NKG2D-NKp46 cell engager molecules and their uses

Abstract

The present invention provides an NK cell engager molecule targeting NKG2D ligand and NKp46 and its use. The cell engager molecule of the present invention comprises at least: a first binding domain, the first binding domain comprises an NKG2D extracellular domain that can specifically bind to the NKG2D ligand; a second binding domain, the second binding domain comprises an NKp46 antigen binding fragment. The cell engager molecule of the present invention can promote the killing effect of natural killer cells on target cells with high expression of NKG2D ligand, and thus can be used as an effective therapeutic agent for related diseases expressing NKG2D ligand.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a cell engager molecule consisting of a NKG2D extracellular domain capable of specifically binding to a NKG2D ligand, a connecting segment and a NKp46 antigen binding fragment and an application thereof. Background Art

[0002] NKG2D is expressed on NK cells, CD8 + On the surface of T cells, activated macrophages and tumor-infiltrating γδT cells, it usually exists as a homodimer formed by itself, and relies on the adaptor protein DAP10 or DAP12 to directly activate NK cells to exert a killing effect, or acts as a co-stimulatory signal to promote the activation of T cells. Its ligands mainly include MICA, MICB, ULBP1-6, etc., which are generally not expressed or expressed at a low level in normal cells; but when cells are under stress conditions such as abnormal transformation, viral infection, and DNA damage, the expression of NKG2D ligands can be significantly upregulated, and it is recognized by the immune system as a sign of abnormal cells. The NKG2D-NKG2D ligand signal chain is an important mechanism for the body's immune system to exert immune surveillance functions. The immunotherapy developed for it should have better safety and effectiveness, and has gradually become the frontier of current immunotherapy strategies. At present, more than 10 CAR-T and CAR-NK designed with NKG2D-NKG2DL as the mechanism of action have entered clinical research, and no serious treatment-related adverse events have been seen. Cell engager molecules constructed based on the NKG2D extracellular domain are also gradually gaining attention in the field of anti-tumor. For example, the T cell bispecific antibody NKG2D-CD3 can inhibit tumor growth and prolong the survival of model mice by targeting tumor cells and immunosuppressive cells in vivo. The NK cell bispecific antibody NKG2D-CD16 can effectively kill tumor cells such as acute myeloid leukemia, lymphoma and soft tissue sarcoma; NKG2D-Fc can significantly improve the tumor immune microenvironment and inhibit tumorigenesis.

[0003] Currently, more cell engager molecules are still to be developed in this field. Summary of the invention

[0004] The object of the present invention is to provide a cell engager molecule containing an extracellular domain of NKG2D and an NKp46 antigen binding fragment and a use thereof.

[0005] In a first aspect of the present invention, a cell engager molecule is provided, wherein the cell engager molecule comprises:

[0006] (a) a first binding domain that specifically binds to a NKG2D ligand; and

[0007] (b) a second binding domain, which specifically binds to NKp46.

[0008] In another preferred embodiment, the first binding domain is derived from the extracellular domain of NKG2D.

[0009] In another preferred example, the amino acid sequence of the first binding domain is as shown in SEQ ID NO:1; or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity with the amino acid sequence shown in SEQ ID NO:1 and can bind to the NKG2D ligand.

[0010] In another preferred example, the second binding domain comprises an antigen-binding fragment specific for NKp46.

[0011] In another preferred embodiment, the anti-NKp46 antigen-binding fragment comprises a structure selected from the group consisting of Fab, Fab', F(ab')2, Fd, Fv, dAb, Fc, complementarity determining region fragment, single-chain antibody, single-domain antibody, or a combination thereof.

[0012] In another preferred embodiment, the anti-NKp46 antigen-binding fragment comprises a structure selected from the following group: a humanized antibody, a chimeric antibody, or a combination thereof.

[0013] In another preferred embodiment, the NKp46 antigen binding fragment comprises: a Fab fragment, a single-chain antibody (scFv), a single-domain antibody, or a combination thereof.

[0014] 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 comprises the following heavy chain complementarity determining region (HCDR):

[0015] 1) HCDR1 having an amino acid sequence as shown in SEQ ID NO: 3;

[0016] 2) HCDR2 having an amino acid sequence as shown in SEQ ID NO: 4;

[0017] 3) HCDR3 having an amino acid sequence as shown in SEQ ID NO: 5;

[0018] Furthermore, the light chain variable region includes the following light chain complementarity determining region (LCDR):

[0019] 1) LCDR1 having an amino acid sequence as shown in SEQ ID NO: 6;

[0020] 2) LCDR2 with the amino acid sequence of YTS;

[0021] 3) LCDR3 having an amino acid sequence as shown in SEQ ID NO: 7,

[0022] The CDRs are based on the IMGT numbering and definition scheme.

[0023] In another preferred example, the amino acid sequence of the heavy chain variable region of the anti-NKp46 Fab fragment is as shown in SEQ ID NO:8, or 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 with the amino acid sequence shown in SEQ ID NO:8.

[0024] In another preferred example, the amino acid sequence of the light chain variable region of the Fab fragment of anti-NKp46 is as shown in SEQ ID NO:9, or 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 with the amino acid sequence shown in SEQ ID NO:9.

[0025] In another preferred embodiment, the amino acid sequence of the Fab fragment of NKp46 is as shown in SEQ ID NO: 2, or 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 with the amino acid sequence shown in SEQ ID NO: 2 (sequence cited from Gauthier, Morel et al. 2019).

[0026] In another preferred embodiment, the amino acid sequence of the anti-NKp46 scFv fragment is as shown in SEQ ID NO: 10, or 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 with the amino acid sequence shown in SEQ ID NO: 10 (sequence cited from the document Gauthier, Morel et al. 2019).

[0027] In another preferred example, the amino acid sequence of the heavy chain variable region of the anti-NKp46 scFv fragment is as shown in SEQ ID NO:8, or 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 with the amino acid sequence shown in SEQ ID NO:8.

[0028] In another preferred example, the amino acid sequence of the light chain variable region of the anti-NKp46 scFv fragment is as shown in SEQ ID NO:9, or 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 with the amino acid sequence shown in SEQ ID NO:9.

[0029] In another preferred embodiment, the heavy chain complementary determining region of the anti-NKp46 single domain antibody comprises:

[0030] 1) HCDR1 having an amino acid sequence as shown in SEQ ID NO: 3;

[0031] 2) HCDR2 having an amino acid sequence as shown in SEQ ID NO: 4;

[0032] 3) HCDR3 with an amino acid sequence as shown in SEQ ID NO:5.

[0033] In another preferred example, the anti-NKp46 single domain antibody amino acid sequence is as shown in SEQ ID NO:11, or 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 with the amino acid sequence shown in SEQ ID NO:11.

[0034] In another preferred embodiment, the second binding domain further comprises: an antigen binding fragment derived from anti-CD16 and / or a polypeptide of IL15 protein.

[0035] In another preferred embodiment, the anti-CD16 antigen-binding fragment comprises a structure selected from the following groups: Fab, Fab', F(ab')2, Fd, Fv, dAb, Fc, complementarity determining region fragment, single-chain antibody, single-domain antibody, or a combination thereof.

[0036] In another preferred embodiment, the anti-CD16 antigen-binding fragment comprises a structure selected from the following group: a humanized antibody, a chimeric antibody, or a combination thereof.

[0037] In another preferred embodiment, the variable region of the anti-CD16 single domain antibody comprises:

[0038] 1) HCDR1 having an amino acid sequence as shown in SEQ ID NO: 12;

[0039] 2) HCDR2 having an amino acid sequence as shown in SEQ ID NO: 13;

[0040] 3) HCDR3 having an amino acid sequence as shown in SEQ ID NO:14.

[0041] In another preferred example, the amino acid sequence of the anti-CD16 single domain antibody is as shown in SEQ ID NO: 15, or 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 with the amino acid sequence shown in SEQ ID NO: 15.

[0042] In another preferred example, the amino acid sequence of the anti-CD16 Fc fragment is as shown 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 sequence identity with the amino acid sequence shown in SEQ ID NO: 16.

[0043] In another preferred example, the amino acid sequence of the IL15 protein is as shown 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 sequence identity with the amino acid sequence shown in SEQ ID NO:17.

[0044] In another preferred embodiment, the cell engager molecule has a structure selected from the following formula (I) or (II) from the N-terminus to the C-terminus:

[0045] S-D1-L1-D2-T (I); or

[0046] S-D2-L1-D1-T (II),

[0047] In the formula,

[0048] Each "-" is independently a connecting peptide or a peptide bond;

[0049] S is none or a signal peptide sequence;

[0050] D1 is the first binding domain;

[0051] L1 is none or a linker peptide;

[0052] D2 is the second binding domain;

[0053] T is no or labeled protein.

[0054] In another preferred embodiment, the second binding domain has a structure selected from the following formula (III) or (IV) from the N-terminus to the C-terminus:

[0055] D3-L2-D4-I (III); or

[0056] D4-L2-D3-I (IV),

[0057] In the formula,

[0058] Each "-" is independently a connecting peptide or a peptide bond;

[0059] D3 is the NKp46 antigen-binding fragment;

[0060] L2 is no or linker peptide;

[0061] D4 is a fragment with no or CD16 antigen binding;

[0062] I is absent or IL15 protein.

[0063] In another preferred embodiment, the S is a signal peptide derived from mammalian CD8α.

[0064] In another preferred embodiment, the connecting peptide is a glycine-serine peptide linker.

[0065] In another preferred embodiment, the connecting peptide is represented by the formula (GGGGS)n, wherein n is 1, 2, 3, 4, 5 or 6, preferably n is 3.

[0066] In another preferred embodiment, the marker protein T is selected from: His tag, FLAG tag.

[0067] In the second aspect of the present invention, a recombinant protein is provided, wherein the recombinant protein comprises the cell engager molecule as described in the first aspect of the present invention.

[0068] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0069] In another preferred embodiment, the recombinant protein specifically binds to NKG2D ligand and NKp46 antigen on the surface of NK cells.

[0070] In another preferred embodiment, the recombinant protein further specifically binds to CD16.

[0071] In the third aspect of the present invention, a polynucleotide is provided, which encodes a polypeptide selected from the group consisting of:

[0072] (1) the cell engager molecule according to the first aspect of the present invention; or

[0073] (2) The recombinant protein as described in the second aspect of the present invention.

[0074] In the fourth aspect of the present invention, a vector is provided, wherein the vector contains the polynucleotide as described in the third aspect of the present invention.

[0075] In another preferred embodiment, the vector includes but is not limited to: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.

[0076] In another preferred embodiment, the vector includes but is not limited to: pCDH, pTOMO, pGEM, pELNS, pMSGV, or a combination thereof.

[0077] In the fifth aspect of the present invention, an engineered host cell is provided, wherein the host cell contains the vector as described in the fourth aspect of the present invention or the polynucleotide as described in the third aspect of the present invention is integrated into its genome.

[0078] In another preferred embodiment, the host cell is an in vivo cell or a cell cultured in vitro that can be transplanted into the body.

[0079] In another preferred embodiment, the immune cells are selected from NK cells.

[0080] In another preferred embodiment, the immune cells are from humans or non-human mammals (such as mice).

[0081] In a sixth aspect of the present invention, an antibody conjugate is provided, wherein the antibody conjugate comprises:

[0082] (a) an antibody portion, the antibody portion being selected from the group consisting of a cell engager molecule as described in the first aspect of the invention; and

[0083] (b) a conjugated moiety conjugated to the antibody portion, wherein the conjugated moiety is selected from the group consisting of a detectable label, a drug, or a combination thereof.

[0084] In another preferred embodiment, the detectable marker comprises a radionuclide.

[0085] In another preferred embodiment, the drugs include toxins, cytokines, and enzymes.

[0086] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (for example, cisplatin) or any form of nanoparticles, etc.

[0087] In another preferred embodiment, the antibody portion and the coupling portion are coupled via a chemical bond or a linker.

[0088] In another preferred embodiment, the immunoconjugate contains: a multivalent (eg, bivalent) cell engager molecule as described in the first aspect of the present invention.

[0089] In another preferred embodiment, the multivalency refers to the presence of multiple repeats of the cell engager molecule as described in the first aspect of the present invention in the amino acid sequence of the immunoconjugate.

[0090] In a seventh aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0091] (a) an active ingredient, wherein the active ingredient is selected from the group consisting of a cell engager molecule as described in the first aspect of the present invention, a recombinant protein as described in the second aspect of the present invention, a host cell as described in the fifth aspect of the present invention, an antibody conjugate as described in the sixth aspect of the present invention, or a combination thereof; and

[0092] (b) one or more pharmaceutically acceptable carriers, diluents, fillers, binders, excipients, or combinations thereof.

[0093] In another preferred embodiment, the pharmaceutical composition is a liquid preparation.

[0094] In another preferred embodiment, the pharmaceutical composition is an injection.

[0095] In another preferred embodiment, the pharmaceutical composition comprises 0.01 to 99.99% of the cell engager molecule as described in the first aspect of the present invention, the recombinant protein as described in the second aspect of the present invention, the host cell as described in the fifth aspect of the present invention, the antibody conjugate as described in the sixth aspect of the present invention, or a combination thereof and 0.01 to 99.99% of a carrier, wherein the percentage is the mass percentage of the pharmaceutical composition.

[0096] In another preferred embodiment, the pharmaceutical composition is used to prevent and / or treat diseases in which NKG2D ligand expression is upregulated.

[0097] In the eighth aspect of the present invention, provided is the use of the cell engager molecule as described in the first aspect of the present invention, or the recombinant protein as described in the second aspect of the present invention, or the host cell as described in the fifth aspect of the present invention, or the antibody conjugate as described in the sixth aspect of the present invention, and / or the pharmaceutical composition as described in the seventh aspect of the present invention in the treatment of diseases related to upregulated expression of NKG2D ligands.

[0098] In another preferred embodiment, the medicine is used for preventing and / or treating diseases.

[0099] In another preferred embodiment, the up-regulated expression refers to the ratio of the expression level of NKG2D ligand (F1) to the expression level of normal cell tissue (F0) (ie, F1 / F0) being ≥1.5, preferably ≥2, and more preferably ≥2.5.

[0100] In another preferred embodiment, the NKG2D ligand includes (but is not limited to) MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

[0101] In another preferred embodiment, the disease includes tumors, autoimmune diseases, transplant rejection, inflammation, aging, and diseases related to aging cell accumulation.

[0102] In another preferred embodiment, the tumor includes blood tumors and solid tumors.

[0103] In another preferred embodiment, the blood tumor is selected from the following group: 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.

[0104] In another preferred embodiment, the solid tumor is selected from the group consisting of lung cancer, ovarian cancer, colorectal cancer, liver cancer, gallbladder cancer, biliary tract cancer, gastric cancer, pancreatic cancer, kidney cancer, prostate cancer, breast cancer, bladder cancer, nasopharyngeal carcinoma, non-small cell lung cancer, glioma, neuroblastoma, melanoma, or a combination thereof.

[0105] In another preferred embodiment, the senescent cell accumulation-related disease is selected from the following group: muscular dystrophy, fatty liver, heart failure, atherosclerosis, diabetes, myocardial hypertrophy, osteoporosis, tissue / organ fibrosis, Alzheimer's disease, Parkinson's syndrome, arthritis, chronic obstructive pulmonary disease and other organ degenerative diseases caused by cell aging, or a combination thereof.

[0106] In another preferred embodiment, the drug is used to inhibit cells with upregulated expression of NKG2D ligands, preferably including: human liver cancer cell line MHCC97H, human liver cancer cell line SMMC7721, human pancreatic cancer cell line ASPC1, or a combination thereof.

[0107] In another preferred embodiment, the senescent cells are selected from the following group: lung cells, fat cells, kidney cells, muscle cells, or a combination thereof.

[0108] In another preferred embodiment, the senescent cells are human embryonic lung cell line HEL1.

[0109] In another preferred embodiment, the senescent cells are naturally or artificially induced to age.

[0110] In another preferred embodiment, the artificial aging induction method comprises: DNA damage-induced aging, overexpression of P16-induced aging, telomere shortening-induced aging, or a combination thereof.

[0111] 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 metabolic syndrome associated with type 2 diabetes.

[0112] In another preferred embodiment, the method further comprises treating the subject with another disease treatment method.

[0113] In another preferred embodiment, the additional disease treatment method is selected from the following group: surgery, radiotherapy, chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, adjuvant therapy, immunotherapy, or a combination thereof.

[0114] In the ninth aspect of the present invention, a method for treating a disease associated with upregulated expression of NKG2D ligands is provided, comprising administering to a subject in need thereof an effective amount of a cell engager molecule as described in the first aspect of the present invention, or a recombinant protein as described in the second aspect of the present invention, or a host cell as described in the fifth aspect of the present invention, or an antibody conjugate as described in the sixth aspect of the present invention, or a pharmaceutical composition as described in the seventh aspect of the present invention, or a combination thereof.

[0115] 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 (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0116] The following drawings are used to illustrate specific embodiments of the present invention and are not used to limit the scope of the present invention defined by the claims.

[0117] Figure 1 NKG2D-NKp46 protein preparation is shown.

[0118] (A) Schematic diagram of the structure of NKG2D-NKp46 and control vectors. (B) 293T cells were transfected with NKG2D-NKp46 and control vectors, 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 NKG2D-NKp46 was incubated with NK92 cells, the binding rate was detected by flow cytometry.

[0119] Figure 2 It was shown that NKG2D-NKp46 protein promoted NK92 cells to kill tumor cells.

[0120] (A) Flow cytometry was used to detect the expression of NKG2D ligands MICA, MICB, ULBP1, ULBP2 and ULBP3 in the hepatoma cell line MHCC97H. (B) NKG2D-NKp46 protein was added to the co-culture system of NK92 cells and MHCC97H cells for 6 hours, and the cell death rate of MHCC97H cells was detected.

[0121] Figure 3 showed that NKG2D ligand expression is upregulated in senescent cells.

[0122] (A) After HEL1-P16 cells were induced to overexpress P16 by tetracycline (DOX), cellular β-gal staining was performed. (B) After HEL1-P16 cells were induced to overexpress P16 by tetracycline, NKG2D ligand expression was detected by quantitative PCR. (C) After HEL1-P16 cells were induced to overexpress P16 by tetracycline (DOX), NKG2D ligand expression was detected by flow cytometry.

[0123] Figure 4 It was shown that NKG2D-NKp46 protein promoted the killing of senescent cells by NK92 cells.

[0124] NKG2D-NKp46 protein was added to the co-culture system of NK92 cells and DOX-induced senescent HEL1-P16 cells for 6 hours, and the death rate of senescent cells was detected. DETAILED DESCRIPTION

[0125] The inventors have conducted extensive and in-depth research and a large number of screenings, and for the first time developed a preparation and application of a bispecific cell engager molecule based on the NKG2D extracellular domain. The experimental results show that the bispecific antibody targeting the NKG2D ligand and the NKp46 antigen of the present invention has a significant killing effect on target cells. The present invention was completed on this basis.

[0126] The bispecific cell engager molecule provided by the present invention consists of three parts: an NKG2D extracellular domain that can specifically bind to an NKG2D ligand, a linker, and an NKp46 antigen binding fragment. The bispecific cell engager can bridge natural killer cells to the vicinity of target cells that highly express NKG2D ligands, thereby killing the target cells, and can therefore be used to treat diseases related to the expression of NKG2D ligands.

[0127] the term

[0128] In order to more easily understand the present invention, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present 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, because such methods and conditions can be changed. It should also be understood that the terms used herein are intended only to describe specific embodiments, and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0129] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. As used herein, the term "about" when used in reference to a specific recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0130] The three-letter and one-letter codes for amino acids used in the present invention are as described in J. biol. chem, 243, p3558 (1968).

[0131] As used herein, the term "treatment" refers to administering an internal or external therapeutic agent to a patient, including antibodies and compositions thereof directed to respiratory syncytial virus fusion protein (preferably pre-fusion F protein) of the present invention, wherein the patient has one or more symptoms of a disease, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, the amount of the therapeutic agent that is effective in alleviating one or more symptoms of the disease (therapeutically effective amount) is administered to the patient.

[0132] As used herein, the term "optional" or "optionally" means that the event or situation described subsequently may occur but need not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable region of a specific sequence may have but need not have, and may be 1, 2 or 3.

[0133] "Sequence identity" as used herein refers to the degree of identity between two nucleic acid or amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions or deletions. The sequence identity between the sequences described herein and the sequences with which they are identical 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%, 100%.

[0134] NKG2D and NKG2D ligands

[0135] NKG2D is expressed on the surface of NK cells, CD8+T cells, activated macrophages and tumor-infiltrating γδT cells. It can directly activate NK cells to exert a killing effect, or act as a co-stimulatory signal to promote the activation of T cells.

[0136] The present invention constructs a cell engager molecule that binds to NKG2D ligands based on the NKG2D extracellular domain. The NKG2D ligands of the present invention include (but are not limited to) MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

[0137] NKp46

[0138] NKp46 belongs to the immunoglobulin superfamily and has two Ig-like domains. NKp46 is the main activating receptor for NK cells to eliminate target cells in the body. Antibodies against NKp46 can effectively trigger the cytotoxic activity and cytokine release of NK cells.

[0139] The present invention constructs a NK cell-specific adaptor molecule using the NKG2D extracellular domain as the target cell binding domain and NKp46 as the NK cell binding domain.

[0140] CD16

[0141] CD16, or FcγRⅢ, is a glycoprotein with a molecular weight of 50,000 to 70,000. It is a member of the Ig superfamily and is mainly expressed on the surface of monocytes and natural killer cells, and participates in antibody-dependent cellular cytotoxicity (ADCC). Using CD16 antibodies to activate NK cells during tumor treatment can promote NK cell infiltration and induce stronger NK cell-mediated ADCC. The present invention uses multispecific antibodies that bind to CD16 to activate and bridge NK cells to promote the killing of senescent cells.

[0142] IL15

[0143] Interleukin-15 (IL15) is a T cell growth factor that activates JAK1 / JAK3 and STAT3 / STAT5, Syk kinase and phospholipase C (PLC)γ, Lck kinase and Shc, thereby activating PI3K / Akt and Ras / Raf / MAPK signaling cascades. Literature shows that IL-15 is involved in regulating NK, memory CD8 + The survival, proliferation and function of immune cells such as T and NKT cells can be inhibited, thereby inhibiting the occurrence and development of tumors.

[0144] The present invention further constructs a multi-specific cell engager comprising an IL15 activation unit based on NKG2D-NKp46 to enhance the killing effect of NK cells on senescent cells.

[0145] Antibody

[0146] The second binding domain of the cell engager molecule of the present invention may include an antigen binding fragment that specifically binds NKp46, and an antigen binding fragment that specifically binds CD16.

[0147] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has 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; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0148] As used herein, the term "variable" means that some parts of the variable region in an antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three fragments called complementary determining regions (CDRs) or hypervariable regions in the variable regions of light and heavy chains. The more conservative part of the variable region is called the framework region (FR). The variable regions of natural heavy and light chains each contain four FR regions, which are roughly in a β-folded configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial β-folded structure. The CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, 647-669 pages (1991)). The constant region does not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody's antibody-dependent cytotoxicity.

[0149] The term "antibody fragment" or "antigen binding fragment" is used to refer 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-linked Fvs (sdFv), fragments comprising VL or VH domains, fragments produced by Fab expression libraries, and anti-idiotypic (anti-Id) antibodies. Regardless of the 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" also includes any synthetic protein or genetically engineered protein comprising an immunoglobulin variable region that acts like an antibody by binding to a specific antigen to form a complex. "Single-chain fragment variable region" or "scFv" refers to a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin. In some aspects, the region domain is connected to a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine for flexibility and serine or threonine for solubility, and can connect 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, this protein still retains the specificity of the original immunoglobulin. Regarding IgG, the standard immunoglobulin molecule contains two identical light chain polypeptides with a molecular weight of about 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 connected to the heavy chain from the mouth of the "Y" and extends through the variable region.

[0150] As mentioned above, the variable region allows the antibody to selectively recognize and specifically bind to the epitope on the antigen. That is, the VL domain and the VH domain of the antibody or the complementary determining region (CDR) subset of the antibody combine to form a variable region that defines a three-dimensional antigen binding site. This quaternary antibody structure forms an 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) on each of the VH and VL chains. In some cases, for example, some immunoglobulin molecules are derived from camelid species or are engineered based on camelid immunoglobulins. Alternatively, an immunoglobulin molecule can be composed of a heavy chain without a light chain or a light chain without a heavy chain.

[0151] As used herein, an antibody, antibody fragment or antibody domain also includes a "variant" thereof, wherein a "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. It should be understood that where sequence identity is expressed in the form of "at least x% identical" or "at least x% identical", such embodiments include any and all numerical percentages equal to or above the lower limit. In addition, it should be understood that where an amino acid sequence is present in the present application, it should be interpreted as additionally disclosing or including an amino acid sequence that has 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.

[0152] The antibody contained in the cell engager molecule of the present invention may be an antibody fragment with immunological activity, such as a Fab or (Fab')2 fragment; an antibody heavy chain; an antibody light chain. The antibody used in the present invention is preferably in the form of a single-chain antibody, which contains an antibody heavy chain variable region, a light chain variable region, but no constant region, and is the smallest antibody fragment with all antigen binding sites. Generally, the Fv antibody also contains a polypeptide linker between the VH and VL domains, and is capable of forming the structure required for antigen binding.

[0153] The terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen. -7 M, for example, less than about 10 -8 M, 10 -9 M or l0 -10 Binds with an affinity (KD) of M or less.

[0154] As used herein, the term "heavy chain variable region" is used interchangeably with "VH".

[0155] As used herein, the term "light chain variable region" is used interchangeably with "VL."

[0156] As used herein, the term "variable region" and "complementarity determining region (CDR)" are used interchangeably.

[0157] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contribute to 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).

[0158] In a preferred example of the present invention, the bispecific cell engager molecule is a single-chain polypeptide, which includes an anti-CD3 single-chain antibody segment, a connecting peptide and a Pep42 ligand segment, wherein the anti-CD3 single-chain antibody is a conventional single-chain antibody in the art, which includes a heavy chain variable region and a light chain variable region.

[0159] Meanwhile, those skilled in the art should understand that although the first binding domain of the BiTE of the present invention is preferably a ligand of uPAR, the first binding domain may also be selected as a specific anti-uPAR antibody as long as it can achieve the cell binding effect of the present invention.

[0160] Bispecific cell engager molecules

[0161] As used herein, the terms "bispecific cell engager molecule", "bispecific cell engager", "cell engager", "bispecific antibody" and "BiTE" are used interchangeably and refer to the cell engager molecule capable of simultaneously binding to NKG2D ligand and NKp46 provided in the first aspect of the present invention.

[0162] Bispecific cell engager molecules are formed by connecting two proteins or polypeptide sequences (antibodies are the most common) that bind to different target proteins. The function of the bispecific cell engager 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 can simultaneously bind to NKG2D ligands and NKp46, connect target cells expressing NKG2D ligands through the NKG2D extracellular domain, and connect natural killer cells through the NKp46 binding domain, thereby effectively bridging natural killer cells with target cells and promoting the killing effect of natural killer cells.

[0163] 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, a bispecific cell engager is a polymer molecule having 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.

[0164] At least two targeting domains of the cell engager molecule of the present invention are optionally connected by a connecting peptide. The preferred connecting peptide sequence is (GGGGS)3, but is not limited thereto.

[0165] In the present invention, the BiTE of the present invention also includes its conservative variants, which refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids of similar or similar properties compared to the amino acid sequence of the BiTE of the present invention. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table 1.

[0166] Table 1

[0167] Initial residue Representative replacement Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0168] Furthermore, the amino acid sequence also includes a sequence formed by adding, deleting, modifying and / or replacing at least one amino acid sequence, preferably an amino acid sequence with a homology or sequence identity of at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.

[0169] Methods for determining sequence homology or identity known to those of ordinary skill in the art include, but are not limited to: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG, ed., 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., ed., Stockton Press, New York, 1991 and Carillo, H. and Lipman, D., SIAM J. Applied Math., 48:1073 (1988). The preferred method for determining identity is to obtain the largest match between the sequences tested. Methods for determining identity are compiled in publicly available computer programs. Preferred computer program methods for determining the 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). The well-known Smith Waterman algorithm can also be used to determine identity.

[0170] The antibody of the present invention may be a single-chain antibody or a double-chain antibody targeting NKG2D ligand and immune cell membrane protein (eg, human NKG2D ligand and CD3).

[0171] In a preferred example of the present invention, the bispecific cell engager molecule is a single-chain antibody, which includes an anti-NKP46 single-chain antibody segment, a connecting peptide and an NKG2D extracellular domain, wherein the anti-NKP46 single-chain antibody is a conventional single-chain antibody in the art, which includes a heavy chain variable region and a light chain variable region.

[0172] In the present invention, the animal is preferably a mammal, such as a mouse and a macaque.

[0173] In the above content of the present invention, the number of added, deleted, modified and / or substituted amino acids is preferably not more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably not more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, more preferably 15-20%.

[0174] In the above content of the present invention, more preferably, the number of the added, deleted, modified and / or substituted amino acids may be 1-7, more preferably 1-5, more preferably 1-3, more preferably 1-2.

[0175] Recombinant protein

[0176] The present invention also provides a recombinant protein, which comprises the cell engager molecule of the present invention.

[0177] The recombinant protein of the present invention may include monomers, dimers, or multimers of the cell engager molecules of the present invention; or a multispecific (eg, trispecific) cell engager.

[0178] The preparation method of the recombinant protein is a conventional preparation method in the art. The preparation method is preferably: obtaining by separation from an expression transformant that recombinantly expresses the protein or obtaining by artificially synthesizing a protein sequence. The separation from the expression transformant that recombinantly expresses the protein is preferably the following method: cloning a nucleic acid molecule encoding the protein and having a point mutation into a recombinant vector, transforming the obtained recombinant vector into a transformant, obtaining a recombinant expression transformant, and culturing the obtained recombinant expression transformant to obtain the recombinant protein.

[0179] Nucleic Acids

[0180] The present invention also provides a polynucleotide molecule encoding the above-mentioned cell adapter molecule. The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide can be the same as the coding region sequence of the cell adapter of the present invention or a degenerate variant. As used herein, "degenerate variant" in the present invention refers to a nucleic acid sequence that encodes an amino acid sequence identical to the polypeptide of the present invention, but has a different coding region sequence.

[0181] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only a mature polypeptide; a coding sequence of a mature polypeptide and various additional coding sequences; a coding sequence of a mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0182] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may include additional coding and / or non-coding sequences.

[0183] The present invention also relates to polynucleotides that hybridize with the above-mentioned 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 with the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) addition of denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably at least 95%. In addition, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the cell engager molecule described in the first aspect of the present invention.

[0184] The full-length nucleotide sequence of the cell adapter molecule of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short. Usually, a long fragment of the sequence can be obtained by synthesizing multiple small fragments first and then connecting them. In addition, the coding sequence and expression tag (such as 6His) can be fused together to form a fusion protein.

[0185] Vectors and host cells

[0186] The present invention also provides a recombinant expression vector comprising the nucleic acid.

[0187] The recombinant expression vector can be obtained by conventional methods in the art, that is, by connecting the nucleic acid molecule of the present invention to various expression vectors. The expression vector is any conventional vector in the art, as long as it can carry the aforementioned nucleic acid molecule. The vector preferably includes various plasmids, cosmids, phages or virus vectors, etc.

[0188] The present invention also provides a recombinant expression transformant comprising the recombinant expression vector.

[0189] Wherein, the preparation method of the recombinant expression transformant is a conventional preparation method in the art, preferably: the above-mentioned recombinant expression vector is transformed into a host cell to obtain it. The host cell is any conventional host cell in the art, as long as it can satisfy the above-mentioned recombinant expression vector to stably replicate itself, and the nucleic acid carried can be effectively expressed. Preferably, the host cell is E.coli TG1 or E.coli BL21 cell, or HEK-293T or CHO cell. The aforementioned recombinant expression plasmid is transformed into a host cell to obtain a preferred recombinant expression transformant of the present invention. Wherein the transformation method is a conventional transformation method in the art, preferably a chemical transformation method, a heat shock method or an electroporation method.

[0190] In a preferred embodiment of the present invention, the available vectors include: pCDH, pTOMO, pGEM, pELNS, pMSGV, or a combination thereof.

[0191] In a preferred embodiment of the present invention, the host cells that can be used include: T cells, NK cells, or a combination thereof.

[0192] Preparation of cell engager molecules

[0193] The method for preparing the sequence of the DNA molecule of the cell engager molecule or its fragment of the present invention is preferably to fuse the coding sequences of the ligand segment and the antibody segment together to form a single-chain antibody. In addition, it can be obtained by conventional techniques, such as PCR amplification or genomic library screening.

[0194] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0195] In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is shorter. Usually, a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.

[0196] At present, the DNA sequence encoding the cell adapter of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0197] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0198] The host cell can be a prokaryotic cell, such as a bacterial cell, a lower eukaryotic cell, such as a yeast cell, or a higher eukaryotic cell, such as a mammalian cell. Preferred cells include (but are not limited to): T cells.

[0199] Typically, the transformed host cells are cultured under conditions suitable for the expression of the antibodies of the present invention, and then purified using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc., conventional separation and purification means well known to those skilled in the art to obtain the antibodies of the present invention.

[0200] The resulting cell engager can be identified by conventional means. For example, its binding specificity can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Its binding affinity can be determined, for example, by Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0201] The cell adapter of the present invention can be expressed in cells, on cell membranes, or secreted outside cells. If necessary, the recombinant protein can be separated and purified by various separation methods using its 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, treatment with protein precipitants (salting out method), centrifugation, osmotic sterilization, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0202] Conventional methods for preparing bispecific cell engager molecules in the art include the following two methods:

[0203] 1. First, the bispecific antibody gene was prepared by PCR, and then the gene was cloned into the expression vector pRB199 and transformed into the Escherichia coli strain BL21 (λDE3) to prepare inclusion bodies. Subsequently, 6Mguanidine-HCl and DET (dithioerythritol) were added to the inclusion bodies for denaturation, and then diluted 100 with the renaturation buffer, mixed rapidly at 4°C, and then incubated at 4°C for 72h to refold the protein. After renaturation, 0.1MTris and 0.5M NaCl were added at a ratio of 1:10 for dialysis, and filtered (0.2μm) after three repetitions, followed by metal ion affinity chromatography. Subsequently, a fast protein liquid chromatography was used (BioLogic DuoFlow 10System; Bio-Rad) for purification, and a histidine tag fusion protein purification column was used for separation. The protein was eluted by imidazole step gradient at a flow rate of 1 ml / min. The product was processed by column (Sartorius Stedim Biotech) to remove proteins with a molecular weight greater than 10,000, dialyzed with PBS, and sterilized by filtration. After the concentration was determined, SDS / PAGE was used for silver staining identification (see PNAS, 2013, 110(1): 270-275);

[0204] 2. Infect CHO cells with lentivirus containing bispecific antibodies, and after culturing for 72 hours after infection, fluorescent expression of CHO cells can be observed. Expand the culture of the successfully infected cell lines. CHO cells that stably express bispecific antibodies can continuously secrete and express. Collect the cell supernatant for protein purification and concentration. Subsequently, a fast protein liquid chromatography (BioLogic DuoFlow 10 System; Bio-Rad) was used for purification, and a histidine tag fusion protein purification column was used for separation. Use five times the volume of the sample's equilibrium buffer to pass through the nickel column at a flow rate of 0.5-1ml / min. After equilibrium, flow the sample through the nickel column at a flow rate of 0.5ml / min. Wash the nickel column with five times the volume of equilibrium buffer to wash away the background protein until the 280nm absorbance of the eluate is 0. The target protein is eluted with imidazole at a flow rate of 0.5ml / min. The protein is then concentrated with an ultrafiltration tube and the salt solution is replaced. After the concentration was determined, western blot was used for identification (see Oncoimmunology, 2015, 4(4): e989776).

[0205] Those skilled in the art can make routine selections or equivalent modifications to the above methods to prepare or produce the bispecific cell engager molecules of the present invention.

[0206] Antibody-drug conjugates (ADCs)

[0207] The term "antibody-drug conjugate (ADC)" used in the present invention refers to a conjugate formed by a cell engager molecule of the present invention and an effector molecule.

[0208] Typically, the antibody-drug conjugate comprises the cell engager molecule and the effector molecule, wherein the cell engager molecule is coupled to the effector molecule, and preferably chemically coupled. Wherein, the effector molecule is preferably a drug with therapeutic activity. In addition, the effector molecule can be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug or a radionuclide.

[0209] The cell engager molecule of the present invention and the effector molecule may be coupled via a coupling agent. Examples of the coupling agent may be any one or more of a non-selective coupling agent, a coupling agent utilizing a carboxyl group, a peptide chain, and a coupling agent utilizing a disulfide bond. The non-selective coupling agent refers to a compound that forms a covalent bond between the effector molecule and the cell engager molecule, such as glutaraldehyde. The coupling agent utilizing a carboxyl group may be any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (the coupling site is an acylhydrazone).

[0210] Certain residues on the cell engager molecule (such as Cys or Lys, etc.) are used to connect to a variety of functional groups, including imaging agents (such as chromophores and fluorescent groups), diagnostic agents (such as MRI contrast agents and radioisotopes), stabilizers (such as ethylene glycol polymers) and therapeutic agents. Cell engager molecules can be coupled to functional agents to form cell engager molecule-functional agent conjugates. Functional agents (such as drugs, detection agents, stabilizers) are coupled (covalently linked) to cell engager molecules. Functional agents can be directly or indirectly connected to cell engager molecules through linkers.

[0211] Cell engager molecules can be coupled to drugs to form antibody drug conjugates (ADCs). Typically, ADCs contain a linker between the drug and the cell engager molecule. The linker can be a degradable or non-degradable linker. Degradable linkers are typically easily degraded in the intracellular environment, such as degradation of the linker at the target site, thereby releasing the drug from the cell engager molecule. Suitable degradable linkers include, for example, enzyme-degradable linkers, including linkers containing peptidyl that can be degraded by intracellular proteases (such as lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronide-containing linkers that can be degraded by glucuronidase. Peptide linkers can include, for example, dipeptides, such as valine-citrulline, phenylalanine-lysine or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (such as linkers that hydrolyze when the pH is less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (such as disulfide linkers). Non-degradable linkers typically release the drug under conditions where the cell engager molecule is hydrolyzed by proteases.

[0212] The drug can be any cytotoxic, cytostatic or immunosuppressive drug. In an embodiment, a linker connects the cell engager molecule and the drug, and the drug has a functional group that can form a bond with the linker. For example, the drug can have an amino group, a carboxyl group, a sulfhydryl group, a hydroxyl group, or a keto group that can form a bond with the linker. In the case where the drug is directly connected to the linker, the drug has a reactive group that reacts before being connected to the cell engager molecule.

[0213] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folate antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. Examples of particularly useful cytotoxic drugs 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.

[0214] 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 process. For example, a cysteine ​​residue is reacted with a reactive moiety of a linker in a first step, and in a subsequent step, a functional group on the linker reacts with a drug to form an ADC.

[0215] Typically, the functional groups on the linker are selected to react specifically with the appropriate reactive groups on the drug moiety. As a non-limiting example, an azide-based moiety can be used to react specifically with a reactive alkynyl group on the drug moiety. The drug is covalently attached to the linker by a 1,3-dipolar cycloaddition between the azide and the alkynyl group. 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 Bioconjugation Technology, 2nd Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will appreciate that when a complementary pair of reactive functional groups is selected for selective reaction of the drug moiety and the linker, each member of the complementary pair can be used for both the linker and the drug.

[0216] Test Uses and Kits

[0217] The bispecific cell engager molecules of the invention or their ADCs can be used in detection applications, for example, for detecting a sample to provide diagnostic information.

[0218] In the present invention, the sample (specimen) used includes cells, tissue samples and biopsy specimens. The term "biopsy" used in the present invention should include all types of biopsies known to those skilled in the art. Therefore, the biopsy used in the present invention may include, for example, a resection sample of a tumor, a tissue sample prepared by an endoscopic method or a puncture or needle biopsy of an organ.

[0219] Samples used in the present invention include fixed or preserved cell or tissue samples.

[0220] The present invention also provides a kit containing the cell adapter of the present invention (or its fragment), 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 fixed to a detection plate.

[0221] Pharmaceutical composition

[0222] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition, which contains the above-mentioned cell engager or its active fragment or its fusion protein or its ADC or corresponding immune cells, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary depending on the nature of the formulated substance and the disease to be treated.

[0223] The prepared pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration. Typically, the administration route of the pharmaceutical composition of the present invention is preferably injection or oral administration. The injection preferably includes intravenous injection, arterial injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in the form of solid, semi-solid or liquid, and can be an aqueous solution, non-aqueous solution or suspension, more preferably tablets, capsules, granules, injections or infusions, etc.

[0224] The cell engager of the present invention may also be expressed in cells by a nucleotide sequence for use in cell therapy.

[0225] The pharmaceutical composition of the present invention is a pharmaceutical composition for preventing and / or treating diseases associated with abnormal expression or function of uPAR and / or CD3.

[0226] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the cell adapter (or its conjugate) of the present invention or a safe and effective amount (1×10 3 -1×10 8 cells / ml, preferably 1×10 4 -1×10 7 The invention relates to engineered immune cells of the invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 5 milligrams / kg body weight per day. In addition, the polypeptide of the present invention can also be used together with other therapeutic agents.

[0227] 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.

[0228] In the present invention, preferably, the pharmaceutical composition of the present invention further comprises one or more pharmaceutical carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and the pharmaceutical carrier can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, preferably including a pharmaceutically acceptable excipient, filler or diluent, etc. More preferably, the pharmaceutical composition comprises 0.01 to 99.99% of the above-mentioned protein and 0.01 to 99.99% of a pharmaceutical carrier, and the percentage is the mass percentage of the pharmaceutical composition.

[0229] In the present invention, preferably, the amount of the pharmaceutical composition administered is 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 determined on an individual basis and will be based in part on considerations of the symptoms to be treated and the results sought. Those skilled in the art can determine the effective amount by using the above factors on an individual basis and using experiments that do not exceed routine.

[0230] When using the conjugate, a safe and effective amount of the conjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms / kg body weight, and in most cases does not exceed about 50 milligrams / kg body weight, preferably the dose is about 10 micrograms / kg body weight to about 20 milligrams / kg body weight. Of course, the specific dose should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

[0231] Therapeutic applications

[0232] The present invention provides uses of NKG2D ligand-targeted cell engager molecules and the pharmaceutical compositions of the present invention for preventing and / or treating diseases expressing NKG2D ligands.

[0233] Diseases that express NKG2D ligands include tumors, autoimmune diseases, transplant rejection, inflammation, aging, and diseases related to the accumulation of senescent cells.

[0234] The tumor expressing 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 carcinoma, leukemia, lymphoma, glioma, neuroblastoma, melanoma or a combination thereof.

[0235] Diseases associated with the accumulation of senescent cells expressing NKG2D ligands are selected from the following groups: muscular dystrophy, fatty liver, heart failure, atherosclerosis, diabetes, myocardial hypertrophy, osteoporosis, tissue / organ fibrosis, Alzheimer's disease, Parkinson's syndrome, arthritis, chronic obstructive pulmonary disease and other organ degenerative diseases caused by cell aging, or a combination thereof.

[0236] The autoimmune disease expressing a 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 metabolic syndrome associated with type 2 diabetes.

[0237] The universal cell engager molecules of the present invention can also be used as a type of vaccine for ex vivo immunization and / or in vivo therapy of mammals. Preferably, the mammal is a human.

[0238] In addition to the use of cell-based vaccines with respect to immune cells ex vivo, the present invention also provides compositions and methods for use in vivo to enhance the immune response to a targeted antigen in a patient.

[0239] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the characteristics of the patient's condition, the type and severity of the disease - although the appropriate dosage can be determined by clinical trials.

[0240] When an "immunologically effective amount", "anti-tumor effective amount", "anti-aging effective amount" or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in age, weight, aging tissue size, degree of aging and condition of the patient (subject).

[0241] Main advantages of the present invention

[0242] The bispecific cell engager molecule constructed by the present invention simultaneously targets NKG2D ligand and NKp46 antigen, and is directly infused into the body, or uses cells in the body (such as NK cells, T lymphocytes, CAR-NK cells, etc.) to carry and continuously express the antibody protein in the body, so that the bispecific cell engager molecule can exert a killing effect in the body. Its main advantages include:

[0243] 1) High targeting: Bispecific cell engager molecules targeting abnormal cells with upregulated expression of NKG2D ligands can effectively bridge target cells and natural killer cells, with stable binding and strong lethality.

[0244] 2) High safety: NKG2D ligand is an important target for natural immune cells to eliminate abnormal cells and tumor cells, and its expression on the surface of normal cells is strictly regulated; the NKG2D-NKG2D ligand signal chain has undergone a long period of natural selection and is highly safe. In addition, a large number of immunotherapies targeting NKG2D ligands are currently undergoing clinical trials, and no serious treatment-related side effects have been found.

[0245] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0246] The sequences involved in the embodiments of the present invention are shown in the following table.

[0247] Table 2 Sequence Listing

[0248]

[0249]

[0250]

[0251] Example 1 Preparation of NKG2D-NKp46 Protein

[0252] 1.1 Vector construction

[0253] Will be like Figure 1 After the target gene nucleotide sequence of the structure shown in A was synthesized, it was cloned into the lentiviral vector pCDH-CMV-MCS-EF1-Puro (see Myeloid Leukemia. Mol Ther, 2016. 24 (9): p. 1615-26.) through the EcoR I and Swa I restriction sites. After the cloned vector was verified to be correct by restriction digestion and sequencing, it was transformed into competent Escherichia coli (Stbl3) and expanded, and then extracted with the endotoxin-free midi extraction kit of QIAGEN and identified by Hind III restriction digestion.

[0254] 1.2 Virus packaging

[0255] HEK-293T cells were cultured in a 15 cm dish for virus packaging. When the confluence of HEK-293T cells reached about 90%, transfection was performed, and 2 ml of OPTIMEM-dissolved plasmid mixture (core plasmid 20 ug, pCMVΔR8.9 10 ug, PMD2.G 4 ug) was prepared; 2 ml of OPTIMEM and 68 ul of lipo8000 were placed in another centrifuge tube. After standing at room temperature for 5 min, the plasmid complex was added to the liposome complex and stood at room temperature for 20 min. The above mixture was added dropwise to HEK-293T cells, and the culture medium was removed after incubation at 37°C for 6 hours. Preheated complete culture medium was added again. After collecting the viral supernatant at 48 hours and 72 hours, centrifuge at 3000 rpm at 4°C for 20 minutes. After filtering with a 0.45 um filter membrane, the virus was concentrated by centrifugation at 25000 rpm at 4°C for 2.5 hours. The concentrated virus was dissolved in 30ul virus lysis buffer overnight, and the virus titer was detected by QPCR.

[0256] 1.3 Protein preparation and purification

[0257] Take the above virus to infect CHO cells, and add 1ug / ml puromycin for selection for 48h after 24h. After the selection, the CHO cells continue to be cultured for 7d, and the culture supernatant is collected and filtered with a 0.22um filter membrane. The antibody with His tag is obtained from the expression supernatant using an affinity chromatography column. The equilibrium buffer is 900ml of 0.5M Nacl and 20mM sodium phosphate buffer, PH=7.4; the elution buffer is 900ml of 0.5M imidazole, 0.5M Nacl, and 20mM sodium phosphate buffer, PH=7.4. The NKG2D-NKp46 bispecific antibody is obtained after passing through a cation exchange column, and finally the solution is replaced with PBS buffer for concentration. The purified NKG2D-NKp46 protein SDS-PAGE electrophoresis is as follows Figure 1 B. The Western Blot results are shown in 1B, and the molecular weight is consistent with the theory.

[0258] 1.4 NKG2D-NKp46 binding assay with NK92 cells

[0259] (1) Resuspend NK92 cells in 200ul 1xPBS (containing 2% FBS);

[0260] (2) Add NKp46 protein at a final concentration of 100 μg / ml to the resuspended cells, mix well, and incubate on ice for 120 min. Vortex the cells every 10 min, centrifuge at 500 g for 5 min, and discard the supernatant;

[0261] (3) Add 1 ml of 1xPBS (containing 2% FBS) to resuspend the cells and centrifuge at 500 g for 5 min;

[0262] (4) Repeat the steps;

[0263] (5) Add anti-His antibody and mix well, place on ice and incubate for 60 min, vortex the cells every 10 min, centrifuge at 500 g for 5 min, and discard the supernatant;

[0264] (6) Add cy3-labeled goat anti-rabbit secondary antibody, incubate at room temperature for 30 min, centrifuge at 500 g for 5 min, and discard the supernatant;

[0265] (7) Add 1 ml of 1xPBS (containing 2% FBS) to resuspend the cells and centrifuge at 500 g for 5 min;

[0266] (8) Repeat step 7;

[0267] (9) Flow cytometry was used to detect the binding rate. The results were as follows: Figure 1 C. The results showed that NKG2D-NKp46 could bind to NK92 cells.

[0268] Example 2 NKG2D-NKp46 protein promotes NK92 cells to kill tumor cells

[0269] 2.1 Detection of NKG2D ligand expression in tumor cells

[0270] (1) MHCC97H cells were collected by trypsin digestion, washed three times with 1xPBS, and resuspended in 200ul 1xPBS (containing 2% FBS) to adjust the cell concentration to 1x10 6 cell / ml;

[0271] (2) Add NKG2D ligand antibody to the resuspended cells, mix well, and incubate on ice for 120 min. Vortex the cells every 10 min, centrifuge at 500 g for 5 min, and discard the supernatant;

[0272] (3) Add 1 ml of 1xPBS (containing 2% FBS) to resuspend the cells and centrifuge at 500 g for 5 min;

[0273] (4) Repeat the steps;

[0274] (5) Flow cytometry was used to detect the expression of NKG2D ligands. Figure 2 As shown in A, the expressions of MICA and ULBP2 were significantly increased in MHCC97H cells.

[0275] 2.2NKG2D-NKp46 protein promotes NK92 cells to kill tumor cells

[0276] MHCC97H cells were co-cultured with NK92 cells at an effector-target ratio of 1:5 (NK92 cells were effector cells, with a concentration of 1*10 5 / mL, 100uL per well; tumor cells are target cells, concentration 2*10 4 / mL, 100uL per well), and added 100ug / ml NKG2D-NKp46 protein, and incubated for 6 hours. The cell killing effect was detected using the Promega fluorescence detection kit. First, the cells were treated with 30ul 1*PLB lysis buffer for 20 minutes, and 30ul substrate was added to each well and immediately detected using a BioTek microplate reader. Cytotoxic killing cells = 1-target cell fluorescence value when containing effector cells / target cell fluorescence value when there are no effector cells. The results are shown in Figure 2 As shown in B, after adding NKG2D-NKp46 protein, the killing of tumor cells by NK92 cells was significantly increased.

[0277] Example 3 Upregulation of NKG2D ligand expression in senescent cells

[0278] 3.1 Construction of a cell senescence model by overexpressing p16 protein using the Tet-on system

[0279] (1) 3×10 5 The cells were plated on 10 cm dishes respectively, and the cell density was about 20% after adherence on the next day;

[0280] (2) After the cells adhered, the Tet-on system overexpressed p16 protein lentivirus was used to infect the cells at a multiplicity of infection (MOI) of 50-100, and polybrene with a stock concentration of 8 mg / mL was added at a ratio of 1:1000 to improve the infection efficiency;

[0281] (3) Second infection with the same amount of virus 24 hours later;

[0282] (4) 4 days after virus infection, puromycin was added at a final concentration of 3 μg / mL for selection;

[0283] (5) The constructed p16 protein-overexpressing cells were transferred to a well plate or culture dish, and after 24 h of adherence, 1 μg / mL dox was added to induce the expression of p16 protein;

[0284] (6) After 8 days of induction, the cells were stained for senescence using the SA-βgal staining kit (CS0030, Sigma). Figure 3 As shown in A, more than 90% of the cells were positive, indicating that the cells were senescent at this time.

[0285] 3.2 Detection of NKG2D ligand transcriptional expression

[0286] (1) After preparing senescent cells as described above, add 1–2 mL of Trizol to a 10 cm dish according to the cell density, place on ice for 5 min, and mix by pipetting with a pipette;

[0287] (2) Pipette 1 mL of lysate from each well into a 1.5 mL EP tube, add 200 μL of chloroform, shake vigorously for 15 seconds, place at room temperature for 5 minutes, and centrifuge (4°C, 12,000 g, 15 minutes);

[0288] (3) Add 450 μL of isopropanol to a new EP tube;

[0289] (4) Carefully aspirate the colorless liquid on the upper layer after centrifugation, add it to an EP tube containing isopropanol, mix well, incubate at room temperature for 10 min, and centrifuge (4°C, 12000g, 10 min);

[0290] (5) Discard the supernatant, add 1 mL of 75% ethanol prepared with RNase-free water to wash the RNA, and centrifuge (4°C, 7500 g, 5 min);

[0291] (6) Carefully remove the supernatant, invert the tube for 5 minutes to dry, and use a pipette to remove the liquid on the wall;

[0292] (7) Add 30 μL RNase Free water to dissolve, place on ice immediately after dissolution, and measure the concentration

[0293] (8) Using the extracted RNA as a template, 2 μg of RNA was reverse transcribed into cDNA using the Thermo Scientific RevertAidTM First StrandcDNA Synthesis Kit. The reaction system is as follows:

[0294] Table 3

[0295]

[0296] (9) Add the reactants to the PCR tube according to the above system, incubate at 65°C for 5 min in a PCR machine, immediately place on ice, and then add the following components to the tube:

[0297] Table 4

[0298]

[0299] Mix gently and centrifuge briefly, then place in a PCR instrument and perform the following reactions: 25°C, 5 min; 42°C, 1 h; 70°C, 5 min;

[0300] (10) Fluorescence real-time quantitative PCR detection of NKG2D ligand expression, the specific operation is based on the Thermo powerupTM The SYBR Green Master Mix (A25742) kit instructions were followed, with the following procedures: 50°C, 2 min; 95°C, 2 min; 95°C, 15 s (40 cycles); 60°C, 1 min (40 cycles); 12°C, forever;

[0301] (11) Export the data in Excel format and calculate the relative expression of NKG2D ligands. The results are as follows Figure 3 As shown in B, the NKG2D ligands MICA and ULBP2 were significantly upregulated.

[0302] 3.2 Detection of NKG2D ligand membrane expression level

[0303] (1) HEIP-P16 cells were collected by trypsin digestion, washed three times with 1xPBS, and resuspended in 200ul 1xPBS (containing 2% FBS) to adjust the cell concentration to 1x10 6 cell / ml;

[0304] (2) Add NKG2D ligand antibody to the resuspended cells, mix well, and incubate on ice for 120 min. Vortex the cells every 10 min, centrifuge at 500 g for 5 min, and discard the supernatant;

[0305] (3) Add 1 ml of 1xPBS (containing 2% FBS) to resuspend the cells and centrifuge at 500 g for 5 min;

[0306] (4) Repeat the steps;

[0307] (5) Flow cytometry was used to detect the expression of NKG2D ligands. Figure 3 As shown in C.

[0308] Example 4 NKG2D-NKp46 protein promotes NK92 cells to kill senescent cells

[0309] Senescent cells induced by P16 overexpression and NK92 cells were inoculated into 96-well plates at a target-effect ratio of 2:1 (senescent cells were target cells and NK92 cells were effector cells) and co-incubated for 8 hours. NKG2D-NKp46 protein (1ng / μl) was added to the experimental group, and irrelevant protein was added to the control group. The number of living cells was first 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 Figure 4 As shown, compared with the control group, NKG2D-NKp46 significantly promoted the killing effect of NK92 cells on senescent cells.

[0310] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A cell engager molecule, characterized in that The cell engager molecules include: (a) a first binding domain, wherein the first binding domain specifically binds to the NKG2D ligand, and the amino acid sequence of the first binding domain is shown in SEQ ID NO: 1; and (b) a second binding domain, wherein the second binding domain specifically binds to NKp46, and the second binding domain comprises an antigen-binding fragment specific for NKp46, wherein the NKp46 antigen-binding fragment is selected from the group consisting of a Fab fragment, a single-chain antibody (scFv), a single-domain antibody, or a combination thereof, Wherein, 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 the following heavy chain complementarity determining region (HCDR): 1) HCDR1 having an amino acid sequence as shown in SEQ ID NO: 3; 2) HCDR2 having an amino acid sequence as shown in SEQ ID NO: 4; 3) HCDR3 having an amino acid sequence as shown in SEQ ID NO: 5; Furthermore, the light chain variable region includes the following light chain complementarity determining region (LCDR): 1) LCDR1 having an amino acid sequence as shown in SEQ ID NO: 6; 2) LCDR2 with the amino acid sequence of YTS; 3) LCDR3 having an amino acid sequence as shown in SEQ ID NO: 7, The CDRs are based on the IMGT numbering and definition scheme; or The heavy chain complementary determining region of the anti-NKp46 single domain antibody comprises: 1) HCDR1 having an amino acid sequence as shown in SEQ ID NO: 3; 2) HCDR2 having an amino acid sequence as shown in SEQ ID NO: 4; 3) HCDR3 with an amino acid sequence as shown in SEQ ID NO:

5.

2. The cell engager molecule according to claim 1, wherein The amino acid sequence of the anti-NKp46 scFv fragment is shown in SEQ ID NO:

10.

3. The cell engager molecule according to claim 1, wherein The amino acid sequence of the heavy chain variable region of the anti-NKp46 Fab fragment is shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

9.

4. A polynucleotide encoding the cell engager molecule according to any one of claims 1 to 3. A vector comprising the polynucleotide according to claim 4.

6. An engineered host cell, the host cell comprising the vector of claim 5 or the polynucleotide of claim 4 integrated into its genome.

7. An antibody conjugate, comprising: (a) an antibody portion selected from the group consisting of a cell engager molecule as described in claim 1; and (b) a conjugated moiety conjugated to the antibody portion, wherein the conjugated moiety is selected from the group consisting of a detectable label, a drug, or a combination thereof.

8. A pharmaceutical composition comprising: (a) an active ingredient selected from the group consisting of a cell engager molecule according to claim 1, a host cell according to claim 6, an antibody conjugate according to claim 7, or a combination thereof; and (b) one or more pharmaceutically acceptable carriers, diluents, fillers, binders, excipients, or combinations thereof.

9. Use of the cell engager molecule according to claim 1, or the host cell according to claim 6, or the antibody conjugate according to claim 7, and / or the pharmaceutical composition according to claim 8 in treating diseases associated with upregulated expression of NKG2D ligands, wherein the disease is selected from tumors, senescence or senescent cell accumulation-related diseases, in, The tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), lung cancer, ovarian cancer, colorectal cancer, liver cancer, gallbladder cancer, biliary tract cancer, gastric cancer, pancreatic cancer, kidney cancer, prostate cancer, breast cancer, bladder cancer, nasopharyngeal carcinoma, glioma, neuroblastoma, melanoma, or a combination thereof; The senescent cell accumulation-related disease is selected from the following group: muscular dystrophy, fatty liver, heart failure, atherosclerosis, diabetes, myocardial hypertrophy, osteoporosis, Alzheimer's disease, Parkinson's syndrome, arthritis, chronic obstructive pulmonary disease, or a combination thereof.

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