WO2022143938A1 - Tumor immunotherapy method based on activation of nk cell - Google Patents

Abstract

Provided is a tumor immunotherapy method based on the activation of an NK cell. Further provided is a use of nucleobindin (NUCB) or a promoter thereof in the preparation of a drug or a preparation for promoting the proliferation of NK cells, treating tumors, and/or killing or inhibiting tumor cells. The NK cell is activated by means of the method, and the obtained NK cell has a broad-spectrum anti-tumor effect without the identification of a tumor-specific antigen, such that the priming of an immune system is fast.

Description

A tumor immunotherapy method that activates NK cells technical field

The present invention relates to the field of biological immunity, in particular, to a tumor immunotherapy method for activating NK cells.

Background technique

With the progress of society and the development of science and technology, the emergence of antibiotics and other drugs, the harm of diseases that seriously threatened human beings in the last century and before, such as infectious diseases, has been significantly reduced. Chronic diseases such as cancer, cardiovascular diseases, neurodegenerative diseases and metabolic diseases have become the main health threats facing human society. Worldwide, 18.1 million new cancer patients were diagnosed in 2018, and 9.6 million people died of cancer. Cancer seriously affects human health and quality of life, and also brings enormous economic pressure to society.

Tumor immunotherapy is undoubtedly the most important step in tumor treatment in recent decades. It won the Nobel Prize in Physiology and Medicine in 2018 and is hailed as the third anti-cancer drug revolution after chemotherapy and targeted therapy. The vast majority of tumor immunotherapy methods, whether immune checkpoint inhibition, chimeric antigen T cells or bispecific antibodies, etc., focus on enhancing the activity of T cells. Although these approaches have achieved great success, only a small percentage of cancer patients benefit from them.

As one of the main members of the innate immune system, natural killer cells (NK cells) play a key role in the body's immune surveillance and early anti-infection and tumor processes. NK cells directly kill tumor cells by releasing lytic granules containing perforin and granzyme, and can also recruit and regulate other innate immunity and acquisition by secreting cytokines and chemokines such as IFN-γ, TNF-α, GM-CSF, etc. The sexual immune system plays an indirect role. These functions are achieved through a variety of germline-encoded activating or inhibitory receptors on the surface of NK cells. But so far, the corresponding ligands for many of these receptors have not been found, which is not conducive to the development of immunotherapeutic methods related to the activation of NK cells.

In conclusion, there is an urgent need in the art to develop a new method for activating NK cells efficiently and rapidly.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a new method for activating NK cells efficiently and rapidly.

In the first aspect of the present invention, there is provided the use of a nucleobindin (Nucleobindin, NUCB) or its promoter for preparing a medicament or a preparation, the medicine or preparation (a) is used for promoting the expansion of NK cells , (b) treating tumors; and/or (c) killing or inhibiting tumor cells.

In another preferred embodiment, the medicament or preparation includes a tumoricidal agent.

In another preferred embodiment, the NUCB protein is of non-viral origin.

In another preferred embodiment, the NUCB protein is derived from mammals, preferably from rodents (rats, mice) and primates (such as humans), more preferably from humans.

In another preferred embodiment, the NUCB protein includes NUCB1 protein and/or NUCB2 protein.

In another preferred embodiment, the amino acid sequence of the NUCB1 protein is shown in SEQ ID NO.:4 or the identity with SEQ ID NO.:4 is ≥85% (preferably ≥90%, more preferably ≥95%) % or ≥98%).

In another preferred embodiment, the amino acid sequence of the NUCB2 protein is shown in SEQ ID NO.: 5 or the identity with SEQ ID NO.: 5 is ≥ 85% (preferably ≥ 90%, more preferably ≥ 95%) % or ≥98%).

In another preferred embodiment, the NUCB1 protein is human NUCB1 (SEQ ID NO.: 6).

In another preferred embodiment, the NUCB2 protein is human NUCB2 (SEQ ID NO.: 7).

In another preferred embodiment, the NUCB protein specifically binds to the NK cell surface receptor LY49H.

In another preferred example, the NUCB protein includes full-length NUCB protein, mature NUCB protein, a key segment of NUCB that binds to LY49H, or a NUCB active fragment containing the key segment. In another preferred embodiment, the key segment of the NUCB protein is the amino acid sequence of positions 40-85 of NUCB1 or NUCB2.

In another preferred embodiment, the sequence of the key segment of NUCB1 is shown in SEQ ID NO.: 1.

In another preferred embodiment, the sequence of the key segment of NUCB2 is shown in SEQ ID NO.: 2.

In another preferred embodiment, the sequence of the key segment of NUCB1 is positions 45-81 of mouse NUCB1 protein (SEQ ID NO. 4).

In another preferred embodiment, the sequence of the key segment of NUCB2 is positions 49-85 of mouse NUCB2 protein (SEQ ID NO. 5).

In another preferred embodiment, the NUCB protein includes a key segment of NUCB that binds to KIR3DS1.

In another preferred embodiment, the sequence of the key segment of NUCB1 is positions 46-82 of human NUCB1 protein (SEQ ID NO. 6).

In another preferred embodiment, the sequence of the key segment of NUCB2 is positions 49-85 of human NUCB2 protein (SEQ ID NO. 7).

In another preferred embodiment, the sequence of the key segment of NUCB1 is positions 45-138, preferably positions 45-63 of human NUCB1 protein (SEQ ID NO. 6).

In another preferred embodiment, the NK cells are NK cells positive for the surface receptor LY49H.

In another preferred embodiment, the promoter includes a promoter-type miRNA, a promoter-type transcriptional regulator, or a promoter-type targeted small molecule compound.

In another preferred embodiment, the tumor is selected from the group consisting of colon cancer, breast cancer, lung cancer, stomach cancer, liver cancer, multiple myeloma, kidney cancer, pancreatic cancer, melanoma, lymphoma, thyroid cancer, or a combination thereof .

In another preferred embodiment, the drug or preparation is administered by a mode of administration selected from the group consisting of intravenous, intratumoral, intracavitary, subcutaneous or hepatic artery administration (eg, injection, drip, etc.).

In another preferred embodiment, the preparation is selected from the group consisting of tablets, capsules, injections, granules, sprays, and freeze-dried preparations.

In another preferred embodiment, the preparation is an injection.

In a second aspect of the present invention, a method for promoting NK cell expansion in vitro is provided, comprising the steps of:

(a) provide a NUCB protein or a promoter thereof; and

(b) culturing NK cells in the presence of the NUCB protein or a promoter thereof, thereby promoting NK cell expansion.

In another preferred embodiment, the NK cells are NK cells positive for the surface receptor LY49H.

In another preferred embodiment, the method is an in vitro method.

In the third aspect of the present invention, an isolated complex is provided, and the complex is a binary complex formed by the combination of NUCB protein and LY49H.

In another preferred embodiment, the NUCB protein includes NUCB1 protein and/or NUCB2 protein.

In another preferred embodiment, the amino acid sequence of the NUCB protein is selected from the following group:

(a) has the amino acid sequence shown in SEQ ID NO.:4 or 5;

(b) The amino acid sequence shown in SEQ ID NO.: 4 or 5 is formed by the substitution, deletion or addition of one or several (such as 1-10) amino acid residues, and the NUCB activity is composed of (a) a derived polypeptide; or

(c) The homology between the amino acid sequence and the amino acid sequence shown in SEQ ID NO.: 4 or 5 is ≥80% (preferably ≥90%, more preferably ≥95% or ≥98%), and has the NUCB activity of polypeptides.

In another preferred embodiment, the amino acid sequence of the LY49H is selected from the following group:

(a) has the amino acid sequence shown in SEQ ID NO.:8;

(b) The amino acid sequence shown in SEQ ID NO.: 8 is formed by the substitution, deletion or addition of one or several (such as 1-10) amino acid residues, and the LY49H activity is formed by (i) ) derived polypeptide; or

(c) The homology between the amino acid sequence and the amino acid sequence shown in SEQ ID NO.: 8 is ≥80% (preferably ≥90%, more preferably ≥95% or ≥98%), and a polypeptide having the LY49H activity .

In the fourth aspect of the present invention, there is provided a use of the complex described in the third aspect of the present invention for screening drugs or compounds that promote NK cell expansion.

In another preferred embodiment, the NK cells are preferably NK cells positive for the surface receptor LY49H.

In a fifth aspect of the present invention, a method for screening a drug or compound for promoting NK cell expansion is provided, comprising the steps of:

(a) In the test group, in the presence of the substance to be tested, NK cells are cultured, and a control group without the substance to be tested is set;

(b) detecting the content H1 of the complex in the test group and comparing it with the complex content H0 in the control group, wherein when H1 is significantly higher than H0, it means that the test substance is a drug that promotes NK cell expansion or compound.

In another preferred embodiment, the significantly higher ratio refers to H1/H0≥2, preferably, ≥3, more preferably, ≥4.

In the sixth aspect of the present invention, there is provided a method of treating tumors, by administering a therapeutically effective amount of NUCB protein or its promoter to a subject in need thereof.

In another preferred embodiment, the subject is a human or a non-human mammal.

In another preferred embodiment, the non-human mammals include rodents (eg, mice, rats, rabbits), primates (eg, monkeys).

In another preferred embodiment, the method further comprises administering NK cells to a subject in need.

In another preferred embodiment, the NK cells are NK cells positive for the surface receptor LY49H.

In another preferred example, the NK cells naturally contain NUCB receptors or obtain NUCB surface receptors through artificial transformation.

In the seventh aspect of the present invention, use of LY49H protein or its active fragment is provided for preparing a pharmaceutical composition for treating diseases related to excessive NUCB protein.

In another preferred embodiment, the sequence of the LY49H protein or its active fragment is selected from the following group:

(a) has the amino acid sequence shown in SEQ ID NO.:9;

(b) The amino acid sequence shown in SEQ ID NO.: 9 is formed by the substitution, deletion or addition of one or several (such as 1-10) amino acid residues, and the LY49H activity is formed by (a) ) derived polypeptide; or

(c) The homology between the amino acid sequence and the amino acid sequence shown in SEQ ID NO.: 9 is ≥80% (preferably ≥90%, more preferably ≥95% or ≥98%), and a polypeptide having the LY49H activity .

In another preferred embodiment, the LY49H protein or its active fragment is the extracellular segment of LY49H (SEQ ID NO.: 9).

In another preferred embodiment, the related diseases with excessive NUCB protein are selected from the group consisting of polycystic ovary syndrome (Polycystic Ovary Syndrome), hypertension (Hypertention), anxiety (Anxiety), epilepsy (Epilepsy) or its combination.

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 in the following (eg, the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, it is not repeated here.

Description of drawings

Figure 1 shows that NUCBs specifically bind LY49H. A. Schematic diagram of the molecular structure of LY49H molecule and NUCBs (where SS is the signal peptide (signal sequence), EF is the EF-hand domain); B. The schematic diagram of the interaction method (LY49 family plasmid and NUCBs expression plasmid were co-transfected into 293T After cells, it is secreted into the cell culture medium); C. Results of co-immunoprecipitation experiment of LY49H molecule and NUCB1 protein, in which NUCB1 protein specifically binds to LY49H in the LY49 family; D. Co-immunoprecipitation experiment of LY49H molecule and NUCB2 protein The result graph of NUCB2 protein specifically binds to LY49H in the LY49 family; E.LY49H binds to NUCBs protein expressed and purified by eukaryotic 293T cells; F.LY49H binds to E. coli expressed and purified NUCBs protein (wherein, lp:Fc is co-immunoprecipitation Fc tag, Input is the input amount of the two proteins in the co-immunoprecipitation experiment, HA is the HA tag, FC is the FC tag, and Flag is the Flag tag).

Figure 2 shows that NUCBs specifically bind to the C-type lectin-like domain of LY49H. Cut-out pattern of the extracellular segment of LY49H; B. The C-type lectin-like domain of LY49H is required for binding to NUCB1; C. The C-type lectin-like domain of LY49H is required for binding to NUCB2; D. LY49 family C-type lectin-like domain sequence alignment; E. LY49 family C-type lectin-like domain sequence association guide tree; F, G. Leucine 196 of the C-type lectin-like domain of LY49H Residues and 216 histidine residues are the key amino acids that determine their binding specificity with NUCB1/NUCB2; H. NUCBs binding site pattern map on LY49H, in which CTLD is a C-type lectin-like domain.

Figure 3 shows that amino acids 40-80 of NUCBs bind LY49H. A. C-terminal truncation pattern of NUCBs protein; B. The three C-terminal truncations of NUCBs do not affect the binding to LY49H; C. N-terminal truncation pattern of NUCBs protein; D. NUCB1 (deficient in 44-80 ) and NUCB2 (44-80 deficiency) significantly affected the binding to LY49H.

Figure 4 shows that overexpression of NUCBs inhibits tumor cell growth in mice. A. Construct the model diagram of MC38 cells overexpressing NUCBs protein; B. Spread the same number of cells, harvest the medium 24 hours later, and detect the overexpression of NUCBs by Western Blot; C. Overexpression of NUCBs protein does not affect the proliferation of MC38 cells; D . MC38 wild-type cells and NUCB1 or NUCB2 overexpressing MC38 cells subcutaneously inoculate C57 mice model diagram; E. NUCB1 and NUCB2 overexpressing MC38 cells in mice subcutaneously grow significantly smaller tumors; F. NUCB1 and NUCB2 overexpressing MC38 Cell-transplanted mice had significantly longer survival.

Figure 5 shows that injection of NUCBs peptides inhibited tumor cell growth. A-C. NUCB1 and NUCB2 protein sequence information, synthetic NUCBs peptide sequence information and control peptide sequence information; D. Schematic diagram of injection of NUCBs peptide; E. Injection of NUCB1 peptide or NUCB2 peptide significantly reduced tumor size ; F. NUCB peptide fragment human-mouse sequence comparison information.

Figure 6 shows the identification of NUCBs knockout mice. A, B. NUCB1 knockout strategy and sequencing identification; C, D. NUCB1 knockout mouse genome PCR and mRNA expression identification (lung tissue); E, F. NUCB2 knockout strategy and sequencing identification; G, H . Genome PCR identification of NUCB2 knockout, WB identification. Among them, marker is the control, and actin is actin.

Figure 7 shows that NUCBs knockout mice promote tumor growth. C. NUCB1&2 knockout male mice promoted tumor growth and shortened the survival time of mice, but single knockout male mice did not change; D-F. NUCB1&2 knockout female mice promoted tumor growth and shortened the mice's survival time The survival time of single knockout female mice did not change (WT: wild type; KO: knockout).

Figure 8 shows that NUCBs promote the expansion of LY49H-positive NK cells. A. Flow cytometric analysis of LY49H-positive NK cells in the gating process; B. NUCB1/2 overexpression reduces tumor size and promotes the expansion of LY49H-positive NK cells in mouse spleen (7 NUCB1/2-overexpressing tumors in 2 were NUCB1-overexpressing tumors, and 5 were NUCB2-overexpressing tumors); C. NUCB1&2 gene knockout increased tumor weight and decreased the number of LY49H-positive NK cells in the spleen of mice; D. Intraperitoneal injection of NUCB2 peptides decreased Tumor volume, increasing the number of LY49H-positive NK cells (LY49H-positive NK cells (%): the proportion of CD45+CD3- cells in splenocytes, as shown in A); E. NUCBs promote LY49H-positive NK cell proliferation pattern.

Figures 9A-C show that NUCB1 protein in humans binds to the activating receptor KIR3DS1 in the KIR family; D. Human NUCB1 is also highly specific compared to other receptors on the surface of human NK cells, such as NKG2D, TIGIT, DNAM1, NKP30 and NKP46 Binding of KIR3DS1; E. The predicted fragmentation of the secondary mechanism of human NUCB1 shows that positions 45-138 of NUCB1 are the segment that mainly binds KIR3DS1; F. Further breakdown of the core amino acid sequence of human NUCB1, showing positions 45-63 of human NUCB1 Amino acids are central to binding KIR3DS1 and KIR3DL1.

Detailed ways

After extensive and in-depth research, the present inventors discovered for the first time that a nucleobindin (NUCB) can actually bind to LY49H, thereby effectively promoting the expansion (or proliferation) of NK cells. Specifically, experiments such as knocking out the NUCB gene from mice, overexpressing the NUCB protein, injecting key peptides and in vitro protein binding have proved that the secreted proteins NUCB1 and NUCB2 are the physiological ligands of the NK cell surface receptor LY49H. The formed binary complex promotes the expansion of LY49H receptor-positive NK cells. Therefore, nucleobindin (NUCB) and its promoter can be used to prepare a drug or preparation for treating tumors and promoting the expansion of NK cells. The present invention has been completed on this basis.

the term

In the present invention, "nucleonectin of the present invention", "nucleonectin", "NUCBs protein", "NUCB protein", "NUCB1 and NUCB2 protein" are used interchangeably, and all refer to NK cell surface receptor LY49H Binds to a secreted protein that promotes NK cell expansion.

Natural Killer Cells (NK Cells)

As one of the main members of the innate immune system, NK cells play a key role in the body's immune surveillance and early anti-infection and tumor processes. Numerous preclinical studies have shown that NK cells play an important role in limiting tumor growth and metastasis. Clinical data also show that NK cell activity is negatively correlated with cancer development, and that infiltration of NK cells in some tumor microenvironments is positively correlated with patient survival. Unlike T cells, NK cells have certain advantages. First, NK cells belong to the innate immune system and are at the forefront of the immune response. Almost all tumor cells or tumor cells that recur and metastasize will be preferentially attacked by NK cells and have broad-spectrum anti-tumor effects; second, no need Tumor-specific antigen recognition or amplification, the immune system activation time is fast; third, the safety is better, and there are fewer restrictions on transplantation.

NK cells directly kill tumor cells by releasing lytic granules containing perforin and granzyme, and can also recruit and regulate other innate immunity and acquisition by secreting cytokines and chemokines such as IFN-γ, TNF-α, GM-CSF, etc. The sexual immune system plays an indirect role. These functions are achieved through a variety of germline-encoded activating or inhibitory receptors on the surface of NK cells. But for many of these receptors, no corresponding ligand has been found so far.

Nucleonectin (NUCB)

NUCBs (Nucleobindins) are a family of calcium-binding secreted proteins, including NUCB1 and NUCB2. The function of NUCB2 protein was first reported in 2006 to suppress appetite and reduce weight, but later studies showed that NUCB2 has no appetite and weight regulation functions under physiological conditions. The biological function of NUCB1 is still unclear. Therefore, the biological functions and mechanisms of the NUCBs family are still unclear. NUCB1 and NUCB2 proteins are evolutionarily conserved and have high homology, such as 87.4% similarity between human and rat NUCB2 protein, and 95.7% similarity between rat and mouse NUCB2 protein.

NUCBs protein expression exists in a variety of tissues and cells. Under conditions such as stress or inflammation, NUCBs proteins are induced to express. It is currently believed that cells of the tumor and microenvironment induce NUCBs expression in a variety of stressful situations, including in tumor situations, to activate NK cells to deal with stressful situations.

It should be understood that there may be an optional tag sequence (such as a 6His tag to facilitate purification) at the N-terminus or C-terminus of the NUCB protein of the present invention. Furthermore, the NUCB proteins of the present invention may include wild type and mutant types, wherein the mutant type retains at least 30%, preferably at least 50% of the corresponding biological activity of the wild type.

It should be understood that although the NUCB proteins of the present invention are derived from mice, those from other animals (such as humans) are highly homologous (such as having more than 85%, such as 85%, 90%, 95% or even 98% sequence) with the mouse NUCB protein Other proteins that are identical) are also contemplated by the present invention. For example, human NUCB1 which is 89% similar to murine NUCB1 (without signal peptide) and human NUCB2 which is 87% similar to murine NUCB2 (without signal peptide). Methods and tools for aligning sequence identity are also well known in the art, such as BLAST.

The sequence of the NUCB protein involved in the present invention is as follows:

Mouse NUCB1 (SEQ ID NO: 4)

Mouse NUCB2 (SEQ ID NO: 5)

Human NUCB1 (SEQ ID NO: 6)

Human NUCB2 (SEQ ID NO: 7)

In addition, the term "derived protein of NUCB" also includes a variant form of the sequence shown in SEQ ID NO: 4 or 5, which has the function of binding LY49H. These variants include (but are not limited to): deletions, insertions and/or substitutions of 1-3 (usually 1-2, more preferably 1) amino acids, as well as C-terminal and/or N-terminal additions or One or several (usually within 3, preferably within 2, more preferably within 1) amino acids are deleted. For example, in the art, substitution with amino acids of similar or similar properties generally does not alter the function of the protein. For another example, addition or deletion of one or several amino acids at the C-terminus and/or N-terminus generally does not alter the structure and function of the protein. Furthermore, the term also includes monomeric and multimeric forms of the polypeptides of the invention. The term also includes linear as well as nonlinear polypeptides (eg, cyclic peptides).

The NUCB-acting derivative proteins of the present invention also include active fragments, derivatives and analogs thereof. As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially retain the function or activity of binding to LY49H. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) in one or more A polypeptide having a substituent group in one amino acid residue, or (iii) a polypeptide formed by fusion of a NUCB protein or a derivative thereof with another compound (such as a compound that prolongs the half-life of a polypeptide, such as polyethylene glycol), or (iv) A polypeptide formed by fusing an additional amino acid sequence with this polypeptide sequence (a fusion protein formed by fusing with a leader sequence, a secretory sequence, or a tag sequence such as 6His). These fragments, derivatives and analogs are well known to those skilled in the art from the teachings herein.

A class of preferred active derivatives refers to those with at most 3, preferably at most 2, more preferably at most 1 amino acids with similar or similar properties compared with the amino acid sequence shown in SEQ ID NO.: 4 or 5. Amino acids are replaced to form polypeptides. These conservatively variant polypeptides are best produced by amino acid substitutions according to Table A.

Table A

initial residue	representative substitution	Preferred substitution
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

The present invention also provides analogs of the NUCB protein. The differences between these analogs and the polypeptides shown in SEQ ID NO: 4 or 5 can be differences in amino acid sequence, differences in modified forms that do not affect the sequence, or both. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), as well as analogs with non-naturally occurring or synthetic amino acids (eg, beta, gamma-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

Modified (generally without altering the primary structure) forms include chemically derivatized forms such as acetylation or carboxylation of the polypeptide in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modifications can be accomplished by exposing the polypeptide to enzymes that perform glycosylation, such as mammalian glycosylases or deglycosylases. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize their solubility properties.

LY49 receptor family

The LY49 receptor family is one of the mouse NK cell receptors, which can transmit activating or inhibitory signals and regulate the killing effect of NK cells. The 10 members of the LY49 receptor family of proteins are LY49A, LY49B, LY49C, LY49D, LY49E, LY49F, LY49G, LY49H, LY49I, LY49Q. Among them, the physiological ligand of NK cell surface receptor LY49H has not been found. Corresponding to the LY49 receptor family in human NK cells is the KIR receptor family, including 8 inhibitory receptors: KIR2DL1-5, KIR3DL1-3, and 6 activating receptors, KIR2DS1-5, KIR3DS1, many of which The receptor has not yet found its ligand.

In the present invention, five truncated plasmids were made on the extracellular end of receptor LY49H, and the binding experiments of these fragments with NUCBs found that the C-type lectin-like domain (CTLD, amino acids 143-266) was Required in conjunction with NUCBs. The amino acid sequence of C-tpye lectin-like domain (C-tpye lectin-like domain) of LY49 family molecules was compared and found that LY49H has three amino acid residues that are unique to LY49H compared to LY49F, LY49C and LY49I, which are the most similar. , specifically L196, H216 and T232.

In a preferred embodiment of the present invention, the amino acid sequence of the LY49H is shown in SEQ ID NO: 8:

Wherein, the underlined part is the CTLD part in LY49H (143-266 of SEQ ID NO:8).

The LY49H extracellular telopeptide segment is SEQ ID NO:9 (segments 67-266 of SEQ ID NO:8):

Accelerators and Formulations

Using the protein of the present invention, through various conventional screening methods, substances that interact with the NUCB protein, especially promoters, can be screened.

The NUCB protein promoter of the present invention, when administered (administered) therapeutically, can promote the expression and/or activity of the NUCB protein, thereby promoting the expansion and/or activity of NK cells, thereby killing tumors. In another preferred embodiment, the NUCB promoter includes a NUCB gene expression product, a promoter-type miRNA, a promoter-type transcriptional regulator, or a promoter-type targeted small molecule compound.

Generally, the NUCB protein of the present invention or its promoter can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, Although pH can vary depending on the nature of the substance being formulated and the condition being treated. The formulated formulation or pharmaceutical composition can be administered by conventional routes including, but not limited to, intratumoral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration.

The present invention also provides a pharmaceutical preparation, which contains a safe and effective amount of the NUCB protein or its promoter of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof. The drug formulation should match the mode of administration. The pharmaceutical preparation of the present invention can be prepared in the form of injection, for example, prepared by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The preparation of the present invention is selected from the group consisting of tablets, capsules, injections, granules, sprays, and freeze-dried preparations. The active ingredient is administered in a therapeutically effective amount, e.g., about 1 microgram to 10 mg/kg body weight per day. The medicaments or preparations of the present invention are administered by means of administration selected from the group consisting of intravenous, intratumoral, intracavitary, subcutaneous or hepatic arterial administration (eg, injection, drip, etc.).

The present invention also provides a pharmaceutical composition, which contains a safe and effective amount of the LY49H protein of the present invention or an active fragment thereof and a pharmaceutically acceptable carrier or excipient, for preparing a kind of disease related to the treatment of excessive NUCB protein . The disease associated with excessive NUCB protein is selected from the group consisting of Polycystic Ovary Syndrome, Hypertention, Anxiety, Epilepsy or a combination thereof.

The pharmaceutical composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such carriers include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof. Usually, the pharmaceutical preparation should match the mode of administration, and the dosage form of the pharmaceutical composition of the present invention is injection, freeze-dried preparation, stem cell preparation, and atomized inhalation preparation. For example, it is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical compositions are preferably manufactured under sterile conditions.

Complex

In the present invention, a complex is provided, which is a binary complex formed by the combination of NUCB protein and LY49H.

The complexes of the present invention can be used to screen for drugs or compounds that promote NK cell expansion, wherein substances that can promote the formation of the complexes have the potential to promote NK cell expansion.

Methods of promoting NK cell expansion

The present invention provides a method for promoting the expansion of NK cells in vitro or in vivo and the long-term existence of NK cells in vivo, comprising:

(1) In vitro: provide a NUCB protein or its promoter; in the presence of the NUCB protein or its promoter, under appropriate culture conditions, it is mixed with NK cells containing its receptor, and the NK cells are greatly expanded. .

(2) In vivo: provide a NUCB protein or its promoter; after the in vivo input of some functional NK cells (such NK cells may naturally have NUCB protein receptors or artificially engineered to contain NUCB protein receptors), then Administration of the NUCB protein or its promoter in vivo promotes the expansion and long-term existence of such infused NK cells in vivo; enhancing the therapeutic efficacy of the infused NK cells.

The main advantages of the present invention include:

(1) The present invention finds for the first time that the secreted proteins NUCB1 and NUCB2 are the physiological ligands of the NK cell surface receptor LY49H.

(2) The method for activating NK cells of the present invention is a brand-new tumor immunotherapy approach.

(3) NK cells are activated by the method of the present invention, and the obtained NK cells have broad-spectrum anti-tumor effects, do not need to recognize tumor-specific antigens, and activate the immune system quickly.

The present invention is further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental method of unreceipted detailed conditions in the following examples, usually according to conventional conditions such as Sambrook et al., molecular cloning: conditions described in laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer the proposed conditions. Percentages and parts are by weight unless otherwise indicated.

General Materials and Methods

1. Gene Cloning

Mouse NUCB1, NUCB2 gene full-length or fragment cloned into pCMV-HA eukaryotic expression vector and pLVX-DsRed-3*Flag-HA-T2A-Puro lentiviral packaging vector, LY49 family gene full-length or fragment cloned into pFUSE-hIgG1 - Fc2 vector. The point mutation of the gene is accomplished by the corresponding site-directed mutagenesis primers.

2. Cell Culture

HEK 293T and MC38 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C with 5% CO2 concentration. The cells were divided into plates 18-24 hours before transfection, and the total amount of plasmid in the 6-well plate was 2 μg/well. When the amount of transfected plasmid was insufficient, LacZ was used to make up. The transfection reagent was Lipo3000 (Invitrogen). Corresponding experiments were performed 24-30 hours after transfection.

3. Co-immunoprecipitation

Collect the culture medium of 293T cells co-transfected with NUCB1 or NUCB2 and LY49 family plasmids, centrifuge at 12,000 rpm for 10 minutes, take the supernatant plus ProteinA/G beads, spin for 2 hours in a cold storage at 4°C to immunoprecipitate the FC-tagged protein, wash 3 times and then aspirate Discard the supernatant, add 40 μL of 2*loading, cook for 10 minutes, and store at -20°C.

4.Western Blot

SDS-PAGE gel with a concentration of 10% was prepared, and protein samples were added for electrophoresis separation. The proteins were electrotransferred to NC (nitrocellulose) membranes and blocked with 0.5% skim milk for 1 hour. After washing 3 times with TBST for 5 min each, the corresponding primary antibody was added and incubated for 2 h at room temperature. After washing 3 times with TBST for 5 minutes each time, the corresponding HRP-conjugated secondary antibody was added and incubated for 1 hour at room temperature. After washing three times, add developer to scan. NUCB2 (N6789) antibody was purchased from Sigma Aldrich company, HA (16B12) antibody was purchased from Covance company, Flag antibody, His antibody were purchased from Sigma Aldrich company, HRP-conjugated Goat anti-human IgG antibody was purchased from Sangon Biotech (Shanghai).

5. Tissue RNA Extraction and Q-PCR

After the mice were euthanized, the corresponding tissues were rapidly dissected out and placed into homogenization tubes containing steel beads and Trizol. After fragmentation, RNA was extracted by the phenol-chloroform-isopropanol method, and reverse transcribed into cDNA using the superscriptTMIII first-strand synthesis system (Invitrogen) kit, using Quantitative SYBR green PCR kit (TaKara) kit and ABI-QuantStudio 6Realtime PCR instrument Do quantitative PCR reaction.

6. Mice

C57BL/6 mice were purchased from Lingchang Company, and were kept in the SPF animal room of the Institute of Biochemical Cells. The lights were turned off for 12 hours (19:00-07:00) and on for 12 hours (07:00-19:00). NUCB1 and NUCB2 single-gene knockout mice and NUCB1 and NUCB2 double-knockout mice were obtained by CRISPR-Cas9 method. Wild-type and corresponding knockout mice. Mice used in all experiments were 7-11 weeks old.

7. Construction of monoclonal strains overexpressing NUCB1 or NUCB2 in MC38 cells

The full-length mouse NUCB1 and NUCB2 genes (including signal peptides) were obtained by PCR amplification, cloned into pLVX-DsRed-3*Flag-HA-T2A-Puro virus packaging vector, and transfected into 293T cells together with the packaging plasmids pSPAX2 and pMD2G. Virus. The obtained virus was used to infect MC38 cells, and single clones were picked to obtain MC38 cells with stable high expression of NUCB1 or NUCB2.

8. Cell Proliferation Assay

The proliferation of MC38 wild-type cells and MC38 cells overexpressing NUCB1 or NUCB2 was detected using the CellTiter-Glo Fluorescent Cell Viability Assay (Promega) kit, and detected according to the instructions.

9. Subcutaneous Xenograft Model

The wild-type cells of MC38 or NUCB1 and NUCB2 overexpressing MC38 cells were cultured after digestion, washed three times with PBS, filtered with 100um filter, adjusted to 5*10 ⁶ /ml cells, and subcutaneously inoculated with 100ul (containing 5*10 ⁵ cells), After the corresponding time, the length and width of the tumor were measured using a vernier caliper, and the tumor volume was calculated by the formula: volume=length*width*width/2. For survival statistics, mice were euthanized when the tumor volume was greater than 2000 mm ³ , which was defined as death.

10. Peptide synthesis

The NUCB1 peptide segment is amino acids 45-81 of the NUCB1 protein, and the NUCB2 peptide segment is the amino acids 49-85 of the NUCB2 protein. The control peptide segment is the same as the NUCB2 peptide segment (49-85), but the sequence is randomly arranged. . Synthesized by Shanghai Jill Biochemical Company, the purity is more than 95%. The specific sequence is:

NUCB1 peptides:

NUCB2 peptides:

Control peptide:

11. Intraperitoneal injection

The purchased 6-week-old wild-type C57 mice were subcutaneously inoculated with 5*10 ⁵ MC38 cells/mice after 1 week of adaptation. After 5 days, the tumor volume was measured and 3 kinds of peptides were injected intraperitoneally, 2 times a day, 100ug each time /Only.

12. Flow Cytometry

The spleen was removed within 5 minutes after the mice were euthanized and placed in pre-cooled phenol red-free DMEM medium. After the red blood cells were lysed, count 106 cells, and follow the procedure: live/dead fixable (Live/Dead Fixable Near-IR Dead Cell Stain Kit, Thermo Fisher), blocking (anti-CD16/32, BioLegend), membrane antibody (anti-CD45, Brilliant Violet) 510, BioLegend; anti-CD3, Brilliant Violet 605, BioLegend; anti-NK1.1, Brilliant Violet 421, BioLegend; anti-LY49H, FITC, eBioscience), on-board analysis (Beckman CytoFlex LX flow cytometer).

13. Statistical analysis of data

Data are presented as mean ± standard error (means ± s.e.m). P values were obtained by t-test (student's test) or ANOVA analysis, and significance was expressed as *: P<0.05, **: P<0.01, ***: P<0.001.

Example 1. Secreted proteins NUCB1 and NUCB2 specifically bind to the NK cell surface receptor LY49H

By mass spectrometry, the inventors found that LY49H may be the binding protein of NUCBs, and cloned 10 members of the LY49 receptor family protein in C57BL/6 mice (LY49A, LY49B, LY49C, LY49D, LY49E, LY49F, LY49G, LY49H, LY49I, LY49Q) into the expression vector PFUSE-hIgG1-Fc2 (IL2ss), because this vector contains the IL2 signal peptide, the protein expressed by the gene cloned into this vector will be secreted into the cell culture medium. Simultaneously clone the protein expression sequences of full-length mouse NUCB1 and NUCB2 into the eukaryotic expression vector pCMV-HA, because the NUCBs gene itself has a secretion signal peptide and will also be secreted into the cell culture medium. At the same time, LY49 family plasmid and NUCBs expression plasmid will be secreted into the cell culture medium after transfection into 293T cells together (Fig. 1A, 1B). This system can well detect the interaction of proteins. The cell culture medium of the co-transfected 293T cells was collected 24-30 hours later, and co-immunoprecipitation found that both NUCB1 and NUCB2 specifically bound LY49H of the LY49 family (Fig. 1C, 1D). Next, eukaryotic 293T cell-expressed (Fig. 1E) and prokaryotic E. coli-expressed (Fig. 1F) NUCB1 and NUCB2 proteins were purified, verifying the direct interaction of NUCB1 and NUCB2 with LY49H.

The experimental results indicated that NUCBs are potential ligands of LY49H.

Example 2. The C-type lectin-like domain of LY49H specifically binds NUCBs

Five truncated plasmids were made on the extracellular end of receptor LY49H, and the binding experiments of these fragments with NUCBs found that its C-type lectin-like domain (CTLD, amino acids 143-266) was necessary for binding to NUCBs ( 2A-C). By comparing the amino acid sequences of the C-type lectin-like domains of LY49 family molecules, it was found that LY49H compared to the most similar LY49F, LY49C and LY49I, there are 3 amino acid residues that are unique to LY49H, specifically L196, H216 and T232 ( Figure 2D, 2E). Point mutations were made to the three amino acid positions of LY49H, respectively, to the amino acid residues corresponding to their similar receptors (L196P, leucine at position 196 was mutated to proline; H216D, histidine at position 216 was mutated). mutated to aspartic acid; T232K, threonine at position 232 was mutated to lysine).

The results showed that L196 and H216 were the key amino acid residues that determined their specific binding to NUCBs (Fig. 2F, 2G). Therefore, as shown in Figure 2H, NUCBs bind to the C-type lectin-like domain of LY49H, where leucine 196 and histidine 216 are key amino acids.

Example 3. Amino acids 40-80 of NUCBs are critical segments for binding LY49H

See Figure 3. As shown in Figure 3A, NUCBs were gradually truncated from the C-terminus to obtain three truncated forms: NUCB1(1-320), NUCB1(1-230), NUCB1(1-160), NUCB2(1-320), NUCB2(1-230), NUCB2(1-160). All of these truncations bound LY49H well (Fig. 3B). Therefore, amino acids 1-160 of NUCBs may be the segment responsible for binding LY49H.

As shown in Figure 3C, the 1-160 positions of the NUCBs were gradually truncated from the N-terminal to obtain NUCB1 (missing 25-44), NUCB1 (missing 44-80), NUCB1 (missing 80-120), and NUCB1 (missing 120-159), respectively. , NUCB2 (missing 24-44), NUCB2 (missing 44-80), NUCB2 (missing 80-125), NUCB2 (missing 125-159).

These truncated to full-length ratios, NUCB1 (44-80 deficiency) and NUCB2 (44-80 deficiency) significantly affected the binding to LY49H (Fig. 3D).

The results showed that amino acids 40-80 of NUCBs were the key segment for binding LY49H.

Example 4. Overexpression of NUCBs inhibits tumor cell growth in mice

MC38 cells were infected with lentivirus, and a single clone was picked to obtain a stable cell line with high expression of NUCB1 or NUCB2. The cell culture medium was collected and it was detected that the stable line cells secreted a large amount of NUCB1 or NUCB2 protein into the cell culture medium (Figure 4A, B). Cell proliferation assay, overexpression of NUCB1 or NUCB2 did not affect the proliferation of the cells themselves (Fig. 4C). 5×10 ⁵ wild-type or NUCBs overexpressing strains were subcutaneously transplanted into C57 male mice and compared with wild-type cells.

Results As shown in Figure 4D-F, the growth rate of MC38 tumors overexpressing NUCB1 or NUCB2 was significantly reduced, and the survival rate of mice was significantly prolonged.

Example 5. Injection of NUCBs protein peptides inhibits tumor growth in mice

According to the amino acid sequence of the NUCBs protein responsible for binding LY49H, the peptide sequence shown in Figure 5A-C and the peptide sequence for the control experiment were synthesized:

NUCB1 peptide (45-81 amino acids):

NUCB2 peptide (49-85 amino acids):

Control peptide:

C57 wild-type mice were inoculated with 5×10 ⁵ MC38 cells subcutaneously and divided into 3 groups, and the polypeptide was injected intraperitoneally after 5 days. The control peptides, NUCB1 peptides and NUCB2 peptides were injected respectively, twice a day in the morning and evening, with a dose of 100ug per animal each time. Tumor size was measured from day 5 and every 2 days.

As shown in Figure 5D,E, after 10 days, injection of NUCB1 peptide or NUCB2 peptide can significantly reduce the tumor size.

Human and mouse NUCBs proteins are highly conserved in evolution. Comparing the functional peptide sequences, the similarity of human and mouse NUCB1 peptides is 97.3%, and the similarity of NUCB2 peptides is 91.9% (Fig. 5F). The results suggest that human NUCB peptides may also have potential tumor-inhibiting effects.

Example 6. Construction of single knockout mice of NUCB1 and NUCB2 and double knockout mice of simultaneous knockout of NUCB1 and NUCB2

The NUCB1 gene of C57BL/6 mice has a total of 13 exons, of which the 2nd to 13th are coding exons. Using the CRISPR-Cas9 method, the sgRNA was designed at the exon 2 encoding signal peptide sequence to achieve the purpose of knockout. Sequencing confirmed that exon 2 of the NUCB1 gene knocked out 10 bases in the signal peptide, achieving the purpose of frameshifting (Fig. 6A,B). Both genomic PCR and real-time PCR at the mRNA level confirmed the knockdown of NUCB1 (Fig. 6C,D).

The NUCB2 gene has a total of 14 exons, of which exons 3 to 14 are coding exons. Using the CRISPR-Cas9 method, two sgRNAs were designed at the exon 3 encoding signal peptide sequence to achieve a certain length of knockout. Sequencing confirmed that 83 bases including part of the signal peptide coding sequence were knocked out in exon 3 of NUCB2 gene, and the purpose of frameshift was also achieved (Fig. 6E, F). The successful construction of NUCB2 knockout mice was confirmed at the genomic and protein levels, respectively (Fig. 6G,H). After obtaining NUCB1 and NUCB2 single-gene-edited mice respectively, the two mice were crossed to obtain double-gene-edited mice, and then NUCB1 and NUCB2 double-gene homozygous knockout mice (NUCB1&2) were obtained.

Example 7. NUCBs knockout mice promote tumor growth

The NUCBs family includes two members with high homology, namely NUCB1 and NUCB2. Biochemical experiments showed that both NUCB1 and NUCB2 could bind LY49H.

Experiments in mice show that cells overexpressing NUCB1 or NUCB2 can significantly inhibit tumor growth; the addition of NUCB1 peptide or NUCB2 peptide can also significantly inhibit tumor growth. Using the obtained NUCBs knockout mice, 5×10 ⁵ MC38 cells were subcutaneously inoculated into NUCB1 and NUCB2 knockout mice alone and NUCB1&2 double knockout mice, respectively.

The results showed that double knockout of NUCB1&2 promoted tumor growth and shortened survival in both male mice (Fig. 7A-C) and female mice (Fig. 7D-F). However, knocking out NUCB1 or NUCB2 alone has no effect, indicating that the two genes in the NUCBs family have a compensatory effect, and silencing one of the functions may be compensated by the other homologous gene.

In summary, the knockout mice further demonstrated the antitumor effect of NUCBs.

Example 8. NUCBs protein promotes the expansion of LY49H positive NK cells

Figure 8A is a flow cytometry flow cytometry flow cytometry analysis of LY49H positive cells. Wild-type C57 male mice were subcutaneously inoculated with MC38 cells and NUCB1/2-overexpressing MC38 cells. On day 20, the tumor volume of the over-expressed cells was much smaller than that of the wild-type. Flow analysis of the spleens showed that the proportion of LY49H-positive NK cells was significantly higher than that of NUCB1/2-overexpressed NK cells. Significantly increased in mice (Figure 8B). At the same time, comparing the wild-type female mice with tumor (MC38) and NUCB1&2 double-knockout female mice on the 15th day, the tumor weight of the knockout mice was significantly higher than that of the wild-type mice. The proportion of positive NK cells was significantly reduced (Fig. 8C). C57 wild-type male mice were subcutaneously inoculated with MC38 cells. From day 2, NUCB2 peptides and peptide solvent PBS were intraperitoneally injected for 8 consecutive days. On day 14, the tumor volume in the NUCB2 peptide group was significantly reduced, and the proportion of LY49H-positive NK cells in the spleen increased. (Fig. 8D). Figure 8E is a graph showing the pattern of NUCBs promoting the proliferation of LY49H-positive NK cells.

The results showed that NUCB protein overexpression reduced tumor size and promoted the expansion of LY49H-positive NK cells in mouse spleen (8B); NUCB1&2 gene knockout increased tumor weight and decreased the number of LY49H-positive NK cells in mouse spleen (8C); Intraperitoneal injection of NUCB2 peptides reduced tumor volume and increased the number of LY49H-positive NK cells (8D).

The positive and negative results indicate that an important physiological role of NUCBs protein binding to LY49H is to promote the expansion of LY49H-positive NK cells, thereby reducing the tumor.

Example 9. NUCB1 in humans binds to NK cell surface receptors KIR3DS1 and KIR3DL1

Figures 9A-C show that NUCB1 protein in humans binds KIR3DS1, an activating receptor in the KIR family (KIR2DS1-5 in B is an activating receptor in the KIR family, KIR2DL1-5 in C, and KIR3DL1-3 are inhibitory receptors in the KIR family ). Compared with other receptors on the surface of human NK cells, such as NKG2D, TIGIT, DNAM1, NKP30 and NKP46, human NUCB1 also binds KIR3DS1 with high specificity (Fig. 9D). Fragmentation according to the secondary mechanism prediction of human NUCB1 showed that positions 45-138 of NUCB1 were the segment that mainly bound KIR3DS1 (Fig. 9E). Further breakdown of the core amino acid sequence revealed that amino acids 45-63 of human NUCB1 are the core that binds KIR3DS1 and KIR3DL1 (Figure 9F). Consistent with positions 44-80 of NUCBs in the previous mouse, and more central.

discuss

Effects and receptors of NUCBs on NK cells in humans

NUCBs are highly conserved between humans and mice in evolution, and have shown significant tumor-treating effects in mice. In the follow-up, on the one hand, the effect and mechanism of NUCBs protein on human NK cells will be detected by the human NK cell line NK92 and the isolation of primary human blood NK cells. Since the corresponding LY49 receptor family in human NK cells is the KIR receptor family, including 8 inhibitory receptors: KIR2DL1-5, KIR3DL1-3, and 6 activating receptors, KIR2DS1-5, KIR3DS1, many of which The receptor has not yet found its ligand. These are the most important potential receptors for NUCBs. In the second aspect, the inventors will firstly verify the KIR receptor family and the NKG2C/CD94 receptor, and find the human NK cell NUCBs receptor. As potential targets of NK cells, the above receptors are of great significance for tumor immunotherapy.

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content 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 appended claims of the present application.

Claims (16)

1. A use of nucleobindin (NUCB) or a promoter thereof, characterized in that it is used to prepare a medicine or a preparation, wherein the medicine or preparation (a) is used to promote the expansion of NK cells, (b) to treat tumors and/or (c) killing or inhibiting tumor cells.
2. The use according to claim 1, wherein the NUCB protein comprises NUCB1 protein and/or NUCB2 protein.
3. The use according to claim 1, wherein the amino acid sequence of the NUCB1 protein is as shown in SEQ ID NO.: 4 or SEQ ID NO. 6 or the same as that of SEQ ID NO.: 4 or SEQ ID NO. 6 Identity > 85% (preferably > 90%, more preferably > 95% or > 98%).
4. The use according to claim 1, wherein the amino acid sequence of the NUCB2 protein is as shown in SEQ ID NO.: 5 or SEQ ID NO. 7 or the same as SEQ ID NO.: 5 or SEQ ID NO. 7 Identity > 85% (preferably > 90%, more preferably > 95% or > 98%).
5. The use of claim 1, wherein the NUCB protein comprises a full-length NUCB protein, a mature NUCB protein, a key segment of NUCB that binds to LY49H, or a NUCB active fragment containing the key segment .
6. The use of claim 1, wherein the NUCB protein specifically binds to the NK cell surface receptor LY49H.
7. A method for promoting NK cell expansion in vitro, comprising the steps of:

   (a) provide a NUCB protein or a promoter thereof; and

   (b) culturing NK cells in the presence of the NUCB protein or its promoter, thereby promoting NK cell expansion;
8. The method of claim 7, wherein the NK cells are NK cells positive for the surface receptor LY49H.
9. An isolated complex, characterized in that the complex is a binary complex formed by the combination of NUCB protein and LY49H.
10. The complex of claim 9, wherein the amino acid sequence of the NUCB protein is selected from the group consisting of:

    (a) has the amino acid sequence shown in SEQ ID NO.:4 or 5;

    (b) The amino acid sequence shown in SEQ ID NO.: 4 or 5 is formed by the substitution, deletion or addition of one or several (such as 1-10) amino acid residues, and the NUCB activity is composed of (a) a derived polypeptide; or

    (c) The homology between the amino acid sequence and the amino acid sequence shown in SEQ ID NO.: 4 or 5 is ≥80% (preferably ≥90%, more preferably ≥95% or ≥98%), and has the NUCB activity of polypeptides.
11. A use of the complex according to claim 9, characterized in that it is used for screening drugs or compounds that promote the expansion of NK cells.
12. A method for screening a drug or compound for promoting NK cell expansion, comprising the steps of:

    (a) In the test group, in the presence of the substance to be tested, NK cells are cultured, and a control group without the substance to be tested is set;

    (b) detecting the content H1 of the complex in the test group and comparing it with the content H0 of the complex in the control group, wherein when H1 is significantly higher than H0, it means that the test substance is a drug that promotes NK cell expansion or compound.
13. A method for treating tumors, characterized in that a therapeutically effective amount of NUCB protein or its promoter is administered to a subject in need thereof.
14. The method of claim 13, wherein the tumor is selected from the group consisting of colon cancer, breast cancer, lung cancer, gastric cancer, liver cancer, multiple myeloma, renal cancer, pancreatic cancer, melanoma, lymphoma, Thyroid cancer, or a combination thereof.
15. A use of LY49H protein or an active fragment thereof, characterized in that it is used to prepare a pharmaceutical composition for treating diseases related to excessive NUCB protein.
16. The method of claim 15, wherein the disease associated with excessive NUCB protein is selected from the group consisting of polycystic ovary syndrome (Polycystic Ovary Syndrome), hypertension (Hypertention), anxiety (Anxiety), Epilepsy or a combination thereof.

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