CN114686429A

CN114686429A - Tumor immunotherapy method for activating NK cells

Info

Publication number: CN114686429A
Application number: CN202011629015.6A
Authority: CN
Inventors: 周云夫; 李林
Original assignee: Center for Excellence in Molecular Cell Science of CAS
Current assignee: Center for Excellence in Molecular Cell Science of CAS
Priority date: 2020-12-31
Filing date: 2020-12-31
Publication date: 2022-07-01
Anticipated expiration: 2040-12-31
Also published as: CN114686429B; WO2022143938A1

Abstract

The invention provides a tumor immunotherapy method for activating NK cells. The invention provides application of Nucleindin (NUCB) or a promoter thereof in preparing a medicament or a preparation, wherein the medicament or the preparation (a) is used for promoting the expansion of NK cells, (b) is used for treating tumors; and/or (c) killing or inhibiting tumor cells. The NK cells are activated by the method, the obtained NK cells have a broad-spectrum anti-tumor effect, tumor specific antigens do not need to be identified, and the immune system is quick in starting time.

Description

Tumor immunotherapy method for activating NK cells

Technical Field

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

Background

With the progress of society and the development of science and technology, the emergence of antibiotics and other medicines seriously threaten human diseases such as infectious diseases in the last century and before, and the harm of the antibiotics and other medicines is remarkably reduced. However, chronic diseases such as cancer, cardiovascular diseases, neurodegenerative diseases and metabolic diseases become major health threats facing human society at present. 1810 million cancer patients are newly added in 2018 all over the world, and 960 million people die of the cancer. Cancer seriously affects human health and life quality and brings great economic pressure to society.

Tumor immunotherapy is undoubtedly the most important step in tumor treatment in recent decades, and obtains the nobel physiological and medical prize in 2018, so that the tumor immunotherapy is known as the third anticancer drug revolution after chemotherapy and targeted therapy. Most tumor immunotherapy approaches, whether they are immunodetection site suppression, chimeric antigen T cells or bispecific antibodies, focus on enhancing T cell activity. Although these methods have met with great success, only a small percentage of patients with cancer benefit from them.

Natural killer cells (NK cells), one of the major members of the innate immune system, play a critical role in the surveillance of body immunity and in early anti-infection and tumor processes. NK cells directly kill tumor cells by releasing lytic granules containing perforin and granzyme, and can indirectly play a role in recruiting and regulating other innate immunity and acquired immunity systems by secreting cytokines and chemokines such as IFN-gamma, TNF-alpha, GM-CSF, and the like. These functions are achieved through activating or inhibitory receptors for various germline codes (germline-encoded) on the surface of NK cells. However, to date, many of these receptors have not found corresponding ligands, which are detrimental to the development of immunotherapeutic approaches associated with activating NK cells.

In view of the above, there is an urgent need in the art to develop a novel method for activating NK cells efficiently and rapidly.

Disclosure of Invention

The invention aims to provide a novel method for efficiently and quickly activating NK cells.

In a first aspect of the present invention, there is provided a use of a nuclear Nexin (NUCB) or a promoter thereof for the manufacture of a medicament or formulation (a) for promoting expansion of NK cells, (b) treating a tumor; and/or (c) killing or inhibiting tumor cells.

In another preferred embodiment, the drug or formulation comprises an antineoplastic agent.

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

In another preferred embodiment, the NUCB protein is derived from a mammal, preferably, from a rodent (rat, mouse) and a primate (e.g., human), more preferably, from a human.

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

In another preferred example, the amino acid sequence of the NUCB1 protein is shown in SEQ ID No.:3 or the identity of the NUCB1 protein and the SEQ ID No.:3 is more than or equal to 85% (preferably more than or equal to 90%, more preferably more than or equal to 95% or more than or equal to 98%).

In another preferred example, the amino acid sequence of the NUCB2 protein is shown in SEQ ID NO. 4 or the identity of the NUCB2 protein and the SEQ ID NO. 4 is more than or equal to 85% (preferably more than or equal to 90%, more preferably more than or equal to 95% or more than or equal to 98%).

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

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

In another preferred embodiment, the NUCB protein specifically binds to NK cell surface receptor LY 49H.

In another preferred embodiment, the NUCB protein comprises a full-length NUCB protein, a mature NUCB protein, a critical segment of NUCB that binds to LY49H, or an active fragment of NUCB comprising the critical segment.

In another preferred example, the key segment of the NUCB protein is the amino acid sequence from positions 40-85 of NUCB1 or NUCB 2.

In another preferred example, the sequence of the key segment of the NUCB1 is shown in SEQ ID No. 1.

In another preferred example, the sequence of the key segment of the NUCB2 is shown in SEQ ID No. 2.

In another preferred example, the NK cell is a NK cell positive for surface receptor LY 49H.

In another preferred embodiment, the promoter comprises a promoting miRNA, a promoting transcription regulatory factor, or a promoting targeting 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 formulation is administered by a mode of administration selected from the group consisting of: intravenous, intratumoral, intracavitary, subcutaneous or hepatic arterial administration (e.g., injection, instillation, etc.).

In another preferred embodiment, the formulation is selected from the group consisting of: tablet, capsule, injection, granule, spray, and lyophilized preparation.

In another preferred embodiment, the formulation is an injection.

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

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

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

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

In a third aspect of the invention, there is provided an isolated complex which is a binary complex of a NUCB protein bound to LY 49H.

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

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

(b) a polypeptide which is formed by substituting, deleting or adding one or more (such as 1-10) amino acid residues of the amino acid sequence shown in SEQ ID NO. 4 or 5, has the NUCB activity and is derived from the (a); or

(c) The homology of the amino acid sequence and the amino acid sequence shown in SEQ ID NO. 4 or 5 is more than or equal to 80 percent (preferably more than or equal to 90 percent, more preferably more than or equal to 95 percent or more than or equal to 98 percent), and the polypeptide has NUCB activity.

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

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

(b) (ii) a polypeptide derived from (i) having the activity of LY49H, which is formed by substituting, deleting or adding one or more (e.g., 1-10) amino acid residues to the amino acid sequence shown in SEQ ID No. 8; or

(c) A polypeptide having the activity of LY49H, wherein the homology of the amino acid sequence with the amino acid sequence shown in SEQ ID NO. 8 is more than or equal to 80% (preferably more than or equal to 90%, more preferably more than or equal to 95% or more than or equal to 98%).

In a fourth aspect of the invention, there is provided a use of the complex according to the third aspect of the invention for screening a drug or a compound that promotes NK cell expansion.

In another preferred embodiment, the NK cell is preferably a NK cell positive for surface receptor LY 49H.

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

(a) culturing NK cells in the presence of a test substance in a test group, and setting a control group without the test substance;

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

In another preferred embodiment, the significantly higher than average is H1/H0 ≧ 2, preferably ≧ 3, more preferably ≧ 4.

In a sixth aspect of the invention, a method of treating a tumor is provided by administering to a subject in need thereof a therapeutically effective amount of a NUCB protein or enhancer thereof.

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

In another preferred embodiment, the non-human mammal includes a rodent (e.g., mouse, rat, rabbit), primate (e.g., monkey).

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

In another preferred embodiment, the NK cell naturally contains a receptor for NUCB or a surface receptor for NUCB obtained by artificial modification.

In a seventh aspect of the invention, there is provided the use of a LY49H protein, or an active fragment thereof, for the manufacture of a pharmaceutical composition for the treatment of a disease associated with NUCB protein hyperactivity.

In another preferred embodiment, the sequence of LY49H protein or an active fragment thereof is selected from the group consisting of:

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

(b) a polypeptide derived from (a) having the activity of LY49H, which is formed by substituting, deleting or adding one or more (e.g., 1-10) amino acid residues to the amino acid sequence shown in SEQ ID No. 9; or

(c) A polypeptide having the activity of LY49H, wherein the homology of the amino acid sequence with the amino acid sequence shown in SEQ ID NO. 9 is more than or equal to 80% (preferably more than or equal to 90%, more preferably more than or equal to 95% or more than or equal to 98%).

In another preferred example, the LY49H protein or active fragment thereof is the extracellular domain of LY49H (SEQ ID No.: 9).

In another preferred embodiment, the disease associated with NUCB protein hyperactivity is selected from the group consisting of: polycystic Ovary Syndrome (Polycystic Ovary Syndrome), hypertension (hypertension), Anxiety (Anxiety), Epilepsy (Epilepsy), or combinations thereof.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

Figure 1 shows that NUCBs specifically bind to LY 49H. LY49H molecule and NUCBs molecule structure pattern map (SS is signal sequence), EF is EF-hand structural domain; B. interaction method pattern (secretion into cell culture medium after co-transfection of LY49 family plasmid and NUCBs expression plasmid into 293T cells); results of co-immunoprecipitation experiments with LYCB 1 protein and LYCB 1 protein, which specifically binds to LY49H in the LY49 family; results of co-immunoprecipitation experiments of LYCB 2 protein and LYCB 2 protein specifically binds to LY49H in the LY49 family; E. LY49H binds to eukaryotic 293T cells expressing purified NUCBs protein; LY49H combined with Escherichia coli to express purified NUCBs protein (wherein lp: Fc is co-immunoprecipitation Fc tag, Input is Input amount of two proteins in the co-immunoprecipitation experiment, HA is HA tag, FC is FC tag, and Flag is Flag tag).

FIG. 2 shows that NUCBs specifically bind to the C-type lectin-like domain (C-type lectin-like domain) of LY 49H. A cutting pattern diagram of LY49H extracellular segment; the C-type lectin-like domain of ly49h is essential for binding NUCB 1; the C-type lectin-like domain of ly49h is essential for binding NUCB 2; aligning sequences of LY49 family C-type lectin-like domains; a tree of guides related to sequences of the LY49 family C-type lectin-like domains; leucine residue 196 and histidine residue 216 of the C-type lectin-like domain of g.ly49h are key amino acids determining their binding specificity to NUCB1/NUCB 2; pattern of binding sites on LY49H for nucbs, wherein CTLD is a C-type lectin-like domain.

FIG. 3 shows amino acids 40-80 of NUCBs binding to LY 49H. A C-terminal truncation pattern map of nucbs protein; the 3C-terminal truncated forms of nucbs did not affect binding to LY 49H; n-terminal truncation pattern maps of nucbs proteins; significant effects of NUCB1 (absence 44-80) and NUCB2 (absence 44-80) were associated with LY 49H.

Figure 4 shows that over-expression of NUCBs inhibits growth of tumor cells in mice. A. Constructing a cell pattern map of over-expression MC38 of the NUCBs protein; B. spreading the same number of cells, collecting the culture medium after 24 hours, and detecting the over-expression of NUCBs by Western Blot; overexpression of the nucbs protein did not affect proliferation of MC38 cells themselves; c57 mouse model images were inoculated subcutaneously with wild type cells of mc38 and cells overexpressing MC38, NUCB1 or NUCB 2; obviously reduced subcutaneous tumor growth in mice of MC38 cells over-expressing NUCB1 and NUCB 2; mice transplanted with MC38 cells over-expressing NUCB1 and NUCB2 had significantly prolonged survival.

FIG. 5 shows that injection of NUCBs peptide fragments inhibits tumor cell growth. A-C.NUCB1 and NUCB2 protein sequence information, synthetic NUCBs peptide sequence information and contrast peptide sequence information; D. experimental pattern diagram of peptide fragment injection of NUCBs; E. the injection of the NUCB1 peptide fragment or the NUCB2 peptide fragment can obviously reduce the size of the tumor; F. comparison information of human and mouse sequences of NUCB peptide fragments.

Figure 6 shows the identification of NUCBs knockout mice. A, B, NUCB1 gene knockout strategy and sequencing identification; identification of C, d.nucb1 knockout mouse genomic PCR and mRNA expression (lung tissue); e, F, NUCB2 gene knockout strategy and sequencing identification; g, H.NUCB2 gene knockout genome PCR identification and WB identification. Wherein marker is a control, and actin is actin.

Figure 7 shows that NUCBs knockout mice promote tumor growth. The NUCB1&2 gene knockout male mouse promotes the growth of tumors, shortens the life cycle of the mouse, but the single knockout gene male mouse has no change; D-F. NUCB1&2 knockout females promoted tumor growth, reducing survival, but single knockout females did not change (WT: wild type; KO: knockout).

Figure 8 shows that NUCBs promote expansion of LY49H positive NK cells. A. A circle gate procedure for flow cytometric analysis of LY49H positive NK cells; overexpression of NUCB1/2 reduces the tumor size and promotes the amplification of mouse spleen LY49H positive NK cells (2 of 7 NUCB1/2 overexpression tumors are NUCB1 overexpression tumors, and 5 tumors are NUCB2 overexpression tumors); the knockout of NUCB1&2 gene increases the weight of tumor, reduces the number of mouse spleen LY49H positive NK cells; D. intraperitoneal injection of NUCB2 peptide reduces tumor volume and increases the number of LY49H positive NK cells (LY49H positive NK cells (%) is the ratio of CD45+ CD3 cells of spleen cells, as shown in A); NuCBs promote LY49H positive NK cell proliferation pattern.

Detailed Description

The present inventors have extensively and intensively studied and, for the first time, have unexpectedly found that a Nucleoprotein (NUCB) can bind to LY49H, thereby effectively promoting NK cell expansion (or proliferation). Specifically, experiments such as knockout of NUCB genes in mice, over-expression of NUCB proteins, injection of key peptide fragments and in-vitro protein binding prove that secretory proteins NUCB1 and NUCB2 are physiological ligands of an NK cell surface receptor LY49H, and a binary complex formed by the NUCB proteins and LY49H promotes the amplification of LY49H receptor positive NK cells. Therefore, the Nucleindin (NUCB) and the promoter thereof can be used for preparing a medicine or a preparation for treating tumors and promoting the expansion of NK cells. The present invention has been completed on the basis of this finding.

Term(s) for

In the present invention, "the ribonucleoproteins", "NUCBs proteins", "NUCB 1 and NUCB2 proteins" of the present invention are used interchangeably and refer to secreted proteins that bind to the NK cell surface receptor LY49H to promote NK cell expansion.

Natural killer cell (NK cell)

NK cells, one of the major members of the innate immune system, play a critical role in immune surveillance in the body and in 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 showed that NK cell activity was negatively correlated with carcinogenesis, and infiltration of NK cells in some tumor microenvironments was positively correlated with patient survival. Unlike T cells, NK cells possess certain advantages. Firstly, NK cells belong to the innate immune system, and walk at the forefront of immune response, almost all tumor cells or tumor cells with recurrent metastasis are attacked by the NK cells preferentially, so that the anti-tumor effect of broad spectrum is achieved; secondly, tumor specific antigen recognition or amplification is not required, and the immune system is started quickly; third, security is better and migration restrictions are less.

NK cells directly kill tumor cells by releasing lytic granules containing perforin and granzyme, and can indirectly play a role in recruiting and regulating other innate immunity and acquired immunity systems by secreting cytokines and chemokines such as IFN-gamma, TNF-alpha, GM-CSF, and the like. These functions are achieved through activating or inhibitory receptors for various germline codes (germline-encoded) on the surface of NK cells. However, many of these receptors have not found corresponding ligands to date.

Nuclear Nexin (NUCB)

NUCBs (nucleobin) are a family of calcium ion-binding secreted proteins, including NUCB1 and NUCB 2. The function of NUCB2 protein was first reported to suppress appetite and reduce body weight in 2006, but later studies showed that NUCB2 was not functional in appetite and body weight regulation under physiological conditions. The biological function of NUCB1 is not known to date. The biological functions and mechanisms of the NUCBs family are not clear. The NUCB1 and NUCB2 proteins are conserved in evolution and have high homology, such as 87.4% similarity of human and rat NUCB2 proteins and 95.7% similarity of rat and mouse NUCB2 proteins.

Expression of NUCBs protein occurs in a variety of tissues and cells. Under the conditions of stress or inflammation and the like, the expression of the NUCBs protein is induced. It is currently believed that cells of tumors and microenvironments induce NUCBs expression under a variety of stress conditions, including in tumor conditions, to activate NK cells to treat the stress conditions.

It will be appreciated that an optional tag sequence (e.g., a 6His tag for ease of purification) may also be present at the N-terminus or C-terminus of the NUCB protein of the invention. Furthermore, the NUCB proteins of the invention may include wild-type and mutant forms, wherein the mutant form retains at least 30%, preferably at least 50%, of the corresponding biological activity of the wild-type form.

It is to be understood that although the NUCB protein of the invention is derived from a mouse, other proteins from other animals (e.g., humans) that are highly homologous (e.g., have greater than 85%, such as 85%, 90%, 95%, or even 98% sequence identity) to the mouse NUCB protein are also within the contemplation of the invention. For example, human-derived NUCB1, which shares 89% similarity with murine-derived NUCB1 (without signal peptide), and human-derived NUCB2, which shares 87% similarity with murine-derived NUCB2 (without signal peptide). Methods and means for aligning sequence identity are also well known in the art, for example BLAST.

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

mouse NUCB1(SEQ ID NO: 4)

MPTSVPRGAPFLLLPPLLMLSAVLAVPVDRAAPPQEDSQATETPDTGLYYH RYLQEVINVLETDGHFREKLQAANAEDIKSGKLSQELDFVSHNVRTKLDELKRQ EVSRLRMLLKAKMDAKQEPNLQVDHMNLLKQFEHLDPQNQHTFEARDLELLI QTATRDLAQYDAAHHEEFKRYEMLKEHERRRYLESLGEEQRKEAERKLQEQQ RRHREHPKVNVPGSQAQLKEVWEELDGLDPNRFNPKTFFILHDINSDGVLDEQE LEALFTKELEKVYDPKNEEDDMREMEEERLRMREHVMKNVDTNQDRLVTLEE FLASTQRKEFGDTGEGWKTVEMSPAYTEEELKRFEEELAAREAELNARAQRLS QETEALGRSQDRLEAQKRELQQAVLQMEQRKQQLQEQSAPPSKPDGQLQFRA DTDDAPVPAPAGDQKDVPASEKKVPEQPPELPQLDSQHL

Mouse NUCB2(SEQ ID NO: 5)

MRWRIIQVQYCFLLVPCMLTALEAVPIDVDKTKVHNTEPVENARIEPPD TGLYYDEYLKQVIEVLETDPHFREKLQKADIEEIRSGRLSQELDLVSHKVRTR LDELKRQEVGRLRMLIKAKLDALQDTGMNHHLLLKQFEHLNHQNPNTFESR DLDMLIKAATADLEQYDRTRHEEFKKYEMMKEHERREYLKTLSEEKRKEEE SKFEEMKRKHEDHPKVNHPGSKDQLKEVWEETDGLDPNDFDPKTFFKLHDV NNDGFLDEQELEALFTRELEKVYNPQNAEDDMIEMEEERLRMREHVMSEID NNKDRLVTLEEFLRATEKKEFLEPDSWETLDQQQLFTEDELKEYESIIAIQEN ELKKRAEELQKQKEDLQRQHDHLEAQKQEYHQAVQHLEQKKLQQGIAPSG PAGELKFEPHT

Human NUCB1(SEQ ID NO: 6)

MPPSGPRGTLLLLPLLLLLLLRAVLAVPLERGAPNKEETPATESPDTGLY YHRYLQEVIDVLETDGHFREKLQAANAEDIKSGKLSRELDFVSHHVRTKLDE LKRQEVSRLRMLLKAKMDAEQDPNVQVDHLNLLKQFEHLDPQNQHTFEAR DLELLIQTATRDLAQYDAAHHEEFKRYEMLKEHERRRYLESLGEEQRKEAE RKLEEQQRRHREHPKVNVPGSQAQLKEVWEELDGLDPNRFNPKTFFILHDIN SDGVLDEQELEALFTKELEKVYDPKNEEDDMREMEEERLRMREHVMKNVD TNQDRLVTLEEFLASTQRKEFGDTGEGWETVEMHPAYTEEELRRFEEELAAR EAELNAKAQRLSQETEALGRSQGRLEAQKRELQQAVLHMEQRKQQQQQQQ GHKAPAAHPEGQLKFHPDTDDVPVPAPAGDQKEVDTSEKKLLERLPEVEVP QHL

Human NUCB2(SEQ ID NO: 7)

MRWRTILLQYCFLLITCLLTALEAVPIDIDKTKVQNIHPVESAKIEPPDTG LYYDEYLKQVIDVLETDKHFREKLQKADIEEIKSGRLSKELDLVSHHVRTKL DELKRQEVGRLRMLIKAKLDSLQDIGMDHQALLKQFDHLNHLNPDKFESTD LDMLIKAATSDLEHYDKTRHEEFKKYEMMKEHERREYLKTLNEEKRKEEES KFEEMKKKHENHPKVNHPGSKDQLKEVWEETDGLDPNDFDPKTFFKLHDV NSDGFLDEQELEALFTKELEKVYDPKNEEDDMVEMEEERLRMREHVMNEV DTNKDRLVTLEEFLKATEKKEFLEPDSWETLDQQQFFTEEELKEYENIIALQE NELKKKADELQKQKEELQRQHDQLEAQKLEYHQVIQQMEQKKLQQGIPPSG PAGELKFEPHI

Furthermore, the term "protein derived from NUCB" also includes the amino acid sequence of SEQ ID NO:4 or 5. These variants include (but are not limited to): deletion, insertion and/or substitution of 1 to 3 (usually 1 to 2, more preferably 1) amino acids, and addition or deletion of one or several (usually up to 3, preferably up to 2, more preferably up to 1) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition or deletion of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the structure and function of the protein. In addition, the term also includes monomeric and multimeric forms of the polypeptides of the invention. The term also includes linear as well as non-linear polypeptides (e.g., cyclic peptides).

The NUCB-acting derivative proteins of the invention also include active fragments, derivatives and analogs thereof. As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that substantially retains the function or activity of binding to LY 49H. The polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, are substituted, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which a NUCB protein or a derived protein thereof is fused to another compound, such as a compound that extends the half-life of the polypeptide, e.g., polyethylene glycol, or (iv) a polypeptide in which an additional amino acid sequence is fused to the polypeptide sequence (fused to a leader sequence, a secretory sequence or a tag sequence such as 6 His). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.

A preferred class of reactive derivatives refers to polypeptides formed by the replacement of at most 3, preferably at most 2, more preferably at most 1 amino acid with an amino acid of similar or analogous nature as compared to the amino acid sequence shown in SEQ ID No. 4 or 5. These conservative variants are preferably produced by amino acid substitutions according to Table A.

TABLE A

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

The invention also provides analogs of the NUCB protein. The analogs may differ from the polypeptide set forth in SEQ ID NO. 4 or 5 by amino acid sequence differences, by modifications that do not affect the sequence, or by both. Analogs also include analogs having residues other than the natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

Modified (generally without altering primary structure) forms include: chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, 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 modification may be accomplished by exposing the polypeptide to an enzyme that effects glycosylation, such as a mammalian glycosylase or deglycosylase. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to increase their resistance to proteolysis or to optimize solubility.

LY49 receptor family

The LY49 receptor family is one of mouse NK cell receptors, can transmit activation or inhibitory signals and regulate NK cell killing effect. The 10 members of the LY49 receptor family proteins are LY49A, LY49B, LY49C, LY49D, LY49E, LY49F, LY49G, LY49H, LY49I, LY 49Q. Among them, a 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, which includes 8 inhibitory receptors: KIR2DL1-5, KIR3DL1-3, and 6 activating receptors, KIR2DS1-5, KIR3DS1, of which many are not currently found to be ligands.

In the present invention, the extracellular end of receptor LY49H was made into 5 truncated forms of plasmids, and the binding of these fragments to NUCBs revealed that the C-type lectin-like domain (CTLD, amino acid 266 at 143-. Alignment of the amino acid sequences of the C-type lectin-like domains (C-tpye lectins-like domains) of the LY49 family of molecules revealed that 3 amino acid residues of LY49H were unique to LY49H, specifically L196, H216 and T232, compared to the most similar amino acid residues of LY49F, LY49C and LY 49I.

In a preferred embodiment of the invention, the amino acid sequence of LY49H is as set forth in SEQ ID NO: 8:

MSEQEVTFPTMRFHKSSGLNSQVRLEGTQRSRKAGLRVCSVPWQLIVIALGI LCSLRLVIVAVFVTKFFQYSQHKQEINETLNHRHNCSNMQRDFNLKEEMLTN KSIDCRPSYELLEYIKREQERWDSETKSVSDSSRDTGRGVKYWFCYGTKCYYFIMNKTTWSGCKANCQHYSVPIVKIEDEDELKFLQRHVILESYWIGLSYDKKKKEWAWIHNGQ SKLDMKIKKMNFTSRGCVFLSKARIEDTDCNTPYYCICGKKLDKFPD

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

The extracellular end peptide fragment of LY49H is SEQ ID NO:9 (fragments 67-266 of SEQ ID NO: 8): KFFQYSQHKQEINETLNHRHNCSNMQRDFNLKEEMLTNKSIDCRPSYELLEY IKREQERWDSETKSVSDSSRDTGRGVKYWFCYGTKCYYFIMNKTTWSGCKA NCQHYSVPIVKIEDEDELKFLQRHVILESYWIGLSYDKKKKEWAWIHNGQSK LDMKIKKMNFTSRGCVFLSKARIEDTDCNTPYYCICGKKLDKFPD

Accelerator and formulation

By utilizing the protein of the invention, substances, particularly promoters and the like, which interact with the NUCB protein can be screened out by various conventional screening methods.

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

Generally, the NUCB protein or enhancer of the invention will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, typically having a pH of from about 5 to about 8, preferably a pH of from about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated preparation or pharmaceutical composition may be administered by conventional routes including, but not limited to: intratumoral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration.

The invention also provides a pharmaceutical preparation, which contains the NUCB protein or the promoter thereof with safe and effective dose and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical preparation of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The formulation of the present invention is selected from the group consisting of: tablet, capsule, injection, granule, spray, and lyophilized preparation. The amount of active ingredient administered is a therapeutically effective amount, for example, from about 1 microgram to 10 milligrams per kilogram of body weight per day. The medicament or the preparation is administered by a mode of administration selected from the group consisting of: intravenous, intratumoral, intracavitary, subcutaneous or hepatic arterial administration (e.g., injection, instillation, etc.).

The invention also provides a pharmaceutical composition containing a safe and effective amount of LY49H protein or its active fragment of the invention and a pharmaceutically acceptable carrier or excipient, for the preparation of a medicament for treating diseases associated with NUCB protein hyperactivity. The diseases related to the NUCB protein overhigh are selected from the following groups: polycystic Ovary Syndrome (Polycystic Ovary Syndrome), hypertension (hypertensition), Anxiety (Anxiety), Epilepsy (epileysy), or combinations 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 vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical preparation should be matched with the administration mode, and the pharmaceutical composition of the invention is in the dosage form of injection, freeze-dried preparation, stem cell preparation and aerosol inhalation preparation. For example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions.

Composite material

In the present invention, a complex is provided which is a binary complex of the NUCB protein bound to LY 49H.

The complex of the present invention can be used for screening drugs or compounds that promote the expansion of NK cells, wherein a substance that promotes the formation of the complex has the potential to promote the expansion of NK cells.

Method for promoting NK cell expansion

The invention provides a method for promoting NK cell amplification in vitro or in vivo and NK cell long-term existence in vivo, which comprises the following steps:

(1) in vitro: providing a NUCB protein or promoter thereof; in the presence of the NUCB protein or the promoter thereof, the NK cells containing the NUCB protein or the promoter thereof are cultured in a mixed way under proper culture conditions, so that the NK cells are promoted to be massively expanded.

(2) In vivo: providing a NUCB protein or promoter thereof; after certain functional NK cells are infused in vivo (the NK cells can naturally have receptors of NUCB proteins or are artificially modified to contain the receptors of the NUCB proteins), the NUCB proteins or the promoters thereof are further administered in vivo to promote the expansion and mass long-term existence of the infused NK cells in vivo; enhancing the therapeutic efficacy of infused NK cells.

The main advantages of the invention include:

(1) the invention discovers that secretory proteins NUCB1 and NUCB2 are physiological ligands of an NK cell surface receptor LY49H for the first time.

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

(3) The NK cells are activated by the method, the obtained NK cells have a broad-spectrum anti-tumor effect, tumor specific antigens do not need to be identified, and the immune system is quick in starting time.

The invention is further illustrated by the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specifying the detailed conditions in the following examples, generally followed by conventional conditions such as Sambrook et al, molecular cloning: conditions described in a Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

Universal materials and methods

1. Cloning of genes

Mouse NUCB1, cloning the full length or fragment of NUCB2 gene to pCMV-HA eukaryotic expression vector and pLVX-DsRed-3 Flag-HA-T2A-Puro slow virus packaging vector, cloning the full length or fragment of LY49 family gene to pFUSE-hIgG1-Fc2 vector. Point mutation of the gene is accomplished by corresponding site-directed mutagenesis primers.

2. Cell culture

HEK 293T, MC38 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37 ℃ with a CO2 concentration of 5%. Cells were plated 18-24 hours prior to transfection with a total plasmid amount of 2. mu.g/well in 6-well plates and LacZ for complementation when the transfection plasmid amount was insufficient. The transfection reagent was Lipo3000 (Invitrogen). Corresponding experiments were performed 24-30 hours after transfection.

3. Co-immunoprecipitation

The 293T cell cotransfected NUCB1 or NUCB2 and LY49 family plasmid culture solution is collected, centrifuged at 12000rpm for 10 minutes, the supernatant plus protein A/G beads is taken, rotated in a cold storage at 4 ℃ for 2 hours to immunoprecipitate FC-labeled protein, the supernatant is removed after washing for 3 times, added with 40 uL of 2-loading and boiled for 10 minutes, and stored at-20 ℃.

4.Western Blot

SDS-PAGE gel with the concentration of 10% is prepared, and protein samples are added for electrophoretic separation. Proteins were transferred to NC (nitrocellulose) membranes in the gel and blocked with 0.5% skim milk for 1 hour. TBST was washed 3 times for 5 minutes each, and the corresponding primary antibody was added and incubated at room temperature for 2 hours. TBST was washed 3 times for 5 min each, and the corresponding HRP-conjugated secondary antibody was added and incubated for 1 hour at room temperature. After three washes, add developer for scanning. NUCB2(N6789) antibody was purchased from Sigma Aldrich, HA (16B12) antibody was purchased from Covance, Flag antibody, His antibody was purchased from Sigma Aldrich, and HRP-conjugated Goat anti-human IgG antibody was purchased from Sangon Biotech (Shanghai).

5. Extraction of tissue RNA and Q-PCR

After euthanizing, the mice were quickly stripped of the corresponding tissue and placed in a homogenizer tube containing steel balls and Trizol. After the RNA is crushed, the RNA is extracted by a phenol-chloroform-isopropanol method, the RNA is reversely transcribed into cDNA by utilizing a superscript TMIII first strand synthesis system (Invitrogen) kit, and Quantitative PCR reaction is carried out by utilizing a Quantitative SYBR green PCR kit (TaKara) kit and an ABI-QuantStudio 6 reaction PCR instrument.

6. Mouse

C57BL/6 mice were purchased from Ling Chang, and were housed in an SPF-rated animal house at Biochemical institute, with lights turned off (19:00-07:00) for 12 hours and turned on (07:00-19:00) for 12 hours. Single-gene knockout mice NUCB1 and NUCB2 and double-knockout mice NUCB1 and NUCB2 are constructed and obtained by a CRISPR-Cas9 method, and are mated with purchased wild mice for 5 generations to purify background, and then the background is used for propagation and amplification to obtain wild mice for experiments and corresponding gene knockout mice. All mice used for the experiment were 7-11 weeks old.

Construction of a monoclonal Strain overexpressing NUCB1 or NUCB2 in MC38 cells

The full length of mouse NUCB1 and NUCB2 genes (containing signal peptide) is obtained by PCR amplification, cloned to pLVX-DsRed-3 × Flag-HA-T2A-Puro virus packaging vector, and transfected to 293T cell packaging lentivirus together with packaging plasmids pSPAX2 and pMD 2G. The obtained virus infects MC38 cells, and single clone is selected to obtain MC38 cells stably and highly expressing NUCB1 or NUCB 2.

8. Cell proliferation assay

Proliferation assays of wild-type cells of MC38 and MC38 overexpressing NUCB1 or NUCB2 cells were performed using the CellTiter-Glo fluorescent cell viability assay (Promega) kit, according to the instructions.

9. Subcutaneous transplantation tumor model

Culturing digested wild type MC38 cells or NUCB1, NUCB2 over-expressing MC38 cells, washing with PBS 3 times, filtering with 100um filter, and adjusting count to 5 × 10⁶Per ml cells, 100ul (5X 10 in content) were inoculated subcutaneously⁵Cells), the length and width of the tumor was measured using a vernier caliper after the corresponding time, and the tumor volume was calculated by the formula: volume length width 2. When the tumor volume is larger than 2000mm in survival statistics³Mice were euthanized, and defined as dead.

10. Polypeptide synthesis

The peptide segment of NUCB1 is amino acids 45-81 of NUCB1 protein, the peptide segment of NUCB2 is amino acids 49-85 of NUCB2 protein, and the comparison peptide segment is consistent with the amino acids of the peptide segment (49-85) of NUCB2, but the sequence is randomly disordered. Synthesized by Shanghai Jier Biochemical company, the purity is more than 95 percent. The specific sequence is as follows:

NUCB1 peptide fragment:

DTGLYYHRYLQEVINVLETDGHFREKLQAANAEDIKS(SEQ ID NO:1)，

NUCB2 peptide fragment:

DTGLYYDEYLKQVIEVLETDPHFREKLQKADIEEIRS(SEQ ID NO:2)，

control peptide stretch:

TGDREKDTYYVQEPKAKLIEQDLERIFSYEEDHLLVI(SEQ ID NO:3)。

11. abdominal injection

Wild type C57 mice, 6 weeks old, were purchased, acclimated for 1 week, and then inoculated subcutaneously with MC38 cells 5 x 10⁵Tumor volume was measured 5 days later and intraperitoneal injections of 3 peptides were started, 2 times per day, 100 ug/mouse.

12. Flow cytometry

The spleens were harvested within 5 minutes after euthanasia and placed in pre-cooled DMEM without phenol red. After red blood cell lysis, 106 cells were counted, according to the protocol: dying activity (Live/Dead Fixable Near-IR Dead Cell Stain Kit, Thermo Fisher), blocking (anti-CD16/32, BioLegend), membrane antibodies (anti-CD45, Brilliant Violet 510, BioLegend; anti-CD3, Brilliant Violet 605, BioLegend; anti-NK1.1, Brilliant Violet 421, BioLegend; anti-LY49H, FITC, eBioscience), on-machine 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 either t-test (student's test) or ANOVA analysis, and significance was expressed as: p < 0.05, x: p < 0.01, x: p is less than 0.001.

Example 1 secretion proteins NUCB1 and NUCB2 specifically bind to NK cell surface receptor LY49H

By mass spectrometry the inventors found that LY49H could be a binding protein for NUCBs, cloning the extracellular domain of 10 members of the LY49 receptor family proteins (LY49A, LY49B, LY49C, LY49D, LY49E, LY49F, LY49G, LY49H, LY49I, LY49Q) in C57BL/6 mice into the expression vector PFUSE-hIgG1-Fc2(IL2ss), since this vector contains the IL2 signal peptide, and the gene expressed protein cloned into this vector will be secreted into the cell culture medium. The protein expression sequences of full-length mice NUCB1 and NUCB2 are cloned into a eukaryotic expression vector pCMV-HA at the same time, because the NUCBs gene HAs a secretion signal peptide per se and is also secreted into a cell culture medium. After the LY49 family plasmid and the NUCBs expression plasmid are transfected into 293T cells together, the plasmids are secreted into a cell culture medium (figures 1A and 1B), and the system can well detect the interaction of proteins. Cell culture media were collected 24-30 hours after co-transfection of 293T cells and co-immunoprecipitation found that both NUCB1 and NUCB2 specifically bound LY49H of the LY49 family (fig. 1C, 1D). Following purification of the NUCB1 and NUCB2 proteins expressed in eukaryotic 293T cells (fig. 1E) and prokaryotic e.coli (fig. 1F), direct interaction of NUCB1 and NUCB2 with LY49H was verified.

Experimental results indicate that NUCBs are potential ligands for LY 49H.

Example 2C-type lectin-like Domain of LY49H specific binding to NUCBs

The extracellular end of receptor LY49H was plasmid in 5 truncated forms, and binding of these fragments to NUCBs was found to be essential for binding to NUCBs (CTLD, amino acids 143-266) (FIGS. 2A-C). Alignment of the amino acid sequences of the C-type lectin-like domains of the LY49 family molecules revealed that 3 amino acid residues of LY49H compared to the most similar ones of LY49F, LY49C, LY49I were unique to LY49H, specifically L196, H216 and T232 (fig. 2D, 2E). The three amino acid sites of LY49H were subjected to point mutation to obtain amino acid residues corresponding to the similar receptors (leucine at position L196P, 196 was mutated to proline; histidine at position H216D, 216 was mutated to aspartic acid; threonine at position T232K, 232 was mutated to lysine).

The results show that L196, H216 are key amino acid residues that determine specific binding to NUCBs (fig. 2F, 2G). Thus, as shown in fig. 2H, NUCBs bind to the C-type lectin-like domain of LY49H, where leucine 196 and histidine 216 are critical amino acids.

Example 3 amino acids 40-80 of NUCBs are key segments that bind to LY49H

See fig. 3. As shown in fig. 3A, tapering truncations of NUCBs from the C-terminus resulted in 3 truncated forms, respectively: NUCB1(1-320), NUCB1(1-230), NUCB1(1-160), NUCB2(1-320), NUCB2(1-230), NUCB2 (1-160). These truncated forms all bind well to LY49H (fig. 3B). Thus, amino acids 1-160 of NUCBs may be the only segment responsible for binding LY 49H.

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

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

The results indicate that amino acids 40-80 of NUCBs are key segments for binding to LY 49H.

Example 4 overexpression of NUCBs protein inhibits tumor cell growth in mice

By infecting MC38 cells with lentivirus, selecting single clone to obtain stable cell strain with high expression of NUCB1 or NUCB2, collecting cell culture solution, and detecting that the stable cell strain secretes NUCB1 or NUCB2 protein in large amount into the cell culture solution (FIG. 4A, B). Cell proliferation assays, overexpression of NUCB1 or NUCB2 did not affect proliferation of the cells themselves (fig. 4C). Will be 5X 10⁵Wild-type or NUCBs overexpressing strains were subcutaneously transplanted into C57 males, compared to wild-type cells.

The results are shown in fig. 4D-F, wherein the growth rate of MC38 tumors overexpressing NUCB1 or NUCB2 is significantly reduced, and the survival rate of mice is significantly prolonged.

Example 5 injection of NUCBs protein peptide fragments inhibits tumor growth in mice

Based on the amino acid sequence of the NUCBs protein specifically responsible for binding to LY49H, peptide sequences as shown in fig. 5A-C and control experiments were synthesized:

NUCB1 peptide segment (amino acids 45-81):

DTGLYYHRYLQEVINVLETDGHFREKLQAANAEDIKS(SEQ ID NO:1)，

NUCB2 peptide segment (amino acids 49-85):

DTGLYYDEYLKQVIEVLETDPHFREKLQKADIEEIRS(SEQ ID NO:2)，

control peptide stretch:

TGDREKDTYYVQEPKAKLIEQDLERIFSYEEDHLLVI(SEQ ID NO:3)。

wild type mice of C57 were all inoculated subcutaneously with 5X 10⁵And divided into 3 groups, and intraperitoneal injection of the polypeptide was started after 5 days. The control peptide fragment, the NUCB1 peptide fragment and the NUCB2 peptide fragment are respectively injected twice a day in the morning and at the evening, and the dosage is 100 ug/mouse. Tumor size was measured starting on day 5 and every 2 days.

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

Human and murine NUCBs proteins were evolutionarily highly conserved, comparing the functional peptide sequences, with 97.3% similarity of the peptide of human murine NUCB1 and 91.9% similarity of the peptide of NUCB2 (fig. 5F). The results suggest that human NUCB peptide fragments may also have potential tumor-inhibiting effects.

Example 6 construction of Single knockout mice NUCB1 and NUCB2 and double knockout mice NUCB1 and NUCB2 knockout simultaneously

The NUCB1 gene of C57BL/6 mice has 13 exons in total, of which the 2 nd to 13 th coding exons. By using a CRISPR-Cas9 method, sgRNA is designed at a position of coding a signal peptide sequence of the No. 2 exon to achieve the purpose of knockout. Sequencing confirmed that 10 bases in the signal peptide were knocked out by exon 2 of NUCB1 gene, achieving the purpose of frame shift (FIG. 6A, B). Both genomic PCR and fluorescent quantitative PCR at mRNA level confirmed knock-out of NUCB1 (fig. 6C, D).

The NUCB2 gene has 14 exons, of which exons 3 to 14 are coding exons. 2 sgRNAs are designed at the position of a signal peptide sequence coded by a No. 3 exon by using a CRISPR-Cas9 method, so that the aim of knocking out with a certain length is fulfilled. Sequencing confirms that 83 bases including part of signal peptide coding sequence are knocked out from exon 3 of NUCB2 gene, and the aim of frame shifting is also achieved (figure 6E, F). The success of the NUCB2 knockout mouse construction was determined at both the genomic and protein levels, respectively (fig. 6G, H). After single-gene editing mice NUCB1 and NUCB2 are respectively taken, the two mice are crossed to obtain double-gene editing mice, and further obtain double-gene homozygous knockout mice NUCB1 and NUCB2 (NUCB1& 2).

Example 7 NUCBs knockout mice promote tumor growth

The NUCBs family includes two members with high homology, namely NUCB1 and NUCB 2. The biochemical experiment shows that: both NUCB1 and NUCB2 can bind to LY 49H.

Mouse experiments show that: the cell over-expression of NUCB1 or NUCB2 has obvious tumor inhibition effect; the added NUCB1 peptide segment or NUCB2 peptide segment can also obviously inhibit the growth of the tumor. Using the NUCBs knockout mice obtained, they were each inoculated subcutaneously with 5X 10⁵In NUCB1 and NUCB2 knockout mice alone and NUCB1&2 double knockout mice.

The results show that: the double knockout NUCB1&2 promoted tumor growth and reduced survival, both in males (fig. 7A-C) and females (fig. 7D-F). However, knockout of NUCB1 or NUCB2 alone was not effective, indicating that two genes of the NUCBs family have compensatory effects, and silencing of one of the functions may be compensated by the other homologous gene.

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

Example 8 NUCBs protein promotes expansion of LY 49H-positive NK cells

Fig. 8A is a flow chart of a circle gate for flow cytometry analysis of LY49H positive cells. Wild type C57 males inoculated MC38 cells and NUCB1/2 overexpressing MC38 cells subcutaneously, and at day 20 the overexpressing cell tumor volume was much smaller than wild type, and spleen flow analysis was collected to find that the proportion of NK cells positive for LY49H was significantly increased in mice overexpressing NUCB1/2 (FIG. 8B). Meanwhile, comparing the tumor-loaded (MC38) wild-type female mice with the NUCB1&2 double knockout female mice at day 15, the tumor weight of the knockout mice was significantly higher than that of the wild-type mice, and flow analysis showed that the proportion of LY49H positive NK cells in the spleens of the knockout mice was significantly reduced (fig. 8C). C57 wild type male mice were inoculated subcutaneously with MC38 cells, intraperitoneally injected with NUCB2 peptide and peptide solvent PBS for 8 consecutive days starting at day 2, and the NUCB2 peptide group had significantly decreased tumor volume and increased proportion of LY49H positive NK cells in the spleen at day 14 (FIG. 8D). FIG. 8E is a graph of the proliferation pattern of LY49H positive NK cells promoted by NUCBs.

The results show that the over-expression of NUCB protein reduces the size of the tumor, and promotes the amplification of mouse spleen LY49H positive NK cells (8B); the NUCB1&2 gene knockout increased tumor weight, decreased the number of mouse spleen LY49H positive NK cells (8C); intraperitoneal injection of NUCB2 peptide reduced tumor volume and increased the number of LY 49H-positive NK cells (8D).

The positive and negative results show that an important physiological effect of the NUCBs protein after being combined with LY49H is to promote the amplification of LY49H positive NK cells and further reduce tumors.

Discussion of the preferred embodiments

NK cell effects and receptors of NUCBs in humans

NUCBs are highly conserved in human mice during evolution, and show a remarkable tumor treatment effect in mice. On the subsequent aspect, the action and mechanism of the NUCBs protein on the human NK cells are detected through the human NK cell line NK92 and the separation of primary human NK cells. Since the family of LY49 receptors in human NK cells corresponds to the KIR receptor family, including 8 inhibitory receptors, KIR2DL1-5, KIR3DL1-3, and 6 activating receptors, KIR2DS1-5, KIR3DS1, many of which have not found their ligands at present. These are the most important receptors for potential NUCBs. In a second subsequent aspect, the inventors will first validate the KIR receptor family and the NKG2C/CD94 receptor, finding the human NK cell NUCBs receptor. The receptors are used as potential targets of NK cells and have important significance on immunotherapy of tumors.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes or modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the appended claims of the present application.

Sequence listing

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Met Leu Leu Lys Ala Lys Met Asp Ala Glu Gln Asp Pro Asn Val Gln

115 120 125

Val Asp His Leu Asn Leu Leu Lys Gln Phe Glu His Leu Asp Pro Gln

130 135 140

Asn Gln His Thr Phe Glu Ala Arg Asp Leu Glu Leu Leu Ile Gln Thr

145 150 155 160

Ala Thr Arg Asp Leu Ala Gln Tyr Asp Ala Ala His His Glu Glu Phe

165 170 175

Lys Arg Tyr Glu Met Leu Lys Glu His Glu Arg Arg Arg Tyr Leu Glu

180 185 190

Ser Leu Gly Glu Glu Gln Arg Lys Glu Ala Glu Arg Lys Leu Glu Glu

195 200 205

Gln Gln Arg Arg His Arg Glu His Pro Lys Val Asn Val Pro Gly Ser

210 215 220

Gln Ala Gln Leu Lys Glu Val Trp Glu Glu Leu Asp Gly Leu Asp Pro

225 230 235 240

Asn Arg Phe Asn Pro Lys Thr Phe Phe Ile Leu His Asp Ile Asn Ser

245 250 255

Asp Gly Val Leu Asp Glu Gln Glu Leu Glu Ala Leu Phe Thr Lys Glu

260 265 270

Leu Glu Lys Val Tyr Asp Pro Lys Asn Glu Glu Asp Asp Met Arg Glu

275 280 285

Met Glu Glu Glu Arg Leu Arg Met Arg Glu His Val Met Lys Asn Val

290 295 300

Asp Thr Asn Gln Asp Arg Leu Val Thr Leu Glu Glu Phe Leu Ala Ser

305 310 315 320

Thr Gln Arg Lys Glu Phe Gly Asp Thr Gly Glu Gly Trp Glu Thr Val

325 330 335

Glu Met His Pro Ala Tyr Thr Glu Glu Glu Leu Arg Arg Phe Glu Glu

340 345 350

Glu Leu Ala Ala Arg Glu Ala Glu Leu Asn Ala Lys Ala Gln Arg Leu

355 360 365

Ser Gln Glu Thr Glu Ala Leu Gly Arg Ser Gln Gly Arg Leu Glu Ala

370 375 380

Gln Lys Arg Glu Leu Gln Gln Ala Val Leu His Met Glu Gln Arg Lys

385 390 395 400

Gln Gln Gln Gln Gln Gln Gln Gly His Lys Ala Pro Ala Ala His Pro

405 410 415

Glu Gly Gln Leu Lys Phe His Pro Asp Thr Asp Asp Val Pro Val Pro

420 425 430

Ala Pro Ala Gly Asp Gln Lys Glu Val Asp Thr Ser Glu Lys Lys Leu

435 440 445

Leu Glu Arg Leu Pro Glu Val Glu Val Pro Gln His Leu

450 455 460

<210> 7

<211> 420

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 7

Met Arg Trp Arg Thr Ile Leu Leu Gln Tyr Cys Phe Leu Leu Ile Thr

1 5 10 15

Cys Leu Leu Thr Ala Leu Glu Ala Val Pro Ile Asp Ile Asp Lys Thr

20 25 30

Lys Val Gln Asn Ile His Pro Val Glu Ser Ala Lys Ile Glu Pro Pro

35 40 45

Asp Thr Gly Leu Tyr Tyr Asp Glu Tyr Leu Lys Gln Val Ile Asp Val

50 55 60

Leu Glu Thr Asp Lys His Phe Arg Glu Lys Leu Gln Lys Ala Asp Ile

65 70 75 80

Glu Glu Ile Lys Ser Gly Arg Leu Ser Lys Glu Leu Asp Leu Val Ser

85 90 95

His His Val Arg Thr Lys Leu Asp Glu Leu Lys Arg Gln Glu Val Gly

100 105 110

Arg Leu Arg Met Leu Ile Lys Ala Lys Leu Asp Ser Leu Gln Asp Ile

115 120 125

Gly Met Asp His Gln Ala Leu Leu Lys Gln Phe Asp His Leu Asn His

130 135 140

Leu Asn Pro Asp Lys Phe Glu Ser Thr Asp Leu Asp Met Leu Ile Lys

145 150 155 160

Ala Ala Thr Ser Asp Leu Glu His Tyr Asp Lys Thr Arg His Glu Glu

165 170 175

Phe Lys Lys Tyr Glu Met Met Lys Glu His Glu Arg Arg Glu Tyr Leu

180 185 190

Lys Thr Leu Asn Glu Glu Lys Arg Lys Glu Glu Glu Ser Lys Phe Glu

195 200 205

Glu Met Lys Lys Lys His Glu Asn His Pro Lys Val Asn His Pro Gly

210 215 220

Ser Lys Asp Gln Leu Lys Glu Val Trp Glu Glu Thr Asp Gly Leu Asp

225 230 235 240

Pro Asn Asp Phe Asp Pro Lys Thr Phe Phe Lys Leu His Asp Val Asn

245 250 255

Ser Asp Gly Phe Leu Asp Glu Gln Glu Leu Glu Ala Leu Phe Thr Lys

260 265 270

Glu Leu Glu Lys Val Tyr Asp Pro Lys Asn Glu Glu Asp Asp Met Val

275 280 285

Glu Met Glu Glu Glu Arg Leu Arg Met Arg Glu His Val Met Asn Glu

290 295 300

Val Asp Thr Asn Lys Asp Arg Leu Val Thr Leu Glu Glu Phe Leu Lys

305 310 315 320

Ala Thr Glu Lys Lys Glu Phe Leu Glu Pro Asp Ser Trp Glu Thr Leu

325 330 335

Asp Gln Gln Gln Phe Phe Thr Glu Glu Glu Leu Lys Glu Tyr Glu Asn

340 345 350

Ile Ile Ala Leu Gln Glu Asn Glu Leu Lys Lys Lys Ala Asp Glu Leu

355 360 365

Gln Lys Gln Lys Glu Glu Leu Gln Arg Gln His Asp Gln Leu Glu Ala

370 375 380

Gln Lys Leu Glu Tyr His Gln Val Ile Gln Gln Met Glu Gln Lys Lys

385 390 395 400

Leu Gln Gln Gly Ile Pro Pro Ser Gly Pro Ala Gly Glu Leu Lys Phe

405 410 415

Glu Pro His Ile

420

<210> 8

<211> 266

<212> PRT

<213> mouse (Mus musculus)

<400> 8

Met Ser Glu Gln Glu Val Thr Phe Pro Thr Met Arg Phe His Lys Ser

1 5 10 15

Ser Gly Leu Asn Ser Gln Val Arg Leu Glu Gly Thr Gln Arg Ser Arg

20 25 30

Lys Ala Gly Leu Arg Val Cys Ser Val Pro Trp Gln Leu Ile Val Ile

35 40 45

Ala Leu Gly Ile Leu Cys Ser Leu Arg Leu Val Ile Val Ala Val Phe

50 55 60

Val Thr Lys Phe Phe Gln Tyr Ser Gln His Lys Gln Glu Ile Asn Glu

65 70 75 80

Thr Leu Asn His Arg His Asn Cys Ser Asn Met Gln Arg Asp Phe Asn

85 90 95

Leu Lys Glu Glu Met Leu Thr Asn Lys Ser Ile Asp Cys Arg Pro Ser

100 105 110

Tyr Glu Leu Leu Glu Tyr Ile Lys Arg Glu Gln Glu Arg Trp Asp Ser

115 120 125

Glu Thr Lys Ser Val Ser Asp Ser Ser Arg Asp Thr Gly Arg Gly Val

130 135 140

Lys Tyr Trp Phe Cys Tyr Gly Thr Lys Cys Tyr Tyr Phe Ile Met Asn

145 150 155 160

Lys Thr Thr Trp Ser Gly Cys Lys Ala Asn Cys Gln His Tyr Ser Val

165 170 175

Pro Ile Val Lys Ile Glu Asp Glu Asp Glu Leu Lys Phe Leu Gln Arg

180 185 190

His Val Ile Leu Glu Ser Tyr Trp Ile Gly Leu Ser Tyr Asp Lys Lys

195 200 205

Lys Lys Glu Trp Ala Trp Ile His Asn Gly Gln Ser Lys Leu Asp Met

210 215 220

Lys Ile Lys Lys Met Asn Phe Thr Ser Arg Gly Cys Val Phe Leu Ser

225 230 235 240

Lys Ala Arg Ile Glu Asp Thr Asp Cys Asn Thr Pro Tyr Tyr Cys Ile

245 250 255

Cys Gly Lys Lys Leu Asp Lys Phe Pro Asp

260 265

<210> 9

<211> 200

<212> PRT

<213> mouse (Mus musculus)

<400> 9

Lys Phe Phe Gln Tyr Ser Gln His Lys Gln Glu Ile Asn Glu Thr Leu

1 5 10 15

Asn His Arg His Asn Cys Ser Asn Met Gln Arg Asp Phe Asn Leu Lys

20 25 30

Glu Glu Met Leu Thr Asn Lys Ser Ile Asp Cys Arg Pro Ser Tyr Glu

35 40 45

Leu Leu Glu Tyr Ile Lys Arg Glu Gln Glu Arg Trp Asp Ser Glu Thr

50 55 60

Lys Ser Val Ser Asp Ser Ser Arg Asp Thr Gly Arg Gly Val Lys Tyr

65 70 75 80

Trp Phe Cys Tyr Gly Thr Lys Cys Tyr Tyr Phe Ile Met Asn Lys Thr

85 90 95

Thr Trp Ser Gly Cys Lys Ala Asn Cys Gln His Tyr Ser Val Pro Ile

100 105 110

Val Lys Ile Glu Asp Glu Asp Glu Leu Lys Phe Leu Gln Arg His Val

115 120 125

Ile Leu Glu Ser Tyr Trp Ile Gly Leu Ser Tyr Asp Lys Lys Lys Lys

130 135 140

Glu Trp Ala Trp Ile His Asn Gly Gln Ser Lys Leu Asp Met Lys Ile

145 150 155 160

Lys Lys Met Asn Phe Thr Ser Arg Gly Cys Val Phe Leu Ser Lys Ala

165 170 175

Arg Ile Glu Asp Thr Asp Cys Asn Thr Pro Tyr Tyr Cys Ile Cys Gly

180 185 190

Lys Lys Leu Asp Lys Phe Pro Asp

195 200

Claims

1. Use of a Nucleindin (NUCB) or a promoter thereof for the manufacture of a medicament or formulation (a) for promoting NK cell expansion, (b) for the treatment of a tumour; and/or (c) killing or inhibiting tumor cells.

2. The use of claim 1, wherein the NUCB protein comprises a NUCB1 protein and/or a NUCB2 protein.

3. The use of claim 1, wherein the NUCB protein specifically binds to NK cell surface receptor LY 49H.

4. A method for promoting NK cell expansion in vitro, comprising the steps of:

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

5. The method of claim 4, wherein said NK cell is a NK cell positive for surface receptor LY 49H.

6. An isolated complex which is a binary complex of a NUCB protein bound to LY 49H.

7. Use of a complex according to claim 6 for screening drugs or compounds that promote NK cell expansion.

8. A method for screening a drug or compound that promotes NK cell expansion, comprising the steps of:

(a) culturing NK cells in the presence of a substance to be tested in the test group, and setting a control group without the substance to be tested;

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

9. A method of treating a tumor comprising administering to a subject in need thereof a therapeutically effective amount of a NUCB protein or enhancer thereof.

10. Use of LY49H protein or an active fragment thereof for the preparation of a pharmaceutical composition for the treatment of a disease associated with NUCB hyperproteinemia.