Use of NKG2D-based cellular adaptor molecules in the elimination of senescent cells

Abstract

The present invention provides the use of NKG2D-CD3 and NKG2D-CD16 cellular adaptor molecules in the elimination of senescent cells and the treatment of diseases associated with the accumulation of senescent cells. The cellular adaptor molecules comprise an NKG2D extracellular domain, a connecting segment, and a CD3 or CD16 binding domain, and by bridging senescent cells and immune cells, efficiently induce the death of senescent cells with high NKG2D ligand expression, making them useful for the elimination of NKG2D ligand-expressing senescent cells and the treatment of aging-related diseases.

Description

[Technical Field]

[0001] The present invention relates to the field of biomedicine, and in particular to the use of NKG2D-CD3 and NKG2D-CD16 cellular adaptor molecules in the elimination of senescent cells and the treatment of associated diseases. [Background technology]

[0002] Cellular senescence is the process by which cells enter a permanent cell cycle arrest in response to various stresses and continues throughout the life cycle of an organism. Cellular senescence and individual aging are related but distinct concepts. Even in aging individuals, the number of senescent cells is very low. If an organism's immune system is strong enough to eliminate senescent cells as needed, they can remain in the body for a while and perform beneficial functions such as promoting tissue regeneration, wound repair, and reducing the incidence of cancer cells. However, if an organism's immune function weakens or sustains persistent damage, the balance between senescent cell generation and elimination is disrupted, and senescent cells begin to accumulate within the organism. Accumulated senescent cells not only directly affect the normal function of tissues and organs, but also continuously secrete numerous inflammatory factors, leading to tissue and organ damage and causing numerous diseases, such as fatty liver, liver fibrosis, pulmonary fibrosis, diabetes, osteoporosis, muscle atrophy, and atherosclerosis. Due to factors such as modern lifestyle changes and poor dietary habits, the incidence of these diseases is gradually increasing among young individuals, attracting societal attention. Preclinical data have shown that the removal of accumulated senescent cells in human disease models, such as mice, rats, and monkeys, can delay, prevent, or alleviate over 40 related diseases. Clinical trials are also underway to treat diseases such as renal fibrosis, diabetes, obesity, renal failure, age-related osteoporosis, and osteoarthritis through the removal of senescent cells. Currently, methods for removing senescent cells have focused primarily on the search for small molecule compounds that can selectively remove senescent cells, such as dasatinib, quercetin, and ABT263. However, these compounds are less effective at removing senescent cells or have significant toxic and adverse effects. For example, ABT-263 can cause transient thrombocytopenia and neutrophil granulocytopenia, while dasatinib can cause severe pulmonary arterial hypertension. Therefore, there is a strong need in this field to develop safer and more effective methods for removing senescent cells. Bispecific antibodies have become one of the hot topics in drug research and development in recent years, with many biotechnology companies working on their development. Breakthroughs have already been achieved in the fields of cancer, autoimmune diseases, and inflammatory diseases.Bispecific cell adaptors are a type of bispecific antibody that selectively recruit immune effector cells (bispecific T cell engagers (BiTEs) for T cells and bispecific NK cell engagers (BiKEs) for NK cells) to target cells, establish an immune synapse, activate immune effector cells, and secrete effector molecules such as telomerase and perforin to eliminate target cells. Bispecific cell adaptors have higher sensitivity and specificity than conventional monoclonal antibodies.

[0003] Therefore, there is a strong demand in this field for the development of technical means for safely and effectively removing senescent cells. Summary of the Invention

[0004] An object of the present invention is to provide a method for specifically and efficiently eliminating senescent cells using NKG2D-CD3 and NKG2D-CD16 cell adaptors.

[0005] In a first aspect of the present invention there is provided the use of an NKG2D ligand and a cellular adaptor to target an immune cell, comprising: (i) Removal of senescent cells; (ii) prevention and / or treatment of diseases associated with the accumulation of senescent cells; (iii) Delay of individual aging For the manufacture of drugs used in the cellular adaptor molecule comprises a first binding domain and a second binding domain; The first binding domain specifically binds to an NKG2D ligand, and the second binding domain specifically binds to a membrane protein on the surface of an immune cell. In another preferred embodiment, the first binding domain is the NKG2D extracellular domain. In another preferred embodiment, the first binding domain comprises a polypeptide set forth in SEQ ID NO: 1, or a polypeptide having 80% or more similarity to the sequence set forth in SEQ ID NO: 1 and capable of binding to an NKG2D ligand. In another preferred embodiment, the second binding domain specifically binds to CD3 or CD16. In another preferred embodiment, 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 either absent or a signal peptide sequence. D1 is the first binding domain. L1 is either absent or a connecting peptide. D2 is the second binding domain. T is either absent or a labeled protein.)

[0006] In another preferred embodiment, the second binding domain has a structure selected from the group consisting of a single domain antibody (sdAb), a single chain antibody (scFv), an antigen-binding fragment (Fab fragment), a ligand, or a multimer thereof, or a combination thereof.

[0007] In another preferred embodiment, the second binding domain is a CD3 binding domain, which can specifically bind to CD3 on the surface of T cells.

[0008] In another preferred embodiment, the CD3 binding domain has the amino acid sequence set forth in SEQ ID NO:2 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 identity to the amino acid sequence set forth in SEQ ID NO:2.

[0009] In another preferred embodiment, the second binding domain is a CD16 binding domain, which can specifically bind to CD16 on the surface of natural killer cells.

[0010] In another preferred embodiment, the CD16 binding domain has an amino acid sequence set forth in SEQ ID NO: 3 or 4, 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 identity to the amino acid sequence set forth in SEQ ID NO: 3.

[0011] In another preferred embodiment, the CD16 binding domain is a human IgG F C fragment.

[0012] In another preferred embodiment, the human IgG F C fragment has the amino acid sequence set forth in SEQ ID NO:5 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 identity to the amino acid sequence set forth in SEQ ID NO:5.

[0013] In another preferred embodiment, the tagged protein T is selected from a His tag and a FLAG tag.

[0014] In another preferred embodiment, the connecting peptide is a glycine-serine connecting peptide.

[0015] In another preferred embodiment, the connecting peptide has the formula (G4S)n, where n is 1, 2, 3, 4, or 5.

[0016] In another preferred embodiment, the senescent cells are selected from the group consisting of lung cells, adipocytes, kidney cells, muscle cells, or combinations thereof.

[0017] In another preferred embodiment, the senescent cells are human fetal lung cells HEL1.

[0018] In another preferred embodiment, the senescent cells are naturally or artificially induced to senesce.

[0019] In another preferred embodiment, the method for artificially inducing senescence includes inducing senescence by DNA damage, inducing senescence by overexpression of P16, inducing senescence by telomere shortening, or a combination thereof.

[0020] In another preferred embodiment, the expression of an NKG2D ligand in the senescent cells is upregulated.

[0021] In another preferred embodiment, the upregulation of NKG2D ligand expression means that the ratio of the expression level of NKG2D ligand in senescent cells (F1) to the expression level in normal cells (F0) (i.e., F1 / F0) is ≧1.5, preferably ≧2, and more preferably ≧2.5.

[0022] In another preferred embodiment, the drug is used for the prevention and / or treatment of diseases caused by the accumulation of senescent cells.

[0023] In another preferred embodiment, the NKG2D ligand includes, but is not limited to, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6. In another preferred embodiment, the geriatric disease is selected from the group consisting of heart failure, atherosclerosis, diabetes, myocardial hypertrophy, osteoporosis, tissue / organ fibrosis, Alzheimer's disease, Parkinson's syndrome, organ degenerative diseases caused by cellular senescence such as arthritis, or a combination thereof.

[0024] In a second aspect of the present invention, there is provided a pharmaceutical composition comprising: (a) NKG2D ligands and cellular adaptor molecules that target immune cells; (b) a drug that eliminates senescent cells other than (a), and (c) A pharmaceutically acceptable carrier, diluent, or excipient.

[0025] In another preferred embodiment, in component (b), the other anti-aging drug includes another drug that can specifically eliminate senescent cells.

[0026] In another preferred embodiment, component (b) comprises a small molecule compound capable of specifically eliminating senescent cells, preferably selected from the group consisting of dasatinib, quercetin, ABT263, ABT737, piperlonguminin, or a combination thereof.

[0027] In a third aspect of the present invention, there is provided a pharmaceutical composition comprising: (a) NKG2D ligands and cellular adaptor molecules that target immune cells; (b) drugs capable of upregulating the expression of NKG2D ligands, and (c) A pharmaceutically acceptable carrier, diluent, or excipient.

[0028] In another preferred embodiment, component (b) comprises an agent capable of upregulating the expression of NKG2D ligands on the cell surface, preferably selected from the group consisting of etoposide, cisplatin, vincristine, taxol, or a combination thereof.

[0029] In another preferred embodiment, the drug composition is a liquid drug composition.

[0030] In another preferred embodiment, the pharmaceutical composition is an injection.

[0031] Of course, it is 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 described here one by one due to space limitations.

[0032] The following drawings are intended to illustrate specific embodiments of the present invention and are not intended to limit the scope of the present invention as defined by the claims. [Brief explanation of the drawings]

[0033] [Figure 1] Figure 1 shows the production of NKG2D-CD3 protein. (A) Schematic diagram of the NKG2D-CD3 and CD19-CD3 vector structures. (B) 293T cells were transfected with the NKG2D-CD3 and CD19-CD3 vectors, and the culture supernatant was collected, purified, and stained with Coomassie Brilliant Blue. (C) Expression of NKG2D-CD3 and CD19-CD3 in the purified protein was detected using a His antibody. [Figure 2] Figure 2 shows the upregulation of NKG2D ligand expression in senescent cells induced by P16 overexpression. (A) After inducing P16 overexpression in HEL1-P16 cells with tetracycline (DOX), the cells were stained for β-gal. (B) After inducing P16 overexpression in HEL1-P16 cells with tetracycline (DOX), the expression of NKG2D ligand was detected by real-time PCR. (C) After inducing P16 overexpression in HEL1-P16 cells with tetracycline (DOX), the expression of NKG2D ligand was detected by flow cytometry. [Figure 3] Figure 3 shows the promotion of NKG2D-CD3 killing of senescent cells induced by overexpression of P16 in T cells. (A) NKG2D-CD3 cells were incubated with T cells and the binding rate was detected by flow cytometry. (B) NKG2D-CD3 cells were incubated with HEL1-P16 cells induced by DOX senescence and the binding rate was detected by flow cytometry. (C) NKG2D-CD3 cells were cocultured with T cells and senescent cells for 8 hours, and the mortality rate of senescent cells was detected. (D) NKG2D-CD3 cells were cocultured with T cells and senescent cells for 8 hours, and the culture supernatant was aspirated and IFN-γ expression was detected by enzyme-linked immunosorbent assay (ELISA). (E) The half maximal inhibitory concentration (IC50) of NKG2D-CD3 cells against senescent cells induced by overexpression of P16 was measured. [Figure 4]Figure 4 shows the upregulation of NKG2D ligand expression in senescent cells induced by DNA damage. (A) HEL1 cells were treated with etoposide to induce DNA damage, and the cells were stained for β-gal. (B) HEL1 cells were treated with etoposide to induce DNA damage, and the expression of the NKG2D ligand MICA was detected by real-time PCR. (B) HEL1 cells were treated with etoposide to induce DNA damage, and the expression of total NKG2D ligands was detected by flow cytometry using the NKG2D-FC fusion protein. [Figure 5] Figure 5 shows the promotion of NKG2D-CD3 T cell killing of senescent cells induced by DNA damage. (A) NKG2D-CD3 and CD19-CD3 antibodies were incubated with HEL1 cells treated with DMSO or etoposide, respectively, and the binding rate was detected by flow cytometry. (B) NKG2D-CD3 or CD19-CD3 antibodies were added to a co-culture system of T cells and HEL1 cells treated with DMSO or etoposide, and the half maximal inhibitory concentration (IC50) of HEL1 cells was determined after 8 hours. [Figure 6] Figure 6 shows the production of NKG2D-CD16 and NKG2D-FC proteins. (A) Schematic diagram of the structures of NKG2D-CD16, NKG2D-FC, and control vectors. (B) NKG2D-CD16, NKG2D-FC, and control vectors were transfected into 293T cells, and the culture supernatant was collected and purified. Expression of NKG2D-NKp46 and control proteins in the purified proteins was detected using a His antibody. [Figure 7]Figure 7 shows the promotion of NK cell killing of senescent cells by NKG2D-CD16 and NKG2D-FC proteins. (A) NKG2D-CD16, NKG2D-FC, and a control vector were incubated with NK cells, respectively, and the binding rates were detected by flow cytometry. (B) NKG2D-CD16 and NKG2D-FC were incubated with young cells and etoposide-induced senescent cells, respectively, and the binding rates were detected by flow cytometry. (C) NKG2D-CD16, NKG2D-FC, and a control vector were cocultured with NK cells and senescent cells, respectively, for 8 h, and the mortality rate of senescent cells was detected. (D) NKG2D-CD16, NKG2D-FC, and a control vector were cocultured with NK cells and senescent cells, respectively, for 8 h, and the supernatants were aspirated, and IFN-γ expression was detected by enzyme-linked immunosorbent assay (ELISA). DETAILED DESCRIPTION OF THE INVENTION

[0034] After extensive and in-depth research and extensive screening, the present inventors have developed, for the first time, a method for eliminating senescent cells and treating age-related diseases in a subject with high specificity and efficiency using bispecific cell adaptor technology.

[0035] The bispecific cellular adaptor molecule provided by the present invention is composed of three parts: an NKG2D extracellular domain that targets NKG2D ligands on the surface of senescent cells, a linking segment, and a CD3 or CD16 domain that binds to immune cells. The adaptor molecule links to senescent cells via the NKG2D extracellular domain and simultaneously bridges the immune cells, thereby bridging effector immune cells to the surface of senescent cells and achieving the purpose of eliminating senescent cells.

[0036] term In the following, some technical and scientific terms are specifically defined to facilitate a better understanding of the present invention. Unless otherwise clearly defined herein, all other technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it is understood that the present invention is not limited to the specific methods and experimental conditions described, since methods and conditions may vary. Furthermore, it is understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting, and the scope of the present invention is limited only by the appended claims.

[0037] 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 with a specific exemplified numerical value, the term "about" means that the value may vary within 1% from the exemplified numerical 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.).

[0038] The three-letter and one-letter abbreviations for amino acids used in the present invention are as described in J. Biol. Chem, 243, p. 3558 (1968).

[0039] As used herein, the term "treatment" refers to the administration of an internal or external therapeutic agent, including any one of the bispecific cell adaptors and compositions thereof, to a patient suffering from one or more disease symptoms, and the therapeutic agent is known to have a therapeutic effect on those symptoms. Typically, the amount of therapeutic agent administered to the patient is sufficient to effectively alleviate one or more disease symptoms (a therapeutically effective amount).

[0040] As used herein, the terms "any" or "optionally" mean that the subsequently described event or circumstance may be present, but is not required. 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 are not necessarily present, and may be 1, 2, or 3.

[0041] "Sequence identity," as used herein, refers to the degree of identity between two nucleic acid or two amino acid sequences when aligned and compared, including appropriate mutations such as substitutions, insertions, or deletions. The sequence identity between a sequence described herein and a sequence having an identity thereto 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%.

[0042] First Binding Domain The first binding domain of the cellular adaptor used in the present invention specifically binds to an NKG2D ligand (NKG2DL). The NKG2D-NKG2DL signal chain is an important mechanism by which the immune system exerts its immune surveillance function. The bispecific cellular adaptor molecules NKG2D-CD3 and NKG2D-CD16, constructed based on the NKG2D extracellular domain, have also attracted attention in the anti-tumor field. Studies have shown that NKG2D-CD3 and NKG2D-CD16 activate T cells and NK cells, respectively, and efficiently kill tumor cells highly expressing NKG2D ligands in vitro, including acute myeloid leukemia, lymphoma, soft tissue sarcoma, and melanoma. In vivo, NKG2D-CD3 can suppress tumor growth and extend the survival of model mice by targeting tumor cells and immunosuppressive cells. NKG2D ligand expression is also significantly upregulated in senescent cells.

[0043] The first binding domain of the cellular adaptor of the present invention is preferably an extracellular domain derived from NKG2D. In a preferred embodiment, the NKG2D extracellular domain comprises the polypeptide set forth in SEQ ID NO: 1 or a polypeptide having 80% or more (preferably 85% or more, more preferably 90% or more) similarity to the sequence set forth in SEQ ID NO: 1 and capable of binding to an NKG2D ligand.

[0044] Second Binding Domain The second binding domain of the cellular adaptor used in the present invention specifically binds to a membrane protein on the surface of an immune cell, preferably CD3 or CD16.

[0045] The second binding domain contained in the cellular adaptor molecule of the present invention may be an antibody, or an antigen-binding fragment of an antibody, or any other polypeptide capable of specifically binding to CD3 or CD16.

[0046] As used herein, the term "antibody" may refer to an immunologically active antibody fragment, such as a Fab or (Fab')2 fragment, an antibody heavy chain, or an antibody light chain. In one preferred embodiment, the antibody used in the present invention is in the form of a single-chain antibody, and the single-chain antibody (scFv) is the smallest antibody fragment that contains the heavy chain variable region and the light chain variable region of an antibody but lacks a constant region and has all of the antigen-binding site. Generally, an Fv antibody further contains a polypeptide linker between the VH and VL domains and is capable of forming the structure necessary for antigen binding. In another preferred embodiment, the antibody used in the present invention is in the form of a single-domain antibody, and the single-domain antibody (VhH) is the smallest antigen-binding fragment that consists of only one heavy chain variable region of a cloned antibody and has complete function.

[0047] 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. Typically, an antibody binds to an epitope of about 10 -7 Less than M, for example, about 10 -8 M, 10 -9 M or 10-10 It binds with an affinity (KD) less than or equal to M.

[0048] In a preferred embodiment of the present invention, the second binding domain is a CD3-binding domain capable of specifically binding to CD3 on the surface of T cells, and the CD3-binding domain has the amino acid sequence set forth in SEQ ID NO:2 or an amino acid sequence that shares at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or identity with the amino acid sequence set forth in SEQ ID NO:2.

[0049] In another preferred embodiment of the present invention, the second binding domain is a CD16-binding domain capable of specifically binding to CD16 on the surface of natural killer cells. In one embodiment, the CD16-binding domain is an anti-CD16 scFv or single-domain antibody and has the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4, or an amino acid sequence that shares at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or identity with the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4. In another embodiment, the CD16-binding domain is a human IgG F C fragment and has the amino acid sequence set forth in SEQ ID NO: 5, or an amino acid sequence that shares at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or identity with the amino acid sequence set forth in SEQ ID NO: 5.

[0050] Bispecific Cell Adaptor Molecules As used herein, the terms "bispecific cellular adaptor molecule," "bispecific cellular adaptor," "cellular adaptor," "BiTE," and "bispecific antibody" are used interchangeably and all refer to a cellular adaptor molecule provided in the first aspect of the invention that is capable of simultaneously binding to an NKG2D ligand and a protein expressed by an immune cell.

[0051] Bispecific cellular adaptor molecules are composed of two linked protein or polypeptide sequences (antibodies are the most common) that bind to two different target proteins. In a preferred embodiment, the function of the BiTEs of the present invention is determined by a fragment of the extracellular domain of the NKG2D receptor and a binding fragment of the CD3 or CD16 protein. The antibodies of the present invention can simultaneously bind to the NKG2D ligand and the membrane protein CD3 or CD16 on the surface of immune cells, and by linking to senescent cells via the ligand fragment and simultaneously linking to immune cells via the membrane protein fragment on the surface of the immune cells, they effectively bridge effector immune cells to senescent cells and more effectively eliminate senescent cells.

[0052] As used herein, the term "bispecific" refers to a molecule comprising binding domains with at least two different binding specificities. Each binding domain can specifically bind to a target molecule. In some embodiments, the bispecific cellular adaptor is a multimeric molecule comprising two or more peptides. In some embodiments, the binding domain comprises an antigen-binding domain, or variable region, or CDR of an antibody. In some embodiments, the binding domain comprises a ligand or fragment thereof that specifically binds to a target protein.

[0053] The at least two targeting domains of the cellular adaptor molecule of the present invention are optionally linked via a linking peptide, with the sequence of a suitable linking peptide being, but not limited to, (G4S)3.

[0054] In one preferred embodiment of the present invention, the bispecific cell adaptor molecule is a single-chain polypeptide comprising an NKG2D extracellular domain as a first binding domain, a connecting segment, and an anti-CD3 or CD16 single-chain antibody as a second binding domain, wherein the anti-CD3 or CD16 single-chain antibody is a conventional single-chain antibody in the art comprising a heavy chain variable region and a light chain variable region.

[0055] At the same time, those skilled in the art will understand that although the first binding domain of the BiTE of the present invention is preferably a fragment of the extracellular domain of NKG2D, the first binding domain may also be any antibody that specifically binds to NKG2D, or an antigen-binding fragment thereof, as long as the cell-binding effect of the present invention is achieved.

[0056] The antibodies of the present invention may be chimeric, humanized, CDR-grafted and / or modified antibodies that target NKG2D ligands and CD3 (eg, human NKG2D ligands and CD3).

[0057] The antibodies of the invention may be chimeric, humanized, CDR-grafted and / or modified antibodies that target NKG2D ligands and CD16 (eg, human NKG2D ligands and CD16).

[0058] In the present invention, BiTEs of the present invention further include conservative variants thereof, and refer to polypeptides in which, compared to the amino acid sequence of a BiTE of the present invention, 10 or fewer, preferably 8 or fewer, more preferably 5 or fewer, and most preferably 3 or fewer amino acids have been substituted with amino acids having similar or close properties. These conservative variant polypeptides are preferably generated by amino acid substitutions as shown in Table 1. [Table 1]

[0059] Furthermore, the above-mentioned amino acid sequence, which has been further subjected to addition, deletion, modification and / or substitution of at least one amino acid sequence, is preferably an amino acid sequence having a homology or sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.

[0060] Methods for determining sequence homology or identity that are well known to those of ordinary skill 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 I, 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, vol. Math., 48:1073 (1988). A preferred method for determining identity is one that obtains the largest match between the sequences being determined. Methods for determining identity are incorporated into publicly available computer programs. Computer programs suitable for determining identity between two sequences include, but are not limited to, the GCG program pack (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., 1990). The BLASTX program (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990) is publicly available from NCBI and other resources. The well-known Smith-Waterman method can also be used to determine identity.

[0061] In the above-mentioned content of the present invention, the number of added, deleted, modified and / or substituted amino acids is preferably 40% or less of the total number of amino acids in the original amino acid sequence, more preferably 35% or less, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, more preferably 15 to 20%.

[0062] 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.

[0063] Drug Composition The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising the above-mentioned cell adaptor or an active fragment thereof, a fusion protein thereof, an ADC thereof, or corresponding immune cells, and a pharmaceutically acceptable carrier.

[0064] In one embodiment of the present invention, there is provided a pharmaceutical composition comprising: (a) NKG2D and cellular adaptor molecules that bind to membrane proteins on the surface of immune cells; (b) a drug that eliminates senescent cells other than (a), and (c) A pharmaceutically acceptable carrier, diluent, or excipient.

[0065] In a preferred embodiment, component (b) comprises a small molecule compound capable of specifically eliminating senescent cells, preferably selected from the group consisting of dasatinib, quercetin, ABT263, ABT737, piperlonguminine, or a combination thereof.

[0066] In another embodiment of the present invention, there is provided a pharmaceutical composition comprising: (a) NKG2D and cellular adaptor molecules that bind to membrane proteins on the surface of immune cells; (b) drugs capable of upregulating the expression of NKG2D ligands, and (c) A pharmaceutically acceptable carrier, diluent, or excipient.

[0067] In a preferred embodiment, component (b) comprises an agent capable of upregulating the expression of NKG2D ligands on the cell surface, preferably selected from the group consisting of etoposide, cisplatin, vincristine, taxol, or combinations thereof.

[0068] These substances are usually formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier, and the pH value depends on the properties of the formulated substances and the disease to be treated, but is usually around 5-8, preferably around 6-8.

[0069] The formulated pharmaceutical composition can be administered by any conventional route, including, but not limited to, intramuscular, intraperitoneal, intravenous, or topical administration. Typically, the pharmaceutical composition of the present invention is administered by injection or oral administration. The injection route preferably includes intravenous, intraarterial, intramuscular, peritoneal, intradermal, or subcutaneous injection. The pharmaceutical composition may be administered in various dosage forms conventional in the art, preferably in solid, semisolid, or liquid form, such as aqueous, non-aqueous, or suspension form, more preferably in the form of tablets, capsules, granules, injections, or infusions.

[0070] The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001 to 99 wt%, preferably 0.01 to 90 wt%, more preferably 0.1 to 80 wt%) of the above-described monoclonal antibody (or conjugate thereof) of the present invention and a pharmaceutically acceptable vehicle or excipient. Such vehicles include, but are not limited to, saline, buffer solution, glucose, water, glycerin, ethanol, and combinations thereof. The pharmaceutical formulation corresponds to the dosage form. The pharmaceutical composition of the present invention may be an injection, which can be prepared by conventional methods using, for example, physiological saline or an aqueous solution containing glucose and other excipients. In the case of an injection or solution, the pharmaceutical composition is 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. The cell adapter of the present invention can also be used in combination with other therapeutic agents.

[0071] In the present invention, the pharmaceutical composition of the present invention preferably further comprises one or more pharmaceutical carriers. The pharmaceutical carriers are conventional pharmaceutical carriers in the art, and may be any suitable physiologically or pharmaceutically acceptable pharmaceutical auxiliary. The pharmaceutical auxiliary is a conventional pharmaceutical auxiliary in the art, preferably including a pharmaceutically acceptable excipient, filler, or diluent. More preferably, the pharmaceutical composition comprises 0.01-99.99% of the protein and 0.01-99.99% of the pharmaceutical carrier, the percentages being mass percentages in the pharmaceutical composition.

[0072] In the present invention, the dosage of the pharmaceutical composition is preferably an effective amount, which is an amount capable of alleviating or slowing the progression of a disease, degenerative or damaging condition. The effective amount may be determined on an individual basis and may take into account, in part, the symptoms to be treated and the results desired. Those skilled in the art can determine the effective amount based on the above factors, such as individual basis, and through routine experimentation.

[0073] When using the pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, and this safe and effective amount is usually at least about 10 μg / kg body weight, and in many cases, not more than about 50 mg / kg body weight, preferably about 10 μg / kg body weight to about 20 mg / kg body weight. Of course, the specific dosage should be determined taking into account factors such as the mode of administration and the patient's health condition, all of which are within the skill of a skilled physician.

[0074] therapeutic use The present invention provides the use of cellular adaptor molecules that target NKG2D ligands and pharmaceutical compositions of the present invention for preventing and / or treating geriatric diseases.

[0075] The present invention further provides use of a cellular adaptor molecule that targets NKG2D and a pharmaceutical composition of the present invention for the manufacture of a drug for use in (i) removing senescent cells, (ii) delaying aging in an individual, and / or (iii) preventing and / or treating geriatric diseases.

[0076] In a preferred embodiment, the NKG2D ligand is upregulated in the senescent cells by 1.5-fold or more compared to normal cells.

[0077] Here, the geriatric disease 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, organ degenerative diseases caused by cellular aging such as arthritis, or a combination thereof.

[0078] The universal cell adaptor molecules of the present invention are also useful as vaccine types for ex vivo immunization and / or in vivo therapy in mammals, preferably humans.

[0079] In addition to cell-based vaccines for in vitro immunization, the present invention also provides compositions and methods for enhancing immune responses to target antigens in patients in vivo.

[0080] The pharmaceutical composition of the present invention can be administered in a form suitable for the disease to be treated (or prevented). The amount and frequency of administration will depend on factors such as the characteristics of the patient's symptoms, the type of disease, and its severity, and the appropriate dosage will be determined by clinical trials. When referring to an "immunologically effective amount," "anti-aging effective amount," "age-disease-inhibitory effective amount," or "therapeutic amount," the exact amount of the composition of the present invention to be administered will be determined by a physician, taking into account individual differences in the patient's (subject's) age, weight, size of aging tissue, degree of aging, and disease.

[0081] Main advantages of this invention The bispecific cellular adaptor molecule constructed in the present invention simultaneously targets NKG2D ligands and immune cells, and can be directly infused into the body or carried by cells in the body (e.g., NK cells, T cells, CAR-T cells, etc.) to continuously express the antibody protein in the body, allowing the bispecific cellular adaptor molecule to exert its killing effect in the body. Its main advantages include the following: 1) High targeting: The bispecific cell adaptor molecule for NKG2D ligand-positive senescent cells can effectively bridge senescent cells and immune cells, with stable binding and strong killing activity. 2) High safety: NKG2D ligands are important targets for natural immune cells to eliminate senescent cells and tumor cells, and their expression on the surface of normal cells is tightly regulated. The NKG2D-NKG2DL signaling chain has been formed through a long period of natural selection and has a high degree of safety. Currently, a large number of immunotherapies targeting NKG2D ligands are undergoing clinical trials, and no severe treatment-related side effects have been observed.

[0082] The present invention will be further described below with reference to 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. Experimental methods for which specific conditions are not specified in the following examples generally follow conventional conditions, such as those described in Sambrook et al., "Molecular Cloning: A Laboratory Manual" (New York: Cold Spring Harbor Laboratory Press, 1989), or the manufacturer's recommended conditions. Unless otherwise specified, percentages and parts are by weight.

[0083] The sequences according to the present invention are as follows: SEQ ID NO: 1 NKG2D extracellular domain FLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV SEQ ID NO: 2 CD3 binding domain (derived from CD3 monoclonal antibody OKT3) DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVE GGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK SEQ ID NO: 3 CD16 binding domain scFv fragment (cited from patent US11167029B2) QVQLVQSGAEVKKPGESLKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGSAYYYDFADYWGQGTLVTVSS GGGGSGGGGSGGGGSSYVLTQPSSVVAPGQTATISCGGHNIGSKNVHWYQQRPGQSPVLVIYQDNKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQVWDNYSVLFGGGTKLTVL SEQ ID NO: 4 CD16 binding domain VhH fragment (cited from GenBank ABS29544.1) EVQLVESGGGFVQAGESLTLSCTSSTLTFTPYRMAWYRQAPGKQRDLVADISSGDGRTTNYADFAKGRFTISRDNIKNTVFLRMTNLKPEDTAVYYCNTFVSFVGIARSWGQGTQVTVSS SEQ ID NO: 5 IgG antibody FC fragment (derived from margetuximab) PKSCDKTHTCPPCPAPELVGGPSVFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTLRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0084] Example 1 Production of NKG2D-CD3 protein 1.1 Vector construction The nucleotide sequence of the target gene, shown in Figure 1A, was synthesized and cloned into the lentiviral vector pCDH-CMV-MCS-EF1-Puro at the EcoR I and Swa I enzyme cleavage sites (see Myeloid Leukemia. Mol Ther. 2016. 24(9):pp.1615-26). The cloned vector was digested and verified to be correct by sequencing. It was then transformed into susceptible E. coli (Stbl3) and expanded. It was then extracted using QIAGEN's endotoxin-free midiprep kit and identified by digestion with Hind III.

[0085] 1.2 Virus packaging HEK-293T cells were cultured in 15 cm Petri dishes and used for virus packaging. When HEK-293T cells reached approximately 90% confluence, they were transfected with 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. In a separate centrifuge tube, 2 ml of OPTIMEM and 68 μl of lipo8000 were prepared. After incubating at room temperature for 5 minutes, the plasmid complex was added to the liposome complex and incubated at room temperature for 20 minutes. The mixture was added dropwise to the HEK-293T cells and incubated at 37°C for 6 hours, after which the medium was removed and replaced with freshly heated complete medium. The viral supernatants were collected at 48 and 72 hours and then centrifuged at 3000 rpm for 20 minutes at 4°C. After filtering through a 0.45 μm filter, the virus was concentrated by centrifugation at 25,000 rpm at 4°C for 2.5 hours. The concentrated virus was dissolved overnight in 30 μl of virus lysis solution, and the virus titer was detected by QPCR. The results showed that the virus titer met the required level.

[0086] 1.3 Protein production and purification CHO cells were infected with the virus and, after 24 hours, 1 μg / ml puromycin was added and screened for 48 hours. The screened CHO cells were then cultured for 7 days, after which the culture supernatant was collected and filtered through a 0.22 μm filter. His-tagged antibodies were isolated from the expression supernatant by affinity chromatography. 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. NKG2D-CD3 bispecific antibodies were isolated by passing through a cation exchange column and finally concentrated by PBS buffer exchange. SDS-PAGE of the purified NKG2D-CD3 protein is shown in Figure 1B. Western blotting results, shown in Figure 1C, confirmed the predicted molecular weight.

[0087] Example 2 Upregulation of NKG2D Ligand Expression in Senescent Cells 2.1 Construction of a cell senescence model using p16 protein overexpression in the Tet-on system (1) 3×10 5 Each cell was placed in a 10 cm petri dish, and the next day, the density of attached cells was about 20%. (2) After the cells attached, they were infected with the Tet-on system p16 protein overexpressing lentivirus at a multiplicity of infection (MOI) of 50-100, and polybrene (8 mg / mL stock solution) was added at a ratio of 1:1000 to improve the infection efficiency. (3) 24 hours later, a secondary infection was carried out with the same amount of virus. (4) Four days after virus infection, puromycin was added at a final concentration of 3 μg / mL for screening. (5) The constructed cells overexpressing p16 protein were transferred to well plates or petri dishes and allowed to attach for 24 hours, after which 1 μg / mL of dox was added to induce p16 protein expression. (6) After 8 days of induction, the cells were stained for senescence using an SA-β gal staining kit (CS0030, Sigma). As shown in Figure 2A, more than 90% of the cells were positive, indicating that the cells had already senesced at this time.

[0088] 2.2 Construction of a cell senescence model induced by DNA-damaging drugs (1) The cells were spread on a 10 cm dish and the density after attachment was about 50%. (2) After 24 hours, etoposide (Sigma, E1383) was added to a final concentration of 50 μM. (3) After 36 h, the medium was replaced with fresh medium. (4) The cells were then cultured, with the medium changed every three days. After eight days, the cells exhibited a senescent phenotype. Senescence was identified using an SA-β-gal staining kit (CS0030, Sigma), and as shown in Figure 4A, over 90% of the cells were positive, indicating that the cells had already undergone senescence.

[0089] 2.3 Detection of transcriptional expression of NKG2D ligands (1) After preparing senescent cells according to the above method, 1 to 2 mL of Trizol was added to a 10 cm dish depending on the cell density, and the dish was placed on ice for 5 minutes and mixed uniformly by blowing with a pipette tip. (2) 1 mL of the digestion solution from each well was aspirated and placed in a 1.5 mL EP tube, and 200 μL of chloroform was added. The tube was vigorously shaken for 15 seconds, left at room temperature, and centrifuged for 5 minutes (4°C, 12,000 g, 15 minutes). (3) 450 μL of isopropanol was placed in a new EP tube. (4) After centrifugation, the upper layer of colorless liquid was carefully aspirated and placed in an EP tube containing isopropanol, mixed uniformly, incubated at room temperature for 10 minutes, and centrifuged (4°C, 12000 g, 10 minutes). (5) The supernatant was discarded, and the RNA was washed with 75% ethanol prepared with 1 mL of RNase-free water and centrifuged (4°C, 7500 g, 5 min). (6) Carefully remove the supernatant, dry the tube upside down for 5 minutes, and then absorb the liquid on the tube wall with a pipette tip. (7) 30 μL of RNase-free water was added to dissolve the sample. After dissolution, the sample was immediately placed on ice and the concentration was measured.

[0090] (8) The extracted RNA was used as a template and purified using Thermo Scientific RevertAid TM 2 μg of RNA was reverse transcribed into cDNA using the First Strand cDNA Synthesis Kit. The reaction system is shown below. [Table 2]

[0091] (9) The reaction mixture was placed in a PCR tube in the above system, placed in a PCR machine at 65°C for 5 minutes, and then immediately placed on ice. The following components were then added to the tube: [Table 3] The mixture was gently mixed to homogenize and centrifuged instantaneously, then placed in a PCR device and the following reactions were carried out: 25°C, 5 min; 42°C, 1 h; 70°C, 5 min.

[0092] (10) NKG2D ligand expression was detected by fluorescent real-time quantitative PCR. The specific procedure was performed using Thermo Scientific™ PowerUp™. TM The reaction was carried out according to the instructions of the SYBR Green Master Mix (A25742) kit, with the following program: 50°C, 2 min; 95°C, 2 min; 95°C, 15 s (40 cycles); 60°C, 1 min (40 cycles); 12°C, storage forever. (11) The data was output in Excel format, and the relative expression levels of NKG2D ligands were calculated. As shown in Figures 2B and 4B, the expression of NKG2D ligands was significantly upregulated.

[0093] 2.4 Detection of membrane expression levels of NKG2D ligands (1) HEIP cells were collected by digestion with trypsin, 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 The concentration was adjusted to give cells / ml. (2) The NKG2D ligand antibody was added to the resuspended cells, mixed uniformly, and incubated on ice for 120 minutes, during which the cells were vortexed once every 10 minutes. The cells were then centrifuged at 500 g for 5 minutes, and the supernatant was discarded. (3) The cells were resuspended in 1 ml of 1×PBS (containing 2% FBS) and centrifuged at 500 g for 5 minutes. (4) The process was repeated. (5) The expression of NKG2D ligands was detected by flow cytometry, as shown in Figures 2C and 4C.

[0094] Example 3 NKG2D-CD3 protein promotes T cell killing of senescent cells 3.1 Isolation of human peripheral blood T cells (1) Transfer human peripheral blood to a 50 mL centrifuge tube and use RosetteSep TM Cocktail was added to the blood (50 μL / mL blood). (2) After thorough mixing, the mixture was incubated at room temperature for 20 minutes. (3) Preparation of dilution solution: 1640 medium and 1x PBS were mixed in a volume ratio of 1:2. (4) A gradient centrifuge tube was prepared and 15 mL of gradient isolation solution, Ficol Lymphoprep, was placed in it. (5) The diluent and the incubated blood sample were mixed in a 1:1 ratio. (6) The diluted blood sample was gently transferred to isolation medium and centrifuged at 1200 g for 20 minutes. (7) After centrifugation, the entire supernatant was quickly transferred to a new centrifuge tube. (8) 25 mL of the diluted solution was mixed uniformly with the supernatant and centrifuged at 300 g for 10 minutes. (9) The above steps were repeated. (10) T cells were resuspended in 2 mL of T cell complete medium, counted, and used in subsequent experiments.

[0095] 3.2 Detection of binding of NKG2D-CD3 to senescent cells and T cells (1) T cells and senescent cells were resuspended in 200 μl of 1×PBS (containing 2% FBS). (2) The resuspended cells were added with NKG2D-CD3 or control antibody at a final concentration of 100 μg / ml, mixed evenly, and incubated on ice for 120 min, during which the cells were vortexed once every 10 min. The cells were then centrifuged at 500 g for 5 min, and the supernatant was discarded. (3) The cells were resuspended in 1 ml of 1×PBS (containing 2% FBS) and centrifuged at 500 g for 5 minutes. (4) The process was repeated. (5) The anti-His antibody was added and mixed uniformly, and the mixture was placed on ice and incubated for 60 minutes, during which the cells were vortexed once every 10 minutes, centrifuged at 500 g for 5 minutes, and the supernatant was discarded. (6) A fluorescently labeled sheep anti-rabbit secondary antibody was added, and the mixture was incubated at room temperature for 30 minutes, then centrifuged at 500 g for 5 minutes, and the supernatant was discarded. (7) The cells were resuspended in 1 ml of 1×PBS (containing 2% FBS) and centrifuged at 500 g for 5 minutes. (8) Step 7 was repeated. (9) The binding rate was detected by flow cytometry, and as shown in Figures 3A, 3B, and 5A, it was shown that NKG2D-CD3 could effectively bind to both senescent cells and T cells.

[0096] 3.3 NKG2D-CD3 protein promotes T cell killing of senescent cells T cells and senescent cells were seeded into 96-well plates at an effector-target ratio of 2:1 (T cells as effector cells, senescent cells as target cells) and then co-incubated with NKG2D-CD3 or CD19-CD3 protein for 8 hours (cell culture medium: Advance 1640 medium (Gibco) + 10% fetal bovine serum (Gibco) + 1% penicillin and streptomycin (Gibco)). Viable cell counts were counted under a microscope, and the T cell killing efficiency was calculated. Killing efficiency = (number of target cells in the blank group - number of target cells in the co-incubated group) / number of target cells in the blank group * 100%. As shown in Figures 3C, 3E, and 5B, NKG2D-CD3 significantly enhanced the killing of T cells against senescent cells compared to the control group, and the effect was dose-dependent. The culture supernatants were collected, and the IFN-γ levels were measured using an ELISA kit. Statistical analysis was performed using GraphPad Prism software. As shown in Figure 3D, the secretion of IFN-γ was significantly increased after the addition of NKG2D-CD3 compared to the control group.

[0097] Example 4 Production of NKG2D-CD16 and NKG2D-FC proteins The nucleotide sequence of the target gene, shown in Figure 6A, was synthesized and cloned into the lentiviral vector pCDH-CMV-MCS-EF1-Puro via the EcoR I and Swa I enzyme cleavage sites. The cloned vector was digested and verified to be accurate by sequencing. It was then transformed into susceptible E. coli (Stbl3) and expanded. The virus was extracted using QIAGEN's endotoxin-free midiprep kit and used for virus packaging, as described in Example 1. The packaged virus was then infected into CHO cells for protein production. The produced NKG2D-CD16 and NKG2D-FC proteins were detected using His-tag antibodies, and the protein sizes were consistent with expectations, as shown in Figure 6B.

[0098] Example 5 NKG2D-CD16 and NKG2D-FC proteins promote NK cell killing of senescent cells 5.1 Detection of association of NKG2D-CD16 and NKG2D-FC with NK cells and senescence (1) NK cells or senescent cells were resuspended in 200 μl of 1×PBS (containing 2% FBS). (2) The resuspended cells were added with NKG2D-CD16 and NKG2D-FC proteins at a final concentration of 100 μg / ml, mixed evenly, and incubated on ice for 120 min, during which the cells were vortexed once every 10 min. The cells were then centrifuged at 500 g for 5 min, and the supernatant was discarded. (3) The cells were resuspended in 1 ml of 1×PBS (containing 2% FBS) and centrifuged at 500 g for 5 minutes. (4) The process was repeated. (5) The anti-His antibody was added and mixed uniformly, and the mixture was placed on ice and incubated for 60 minutes, during which the cells were vortexed once every 10 minutes, centrifuged at 500 g for 5 minutes, and the supernatant was discarded. (6) A fluorescently labeled sheep anti-rabbit secondary antibody was added, and the mixture was incubated at room temperature for 30 minutes, then centrifuged at 500 g for 5 minutes, and the supernatant was discarded. (7) The cells were resuspended in 1 ml of 1×PBS (containing 2% FBS) and centrifuged at 500 g for 5 minutes. (8) Step 7 was repeated. (9) The binding rates were detected by flow cytometry, as shown in Figures 7A and 7B. The results showed that both NKG2D-CD16 and NKG2D-FC could bind to NK cells or senescent cells.

[0099] 5.2 NKG2D-CD16 and NKG2D-FC promote NK cell killing of senescent cells P16-overexpressing senescent cells and NK cells were seeded into 96-well plates at an effector-target ratio of 2:1 (senescent cells served as targets, NK cells served as effectors) and then co-incubated with different concentrations of NKG2D-CD16 or NKG2D-FC protein for 8 hours. The viable cell counts were 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-incubated group) / number of target cells in the blank group. The results are shown in Figure 7C. Compared to the control group, NKG2D-CD16 and NKG2D-FC significantly enhanced the killing of NK cells against senescent cells. Culture supernatants were collected, and IFN-γ levels were measured using an ELISA kit. Statistical analysis was performed using GraphPad Prism software. The results are shown in Figure 7D. IFN-γ secretion was significantly increased after the addition of NKG2D-CD16, NKG2D-CD16, or NKG2D-FC compared to the control group.

[0100] All documents related to the present invention are incorporated herein by reference as if each document were individually incorporated by reference. After reading the above content of the present invention, it should be understood that those skilled in the art can make various changes and modifications to the present invention, and that equivalents thereof are within the scope of the claims of the present invention.

Claims

1. 1. Use of an NKG2D ligand and a cellular adaptor to target immune cells, comprising: (i) removal of senescent cells; (ii) prevention and / or treatment of diseases associated with the accumulation of senescent cells; (iii) Slowing individual aging For the manufacture of drugs used in the cellular adaptor molecule comprises a first binding domain and a second binding domain; wherein the first binding domain specifically binds to an NKG2D ligand, and the second binding domain specifically binds to a membrane protein on the surface of an immune cell;

2. The use according to claim 1, characterized in that the first binding domain is the NKG2D extracellular domain.

3. The use according to claim 1, characterized in that the first binding domain comprises a polypeptide whose amino acid sequence is set forth in SEQ ID NO: 1, or a polypeptide whose amino acid sequence has 80% or more similarity to the sequence set forth in SEQ ID NO: 1 and is capable of binding to an NKG2D ligand.

4. The use according to claim 1, characterized in that the second binding domain specifically binds to CD3 or CD16.

5. 2. The use according to claim 1, wherein the second binding domain has a structure selected from the group consisting of a single domain antibody (sdAb), a single chain antibody (scFv), an antigen-binding fragment (Fab fragment), a ligand, or a multimer thereof, or a combination thereof.

6. The use according to claim 1, characterized in that the second binding domain is a CD3 binding domain, which is capable of specifically binding to CD3 on the surface of T cells.

7. The use according to claim 6, characterized in that the CD3 binding domain has an amino acid sequence set forth in SEQ ID NO:2 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 to the amino acid sequence set forth in SEQ ID NO:

2.

8. The use according to claim 1, characterized in that the second binding domain is a CD16 binding domain, and the CD16 binding domain can specifically bind to CD16 on the surface of natural killer cells.

9. The use described in claim 8, characterized in that the CD16 binding domain has an amino acid sequence set forth in SEQ ID NO: 3 or 4, 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 to the amino acid sequence set forth in SEQ ID NO: 3 or 4.

10. The use according to claim 8, characterized in that the CD16 binding domain is a human IgG F C fragment.

11. The use described in claim 10, characterized in that the human IgG FC fragment has an amino acid sequence set forth in SEQ ID NO: 5 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 to the amino acid sequence set forth in SEQ ID NO:

5.

12. The use according to claim 1, characterized in that the cell adaptor molecule has a structure selected from the following formula (I) or (II) from the N-terminus to the C-terminus: S-D １ -L １ -D ２ -T (I) or S-D ２ -L １ -D １ -T (II) (wherein Each "-" is independently a connecting peptide or a peptide bond. The S is either absent or a signal peptide sequence. D １ is the first binding domain. L １ It is either a nucleotide or a connecting peptide. D ２ is the second binding domain. T is either absent or a labeled protein.)

13. The use according to claim 1, characterized in that the senescent cells are selected from the group consisting of lung cells, adipocytes, kidney cells, muscle cells, or combinations thereof.

14. The use according to claim 1, characterized in that the expression of NKG2D ligands in said senescent cells is upregulated.

15. The use according to claim 1, characterized in that the upregulation of the expression of NKG2D ligand means that the ratio of the expression level of NKG2D ligand in senescent cells (F1) to the expression level in normal cells (F0) (i.e., F1 / F0) is ≧1.5, preferably ≧2, more preferably ≧2.

5.

16. The use according to claim 1, characterized in that the NKG2D ligand is selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, or a combination thereof.

17. 2. The use according to claim 1, wherein the geriatric disease is selected from the group consisting of heart failure, atherosclerosis, diabetes, myocardial hypertrophy, osteoporosis, tissue / organ fibrosis, Alzheimer's disease, Parkinsonism, arthritis, or a combination thereof.

18. A pharmaceutical composition comprising: (a) NKG2D and cellular adaptor molecules that target immune cells; (b) a drug for eliminating senescent cells other than (a), and (c) A pharmaceutically acceptable carrier, diluent or excipient.

19. A pharmaceutical composition comprising: (a) NKG2D and cellular adaptor molecules that target immune cells; (b) a drug capable of upregulating the expression of an NKG2D ligand; and (c) A pharmaceutically acceptable carrier, diluent or excipient.

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