kidney cells, muscle cells, or combinations thereof.

[0071] In another preferred embodiment, the senescent cells are human embryonic lung cell line HEL1.

[0072] In another preferred embodiment, the senescent cells are induced to senesce naturally or artificially.

[0073] In another preferred embodiment, the method for inducing artificial aging includes DNA damage-induced aging, P16 overexpression-induced aging, telomere shortening-induced aging, or a combination thereof.

[0074] In another preferred embodiment, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, colitis, celiac disease, multiple sclerosis, alopecia areata, type 1 diabetes, chronic obstructive pulmonary disease, atherosclerosis, or type 2 diabetes-associated metabolic syndrome.

[0075] In another preferred embodiment, the method includes treating the subject with an additional disease therapy.

[0076] In another preferred embodiment, the additional disease treatment is selected from the group consisting of surgery, radiation therapy, chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, adjuvant therapy, immunotherapy, or a combination thereof.

[0077] A ninth aspect of the present invention provides a method of treating a disease associated with upregulated expression of an NKG2D ligand, comprising administering to a subject in need thereof an effective amount of a cellular adaptor molecule according to the first aspect of the invention, or a recombinant protein according to the second aspect of the invention, or a host cell according to the fifth aspect of the invention, or an antibody conjugate according to the sixth aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention, or a combination thereof. [Effects of the Invention]

[0078] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (e.g., in the Examples) can be combined with each other to form new or preferred technical solutions, which will not be repeated here due to space limitations. [Brief explanation of the drawings]

[0079] The following drawings are used to illustrate specific embodiments of the present invention and are not intended to limit the scope of the present invention, which is defined by the claims.

[0080] [Figure 1]NKG2D-NKp46 protein preparation. (A) Schematic diagram of the NKG2D-NKp46 and control vector structures. (B) NKG2D-NKp46 and control vectors were transfected into 293T cells, and the culture supernatant was collected and purified. The expression of NKG2D-NKp46 and control proteins in the purified proteins was detected using His antibody. (C) After incubation of NKG2D-NKp46 and NK cells, the binding rate was detected by flow cytometry. [Figure 2] This demonstrates that NKG2D-NKp46 protein promotes NK cells to kill tumor cells. (A) Flow cytometry was used to detect the expression of NKG2D ligands MICA, MICB, ULBP1, ULBP2, and ULBP3 in the liver cancer cell line MHCC97H. (B) After co-incubation of NKG2D-NKp46 and MHCC97H cells, the binding rate was detected by flow cytometry. (C) Eight hours after addition of NKG2D-NKp46 protein to the co-culture of NK cells and MHCC97H cells, the mortality rate of MHCC97H cells was detected. (D) Eight hours after addition of NKG2D-NKp46 protein to the co-culture of NK cells and MHCC97H cells, the supernatant of the culture was aspirated and the expression of IFN-γ was detected by enzyme-linked immunosorbent assay (ELISA). [Figure 3] NKG2D ligand expression is upregulated in senescent cells. (A) HEL1-P16 cells were treated with tetracycline (DOX) to induce P16 overexpression, and then stained with β-gal. (B) HEL1-P16 cells were treated with tetracycline to induce P16 overexpression, and then NKG2D ligand expression was detected by quantitative PCR. (C) HEL1-P16 cells were treated with tetracycline (DOX) to induce P16 overexpression, and then NKG2D ligand expression was detected by flow cytometry. [Figure 4]NKG2D-NKp46 protein promotes NK cell killing by senescent cells. (A) After co-incubation of NKG2D-NKp46 and senescent cells, the binding rate was detected by flow cytometry. (B) Eight hours after adding NKG2D-NKp46 protein to a co-culture of NK cells and DOX-induced senescent HEL1-P16 cells, the mortality rate of senescent cells was detected using a microscope. (C) Different concentrations of NKG2D-NKp46 protein were added to a co-culture of NK cells and DOX-induced senescent HEL1-P16 cells to detect the lysis rate of senescent cells. (D) Eight hours after adding NKG2D-NKp46 protein (5 μg / ml) to a co-culture of NK cells and senescent HEL1-P16 cells, the supernatant of the culture was aspirated and IFN-γ expression was detected by enzyme-linked immunosorbent assay (ELISA). [Figure 5] Figure 1 shows the effect of proteins containing NKp46 scFv in promoting NK cell killing. (A) Schematic diagram of the structures of proteins containing NKp46 scFv and a control vector. Here, the scFc structure is CH2-CH3-CH2-CH3. CH2 and CH3 are derived from human IgG1 antibody. NKp46 scFv is a single-chain antibody of NKp46. (B) Measurement of SMMC7721 cell lysis rate 24 hours after adding proteins containing NKp46 scFv to a co-culture system of resting NK cells and SMMC7721 cells, respectively. (C) Measurement of cytolysis rate 24 hours after adding proteins containing NKp46 scFv to a co-culture system of resting NK cells and DOX-induced senescent HEL1-P16 cells, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0081] As a result of extensive and intensive research and extensive screening, the present inventors have developed the first bispecific cell adaptor molecule constructed based on the extracellular domain of NKG2D and its application. Experimental results show that the bispecific antibody targeting the NKG2D ligand and the NKp46 antigen described in this invention exhibits a significant killing effect on target cells. Based on this, the present invention has been completed.

[0082] The bispecific cell adaptor molecule provided by the present invention is composed of three parts: an extracellular domain of NKG2D capable of specifically binding to an NKG2D ligand, a connecting segment, and an antigen-binding fragment of NKp46. The bispecific cell adaptor can bridge natural killer cells to target cells with high expression of an NKG2D ligand to kill the target cells, and can therefore be used to treat diseases associated with expression of an NKG2D ligand.

[0083] term In order to make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless expressly defined herein, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which this invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. It should also be understood that the terms used herein are intended only to describe specific embodiments and are not intended to be limiting, and the scope of the present invention is limited only by the appended claims.

[0084] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. As used herein, when used in connection with a specifically stated numerical value, the term "about" means that the value can vary by no more than 1% from the stated value. For example, as used herein, the term "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0085] The three-letter and one-letter codes for amino acids used in the present invention are as described in J. Biol. Chem, 243, p. 3558 (1968).

[0086] As used herein, the term "treatment" refers to the administration of an internal or external therapeutic agent, including antibodies and compositions thereof against a respiratory syncytial virus fusion protein (preferably a pre-fusion F protein) of the present invention, to a patient suffering from one or more disease symptoms for which the therapeutic agent is known to have a therapeutic effect. Typically, the patient is administered an amount of therapeutic agent that effectively alleviates one or more disease symptoms (a therapeutically effective amount).

[0087] As used herein, the term "optional" or "optionally" means that the following event or circumstance may occur, but need not occur. For example, "optionally comprising 1 to 3 antibody heavy chain variable regions" means that antibody heavy chain variable regions of a particular sequence may be present, but do not necessarily have to be present, and may be 1, 2, or 3.

[0088] "Sequence identity" as used herein refers to the degree of identity between two nucleic acid or two amino acid sequences when compared optimally under the conditions of appropriate mutations such as exchanges, insertions, or deletions. The sequence identity between a sequence described herein and a sequence having the same identity may be at least 85%, 90%, or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0089] NKG2D and NKG2D ligands NKG2D is a cytotoxic factor that binds to NK cells and CD8 + It is expressed on the surface of T cells, activated macrophages, and tumor-infiltrating γδT cells, and either directly activates NK cells to exert its killing effect or acts as a costimulatory signal to promote T cell activation.

[0090] The present invention constructs cellular adaptor molecules that bind to NKG2D ligands based on the extracellular domain of NKG2D. NKG2D ligands described in the present invention include, but are not limited to, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

[0091] NKp46 NKp46 belongs to the immunoglobulin superfamily and has two Ig-like domains. NKp46 is the main activating receptor for NK cells in the body to eliminate target cells, and antibodies against NKp46 can effectively induce NK cell cytotoxicity and cytokine release. The present invention constructs an NK cell-specific adaptor molecule using the extracellular domain of NKG2D as the target cell binding domain and NKp46 as the NK cell binding domain.

[0092] CD16 CD16, or FcγRIII, is a glycoprotein with a molecular weight of 50,000-70,000 and a member of the Ig superfamily. It is primarily expressed on the surface of monocytes and natural killer cells and is involved in antibody-dependent cellular cytotoxicity (ADCC). Activating NK cells using a CD16 antibody during tumor therapy promotes NK cell infiltration and induces a more potent NK cell-mediated ADCC effect. The present invention utilizes a multispecific antibody that binds to CD16 to activate and crosslink NK cells, promoting the killing of senescent cells.

[0093] IL15 Interleukin-15 (IL15) is a T cell growth factor that activates the PI3K / Akt and Ras / Raf / MAPK signaling cascades by activating JAK1 / JAK3 and STAT3 / STAT5, Syk catalyzer and phospholipase C (PLC)γ, Lck catalyzer and Shc. According to literature, IL-15 promotes the proliferation of NK, memory CD8 + It inhibits the development and progression of tumors by regulating the survival, proliferation, and function of immune cells such as T and NKT.

[0094] The present invention constructs a multispecific cell adaptor based on NKG2D-NKp46 containing an IL15 activation unit to enhance the killing effect of NK cells against senescent cells.

[0095] antibody The second binding domain of the cellular adaptor molecule of the present invention can comprise an antibody or antigen-binding fragment that specifically binds to NKp46 and / or an antibody or antigen-binding fragment that specifically binds to CD16.

[0096] As used herein, the term "antibody" or "immunoglobulin" refers to a heterotetrameric glycoprotein of approximately 150,000 daltons composed of two identical light chains (L) and two identical heavy chains (H) with identical structural characteristics. Each light chain is linked to a heavy chain by one covalent disulfide bond, and the number of disulfide bonds varies among heavy chains of different immunoglobulin isotypes. Each heavy and light chain also contains regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end, with the light chain constant region facing the first constant region of the heavy chain and the light chain variable region facing the heavy chain variable region. Certain amino acid residues form an interface between the light and heavy chain variable regions.

[0097] As used herein, the term "variable" refers to differences in the sequences of certain portions of the variable regions in antibodies, which determines the binding and specificity of various specific antibodies for a particular antigen. However, variability is not evenly distributed throughout antibody variable regions. It is concentrated in three fragments called the complementarity-determining regions (CDRs) or hypervariable regions of the light and heavy chain variable regions. The more conserved portions of the variable regions are called framework regions (FRs). Natural heavy and light chain variable regions each contain four FR regions, which are usually in a β-folded configuration connected by the three CDRs that form a connecting ring, and can occasionally form a partial β-folded structure. The CDRs of each chain are closely juxtaposed by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publication No. 91-3242, Vol. I, pp. 647-669 (1991)). The constant regions are directly involved in binding the antibody to an antigen, but they also exhibit various effector functions, such as those involved in antibody-dependent cellular toxicity of the antibody.

[0098] The term "antibody fragment" or "antigen-binding fragment" refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, single-chain Fvs (scFv), single-chain antibodies, disulfide-bonded Fvs (sdFv), VL or VH domain-containing fragments, fragments produced by Fab expression libraries, and anti-idiotypic (anti-Id) antibodies. Regardless of structure, antibody fragments bind to the same antigen recognized by the intact antibody. The term "antibody fragment" includes DART and diabodies. The term "antibody fragment" further includes any synthetic or genetically engineered protein containing an immunoglobulin variable region that acts like an antibody by binding to a specific antigen to form a complex. A "single-chain fragment variable region" or "scFv" refers to a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin. In some embodiments, the variable domains are connected to a short linker peptide of 10 to about 25 amino acids. The linker is glycine-rich for flexibility and serine- or threonine-rich for solubility, and connects the N-terminus of VH or the C-terminus of VL, or vice versa. Despite the removal of the constant region and the introduction of the linker, such proteins still retain the specificity of the original immunoglobulin. For IgG, a typical immunoglobulin molecule contains two identical light chain polypeptides with a molecular weight of approximately 23,000 daltons and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000. The four chains are usually connected by disulfide bonds in a "Y" configuration, where the light chain is attached to the heavy chain at the bracket of the "Y" configuration and extends the variable region.

[0099] As mentioned above, the variable region enables an antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, the VL domain and VH domain of an antibody, or a subset of the antibody's complementarity-determining regions (CDRs), combine to form the variable region that defines a three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding site present at the end of each arm of each Y configuration. More specifically, the antigen-binding site is defined by three CDRs (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) in each of the VH and VL chains. In certain cases, for example, a particular immunoglobulin molecule is derived from a camelid species or is engineered based on a camelid mp immunoglobulin. Alternatively, the immunoglobulin molecule can be composed of only heavy chains and no light chains, or only light chains and no heavy chains.

[0100] As used herein, an antibody, antigen-binding fragment, or antibody domain further includes "variants" thereof, where "variant" refers to an antibody, antibody fragment, or antibody domain that (1) has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the original antibody, antibody fragment, or antibody domain, and (2) specifically binds to the same target as the original antibody, antibody fragment, or antibody domain. When sequence identity is expressed in the form of "at least x% identical" or "at least x% identity," it is understood that such embodiments include any numerical percentage above the lower limit. Furthermore, it is understood that when an amino acid sequence is present in this application, it should be construed as also disclosing or including sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence.

[0101] The antibody contained in the cell adaptor molecule of the present invention may be an antibody fragment having immunological activity, such as a Fab or (Fab')2 fragment, an antibody heavy chain, or an antibody light chain. The antibody used in the present invention is preferably in the form of a single-chain antibody, and the single-chain antibody (scFv) is the minimum antibody fragment that contains the heavy chain variable region and light chain variable region of an antibody but does not contain the constant region and has the entire antigen-binding site. Generally, an Fv antibody further contains a polypeptide linker between the VH domain and the VL domain and is capable of forming the structure necessary for antigen binding.

[0102] The terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to an antibody binding to a predetermined epitope on an antigen. Typically, an antibody binds to an epitope of about 10 -7 Less than M, e.g., about 10 -8 Under M, 10 -9 Less than M or 10 -10 It binds with an affinity (KD) of less than M or less.

[0103] As used herein, the terms "heavy chain variable region" and "VH" are used interchangeably.

[0104] As used herein, the terms "light chain variable region" and "VL" are used interchangeably.

[0105] As used herein, "variable region" and "complementarity determining region (CDR)" are used interchangeably.

[0106] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily mediates antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242.

[0107] At the same time, those skilled in the art should understand that the first binding domain of the BiTE of the present invention is preferably the extracellular domain of NKG2D, but the first binding domain can be selected as a specific anti-NKG2D ligand antibody as long as it can achieve the cell binding effect of the present invention.

[0108] Bispecific Cell Adaptor Molecules As used herein, "bispecific cellular adaptor molecule", "bispecific cellular adaptor", "cellular adaptor", "bispecific antibody", "BiTE" are used interchangeably and refer to a cellular adaptor molecule capable of simultaneously binding to an NKG2D ligand and NKp46 as provided by the first aspect of the invention.

[0109] Bispecific cellular adaptor molecules are connected by two protein or polypeptide sequences (antibodies are the most common) that bind to different target proteins. The function of the bispecific cellular adaptor molecules of the present invention is determined by the specific gene sequences of the NKG2D receptor extracellular domain and the NKp46 antigen-binding domain. The antibodies of the present invention simultaneously bind to an NKG2D ligand and NKp46, connect to target cells expressing an NKG2D ligand via the NKG2D extracellular domain, and connect to natural killer cells via the NKp46-binding domain, thereby effectively cross-linking the natural killer cells and target cells and promoting the killing effect of the natural killer cells.

[0110] As used herein, the term "bispecific" refers to a molecule comprising at least two binding domains with different binding specificities. Each binding domain is capable of specifically binding to a target molecule. In some embodiments, the bispecific cellular adaptor is a polymer molecule comprising two or more peptides. In some embodiments, the binding domain comprises a single domain antibody, an antigen-binding fragment of an antibody, a single-chain variable fragment, or a variable region, or a CDR, or a combination thereof, that specifically binds to a target protein. In some embodiments, the binding domain comprises a ligand or a fragment thereof that specifically binds to a target protein. In some embodiments, the binding domain comprises a combination of the above structures.

[0111] The at least two targeting domains of the cellular adaptor molecule of the present invention can be optionally linked by a connecting peptide or a single-chain Fc fragment. A preferred connecting peptide sequence is, but is not limited to, (G4S)3.

[0112] In the present invention, BiTEs of the present invention further include conservative variants thereof, and refer to polypeptides formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids having similar or similar properties compared to the amino acid sequence of a BiTE of the present invention. These conservative variant polypeptides are most preferably generated by replacing amino acids according to Table 1. [Table 1]

[0113] The amino acid sequence also includes sequences formed by adding, deleting, modifying and / or substituting at least one amino acid sequence, and is preferably an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% homology or sequence identity.

[0114] Methods for measuring sequence homology or identity that are well known to those skilled in the art include those described in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.M. and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991, and Carillo, H. and Lipman, D., SIAM J. Applied Biology, 1999. Math., 48:1073 (1988). A preferred method for determining identity is the method that gives the largest match between the sequences tested. Methods for determining identity are codified in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to, the GCG program package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S., F. et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). Identity can also be determined using the well-known Smith-Waterman algorithm.

[0115] The antibodies of the present invention can be single-chain or dual-chain antibodies that target NKG2D ligands and immune cell membrane proteins (eg, human NKG2D ligands and NKP46).

[0116] In a preferred embodiment of the present invention, the bispecific cellular adaptor molecule is a single chain antibody comprising an anti-NKP46 single chain antibody section, a connecting peptide and the extracellular domain of NKG2D, wherein the anti-NKP46 single chain antibody is a conventional single chain antibody in the art comprising a heavy chain variable region and a light chain variable region.

[0117] In the above-described content of the present invention, the number of added, deleted, modified and / or substituted amino acids is preferably 40% or less, more preferably 35% or less, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, and more preferably 15 to 20% of the total number of amino acids in the initial amino acid sequence.

[0118] In the above content of the present invention, more preferably, the number of added, deleted, modified and / or substituted amino acids may be 1 to 7, more preferably 1 to 5, more preferably 1 to 3, and more preferably 1 to 2.

[0119] Recombinant proteins The present invention further provides recombinant proteins comprising the cellular adapter molecules of the present invention.

[0120] The recombinant proteins of the present invention can comprise monomers, dimers, or multimers of the cellular adaptor molecules of the present invention, or multispecific (eg, trispecific) cellular adaptors.

[0121] The method for preparing the recombinant protein is a conventional method in the art. Preferably, the preparation method involves isolating the protein from an expression transformant that recombinantly expresses the protein, or artificially synthesizing the protein sequence. A preferred method for isolating the protein from an expression transformant that recombinantly expresses the protein involves cloning a nucleic acid molecule encoding the protein and having a point mutation into a recombinant vector, transforming the resulting recombinant vector into a transformant to obtain a recombinant expression transformant, and culturing the resulting recombinant expression transformant, thereby isolating and purifying the recombinant protein.

[0122] nucleic acid The present invention further provides a polynucleotide molecule encoding the above-mentioned cellular adapter molecule. The polynucleotide of the present invention may be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be the same as the coding region sequence of the cellular adapter of the present invention, or may be a degenerate variant. As used herein, the term "degenerate variant" refers to a nucleic acid sequence that encodes the same amino acid sequence as the polypeptide of the present invention but has a difference in its coding region sequence.

[0123] Polynucleotides encoding mature polypeptides of the present invention include coding sequences encoding only the mature polypeptide, coding sequences for the mature polypeptide and various additional coding sequences, coding sequences for the mature polypeptide (and optionally additional coding sequences) and non-coding sequences.

[0124] The term "polynucleotide encoding a polypeptide" may be a polynucleotide that encodes the polypeptide, or it may be a polynucleotide that further comprises additional coding and / or non-coding sequences.

[0125] The present invention further relates to polynucleotides that hybridize to the above sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize to the above polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refers to (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, and 60°C, or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, and 42°C, or (3) hybridization that occurs only when the identity between the two sequences is at least 90%, more preferably 95%. Furthermore, polypeptides encoded by hybridizable polynucleotides have the same biological functions and activities as the cellular adapter molecules described in the first aspect of the present invention. The full-length nucleotide sequence of the cell adapter molecule of the present invention or a fragment thereof can usually be obtained by PCR amplification, recombinant methods, or artificial synthesis. Especially when the fragment is short, a viable method is to synthesize the relevant sequence using artificial synthesis. Generally, multiple small fragments are first synthesized and then linked to obtain a very long fragment sequence. Furthermore, the coding sequence and an expression tag (e.g., 6His) can also be fused to form a fusion protein.

[0126] Vectors and host cells The present invention further provides a recombinant expression vector comprising the nucleic acid.

[0127] The recombinant expression vector can be constructed by connecting the nucleic acid molecule of the present invention to various expression vectors according to conventional methods in the art. The expression vector can be any conventional vector in the art, as long as it can carry the nucleic acid molecule. The vector preferably includes various plasmids, cosmids, phages, or virus vectors.

[0128] The present invention further provides a recombinant expression transformant comprising the above-mentioned recombinant expression vector.

[0129] The recombinant expression transformant can be prepared by a conventional method in the art, preferably by transforming the recombinant expression vector into a host cell. The host cell can be any of a variety of conventional host cells in the art, as long as the recombinant expression vector can stably replicate and the nucleic acid carried therein can be effectively expressed. Preferably, the host cell is E. coli TG1 or E. coli BL21 cells, or HEK-293T or CHO cells. The preferred recombinant expression transformant of the present invention can be obtained by transforming the recombinant expression plasmid into a host cell. The transformation method can be a conventional method in the art, preferably chemical transformation, heat shock, or electroporation.

[0130] In a preferred embodiment of the present invention, the vectors available include pCDH, pTOMO, pGEM, pELNS, pMSGV, or a combination thereof. In a preferred embodiment of the present invention, the available host cells include T cells, NK cells, or a combination thereof.

[0131] Preparation of cellular adaptor molecules The method for preparing the DNA molecule sequence of the cellular adapter molecule or a fragment thereof of the present invention, preferably by fusing the coding sequence of the ligand section and the coding sequence of the antibody section to form a single chain antibody, can be obtained using conventional techniques such as PCR amplification or genomic library screening.

[0132] Once the relevant sequence is obtained, recombinant methods can be used to obtain large quantities of the relevant sequence, typically by cloning it into a vector, then transforming it into cells, and then isolating the relevant sequence from the host cells grown by conventional methods.

[0133] Additionally, related sequences can be synthesized by artificial synthesis, especially when the fragments are relatively short in length, usually by first synthesizing several smaller fragments and then joining them together to obtain fragments of much longer sequences.

[0134] Currently, DNA sequences encoding the cell adapters of the present invention (or fragments or derivatives thereof) can be obtained entirely by chemical synthesis. These DNA sequences can then be introduced into a variety of existing DNA molecules (or vectors, etc.) and cells known in the art. Furthermore, mutations can be introduced into the protein sequences of the present invention by chemical synthesis.

[0135] The present invention further relates to vectors containing the appropriate DNA sequences and appropriate promoter or control sequences, which can be used to transform appropriate host cells so as to express the proteins.

[0136] The host cell can be a prokaryotic cell, such as, for example, a bacterial cell, or a lower eukaryotic cell, such as, for example, a yeast cell, or a higher eukaryotic cell, such as, for example, a mammalian cell. Preferred cells include, but are not limited to, T cells.

[0137] Typically, the obtained host cells can be cultured and transformed under conditions suitable for expression of the antibodies of the invention, and the antibodies of the invention can then be produced and obtained using conventional separation and purification means well known to those skilled in the art, such as conventional immunoglobulin purification steps, e.g., protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography.

[0138] The resulting cellular adapters can be identified using conventional means. For example, their binding specificity can be measured by immunoprecipitation or in vitro binding assays (e.g., radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). Their binding affinity can be measured, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0139] The cell adapters of the present invention can be expressed intracellularly or on the cell membrane, or can be secreted extracellularly. If necessary, recombinant proteins can be isolated and purified by various separation methods that utilize their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation, treatment with protein precipitants (salting out), centrifugation, osmotic shock, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0140] Conventional methods for preparing bispecific cell adaptor molecules in the art include the following two methods. 1. First, the bispecific antibody gene was prepared by PCR, then cloned into the expression vector pRB199 and transformed into E. coli strain BL21(λDE3) to prepare inclusion bodies. Subsequently, the inclusion bodies were denatured by adding 6 M guanidine-HCl and dithioerythritol (DET), then diluted 100-fold with renaturation buffer, mixed quickly at 4°C, and incubated at 4°C for 72 hours to allow the protein to refold. After renaturation, the protein was dialyzed against 0.1 M Tris and 0.5 M NaCl at a 1:10 ratio, repeated three times, filtered (0.2 μm), and then subjected to metal ion affinity chromatography. Subsequently, purification was performed using fast protein liquid chromatography (BioLogic DuoFlow 10 System, Bio-Rad) and separation was performed using a histidine-tagged fusion protein purification column. The protein was eluted with an imidazole step gradient at a flow rate of 1 mL / min. The product is processed through a column (Sartorius Stedim Biotech) to remove proteins with a molecular weight above 10,000, dialyzed against PBS, and sterilized by filtration. The concentration is measured and then identified using SDS / PAGE with silver staining (see PNAS, 2013, 110(1):270-275).

[0141] 2. CHO cells were infected with lentivirus containing the bispecific antibody and cultured for 72 hours post-infection, revealing fluorescent expression in the CHO cells. The successfully infected cell line was expanded. CHO cells stably expressing the bispecific antibody were capable of continuous secretion and expression. The cell supernatant was collected and the protein purified and concentrated. Purification was then performed using high-speed protein liquid chromatography (BioLogic DuoFlow 10 System, Bio-Rad) and separation was performed using a histidine-tagged fusion protein purification column. Five volumes of the sample were passed through a nickel column with equilibration buffer at 0.5–1 ml / min. After equilibration, the sample was passed through the nickel column at a flow rate of 0.5 ml / min. The nickel column was washed with five volumes of equilibration buffer to remove background proteins until the absorbance of the eluate at 280 nm reached zero. The target protein was then eluted with imidazole at a flow rate of 0.5 ml / min. The protein was then concentrated and the salt solution was exchanged using an ultrafiltration tube. The concentration is measured and then identified by Western blot analysis (see Oncoimmunology, 2015, 4(4):e989776).

[0142] Those skilled in the art will perform routine selection or equivalent modifications to the above methods to prepare or produce the bispecific cell adaptor molecules of the present invention.

[0143] Antibody-drug conjugates (ADCs) As used herein, the term "antibody-drug conjugate (ADC)" refers to a conjugate formed from a cellular adaptor molecule and an effector molecule of the present invention.

[0144] Typically, the antibody-drug conjugate comprises the cellular adaptor molecule and an effector molecule, wherein the cellular adaptor molecule is coupled, preferably chemically coupled, to the effector molecule, and the effector molecule is preferably a drug having therapeutic activity. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug, or a radionuclide.

[0145] The cell adaptor molecule of the present invention and the effector molecule can be coupled via a coupling agent. Examples of the coupling agent include any one or more of a non-selective coupling agent, a coupling agent using a carboxy group, a peptide chain, or a disulfide bond. The non-selective coupling agent refers to a compound that forms a covalent bond between the effector molecule and the cell adaptor molecule, such as glutaraldehyde. The coupling agent using a carboxy group can be any one or more of an aconitic anhydride coupling agent (e.g., aconitic anhydride) or an acylhydrazone coupling agent (the coupling site is an acylhydrazone).

[0146] Specific residues (e.g., Cys or Lys) on the cellular adaptor molecule are used to connect with various functional groups, including imaging reagents (e.g., chromophores and fluorescent groups), diagnostic reagents (e.g., MRI comparison agents and radioisotopes), stabilizers (e.g., glycol polymers), and therapeutic agents. The cellular adaptor molecule can be coupled to a functional agent to form a cellular adaptor molecule-functional agent conjugate. The functional agent (e.g., drug, detection reagent, stabilizer) is coupled (covalently bound) to the cellular adaptor molecule. The functional agent can be connected to the cellular adaptor molecule directly or indirectly via a linker.

[0147] Cell adaptor molecules can be coupled to drugs to form antibody-drug conjugates (ADCs). Typically, ADCs include a linker positioned between the drug and the cell adaptor molecule. The linker can be degradable or non-degradable. Degradable linkers are typically susceptible to degradation in the intracellular environment, e.g., degradation of the linker at the target site releases the drug from the cell adaptor molecule. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptide-group-containing linkers that are degraded by intracellular proteases (e.g., lysosomal or endosomal proteases) or sugar linkers, such as glucuronic acid-containing linkers, that can be degraded by glucuronidase. Peptidyl linkers can include dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that are hydrolyzed when the pH is below 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide bond linkers). Non-degradable linkers typically release the drug under conditions where the cellular adaptor molecule is hydrolyzed by proteases. The drug can be any cytotoxic drug, cell growth inhibitory drug, or immunosuppressive drug. In embodiments, a linker connects the cellular adaptor molecule and the drug, and the drug has a functional group capable of forming a bond with the linker. For example, the drug can include an amino group, a carboxy group, a sulfhydryl group, a hydroxy group, or a keto group capable of forming a bond with the linker. When the drug is directly connected to the linker, the drug has an active group that reacts before connecting to the cellular adaptor molecule.

[0148] Useful drug classes include, for example, antitubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, antifolates, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. Examples of particularly useful cytotoxic drug classes include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors; typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines, and oxazolidinobenzodiazepines), and vinca alkaloids. In the present invention, drug-linkers can be used to form ADCs in one simple step. In other embodiments, bifunctional linker compounds can be used to form ADCs in a two-step or multi-step manner. For example, a cysteine ​​residue can be reacted with a reactive active moiety on the linker in a first step, and a functional group on the linker can be reacted with the drug in a subsequent step to form the ADC.

[0149] Typically, functional groups on the linker are selected to react specifically with appropriate reactive activity on the drug moiety. As a non-limiting example, azide-based moieties can be used to react specifically with reactive alkynyl groups on the drug moiety. The drug is covalently attached to the linker via 1,3-dipolar cycloaddition between the azide and alkyne groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides), isocyanates and isothiocyanates (suitable for reaction with amines and alcohols), and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other attachment strategies, such as those described in "Biocoupling Techniques," 2nd Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will recognize that when selecting a complementary pair of reactive functional groups for selective reaction of a drug moiety and a linker, each member of the complementary pair can be used as both a linker and a drug.

[0150] Detection Applications and Kits The bispecific cellular adapter molecules of the invention or ADCs thereof can be used in detection applications, for example, to detect specimens and provide diagnostic information.

[0151] In the present invention, specimens (samples) used include cells, tissue specimens, and biopsy specimens. The term "biopsy" used in the present invention includes all types of biopsies known to those skilled in the art. Therefore, biopsies used in the present invention can include, for example, tumor resection specimens, tissue specimens prepared by endoscopic methods, or organ puncture or needle biopsy.

[0152] Specimens for use in the present invention include fixed or preserved cell or tissue specimens.

[0153] The present invention further provides a kit comprising the cell adapter of the present invention (or a fragment thereof), and in a preferred embodiment of the present invention, the kit further comprises a container, instructions for use, a buffer, etc. In a preferred embodiment, the cell adapter of the present invention can be immobilized on a detection plate.

[0154] Pharmaceutical Composition The present invention further provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition, which comprises the above-mentioned cell adaptor, or an active fragment thereof, or a fusion protein thereof, or an ADC thereof, or corresponding immune cells, and a pharmaceutically acceptable carrier. Typically, these substances can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous vector medium, wherein the pH is usually about 5 to 8, preferably about 6 to 8, depending on the nature of the formulated substance and the disease to be treated.

[0155] The formulated pharmaceutical composition can be administered by a conventional route, including (but not limited to) intratumoral, intraperitoneal, intravenous, or topical administration. Typically, the administration route of the pharmaceutical composition according to the present invention is preferably injection or oral administration. The injection administration preferably includes routes such as intravenous injection, intraarterial injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection. The pharmaceutical composition may be in various dosage forms conventional in the art, preferably in the form of a solid, semisolid, or liquid, such as an aqueous solution, a non-aqueous solution, or a suspension, more preferably a tablet, capsule, granule, injection, or infusion.

[0156] The cell adapters of the present invention can also be expressed intracellularly by nucleotide sequences for use in cell therapy.

[0157] The pharmaceutical composition according to the present invention is a pharmaceutical composition for preventing and / or treating diseases associated with abnormalities in the expression or function of an NKG2D ligand.

[0158] The pharmaceutical composition of the present invention contains a safe and effective amount (for example, 0.001 to 99 wt %, preferably 0.01 to 90 wt %, more preferably 0.1 to 80 wt %) of the above-mentioned cell adaptor of the present invention (or a conjugate thereof) or a safe and effective amount (1 × 10 3 ~1×10 8 cells / ml, more preferably 1 x 10 4 ~1×10 7 The modified immune cells are administered in a concentration of 1000 mg / mL (1000 cells / mL) and a pharmaceutically acceptable carrier or excipient. Such vectors include, but are not limited to, saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The drug formulation must be consistent with the method of administration. The pharmaceutical compositions of the present invention can be prepared in injection form by conventional methods, for example, using saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions must be prepared under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 μg / kg body weight to about 5 mg / kg body weight daily. Furthermore, the polypeptides of the present invention can also be used in combination with other therapeutic agents.

[0159] In a preferred embodiment of the present invention, the polypeptide of the present invention can be used in combination with other therapeutic agents for treating and / or preventing cancer and / or cancer metastasis.

[0160] In the present invention, preferably, the pharmaceutical composition described in the present invention further comprises one or more pharmaceutically acceptable carriers. The pharmaceutically acceptable carriers are conventional pharmaceutically acceptable carriers in the art, and may be any suitable physiologically or pharmaceutically acceptable adjuvants. The adjuvants are conventional pharmaceutically acceptable adjuvants in the art, and include pharmaceutically acceptable excipients, fillers, diluents, etc. More preferably, the pharmaceutical composition comprises 0.01 to 99.99% of the protein and 0.01 to 99.99% of the pharmaceutically acceptable carrier, the percentages being mass percentages of the pharmaceutical composition.

[0161] In the present invention, the dosage of the pharmaceutical composition is preferably an effective amount, which is an amount that can alleviate or delay the progression of a disease, degenerative or damaging condition. The effective amount can be measured individually and is determined taking into account the symptoms to be treated and the desired results. Those skilled in the art can determine the effective amount using the above factors, such as on an individual basis, and only conventional experimentation.

[0162] When the conjugate is used, a safe and effective amount of the conjugate is administered to a mammal, where the safe and effective amount is usually at least about 10 μg / kg body weight, and in most cases does not exceed about 50 mg / kg body weight, and preferably the dosage is about 10 μg / kg body weight to about 20 mg / kg body weight. Of course, the specific dosage should take into account factors such as the route of administration and the patient's health condition, all of which are within the skill of a skilled physician.

[0163] Therapeutic applications The present invention provides uses of NKG2D ligand target cell adaptor molecules and pharmaceutical compositions of the present invention for preventing and / or treating diseases in which an NKG2D ligand is expressed.

[0164] Here, diseases expressing NKG2D ligands include tumors, autoimmune diseases, transplant rejection, inflammation, aging, and diseases associated with the accumulation of senescent cells.

[0165] The tumor expressing an NKG2D ligand is selected from the group consisting of lung cancer, ovarian cancer, colon cancer, liver cancer, gastric cancer, pancreatic cancer, kidney cancer, prostate cancer, breast cancer, bladder cancer, nasopharyngeal cancer, leukemia, lymphoma, glioblastoma, neuroblastoma, melanoma, or a combination thereof.

[0166] The disease associated with the accumulation of senescent cells expressing NKG2D ligands is selected from the group consisting of muscular dystrophy, fatty liver, heart failure, atherosclerosis, diabetes, myocardial hypertrophy, osteoporosis, tissue / organ fibrosis, Alzheimer's disease, Parkinson's syndrome, arthritis, organ degenerative diseases caused by cellular senescence such as chronic obstructive pulmonary disease, or a combination thereof.

[0167] The autoimmune disease expressing an NKG2D ligand is selected from the group consisting of rheumatoid arthritis, colitis, celiac disease, multiple sclerosis, alopecia areata, type 1 diabetes, chronic obstructive pulmonary disease, atherosclerosis, or type 2 diabetes-associated metabolic syndrome.

[0168] The general cell adaptor molecules of the present invention can also be used as a class of vaccines for ex vivo immunization and / or in vivo treatment of mammals, preferably humans.

[0169] In addition to the use of cell-based vaccines to target immune cells ex vivo, the present invention also provides compositions and methods for use in vivo to enhance the immune response to a target antigen in a patient.

[0170] The pharmaceutical composition of the present invention can be administered in a manner appropriate for the disease to be treated (or prevented). The number and frequency of administration are determined by factors such as the characteristics of the patient's symptoms, the type and severity of the disease, etc., and the appropriate dosage can be determined by clinical trials.

[0171] When an "immunologically effective amount," "antitumor effective amount," "anti-aging effective amount," or "therapeutic amount" is indicated, the exact dose of the composition of the present invention to be administered can be determined by a physician taking into account individual differences in the age, weight, size of aging tissue, degree of aging, and disease symptoms of the patient (subject).

[0172] Main advantages of this invention The bispecific cellular adaptor molecule constructed according to the present invention simultaneously targets the NKG2D ligand and the NKp46 antigen, and can exert its killing effect in vivo by being directly injected into the body or by using cells within the body (e.g., NK cells, T lymphocytes, CAR-NK cells, etc.) to deliver and sustainably express the antibody protein. Its main advantages are as follows: 1) High targeting: Bispecific cell adaptor molecules targeting abnormal cells with upregulated NKG2D ligand expression can effectively bridge target cells and natural killer cells, with stable binding and strong killing activity. 2) High safety: NKG2D ligands are important targets for innate immune cells to eliminate abnormal and tumor cells. Their expression on the surface of normal cells is strictly regulated, and the NKG2D-NKG2D ligand signaling chain has undergone a long period of natural selection, resulting in a high level of safety. Furthermore, numerous immunotherapies targeting NKG2D ligands are currently undergoing clinical trials, and no serious side effects related to the treatment have been observed.

[0173] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are used only to illustrate the present invention and do not limit the scope of the present invention. In the following examples, experimental methods without specific conditions are generally in accordance with conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions suggested by manufacturers. Unless otherwise specified, percentages and parts are calculated as weight percentages and weight parts.

[0174] The sequences included in the examples of the present invention are shown in the following table. [Table 2] TIFF2025531169000003.tif254170TIFF2025531169000004.tif118170

[0175] The Fab sequence of the NKp46 antibody is taken from the publication "Multifunctional Natural Killer Cell Engagers Targeting NKp46 Trigger Protective Tumor Immunity." The CD16 VHH antibody sequence is taken from GenBank ABS29544.1. The F C fragment sequence of the IgG antibody is derived from magituximab.

[0176] Example 1. Preparation of NKG2D-NKp46 protein 1.1. Vector Construction The nucleotide sequence of the target gene, structured as shown in Figure 1A, was synthesized and then cloned into the lentiviral vector pCDH-CMV-MCS-EF1-Puro via the EcoR I and Swa I restriction sites (see Myeloid Leukemia. Mol Ther. 2016.24(9):pp.1615-26). The cloned vector was verified to be correct by restriction digestion and sequencing, then transformed into susceptible E. coli (Stbl3) and grown. It was then extracted using QIAGEN's endotoxin-free midstream extraction kit and identified by Hind III restriction digestion.

[0177] 1.2.Virus packaging HEK-293T cells were cultured in 15 cm culture dishes to package the virus. When HEK-293T cells reached approximately 90% confluence, a plasmid mixture (20 μg of core plasmid, 10 μg of pCMVΔR8.9, and 4 μg of PMD2.G) dissolved in 2 ml of OPTIMEM was prepared. In a separate centrifuge tube, 2 ml of OPTIMEM and 68 μl of Lipo8000 were added. After allowing to stand at room temperature for 5 minutes, the plasmid complex was added to the liposome complex and allowed to stand at room temperature for 20 minutes. The mixture was then added dropwise to the HEK-293T cells and incubated at 37°C for 6 hours, after which the medium was removed. Preheated complete medium was added beforehand. After 48 and 72 hours, the viral supernatant was collected and centrifuged at 3,000 rpm at 4°C for 20 minutes. The virus was concentrated by filtering through a 0.45 μm filter and centrifuging at 25,000 rpm at 4°C for 2.5 hours. The concentrated virus is lysed overnight in 30 ul of virus lysis solution, and the virus titer is then detected by QPCR.

[0178] 1.3. Protein preparation and purification The virus was used to infect CHO cells, and after 24 hours, 1 μg / ml puromycin was added and the cells were screened for 48 hours. After the screening, the CHO cells were cultured for 7 days, and the culture supernatant was collected and filtered through a 0.22 μm filter. His-tagged antibodies were isolated from the expressed supernatant using an affinity chromatography column. The equilibration buffer was 900 ml of 0.5 M NaCl, 20 mM sodium phosphate buffer, pH 7.4, and the elution buffer was 900 ml of 0.5 M imidazole, 0.5 M NaCl, 20 mM sodium phosphate buffer, pH 7.4. After passing through a cation exchange column, the NKG2D-NKp46 bispecific antibody was obtained, and finally, the solution was replaced with PBS buffer and concentrated. SDS-PAGE electrophoresis of the purified NKG2D-NKp46 protein is shown in Figure 1B. Western blot results are shown in Figure 1B, and the molecular weight is consistent with theory.

[0179] 1.4. Detection of NKG2D-NKp46 binding to NK cells and target cells (1) Resuspend NK cells or target cells in 200 μl of 1x PBS (containing 2% FBS). (2) Add NKG2D-NKp46 protein to the resuspended cells at a final concentration of 100 μg / ml, mix evenly, and incubate on ice for 120 minutes, vortexing the cells every 10 minutes during this period, centrifuging at 500 g for 5 minutes, and discarding the supernatant. (3) Add 1 ml of 1x PBS (containing 2% FBS) to resuspend the cells, then centrifuge at 500 g for 5 minutes. (4) Repeat the steps, (5) Add anti-His antibody and mix evenly, place on ice and incubate for 60 minutes, vortex the cells every 10 minutes during this period, centrifuge at 500 g for 5 minutes, and discard the supernatant; (6) Add fluorescently labeled goat anti-rabbit secondary antibody, incubate at room temperature for 30 minutes, then centrifuge at 500 g for 5 minutes, discard the supernatant, (7) Add 1 ml of 1x PBS (containing 2% FBS) to resuspend the cells, then centrifuge at 500 g for 5 minutes. (8) Repeat step 7. (9) The binding rate was detected by flow cytometry, and the results are shown in Figures 1C, 2B, and 4A. The results indicate that NKG2D-NKp46 can bind to NK cells and target cells.

[0180] Example 2. NKG2D-NKp46 Protein Promotes NK Cell Killing of Tumor Cells 2.1. Detection of NKG2D Ligand Expression on Tumor Cells (1) MHCC97H cells were harvested by trypsin digestion, washed three times with 1x PBS, and then resuspended in 200 μl of 1x PBS (containing 2% FBS) to a cell concentration of 1x10 6 Adjust to cells / ml (2) Add NKG2D ligand antibody to the resuspended cells, mix evenly, and incubate on ice for 120 minutes, vortexing the cells every 10 minutes during this period, centrifuging at 500 g for 5 minutes, and discarding the supernatant. (3) Add 1 ml of 1x PBS (containing 2% FBS) to resuspend the cells, then centrifuge at 500 g for 5 minutes. (4) Repeat the steps, (5) NKG2D ligand expression was detected by flow cytometry, and the results were as shown in Figure 2A, which showed that the expression of MICA and ULBP2 in MHCC97H cells was significantly increased.

[0181] 2.2. NKG2D-NKp46 protein promotes NK cell killing of tumor cells MHCC97H cells were co-incubated with NK cells at an effector:target ratio of 1:5 (NK cells were effector cells, and the concentration was 1 × 10 5 / mL, 100uL per well, tumor cells are target cells, concentration is 2x10 4 The cells were incubated with 100µg / ml NKG2D-NKp46 protein (100µL / mL per well) for 8 hours. The cell killing effect was detected using a Promega fluorescent detection kit. First, the cells were treated with 30µl of 1x PLB lysis solution for 20 minutes, and then 30µl of substrate was added to each well. Immediately, detection was performed using a BioTek microplate reader. Cytotoxicity of killed cells = 1 - target cell fluorescence value with effector cells / target cell fluorescence value without effector cells. The results are shown in Figure 2C. After the addition of NKG2D-NKp46 protein, NK cell killing of tumor cells was significantly increased. The supernatants from the 1ng / ml NKG2D-NKp46 cultures were collected, and IFN-γ concentrations were measured using an ELISA kit. Statistical analysis was performed using GraphPad Prism software. The results are shown in FIG. 2D, and show that IFN-γ secretion is significantly increased after the addition of NKG2D-NKp46 compared to the control group.

[0182] Example 3. Upregulation of NKG2D Ligand Expression in Senescent Cells 3.1. Construction of a cellular senescence model by overexpressing p16 protein using the Tet-on system (1) 3 × 105 The cells were plated on a 10 cm dish, and the cell density after adhesion the next day was about 20%. (2) After cell attachment, the cells were infected with a lentivirus overexpressing p16 protein in the Tet-on system at a multiplicity of infection (MOI) of 50-100, and polybrene (stock solution concentration: 8 mg / mL) was added at a ratio of 1:1000 to improve infection efficiency. (3) A secondary infection was carried out with the same viral load 24 hours later. (4) Four days after virus infection, puromycin was added to a final concentration of 3 μg / mL for screening. (5) The constructed cells overexpressing p16 protein were transferred to a well plate or culture dish, allowed to adhere for 24 hours, and then 1 μg / mL of doxorubicin was added to induce the expression of p16 protein. (6) Eight days after induction, the cells were stained for senescence using an SA-β-gal staining kit (CS0030, Sigma). The results are shown in Figure 3A. More than 90% of the cells were positive, indicating that the cells had senesced at this time point.

[0183] 3.2. Detection of NKG2D Ligand Transcript Expression (1) After preparing senescent cells according to the above method, add 1-2 mL of Trizol to a 10 cm dish according to the cell density, place on ice for 5 minutes, and then mix thoroughly by pipetting. (2) 1 mL of the lysate was aspirated from each well and added to a 1.5 mL EP tube, 200 μL of chloroform was added, the tube was shaken vigorously for 15 seconds, left at room temperature for 5 minutes, and then centrifuged (4°C, 12,000 g, 15 minutes). (3) Add 450 μL of isopropanol to a new EP tube. (4) The upper colorless liquid after centrifugation was carefully aspirated and added to the EP tube containing isopropanol, mixed uniformly, incubated at room temperature for 10 minutes, and centrifuged (4°C, 12000 g, 10 minutes). (5) Discard the supernatant, add 1 mL of 75% ethanol prepared with RNase-free water to wash the RNA, and then centrifuge (4°C, 7500 g, 5 minutes). (6) Carefully remove the supernatant, invert the tube and dry it for 5 minutes, then aspirate the liquid on the tube wall with a pipette. (7) Dissolve in 30 μL of RNase-free water, place on ice immediately after dissolution, and measure the concentration. (8) Using the extracted RNA as a template, 2 μg of RNA was reverse transcribed into cDNA using the Thermo Scientific RevertAid™ First Strand cDNA Synthesis Kit. The reaction system was as follows: [Table 3]

[0184] (9) Add the reactants to a PCR tube according to the above system, incubate at 65°C for 5 minutes in a PCR machine, and immediately place on ice. Then add the following components to the tube: [Table 4] Mix gently until uniform, then instantaneously centrifuge, place in a PCR machine, and carry out the following reactions: 25°C for 5 minutes, 42°C for 1 hour, and 70°C for 5 minutes.

[0185] (10) NKG2D ligand expression was detected by fluorescent real-time quantitative PCR. The specific procedure was performed according to the instructions of the Thermo Powerup™ SYBR Green Master Mix (A25742) kit, with the following cycles: 50°C for 2 minutes, 95°C for 2 minutes, 95°C for 15 seconds (40 cycles), 60°C for 1 minute (40 cycles), 12°C, forever. (11) The data was exported in Excel format, and the relative expression levels of NKG2D ligands were calculated. The results are shown in Figure 3B. The NKG2D ligands MICA and ULBP2 were both significantly upregulated.

[0186] 3.2. Detection of NKG2D Ligand Membrane Expression Levels (1) HEIP-P16 cells were harvested by trypsin digestion, washed three times with 1x PBS, and then resuspended in 200 μl of 1x PBS (containing 2% FBS) to a cell concentration of 1x10 6 Adjust to cells / ml (2) Add NKG2D ligand antibody to the resuspended cells, mix evenly, and incubate on ice for 120 minutes, vortexing the cells every 10 minutes during this period, centrifuging at 500 g for 5 minutes, and discarding the supernatant. (3) Add 1 ml of 1x PBS (containing 2% FBS) to resuspend the cells, then centrifuge at 500 g for 5 minutes. (4) Repeat the steps, (5) NKG2D ligand expression was detected by flow cytometry, and the results are shown in Figure 3C.

[0187] Example 4. NKG2D-NKp46 protein promotes NK cell killing of senescent cells Senescent cells induced by P16 overexpression and NK cells were seeded into 96-well plates at an effector:target ratio of 2:1 (senescent cells as target cells, NK cells as effector cells) and co-incubated for 8 hours. NKG2D-NKp46 protein was added to the experimental group, while an irrelevant protein was added to the control group. The number of viable cells was counted under a microscope and the T cell killing rate was calculated. Killing efficiency = (number of target cells in the blank group - number of target cells in the co-culture group) / number of target cells in the blank group. The results are shown in Figures 4B and 4C. Compared to the control group, NKG2D-NKp46 significantly enhanced the killing effect of NK cells against senescent cells. Supernatants from 1 ng / ml NKG2D-NKp46 cultures were collected, and IFN-γ concentrations were detected using an ELISA kit. Statistical analysis was performed using GraphPad Prism software. The results are shown in FIG. 4D, and show that IFN-γ secretion is significantly increased after the addition of NKG2D-NKp46 compared to the control group.

[0188] Example 5. Proteins containing NKp46 scFv that enhance the killing effect of NK cells Resting NK cells, P16-overexpressing senescent cells, or the liver cancer cell line SMMC7721 were seeded into 96-well plates at an effector:target ratio of 10:1. Then, control, NKG2D-NKp46 scFv, NKG2D-scFc-NKp46 scFv, and NKG2D-scFc-NKp46 scFv-IL15 proteins were added, respectively, as shown in Figure 5A. Here, the structure of the scFc is CH2-CH3-CH2-CH3. CH2 and CH3 are derived from human IgG1 antibody. NKp46 scFv is a single-chain antibody of NKp46. After 24 hours of treatment, the number of viable cells was counted under a microscope, and the NK cell killing rate was calculated. Killing efficiency = (number of target cells in the blank group - number of target cells in the co-culture group) / number of target cells in the blank group. The results, shown in Figures 5B and 5C, demonstrate that compared with the control group, the protein containing NKp46 scFv significantly enhanced the killing effect of resting NK cells against target cells, an effect further enhanced by the addition of IL-15.

[0189] All documents mentioned in this application are incorporated by reference in this application as if each document were incorporated by reference individually. Furthermore, after reading the above teachings of the present invention, those skilled in the art will be able to make various changes or modifications to the present invention, and these equivalents will also fall within the scope defined by the appended claims of this application.

Claims

1. A cellular adaptor molecule, The cellular adaptor molecule (a) a first binding domain that specifically binds to an NKG2D ligand; and (b) a second binding domain that specifically binds to NKp46.

2. The first binding domain is derived from the extracellular domain of NKG2D. The cell adaptor molecule of claim 1 .

3. The amino acid sequence of the first binding domain is as set forth in SEQ ID NO: 1 or has at least 95% sequence homology or sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 and is capable of binding to an NKG2D ligand. The cell adaptor molecule of claim 1 .

4. The second binding domain comprises an antigen-binding fragment specific to anti-NKp46. The cell adaptor molecule of claim 1 .

5. The antigen-binding fragment of NKp46 comprises a Fab fragment, a single-chain antibody (scFv), a single-domain antibody, or a combination thereof. The cell adaptor molecule of claim 4 .

6. The anti-NKp46 Fab fragment and / or single chain antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: 1) HCDR1 as shown in the amino acid sequence SEQ ID NO:3; 2) HCDR2 as shown in the amino acid sequence SEQ ID NO:4; 3) comprising a heavy chain complementarity determining region (HCDR) of HCDR3 as set forth in the amino acid sequence SEQ ID NO: 5; In addition, the light chain variable region is 1) LCDR1 as shown in the amino acid sequence SEQ ID NO:6; 2) LCDR2 whose amino acid sequence is YTS; 3) comprising the light chain complementarity determining region (LCDR) of LCDR3 as shown in the amino acid sequence SEQ ID NO: 7; The cell adaptor molecule of claim 5 .

7. The heavy chain complementarity determining region of the anti-NKp46 single domain antibody is 1) HCDR1 as shown in the amino acid sequence SEQ ID NO:3; 2) HCDR2 as shown in the amino acid sequence SEQ ID NO:4; 3) It is characterized by containing HCDR3 as shown in the amino acid sequence SEQ ID NO:

5. The cell adaptor molecule of claim 5 .

8. The second binding domain further comprises an antigen-binding fragment derived from anti-CD16 and / or a polypeptide of an IL15 protein. The cell adaptor molecule of claim 1 .

9. A recombinant protein comprising: The recombinant protein is characterized in that it comprises a cell adaptor molecule according to any one of claims 1 to 8.

10. A polynucleotide comprising: The polynucleotide is (1) A cell adaptor molecule according to any one of claims 1 to 8, or (2) The polynucleotide, characterized in that it encodes a polypeptide selected from the group consisting of the recombinant proteins of claim 9.

11. A vector comprising: The vector, characterized in that it comprises the polynucleotide of claim 10.

12. 1. A modified host cell comprising: The modified host cell, characterized in that the host cell contains the vector of claim 11 or has the polynucleotide of claim 10 integrated into its genome.

13. 1. An antibody conjugate comprising: The antibody conjugate comprises: (a) an antibody moiety selected from the group consisting of the cellular adaptor molecule of claim 1; and (b) a coupling moiety coupled to the antibody moiety selected from the group consisting of a detectable marker, a drug, or a combination thereof.

14. 1. A pharmaceutical composition comprising: The pharmaceutical composition comprises: (a) an active ingredient selected from the group consisting of the cell adaptor molecule of claim 1, the recombinant protein of claim 9, the host cell of claim 12, the antibody conjugate of claim 13, or a combination thereof; and (b) one or more pharmaceutically acceptable carriers, diluents, fillers, binders, excipients, or combinations thereof.

15. Use of a cellular adapter molecule according to claim 1, or a recombinant protein according to claim 9, or a host cell according to claim 12, or an antibody conjugate according to claim 13, and / or a pharmaceutical composition according to claim 14 in the preparation of a drug for treating a disease associated with upregulation of expression of an NKG2D ligand.

16. The upregulation of expression is characterized in that the ratio of the expression level of NKG2D ligand (F1) to the expression level of normal cell tissue (F0) (i.e., F1 / F0) is ≧1.5, preferably ≧2, and more preferably ≧2.

5.

16. The use according to claim 15.

17. The NKG2D ligand is selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, or a combination thereof.

16. The use according to claim 15.

18. The diseases include those associated with tumors, autoimmune diseases, transplant rejection, inflammation, aging, and accumulation of senescent cells.

16. The use according to claim 15.

19. The tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), lung cancer, ovarian cancer, colorectal cancer, liver cancer, gallbladder cancer, bile duct cancer, gastric cancer, pancreatic cancer, kidney cancer, prostate cancer, breast cancer, bladder cancer, nasopharyngeal cancer, non-small cell lung cancer, glioblastoma, neuroblastoma, melanoma, or a combination thereof.

19. The use according to claim 18.

20. The disease associated with the accumulation of senescent cells is selected from the group consisting of organ degenerative diseases caused by cellular senescence, such as muscular dystrophy, fatty liver, heart failure, atherosclerosis, diabetes, myocardial hypertrophy, osteoporosis, tissue / organ fibrosis, Alzheimer's disease, Parkinson's syndrome, arthritis, and chronic obstructive pulmonary disease, or a combination thereof.

19. The use according to claim 18.

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